Plant Bioactive Molecules 1-5275-1314-9, 978-1-5275-1314-3

Table of contents :
Table of Contents......Page 6
Preface......Page 13
UNIT I: Biodiversity and the Sites of Synthesis, Functional roles, Phytochemistry and Chemotaxonomy of Bioactive Plant Molecules......Page 16
1.1. Biodiversity......Page 17
1.1.1. Distribution of Biodiversity......Page 20
1.1.2. Actions to Sustain Biodiversity......Page 21
1.2. Sustainability......Page 29
1.2.1. Mineral Nutrition and Soil......Page 33
1.2.2. Pests and Pathogens......Page 34
1.2.3. Biotechnology and Sustainability......Page 35
1.2.4. Extraction of Phytochemicals......Page 39
1.2.5. Toward what future?......Page 40
1.3. Quantifying Biodiversity......Page 41
1.4.1. Taxonomy......Page 43
1.4.3. Character......Page 44
1.4.4. Data Analysis......Page 45
Suggested Reading......Page 48
2.1. Secretion......Page 50
2.2. Glandular Trichomes......Page 53
2.2.1. Glandular Trichomes of the Lamiaceae Family......Page 55
2.2.2. Glandular Trichomes of the Asteraceae Family......Page 61
2.2.3. Glandular Trichomes of the Geraniaceae F......Page 62
2.2.4. Glandular Trichomes of the Moraceae Family......Page 64
2.2.5. Glandular Trichomes of the Cannabaceae Family......Page 65
2.2.6. Glandular Trichomes of the Solanaceae Family......Page 66
2.3. Secretory Cavities and Resin Ducts......Page 68
2.4. Lysigenous Cavities......Page 70
2.5. Oil-bearing Cells and Secretory Cells associated with Bacteria......Page 72
2.6. Laticifers......Page 74
Suggested Reading......Page 76
3.1. Primary and Secondary Metabolites......Page 81
3.2. Phenotypic Plasticity......Page 85
3.2.1. Chemical Defence in Prehistory......Page 87
3.2.2. Chemical Ecology......Page 88
3.2.3. Coevolution......Page 89
3.2.4. Constitutive Chemical Defence......Page 96
3.2.5. Induced Chemical Defence......Page 99
3.2.6. Theories on Defence from Herbivores......Page 117
3.2.7. Allelopathy......Page 120
3.2.8. Chemical Defence from Microorganisms......Page 126
3.3.1. Plant Defence from Ultraviolet Radiation......Page 129
Suggested Reading......Page 130
Chapter Four......Page 140
4.1. Dietary and Food Supplements......Page 141
4.1.1. Functional Foods......Page 143
4.2. Plant Bioactive Molecules and the Treatment of Diseases......Page 147
4.2.1. Interaction between Bioactive Plant Molecules and Drugs......Page 149
4.2.2. Herbal Regulatory......Page 167
4.2.3. Ethnofarmacognosy......Page 172
4.3.1. Effect on Cell Division......Page 173
4.3.2. Effect of Plant Bioactive Molecules on Cell Membranes, Channels and Receptors......Page 0
4.3.3. Immunomodulatory Effect of Plant Bioactive Molecules......Page 190
4.3.4. Toxic Effect of Plant Bioactive Molecules......Page 194
4.3.5. Plant Bioactive Molecules against Uropatogenic Escherichia Coli......Page 210
4.3.6. Plant Bioactive Molecules for Brain and Mental Disorders......Page 212
Suggested Reading......Page 215
5.1. Overview on Chemotaxonomy......Page 224
5.2. Chemotaxonomy of Phenolic Compounds......Page 225
5.2.1. Asteraceae......Page 226
5.2.2. Lamiaceae......Page 227
5.2.3. Leguminosae......Page 228
5.2.4. Other Plant Families......Page 229
5.3.1 Monoterpenes......Page 231
5.3.2. Sesquiterpenes......Page 233
5.3.3. Diterpenes......Page 236
5.3.4. Triterpenes......Page 237
5.3.5. Tetraterpenes......Page 239
5.4.1. Alkaloids......Page 241
5.4.3. Cyanogenic Glycosides......Page 244
5.5. Chemotaxonomic Significance of Fatty Acids......Page 245
5.7. Correlation between Micromolecular and Macromolecular Data......Page 248
5.7.1. Using the 5S-Rrna Gene for the DNA Fingerprinting of Plants Producing Bioactive Molecules......Page 249
Suggested Reading......Page 256
Unit II: Biochemistry of Bioactive Plant Molecules......Page 266
6.1.1. The Shikimate Pathway and the Biosynthesis of Chorismate......Page 267
6.1.2. Aromatic Amino Acid Biosynthesis......Page 269
6.1.3. Phenylpropanoid and Lignin Biosynthesis......Page 271
6.1.4. Other Chorismate Derivatives......Page 274
6.1.5. Benzoic Acid Derivatives......Page 275
6.1.6. Coumarins and Furanocoumarins......Page 277
6.1.7. Biosynthesis of Stilbenes......Page 280
6.2. The Biosynthesis of Complex Phenolics......Page 281
6.2.1. The Biosynthesis of Flavonoids......Page 282
6.3. Polymeric phenolic compounds......Page 284
6.3.1. The Biosynthesis of Hydrolysable Tannins......Page 285
6.3.2. The Biosynthesis of Condensed Tannins......Page 286
Suggested Reading......Page 288
7.1. Two Biosynthetic Pathways produce all Plant Terpenoids......Page 290
7.1.1. The Mevalonic Acid Pathway......Page 291
7.1.2. The Methylerythritol 4-Phosphate Pathway......Page 292
7.1.3. Comparing the Two Pathways......Page 293
7.2. Hemiterpenes......Page 294
7.3. Monoterpenes......Page 295
7.4. Sesquiterpenes......Page 298
7.5. Diterpenes......Page 302
7.7. Triterpenes......Page 305
7.7.1. Ecdysteroids......Page 307
7.7.2. Saponins......Page 308
7.7.3. Limonoids......Page 310
7.7.5. Cardenolides and Bufadienolides......Page 311
7.9. Tetraterpenes......Page 313
7.9.1. Carotenoids......Page 314
Suggested Reading......Page 319
8.1. Biosynthesis of Oxylipins......Page 323
8.2. Biosynthesis of Green Leaf Volatiles (Glvs)......Page 324
8.2.3. Biochemical Pathway to GLV Production......Page 325
8.3. Biochemical Pathway to Jasmonates......Page 329
Suggested Reading......Page 331
9.1. Biosynthesis and Catabolism of Cyanogenic Glycosides......Page 334
9.2. Biosynthesis and Catabolism of Glucosinolates......Page 336
9.3. Biosynthesis of Alkaloids......Page 339
9.3.1. Biosynthesis of Piperidine Alkaloids......Page 340
9.3.2. Biosynthesis of Tropane Alkaloids......Page 341
9.3.3. Biosynthesis of Benzylisoquinoline Alkaloids......Page 344
9.3.4. Biosynthesis of Indole Alkaloids......Page 347
9.3.5. Biosynthesis of Purine Alkaloids......Page 353
9.3.6. Biosynthesis of Other Alkaloids......Page 354
9.4. Biosynthesis of Betalains......Page 357
Suggested Reading......Page 358
Unit III: Biotechnology of Bioactive Plant Molecules......Page 362
Chapter Ten......Page 363
10.1. Interaction between the Primary and Secondary Metabolisms......Page 364
10.1.2. Nitrogen as a Nutritional Source......Page 366
10.1.4. The Culture Cycle......Page 367
10.2. Cell and Tissue Cultures......Page 369
10.3. Bioactive Molecules from Cell Cultures......Page 372
10.4. Bioactive Molecules from Tissue and Organ Cultures......Page 375
10.4.1. Root Cultures......Page 376
10.4.2. Shoot and Bud Cultures......Page 379
10.5. In Vitro Turnover, Regulation and Storage of Plant Bioactive Metabolites......Page 380
10.5.1. Metabolic Turnover......Page 381
10.5.2. Transport and Storage of Bioactive Molecules......Page 383
10.5.3. Regulation of Secondary Metabolism in Cell Cultures......Page 384
10.6. The Search For and Selection of Cells with a High Production of Plant Bioactive Molecules......Page 386
10.7. Elicitation of In Vitro Production of Plant Bioactive Molecules......Page 389
10.8. In Vitro Production of Plant Bioactive Molecules of Economic Importance......Page 393
Suggested Reading......Page 397
11.1. Plant Biotechnology......Page 399
11.2. Biotransformation of Plant Bioactive Molecules......Page 401
11.3. Bioreactors and Fermenters......Page 406
11.3.1. Photobioreactors......Page 408
11.4. Immobilized Plant Cell Cultures......Page 411
11.4.1. Plant Cell Immobilization Techniques......Page 413
11.4.2. Viability of Cells......Page 414
11.4.3. Biosynthetic Capacity......Page 415
11.4.4. Release of Bioactive Molecules......Page 416
11.5. Cryopreservation......Page 417
Suggested Reading......Page 420
12.1. Transgenic Plants......Page 422
12.2. Genetic Manipulation and the Regulation of Gene Expression......Page 425
12.3.1. Terpene Engineering......Page 428
12.3.2. Phenolic Compounds Engineering......Page 431
12.3.3. Alkaloid Engineering......Page 433
12.4. Plant Molecular Pharming......Page 435
12.5. Food Safety, Recombinant DNA and Bioethics......Page 440
Suggested Reading......Page 445

Citation preview

Plant Bioactive Molecules

Plant Bioactive Molecules By

Massimo Maffei

Plant Bioactive Molecules By Massimo Maffei This book first published 2018 Cambridge Scholars Publishing Lady Stephenson Library, Newcastle upon Tyne, NE6 2PA, UK British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library Copyright © 2018 by Massimo Maffei All rights for this book reserved. No part of this book may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, without the prior permission of the copyright owner. ISBN (10): 1-5275-1314-9 ISBN (13): 978-1-5275-1314-3

TABLE OF CONTENTS

Preface . ...................................................................................................... xii UNIT I: Biodiversity and the Sites of Synthesis, Functional roles, Phytochemistry and Chemotaxonomy of Bioactive Plant Molecules Chapter One . ................................................................................................ 2 Biodiversity and its Distribution, and Characterization of Bioactive Plant Molecules 1.1. Biodiversity . .................................................................................... 2 1.1.1. Distribution of Biodiversity. ................................................... 5 1.1.2. Actions to Sustain Biodiversity .............................................. 6 1.2. Sustainability . ............................................................................... 14 1.2.1. Mineral Nutrition and Soil . .................................................. 18 1.2.2. Pests and Pathogens. ............................................................. 19 1.2.3. Biotechnology and Sustainability ......................................... 20 1.2.4. Extraction of Phytochemicals . .............................................. 24 1.2.5. Toward what Future?. ........................................................... 25 1.3. Quantifying Biodiversity . ............................................................. 26 1.4. Classification and Characterization of Natural Compounds ......... 28 1.4.1. Taxonomy . ........................................................................... 28 1.4.2. Evolution . ............................................................................. 29 1.4.3. Character . ............................................................................. 29 1.4.4. Data Analysis . ...................................................................... 30 1.4.4.1. Morphological Data . .................................................... 30 1.4.4.2. Anatomical Data . ......................................................... 30 1.4.4.3 Palynological Data . ....................................................... 31 1.4.4.4. Cytological Data . ......................................................... 31 1.4.4.5. Cytogenetic and Genetic Data ..................................... 31 1.4.4.6. Chemical Data . ............................................................ 32 1.4.4.7. Ecological Data . ........................................................... 32 Suggested Reading . ............................................................................... 33

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Chapter Two . ............................................................................................. 35 Sites of Synthesis and Storage of Bioactive Plant Molecules 2.1. Secretion . ...................................................................................... 35 2.2. Glandular Trichomes . ................................................................... 38 2.2.1. Glandular Trichomes of the Lamiaceae Family ................... 40 2.2.2. Glandular Trichomes of the Asteraceae Family ................... 46 2.2.3. Glandular Trichomes of the Geraniaceae Family ................. 47 2.2.4. Glandular Trichomes of the Moraceae Family ..................... 49 2.2.5. Glandular Trichomes of the Cannabaceae Family................ 50 2.2.6. Glandular Trichomes of the Solanaceae Family ................... 51 2.3. Secretory Cavities and Resin Ducts .............................................. 53 2.4. Lysigenous Cavities . ..................................................................... 55 2.5. Oil-bearing Cells and Secretory Cells associated with Bacteria ... 57  2.6. Laticifers . ...................................................................................... 59 Suggested Reading . .............................................................................. 61 Chapter Three . ........................................................................................... 66 Functional Role of Bioactive Plant Molecules 3.1. Primary and Secondary Metabolites ............................................. 66 3.2. Phenotypic Plasticity . .................................................................... 70 3.2. Chemical Defence from Biotic Stress . .......................................... 72 3.2.1. Chemical Defence in Prehistory . .......................................... 72 3.2.2. Chemical Ecology . ............................................................... 73 3.2.3. Coevolution . ......................................................................... 74 3.2.3.1. Plant–herbivore Coevolution . ...................................... 77 3.2.3.2. Plant–microbial Coevolution . ...................................... 80 3.2.4. Constitutive Chemical Defence . ........................................... 81 3.2.5. Induced Chemical Defence. .................................................. 84 3.2.5.1. Signal Transduction Pathway and Early Events .......... 86 3.2.5.2. The Sensitivity of the Plasma Membrane and the Role of Symplastic Signaling ................................. 87 3.2.5.3. Calcium and other ions act as Second Messengers in Plant–insect Interactions . ................................................ 90 3.2.5.4. Oxidizing Chemical Defences: Reactive Oxygen (ROS) and Nitrogen (RNS) Species . .............................................. 91 3.2.5.5. Priming . ....................................................................... 94 3.2.5.6. Plant–plant Communication: The Chemical Language . .................................................... 95 3.2.5.7. Tritrophic and Multitrophic Interactions ..................... 99 3.2.6. Theories on Defence from Herbivores ............................... 102 3.2.7. Allelopathy . ........................................................................ 105

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3.2.7.1. Parasitic Plants and Allelochemicals ......................... 109 3.2.8. Chemical Defence from Microorganisms........................... 111 3.3. Chemical Defence from Abiotic Stress . ...................................... 114 3.3.1. Plant Defence from Ultraviolet Radiation .......................... 114 3.3.2. Plant Volatiles and Response to Extreme Climatic Conditions. ..................................................................... 115 Suggested Reading . ............................................................................ 115 Chapter Four . ........................................................................................... 125 Bioactive Plant Molecules in Foods, Drugs and Dietary Supplements 4.1. Dietary and Food Supplements . .................................................. 126 4.1.1. Functional Foods . ............................................................... 128 4.1.1.1. Functional Foods or Phytopharmaceuticals? ............. 130 4.2. Plant Bioactive Molecules and the Treatment of Diseases ......... 132 4.2.1. Interaction between Bioactive Plant Molecules and Drugs 134



4.2.1.1. Interaction between Ginkgo Extracts and Drugs ....... 141 4.2.1.2. Interaction between Ginseng Extracts and Drugs ...... 144 4.2.1.3. Interaction between St John’s wort Extracts and Drugs . ......................................................................... 147 4.2.1.4. Interaction between Echinacea Extracts and Drugs ... 150



4.2.2. Herbal Regulatory: Monographs . ....................................... 152 4.2.2.1. ESCOP Monographs . ................................................. 153 4.2.2.2. WHO Monographs. .................................................... 154 4.2.2.3. German Commission E . ............................................. 155 4.2.2.4. USP . ........................................................................... 155 4.2.2.5. European Pharmacopoeia . ......................................... 157 4.2.3. Ethnofarmacognosy: The Root of Popular Culture ............ 157 4.3. Mode and Action of Plant Bioactive Molecules ......................... 158 4.3.1. Effect on Cell Division . ...................................................... 158 4.3.1.1. Plant Bioactive Molecules Targeting Cell Cycle ....... 159 4.3.1.2. Plant Bioactive Molecules Targeting DNA Synthesis. ................................................................. 160 4.3.1.3. Plant Bioactive Molecules Targeting Cytoskeleton and Mitosis . ....................................................................... 163 4.3.1.4. Plant Bioactive Molecules Targeting Apoptosis ........ 167 4.3.2. Effect of Plant Bioactive Molecules on Cell Membranes, Channels and Receptors. ......................................................... 171 4.3.3. Immunomodulatory Effect of Plant Bioactive Molecules .. 175



4.3.4. Toxic Effect of Plant Bioactive Molecules......................... 179 4.3.4.1. Kidney Injury . ............................................................ 179 4.3.4.2. Liver Injury . ............................................................... 180

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4.3.4.3. Cardiotoxicity . ........................................................... 184 4.3.4.4. Neurotoxicity . ............................................................ 190 4.3.4.5. Genotoxicity . ............................................................. 193 4.3.5. Plant Bioactive Molecules against Uropatogenic Escherichia coli . ..................................................................... 195 4.3.6. Plant Bioactive Molecules for Brain and Mental Disorders. ............................................................. 197 Suggested Reading . ............................................................................ 200 Chapter Five . ........................................................................................... 209 Chemotaxonomic Significance of Plant Bioactive Molecules 5.1. Overview on Chemotaxonomy . .................................................. 209 5.2. Chemotaxonomy of Phenolic Compounds.................................. 210 5.2.1. Asteraceae . ......................................................................... 211 5.2.2. Lamiaceae. .......................................................................... 212 5.2.3. Leguminosae . ..................................................................... 213 5.2.4. Other Plant Families . .......................................................... 214 5.3. Chemotaxonomy of Terpenoids . ................................................. 216 5.3.1 Monoterpenes . ..................................................................... 216 5.3.2. Sesquiterpenes . ................................................................... 218 5.3.3. Diterpenes . ......................................................................... 221 5.3.4. Triterpenes. ......................................................................... 222 5.3.5. Tetraterpenes . ..................................................................... 224 5.4. Chemotaxonomy of Secondary Products Containing Nitrogen .. 226 5.4.1. Alkaloids . ........................................................................... 226 5.4.2. Glucosinolates . ................................................................... 229 5.4.3. Cyanogenic Glycosides . ..................................................... 229 5.4.4 Non-protein Amino Acids . .................................................. 230 5.5. Chemotaxonomic Significance of Fatty Acids............................ 230 5.6. Chemotaxonomic Significance of Surface Alkanes .................... 233 5.7. Correlation between Micromolecular and Macromolecular Data . ........................................................... 233 5.7.1. Using the 5S-rRNA Gene for the DNA Fingerprinting of Plants Producing Bioactive Molecules ................................... 234 5.7.1.1. Molecular and Chemical Correlation in the Gymnosperms . ........................................................ 234 5.7.1.2. Molecular and Chemical Correlation in the Angiosperms . .......................................................... 235 Suggested Reading . ............................................................................ 241

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Unit II: Biochemistry of Bioactive Plant Molecules Chapter Six . ............................................................................................. 252 The Shikimate Pathway: Aromatic Amino Acids and Phenolic Compounds 6.1. The Biosynthesis of Simple Phenolics ........................................ 252 6.1.1. The Shikimate Pathway and the Biosynthesis of Chorismate . ........................................................................ 252 6.1.2. Aromatic Amino Acid Biosynthesis . .................................. 254 6.1.3. Phenylpropanoid and Lignin Biosynthesis ......................... 256 6.1.4. Other Chorismate Derivatives . ........................................... 259 6.1.5. Benzoic Acid Derivatives . .................................................. 260 6.1.6. Coumarins and Furanocoumarins ....................................... 262 6.1.7. Biosynthesis of Stilbenes. ................................................... 265 6.2. The Biosynthesis of Complex Phenolics..................................... 266 6.2.1. The Biosynthesis of Flavonoids . ........................................ 267 6.3. Polymeric Phenolic Compounds . ................................................ 269 6.3.1. The Biosynthesis of Hhydrolysable Tannins ...................... 270 6.3.2. The Biosynthesis of Condensed Tannins............................ 271 Suggested Reading . ............................................................................ 273 Chapter Seven. ......................................................................................... 275 The Biosynthesis of Terpenoids 7.1. Two Biosynthetic Pathways Produce all Plant Terpenoids ......... 275 7.1.1. The Mevalonic acid Pathway . ............................................ 276 7.1.2. The Methylerythritol 4-phosphate Pathway ....................... 277 7.1.3. Comparing the Two Pathways............................................ 278 7.2. Hemiterpenes . ............................................................................. 279 7.3. Monoterpenes. ............................................................................. 280 7.4. Sesquiterpenes . ........................................................................... 283 7.5. Diterpenes . .................................................................................. 287 7.6. Sesterterpenes . ............................................................................ 290 7.7. Triterpenes . ................................................................................. 290 7.7.1. Ecdysteroids . ...................................................................... 292 7.7.2. Saponins . ............................................................................ 293 7.7.3. Limonoids . ......................................................................... 295 7.7.4. Quassinoids . ....................................................................... 296 7.7.5. Cardenolides and Bufadienolides ....................................... 296 7.8. Sesquarterpenes. .......................................................................... 298 7.9. Tetraterpenes . .............................................................................. 298 7.9.1. Carotenoids. ........................................................................ 299 7.9.1.1. Abscisic Acid . ............................................................ 302

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7.9.1.2. Strigolactones . ........................................................... 302 7.10. Polyterpenes . ............................................................................. 303 Suggested Reading . ............................................................................ 304 Chapter Eight . .......................................................................................... 308 Oxylipin Biosynthetic Pathway 8.1. Biosynthesis of Oxylipins . .......................................................... 308 8.2. Biosynthesis of Green Leaf Volatiles (GLVs) ............................ 309 8.2.2. Site of Synthesis of GLVs . ................................................. 310 8.2.3. Biochemical Pathway to GLV Production ......................... 310 8.3. Biochemical Pathway to Jasmonates .......................................... 314 Suggested Reading . ............................................................................. 316 Chapter Nine. ........................................................................................... 319 Biosynthesis of Bioactive Nitrogen-containing Molecules 9.1. Biosynthesis and Catabolism of Cyanogenic Glycosides ........... 319 9.2. Biosynthesis and Catabolism of Glucosinolates ......................... 321 9.3. Biosynthesis of Alkaloids . .......................................................... 324 9.3.1. Biosynthesis of Piperidine Alkaloids ................................. 325 9.3.2. Biosynthesis of Tropane Alkaloids .................................... 326 9.3.3. Biosynthesis of Benzylisoquinoline Alkaloids ................... 329 9.3.4. Biosynthesis of Indole Alkaloids........................................ 332 9.3.4.1. Biosynthesis of Quinoline Alkaloids ......................... 335 9.3.4.2. Biosynthesis of Pyrroloindole Alkaloids ................... 336 9.3.4.3. Biosynthesis of Ergot Alkaloids ................................ 337 9.3.5. Biosynthesis of Purine Alkaloids ....................................... 338 9.3.6. Biosynthesis of other Alkaloids.......................................... 339 9.4. Biosynthesis of Betalains . ........................................................... 342 Suggested Reading . ............................................................................ 343 Unit III: Biotechnology of Bioactive Plant Molecules Chapter Ten . ............................................................................................ 248 In Vitro Production of Bioactive Plant Molecules 10.1. Interaction between the Primary and Secondary Metabolisms . 349  10.1.1. Carbon as a Nutritional Source......................................... 351 10.1.2. Nitrogen as a Nutritional Source ...................................... 351 10.1.3. Other Nutritive Elements. ................................................. 352 10.1.4. The Culture Cycle . ........................................................... 352 10.2. Cell and Tissue Cultures . .......................................................... 354 10.3. Bioactive Molecules from Cell Cultures ................................... 357

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10.4. Bioactive Molecules from Tissue and Organ Cultures ............. 360 10.4.1. Root Cultures. ................................................................... 361 10.4.2. Shoot and Bud Cultures .................................................... 364 10.5. In vitro Turnover, Regulation and Storage of Plant Bioactive Metabolites . .................................................... 365 10.5.1. Metabolic Turnover . ......................................................... 366 10.5.2. Transport and Storage of Bioactive Molecules ................ 368 10.5.3. Regulation of Secondary Metabolism in Cell Cultures .... 369  10.6. The Search for and Selection of Cells with a High Production of Plant Bioactive Molecules . ...................................................... 371 10.7. Elicitation of in vitro Production of Plant Bioactive Molecules 374  10.8. In vitro Production of Plant Bioactive Molecules of Economic Importance . ............................................................. 378 Suggested Reading . ............................................................................ 382 Chapter Eleven . ....................................................................................... 384 Biotechnology of Bioactive Plant Molecules 11.1. Plant Biotechnology . ................................................................. 384 11.2. Biotransformation of Plant Bioactive Molecules ...................... 386 11.3. Bioreactors and Fermenters . ..................................................... 391 11.3.1. Photobioreactors . .............................................................. 393 11.4. Immobilized Plant Cell Cultures . .............................................. 396 11.4.1. Plant Cell Immobilization Techniques ............................. 398 11.4.2. Viability of Cells . ............................................................. 399 11.4.3. Biosynthetic Capacity. ...................................................... 400 11.4.4. Release of Bioactive Molecules ....................................... 402 11.5. Cryopreservation . ...................................................................... 402 Suggested Reading . ............................................................................ 405 Chapter Twelve . ...................................................................................... 407 Genetic Engineering of Bioactive Plant Molecules 12.1. Transgenic Plants . ..................................................................... 407 12.2. Genetic Manipulation and the Regulation of Gene Expression 410



12.3. Molecular Engineering and the Production of Plant Bioactive Molecules . ...................................................... 413 12.3.1. Terpene Engineering . ....................................................... 413 12.3.2. Phenolic Compounds Engineering ................................... 416 12.3.3. Alkaloid Engineering . ...................................................... 418 12.4. Plant Molecular Pharming . ....................................................... 420 12.5. Food Safety, Recombinant DNA and Bioethics........................ 425 Suggested Reading . ............................................................................ 430

PREFACE

Plants have always been a source of nourishment and care for living beings. Their dual task as producers of nutrients and drugs played a fundamental role in the evolution (and co-evolution) of herbivorous and omnivorous organisms. The so-called secondary (or special) metabolites are molecules with welldefined functional roles, aimed primarily at defending plants from abiotic (temperature, light, water availability, etc.) and biotic (attacks of herbivores, fungi, bacteria and viruses) stress. The complexity of the molecular structures produced by plants is only equal to their versatility and biodiversity, while the harmonious interweaving of biosynthetic and metabolic pathways offers a perfect picture of the adaptive plasticity of plants as environmental conditions change. This book is divided into three units to offer the reader a general, biochemical and biotechnological framework of bioactive plant molecules. The first unit analyses the concepts of biodiversity and sustainability and the functional roles of bioactive molecules, exploring the sites of synthesis and accumulation, the strategies adopted by plants to defend themselves from stress and the use of bioactive molecules as food supplements and as a source for natural medicines to combat diseases. The first unit also includes chemotaxonomy, where bioactive molecules and other secondary products play a fundamental role in support of the identification of plant species. The second unit describes plant biochemistry with a detailed discussion on the main biosynthetic pathways leading to the synthesis of aromatic compounds (phenols and flavonoids) and terpenes (from volatile substances to phytosterols, to antioxidant molecules such as carotenoids and astaxanthin) to conclude with the biosynthetic pathways leading to the synthesis of nitrogen-containing bioactive molecules, including alkaloids, glucosinolates and cyanogenic glucosides. In this unit, one chapter is also dedicated to oxylipins, describing the biochemistry of jasmonates and

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green leaf volatiles, substances typical of plant reactions to biotic stress and mechanical damage. The third and last unit deals with plant biotechnology and the production of bioactive molecules both in vivo and in vitro. The main techniques are described, such as cell and tissue cultures and root and shoot cultures, with particular attention to the in vitro production of bioactive molecules of industrial interest. In addition to the defining of plant biotechnology, a chapter deals with its technological aspects by describing bioreactors, photobioreactors and cryopreservation techniques. The unit concludes with a chapter dedicated to genetic engineering for the production of bioactive molecules, where in addition to the definition of transgenic plants ethical problems, risks and benefits of using recombinant DNA in genetically modified organisms (GMOs) are discussed. Several examples of terpene, phenolic compound and alkaloid engineering are presented along with methods and techniques for industrial application. Molecular pharming is also described, revealing its peculiarities and potential, with examples of bioactive molecules produced to treat infectious diseases and to improve the quality of human life. Finally, a paragraph is dedicated to food safety issues and bioethical considerations. I wrote this book for science students of university undergraduate and graduate courses, but the language used (especially in the first and third unit) is simple enough to be understood by all people who are interested in bioactive natural molecules. Writing a book on these issues is always a challenge, especially due to the continuous stream of new notions being published every day across hundreds of international scientific journals. The intent was to collect most of the recent notions, being fully aware of the limits imposed by the vastness of the subject. I wish you a very good reading. Massimo Maffei

UNIT I BIODIVERSITY AND THE SITES OF SYNTHESIS, FUNCTIONAL ROLES, PHYTOCHEMISTRY AND CHEMOTAXONOMY OF BIOACTIVE PLANT MOLECULES

CHAPTER ONE BIODIVERSITY AND ITS DISTRIBUTION, AND CHARACTERIZATION OF BIOACTIVE PLANT MOLECULES

Biodiversity, or biological diversity, is a global concept of biology that includes the analysis and description of variability in living forms, whether it is related to microbes, plants or animals present in aquatic and terrestrial ecosystems. This concept can be further extended to molecules produced by living organisms, regardless of their function or biosynthetic pathway. In the biosphere, there are many areas of biodiversity and the most common are insects, ranging from 2 to 5 million species, angiosperms, with more than 275,000 species known, and the broad area of secondary (or specialized) metabolites, which exceeds 100,000 known molecular structures. Biodiversity is present below ground, where billions of microorganisms live, and above ground, where weeds and spontaneous plants seem to confirm the concept that nature ultimately prevails. There are about 275,000 plant species on our planet and about 33,000, or 12.5%, are threatened with extinction. This is a sad reality that, alongside 11% of bird species being endangered, shows how biodiversity is imperilled, especially in those areas where human intervention is devastating. Plants, which comprise about 370 families, are distributed all over the world in all its almost 200 countries, but 91% of plant families are concentrated in only one country, linking the potential danger of their extinction to national, social and economic conditions.

1.1. Biodiversity The concept of biodiversity includes diversity within species, between species and among ecosystems. Molecular plant biodiversity is a topic of

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great interest because it reflects the impressive diversity in the chemical structures produced by individual species. The concept can be extended to several ecosystems and resized to a smaller scale such as a given nation, a park, a group of plants and even one species. The term “biodiversity” became popular after the signing by 168 countries of the “Convention on Biological Diversity” (see below). Today, “biodiversity” is a term familiar to many: almost no research programme with an ecological intent can avoid considering biodiversity. Similarly to the term “ecology”, coined more than 60 years ago, the term “biodiversity” has been used by several social groups with different aims and goals. The most recent interpretations of the term “biodiversity” are not limited to the concept of “species richness”, but rather are also related to varieties, races, life forms and genotypes, as well as types of landscape, habitats and structural elements (e.g., shrubs, stone walls, bushes, ponds). Biodiversity is assessed by the classification criteria used in taxonomy. Biosystematics, which includes taxonomy, is a powerful tool for studying biodiversity and makes use of biological disciplines such as evolution, phylogeny, genetics and phytogeography. Another important component in the study of biodiversity is the evaluation of the genetic diversity which, within species, allows a certain individual to evolve under environmental pressures and natural selection. The variability we observe among individuals (the phenotype) is partly the result of the interaction between genetic differences (the genotype) and the surrounding environment. In the specific case of many secondary metabolites such as monoterpenes, the genotypic expression can be influenced by several factors, both biotic (such as herbivore attack) and abiotic (such as environmental changes). Biodiversity is mainly based on speciation – the formation of a new species – which follows three basic steps: (i) it begins with the existence of a species; (ii) is associated with genetic changes; and (iii) closely depends on the ecological context. Taxonomic groups and eco-regions shape the “lenses” with which biodiversity is valued and preserved. According to some authors, the design of effective conservation strategies requires the examination of groups of eco-regional or biome-specific indicators, rather than a tight set of global indicator groups.

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Locations of and threats to biodiversity are distributed unevenly, so prioritizing is essential to the minimization of biodiversity loss. To meet this goal, biodiversity conservation organizations have put forward real models of global priority. Most models give priority to heavily irreplaceable regions; some others are reactive (favouring high vulnerability) while others again are proactive (with less priority given to vulnerability). Plants, as we will discuss in the next paragraph, are not uniformly distributed on our planet and this is primarily due to historical, causal and functional reasons. Historical causes are studied by biogeography, which assesses the nature of events that occurred during the various historical eras. The drift of continents and the impossibility of plants crossing the oceans that formed from the separation of the land that emerged is a compelling example. Physical isolation caused the independent evolution of the various species and greatly contributed to the facilitating of biodiversity. Climatic zones, variable soil nature, altitude and selective pressures imposed by herbivores as well as natural disasters have contributed greatly to the variability of biological forms present on our planet. The formation of new species following spatial separation (allopatric speciation) is particularly common in animals, but as we will see, it also occurs in the plant kingdom. The loss of genetic variability in a population reduces its ability to respond to the environment and reduces the possibility of rehabilitating a given habitat. The number of species present is therefore directly proportional to the ability of certain ecosystems to withstand environmental adversities. How does human action affect ecosystems and biodiversity? Humans have been surrounded by biological diversity for millions of years and have made biodiversity one of the main resources by which to nurture, heal, build, gain energy and much more. Optimally, the resources used by humans are renewable, but often the abuse or misuse of these resources can lead to their extinction. It is evident that the expansion of the ecological niche partly occupied by humans some tens of thousands of years ago has inevitably reduced the niche occupied by animals and plants and inevitably reduced biodiversity. Humans are one of the main causes of extinction. For instance, it is estimated that in the past 400 years more than 600 plant species have become extinct due to human intervention. The extinction of species is also a natural factor, and the most obvious example is the extinction of dinosaurs, which dominated Earth millions of

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years before the appearance of humans. The extinction of a species can occur because of natural catastrophes, such as volcanic eruptions or tsunamis, or simply because of the genetic inability to adapt to environmental changes, even in the long run. Small populations are more likely to be at risk of extinction, but some of the most significant current causes are environmental degradation, the over-exploitation of natural resources and the introduction of exotic species. Yet humans rely for most of their activities on biodiversity. For instance, the bioactive compounds of one prescription drug out of four are made from plant extracts and the pharmaceutical and biotechnology industries are increasingly searching for natural areas exhibiting the greatest expression of biodiversity, where thousands of species are still to be discovered or analysed. Some industries are paying property rights in selected countries to preserve seriously endangered areas. However, while there are hundreds of thousands of potentially usable species, the plant kingdom is also characterized by a remarkable biochemical redundancy. However, the increasing population and the continuous search for cultivatable land lead to the progressive and seemingly unstoppable destruction of rainforests, with irreversible consequences for biodiversity and unimaginable losses of unknown resources. The rise of biotechnology has recently led some anthropologists towards the ethically and philosophically stimulating field of bioprospecting, the searching for genetic and biochemical resources of commercial value. It is an innovative arena that can help produce new therapies while preserving traditional medical systems and biological and cultural diversity by showing their medical, economic and social value, and by bringing biotechnology and other benefits to poor countries which are rich in biodiversity but poor in technology.

1.1.1. Distribution of Biodiversity Medicinal plants are undoubtedly one of the most fascinating categories of plants in large part because they are sources of bioactive molecules. About 80% of the nearly 30,000 known natural products derive from plants, and in addition, some specialized metabolites are unique to the plant kingdom, not being produced by microbes or animals. The distribution of medicinal plants can be classified geographically based on the centres of origin of certain species:

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x North American: Echinacea angustifolia, Hamamelis virginiana, Sassafras officinale, Lobelia inflata, Hydrastis canadensis and Podophyllum peltatum; x South and Central American: Vanilla planifolia, Carica papaya, Aloe vera, Erythroxylon coca, Ilex paraguariensis, Theobroma cacao, Dioscorea composita and Echinocactus williamsii; x Mediterranean: most of the Lamiaceae, Valeriana officinalis, Digitalis purpurea, Crocus sativus, Laurus nobilis, Foeniculum vulgare, Glycyrrhiza glabra, Colchicum autumnale and Atropa belladonna; x African: Acacia senegal, Ricinus communis, Cassia acutifolia, Datura stramonium, Rauwolfia vomitori and Physostigma vevenosum; x Madagascan: Eugenia caryophyllata, Catharanthus roseus and Piper nigrum; x Indian: Rauwolfia serpentina, Datura spp., Cannabis sativa var. indica, Curcuma longa, Strychnos nux-vomica, Cinnamomum zeilanicum, Cassia angustifolia, Zingiber officinale and Dioscorea spp; x Asian: Papaver somniferum, Panax ginseng, Rheum spp., Cinnamomum camphora, Thea sinensis and Vaccinium myrtillus; x Indonesian: Myristica fragrans, Illicium verum, Eugenia caryophyllata and Piper methysticum; x Australian: Eucalyptus spp. and Duboisia myoporoides. Certainly one of the most important centres of origin of aromatic and medicinal plants is the Mediterranean basin. This area is difficult to define because it does not coincide with any political boundaries and is characterized by nations of different ethnic groups and climates. According to some authors, the importance of the Mediterranean region derives from a number of considerations, including the high variability of soil and climatic conditions, which have favoured the high biodiversity of plant species, and the fact that it contains high proportions of annual species belonging particularly to the Caryophyllaceae, Brassicaceae, Asteraceae and Apiaceae.

1.1.2. Actions to Sustain Biodiversity What are humans doing to prevent the progressive depletion of biological diversity? One of the firmest answers to this question was provided by the

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Council of Europe’s Convention on the Conservation of European Wildlife and Natural Habitats (1979), or the Bern Convention, that was the first international treaty to protect both species and habitats and to get countries together to decide how to act on nature conservation. The Convention aimed to ensure conservation of wild flora and fauna and their habitats. Special attention was given to endangered and vulnerable species, including selected endangered and vulnerable migratory species. Another important step was the Convention on Biological Diversity (CBD), which was signed by a number of nations during the United Nations Environment Conference held in Rio de Janeiro in June 1992 and that became effective on 29 December 1993. The objectives of the Convention are the conservation of biodiversity, the sustainable use of its components and the true sharing of benefits coming from the exploitation of genetic resources, including the access to such resources and the transfer of relevant technologies. The countries that signed the Convention have the sovereign right to exploit the resources of their territory and the duty to make sure that activities within their jurisdiction do not harm the environment of other neighbouring states. The main task of each state will be to identify those components of biodiversity that need to be conserved and used in a sustainable manner and to monitor identified areas by giving priority to those with the greatest need for immediate intervention. An important task that each country will have to carry out is to respect, retain and maintain the knowledge, innovations and practices adopted by indigenous and local communities by promoting those lifestyles inherent to conservation and sustainable use of biodiversity and by promoting the application of such knowledge, always upon the consent of the people who hold the rights. In the case of developing and underdeveloped countries, actions to preserve biodiversity in situ will be particularly important. Nevertheless, the ex situ conservation of biodiversity components should be likewise favoured, preferably in the country of origin of such resources. An interesting aspect of the CBD is education and educational programmes. However, these programmes are not aimed at the education of indigenous populations on the use of biodiversity because indigenous peoples have a cultural heritage that needs to be valued. Instead, attention should be paid to educating people scientifically and technically on species identification, on in situ and ex situ conservation, and on the sustainable use of biodiversity and its components. Biodiversity conservation research should be promoted and encouraged. Only through the conservation of biological complexity will it be possible to obtain the best results in the

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search for bioactive molecules for medical and pharmaceutical applications. Priority policies for the countries that undersigned the CBD will be environmental risk assessment and management of potentially biodiversity-threatening practices as well as the immediate reporting of any ecological disturbances to avoid disasters in neighbouring areas. An important innovation is the concept that the genetic resources of a country remain under its absolute jurisdiction and that these resources are subject to national legislation. At the same time, however, each country will have to provide the countries that contract with it free access to genetic resources to be used for environmental protection purposes. In any case and always, the exploitation of genetic resources by a second country will have to be authorized by the government of the first country. Agreements between research institutes or individuals who are nonrepresentative of their own country will not be sufficient. In many cases, the escape of genetic resources from a country has been caused by personal and non-governmental contacts with universities, research institutes, industries or corporations. Now, with the CBD, member countries can enact biodiversity protection laws that can pursue those who take a free initiative in managing genetic resources. Therefore, member countries will have to monitor ecosystems and habitats that contain high density, high endemic or endangered species of social, economic, cultural and scientific importance, or which are representative, unique or associated with evolutionary or other biological processes. At present, relatively few countries have set clear and well defined priorities to be applied for biodiversity management. When applied, they have suffered from a lack of population participation and they have often ignored those social, economic and institutional factors that play an important role in deciding how to handle a conservation problem. Economic incentive policies have been followed by many countries, both in the developed and developing economies. Their application has certainly sensitized populations to the problem of biodiversity conservation with the hope of assimilating the concept of sustainability of genetic resources in their minds. However, incentives are not enough to preserve biodiversity. People’s activities need to be regulated, especially when biodiversity conservation becomes a social factor. Legislationrelated laws and traditions have proven incredibly effective in preserving and managing biodiversity for hundreds of years in some African and Asian countries.

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Table 1.1 lists the key terms of the CBD. Table 1.1 – Glossary of terms used in the Convention on Biological Diversity Biological diversity – the variability among living organisms from all sources including, inter alia, terrestrial, marine and other aquatic ecosystems and the ecological complexes of which they are part: this includes diversity within species, between species and ecosystems. Biological resources – includes genetic resources, organisms or parts thereof, populations, or any other biotic component of the ecosystems with actual or potential use or value for humanity. Biotechnology – any technological application that uses biological systems, living organisms, or derivatives thereof, to make or modify products or processes for specific use. Country of origin of genetic resources – the country which possesses those genetic resources in in situ conditions Country providing genetic resources – the country supplying genetic resources collected from in situ sources, including populations of both wild and domesticated species, or taken from ex situ sources, which may or may not have originated in that country. Domesticated or cultivated species – species in which the evolutionary process has been influenced by humans to meet their needs. Ecosystem – a dynamic complex of plant, animal and microorganism communities and their non-living environment interacting as a functional unit. Ex situ conservation – the conservation of components of biological diversity outside their natural habitats. Genetic material – any material of plant, animal, microbial or other origin containing functional units of heredity. Genetic resources – genetic material of actual or potential value. Habitat – the place or type of site where an organism or population naturally occurs In situ conditions – conditions where genetic resources exist within ecosystems and natural habitats, and in the case of domesticated or cultivated species, in the surroundings where they have developed their distinctive properties. In situ conservation – the conservation of ecosystems and natural habitats and the maintenance and recovery of viable populations of species in their natural surroundings and, in the case of domesticated or cultivated species, in the surroundings where they have developed their distinctive properties.

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Protected area – a geographically defined area which is designated or regulated and managed to achieve specific conservation objectives. Regional economic integration organization – an organization constituted by sovereign States of a given region, to which its member States have transferred competence in respect of matters governed by this Convention and which has been duly authorized, in accordance with its internal procedures, to sign, ratify, accept, approve or accede to it. Sustainable use – the use of components of biological diversity in a way and at a rate that does not lead to the long-term decline of biological diversity, thereby maintaining its potential to meet the needs and aspirations of present and future generations. Technology – includes biotechnology. In 1994, the UK Government published “Biodiversity: the UK Action Plan”, and a UK Plant Conservation Strategy was presented along with this action plan. The Strategy is a framework for the conservation of the native flora of Great Britain and Northern Ireland, officially approved by the statutory conservation agencies (including the Joint Nature Conservation Committee, English Nature, the Countryside Council for Wales, Scottish Natural Heritage and the Department of the Environment for Northern Ireland). The aim of the Strategy is to maintain the character and diversity of the natural flora of the UK and to ensure the viability of species. The Global Plan of Action for the Conservation and Sustainable Utilization of Plant Genetic Resources for Food and Agriculture was formally adopted by representatives of 150 countries during the Fourth International Technical Conference on Plant Genetic Resources, which was held in Leipzig, Germany, from 17 to 23 June 1996. The Conference also adopted the Leipzig Declaration, which focuses on the importance of plant genetic resources for world food security, and commits countries to implementing the Plan. The FAO (Food and Agriculture Organization of the United Nations) is committed to carrying out the Global Plan of Action, under the guidance of the intergovernmental Commission on Genetic Resources for Food and Agriculture, as part of the FAO Global System for the Conservation and Utilization of Plant Genetic Resources. The Nagoya Protocol on Access to Genetic Resources and the Fair and Equitable Sharing of Benefits Arising from their Utilization (ABS, from Access and Benefit Sharing) of the Convention on Biological Diversity is a supplementary agreement to the CBD. It provides a transparent legal

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framework for the effective implementation of one of the three objectives of the CBD: the fair and equitable sharing of benefits arising out of the utilization of genetic resources. A significant innovation of the Protocol is the specific obligation of the party to support compliance with national legislation – or regulatory requirements – providing genetic resources and mutually agreed contractual obligations. These compliance provisions, as well as the provisions that lay down the most predictable conditions for the access to genetic resources, help to ensure the sharing of benefits when genetic resources are taken up by those who provide them. In addition, the provisions of the Protocol will strengthen the ability of communities to benefit from the use of their knowledge, innovations and practices. By promoting the use of genetic resources and related traditional knowledge, and by strengthening the opportunities for fair and equitable sharing of the benefits of using them, the Protocol aims to create incentives to preserve biodiversity, use its components in a sustainable way and to further improve the contribution of biological diversity to sustainable development and human well-being. The Conference of Participants to the CBD held its twelfth meeting in Pyeongchang, Republic of Korea, (6–17 October 2014) and its thirteenth meeting in Cancun, Mexico (4–17 December 2016). It was recognized by the majority of participants that the actual trend could not continue and that the post-2016 agenda should support a transformational approach based on consumption and production within the planetary boundaries, integrating biodiversity in all sectors, building synergies and working through “innovative” partnerships with the participation of all ministries and academia, civil society and the private sector for a sustainable future. The 2011–2020 Strategic Plan for Biodiversity is a decennial framework action brought by all countries and stakeholders to save biodiversity and improve people’s benefits. The Strategic Plan is based on a shared vision, mission, strategic goals and 20 ambitious goals still attainable, collectively known as Aichi’s goals. The Strategic Plan serves as a flexible framework for defining national and regional objectives and promotes the coherent and effective implementation of the three objectives of the CBD. Table 1.2 lists Aichi’s goals. (For further information and developments of the CBD link to http://www.cbd.int.)

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Table 1.2. The Aichi Biodiversity Targets Strategic Goal A: Address the underlying causes of biodiversity loss by mainstreaming biodiversity across government and society 1. By 2020, at the latest, people are aware of the values of biodiversity and the steps they can take to conserve and use it sustainably 2. By 2020, at the latest, biodiversity values have been integrated into national and local development and poverty reduction strategies and planning processes and are being incorporated into national accounting, as appropriate, and reporting systems. 3. By 2020, at the latest, incentives, including subsidies, harmful to biodiversity are eliminated, avoid negative impacts, and positive incentives for the conservation and sustainable use of biodiversity are developed and applied, consistent and in harmony with the Convention and other relevant international obligations, taking into account national socio economic conditions 4. By 2020, at the latest, Governments, business and stakeholders at all levels have taken steps to achieve or have implemented plans for sustainable production and consumption and have kept the impacts of use of natural resources well within safe ecological limits. Strategic Goal B: Reduce the direct pressures on biodiversity and promote sustainable use 5. By 2020, the rate of loss of all natural habitats, including forests, is at least halved and where feasible brought close to zero, and degradation and fragmentation is significantly reduced. 6. By 2020 all fish and invertebrate stocks and aquatic plants are managed and harvested sustainably, legally and applying ecosystem based approaches, so that overfishing is avoided, recovery plans and measures are in place for all depleted species, fisheries have no significant adverse impacts on threatened species and vulnerable ecosystems and the impacts of fisheries on stocks, species and ecosystems are within safe ecological limits. 7. By 2020 areas under agriculture, aquaculture and forestry are managed sustainably, ensuring conservation of biodiversity. 8. By 2020, pollution, including from excess nutrients, has been brought to levels that are not detrimental to ecosystem function and biodiversity. 9. By 2020, invasive alien species and pathways are identified and prioritized, priority species are controlled or eradicated, and measures are in place to manage pathways to prevent their introduction and establishment.

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10. By 2015, the multiple anthropogenic pressures on coral reefs, and other vulnerable ecosystems impacted by climate change or ocean acidification are minimized, so as to maintain their integrity and functioning. Strategic Goal C: Improve the status of biodiversity by safeguarding ecosystems, species and genetic diversity 11. By 2020, at least 17 per cent of terrestrial and inland water, and 10 per cent of coastal and marine areas, especially areas of particular importance for biodiversity and ecosystem services, are conserved through effectively and equitably managed, ecologically representative and well connected systems of protected areas and other effective area-based conservation measures, and integrated into the wider landscape and seascapes. 12. By 2020 the extinction of known threatened species has been prevented and their conservation status, particularly of those most in decline, has been improved and sustained. 13. By 2020, the genetic diversity of cultivated plants and farmed and domesticated animals and of wild relatives, including other socioeconomically as well as culturally valuable species, is maintained, and strategies have been developed and implemented for minimizing genetic erosion and safeguarding their genetic diversity. Strategic Goal D: Enhance the benefits to all from biodiversity and ecosystem services 14. By 2020, ecosystems that provide essential services, including services related to water, and contribute to health, livelihoods and wellbeing, are restored and safeguarded, taking into account the needs of women, indigenous and local communities, and the poor and vulnerable. 15. By 2020, ecosystem resilience and the contribution of biodiversity to carbon stocks has been enhanced, through conservation and restoration, including restoration of at least 15 per cent of degraded ecosystems, thereby contributing to climate change mitigation and adaptation and to combating desertification. 16. By 2015, the Nagoya Protocol on Access to Genetic Resources and the Fair and Equitable Sharing of Benefits Arising from their Utilization is in force and operational, consistent with national legislation. Strategic Goal E: Enhance implementation through participatory planning, knowledge management and capacity building 17. By 2015 each Party has developed, adopted as a policy instrument, and has commenced implementing an effective, participatory and updated national biodiversity strategy and action plan.

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18. By 2020, the traditional knowledge, innovations and practices of indigenous and local communities relevant for the conservation and sustainable use of biodiversity, and their customary use of biological resources, are respected, subject to national legislation and relevant international obligations, and fully integrated and reflected in the implementation of the Convention with the full and effective participation of indigenous and local communities, at all relevant levels. 19. By 2020, knowledge, the science base and technologies relating to biodiversity, its values, functioning, status and trends, and the consequences of its loss, are improved, widely shared and transferred, and applied. 20. By 2020, at the latest, the mobilization of financial resources for effectively implementing the Strategic Plan 2011–2020 from all sources and in accordance with the consolidated and agreed process in the Strategy for Resource Mobilization should increase substantially from the current levels. This target will be subject to changes contingent to resources needs assessments to be developed and reported by Parties A new concept emerging from the conferences dedicated to the study of methods and strategies for the conservation of biodiversity is sustainability. We will discuss this concept in the next section.

1.2. Sustainability Sustainability is a general concept applicable to social, economic, environmental and agricultural considerations. Although these four categories have many common points, the greatest overlap is between economic, environmental and agricultural sustainability. In very general terms, the term sustainability encompasses the use of components of biological diversity in a way and at a rate that does not lead to the longterm decline of natural resources, thereby maintaining its potential to meet the needs and aspirations of present and future generations. From a social point of view, the main objective of sustainability is to reduce poverty, and some social scientists place social sustainability above any other sustainability. The reduction of poverty can only be achieved through qualitative development, fair distribution of wealth and community strength, rather than demographic growth control. Countries that fully and successfully adopt the criteria of social sustainability are

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those with a more “peaceful” lifestyle, compared to nations suffering from economic and social insecurity. Once social sustainability is achieved, populations can easily move towards commitment to environmental sustainability, thus achieving a sustainable development. Sustainable development integrates all four categories of sustainability mentioned above and is defined by the WWF as “the enhancement of the quality of human life combined with the ability to support ecosystems”. Sustainable environmental development implies in any case more sustainability for production and consumption than for the growth of a sustainable economy. It follows that a priority for sustainable development is the increase of human well-being in terms of reduction of poverty, illiteracy, hunger, disease and inequality. The key task of environmental sustainability is to sustain global life-support systems indefinitely (this referring principally to those systems maintaining human life). Protecting human life is the main anthropocentric reason humans seek environmental sustainability. Human life depends on species for food, shelter, breathable air, plant pollination, waste assimilation and other environmental life-support services. It is difficult to predict what choices will be required to sustainably maintain the environment, but surely we will not be able to sustain less than what remains of the existing environment. In other words, we can define environmental sustainability as “maintenance of Nature capital”. The fundamental point is that environmental sustainability is a natural science concept and obeys biophysical laws. This general definition seems to be robust irrespective of country, sector or future epoch. While, on the one hand, the application of sustainability principles to intensively cultivated areas in countries with high industrial and socioeconomic development is aimed at improving living conditions and controlling the environmental impact of humans, on the other hand, it serves to increase the long-term biological potential in those marginal lands that suffer from poor fertility and depletion of organic matter. Sustainable development of agriculture requires an in-depth study of some key themes that include the knowledge and management of the rhizosphere (the space around the roots of plants), the evaluation of the benefits and risks of modifying these processes and the contribution and the limits of biotechnology to improve the productivity of transgenic plants (see also Chapter 12). Farmers who practice sustainable agriculture must rely on a continuous network of information, new technologies and innovations that are instrumental to succeeding in the management of their farmland. However,

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it remains to be determined whether the current agricultural extension scheme is capable of achieving sustainable farming. The role of the agricultural extension is to facilitate the learning process. This involves facilitating: x the process of community development and innovation; x the process of collective and individual learning of innovation (technical and social) to improve the community’s ability to innovate; and x the management of rural knowledge. Sustainable agricultural production is necessary to ensure both global food safety and environmental safety. Conservation agriculture is gaining popularity around the world for such sustainable strategies as permanent soil cover, soil low-sturgeon, crop rotation and integrated pest management. The control of weeds is the biggest challenge in the adoption of conservation agriculture. Systems with a high sustainability make the best use of environmental resources and services. The key principles for sustainability are: x integrating biological and ecological processes such as nutrient cycling, nitrogen fixation, soil regeneration, allelopathy, competition, predation and parasitism into food production processes; x minimizing the use of non-renewable inputs that cause harm to the environment or to the health of farmers and consumers; x making productive use of the knowledge and skills of farmers, thus improving their self-reliance and substituting human capital for costly external inputs; and x making productive use of people’s collective capacities to work together to solve common agricultural and natural resource problems, such as for pest, watershed, irrigation, forest and credit management. What is the meaning of sustainability with regard to natural resources? What is to be sustained, for how long and for whom? Many difficulties arising when trying to define sustainability become more apparent if we consider the concept of unsustainability, which is the complementary side of sustainability. However, in this way only the limits of uncertainty are seen and this does not contribute to the development of models and

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methods able to provide a system that sustains itself without degrading the land, the environment and the population. Sustainable agricultural systems reduce the costs of purchased inputs and provide a sustained level of production and profit from farming. Among the various objectives of sustainable agriculture, the major one is the reduction of inputs into crop production, thus moving world agriculture closer to the goals of profitability, competitiveness and environmental stewardship. It becomes more and more evident that to achieve such a goal we should: protect plants from disease and pests; consider the yield limitations of the agrosystem; use forage legumes to improve the soil; use pathogen-free seeds and pest-resistant crops; maximize the benefits of beneficial organisms; reduce the use of pesticides and inorganic fertilizers; preserve the organic matter of the soil; make the most efficient use of non-renewable resources; utilize renewable energy sources such as biological, geothermal, hydroelectric, solar or wind; and x conserve all resources and minimize waste and environmental damage. x x x x x x x x x

All around the world, the cultivation and the production of phytochemicals is limited by agricultural and environmental factors, the presence of specific pathogens and by differences in comparative costs. In developed countries, the continuous struggle to try to solve these problems led to the development of high-input agro-industrial methods with a considerable use of fossil fuel-derived energy, inorganic fertilizers and pesticides. These agricultural methods, which were the fundamentals for the so called “Green Revolution”, need now to be improved to maintain high yield crop production while protecting the environment and human health. In general, the use of inputs eventually leads to increased costs, both of cultivation and for phytochemicals extraction, thus leading to increased cost/quality ratios. All these practices cause alteration of the ecosystem, and the increase of harvestable biomass generates costs to both humans and the environment. The situation is even more critical in developing countries, where the cost/quality ratio is kept low at the expense of human work and health;

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moreover, the use of organic fuel relies mainly on deforestation. In underdeveloped countries, cultivation of plants that produce phytochemicals is non affordable; in such locations, plants are harvested from the wild, with the risk of endangering the preservation and the biodiversity of natural resources. The challenge for the 21st century burdens also on developed countries’ shoulders, for they need to reduce fossil fuel energy and inputs in order to obtain sustainable agriculture, and plants producing phytochemicals are just one of the several aspects of the global issue. However, the complexity of sustainability in phytochemical production does not rely only on agricultural practices. The extraction of natural compounds from plants and their processing requires energy and technology, which weigh heavily on both the cost/quality ratio and the environment. Once again, the evident contrast between developed and developing countries makes the difference in comparative costs. We will now discuss the application of sustainability in agro-industry with regard to the production of phytochemicals, by considering separately the several aspects of the problem.

1.2.1. Mineral Nutrition and Soil In monocultures for phytochemical production there is the potential hazard of the suppression of soil fertility, productivity, structure and microbial activity. One of the renewable resources on which sustainable agriculture relies is biologically fixed nitrogen. This can be obtained through the use of plants like the legumes in which the biological nitrogen fixation (BNF) occurs in roots via symbiotic bacteria (diazotrophs). Other than BNF, legumes give other benefits to the soil through rhizodeposition, secretion via roots of a complex mixture of substances that provide improved nutrient availability, improved structure, reduced pest and disease levels and hormonal effects. Besides crop rotation, intercropping gives economic and environmental benefits to crops. For instance, peppermint plants intercropped with soybean require a lesser amount of fertilizers and show a general increase in monoterpene hydrocarbons and oxygenated compounds such as 1,8-cineole, menthone and menthofuran, whereas the percentage of menthol, isomenthone and menthyl acetate decreases (see Chapter 7 for chemical structures). In general, intercropping increases the total amount of phytochemicals produced per surface unit. Moreover, the legume/phytochemical-producing plant intercropping practice can be used

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in poor fertility soils, to improve the soil structure and fertility, to increase the soil aggregate stability and to promote soil microbial diversity. A healthy soil is the basis of sustainability and proper soil management can help prevent some pest problems. In the cultivation of plants for phytochemicals production, soil impairments result in a reduced production of biomass and phytochemical productivity, while enhancing the required inputs of water, nutrients, pesticides and energy. Depletion of soil destroys natural predators of crop pests and increases the spreading of pests.

1.2.2. Pests and Pathogens Many plants that produce phytochemicals are affected by a number of pests and pathogens. The continuous and unsustainable use of pesticides and fertilizers increases pest vigour and depletes pest resistance, with increasing demands for inputs. Besides pathogens, weeds affect phytochemical yield by reducing the nitrogen and water availability to producing plants. The struggle against disease and weeds has been brought by the use of pesticides and herbicides, on one hand, and of selected pest resistant crops, on the other. In peppermint, the “Murray Mitcham” cultivar is widely grown because of its resistance to the pathogen Verticillium dahliae and the search for new and pest resistant varieties continues. The finding of new resistant strains gives the opportunity to study the mechanisms of pest resistance. The virulence of a pathogen and the resistance of a plant are reciprocal concepts, and usually before a pathogen can succeed in infection, it must overcome the plant’s defensive barriers. Two other important noteworthy mechanisms of defence are: systemic acquired resistance (SAR), in which the resistance to a pathogen attack is expressed locally at the site of infection but also systemically, in tissues far away from the initial infection; and hypersensitive response (HR), which leads to a rapid cell (and pathogen) death while inducing a series of biochemical responses such as the synthesis of phytoalexins, lignin and/or hydroxyproline-rich proteins, hydrolytic enzymes and antimicrobial polypeptides. The selection of resistant plants producing phytochemicals and the transfer of resistance to high quality/high yield not-resistant strains is one of the goals of sustainable agriculture. This can be achieved by the

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use of naturally evolved defence mechanisms while maintaining ecosystem equilibrium. The use of pesticides is not banned by sustainable agricultural programmes. However, they have to be used only when the economic threshold of damage is reached, the point when the damage caused by the pest exceeds the costs of chemical control. The inappropriate use of pesticides is not only potentially harmful to the environment, but forces farmers to depend on them eventually weakening agriculture’s economic sustainability. Sustainable approaches are the least toxic and least energy intensive, while maintaining productivity and profitability. Besides pesticides, biological control of pests is growing rapidly in the modern agricultural practice of biological struggle. Integrated pest management (lPM) is a sustainable approach to managing pests by combining regular scouting and biocontrol use of living organisms to fight pests with cultural, physical and chemical tools to minimize economic, health and environmental risks. This practice is based on the antagonistic interaction between a non-pathogenic organism and a plant pathogen. Plants producing phytochemicals also host many arthropods, both harmful and beneficial. The adequate sustainable management of insects and mites is based on several IPM practices such as the identification of beneficial and harmful organisms, the protection of beneficial insects and mites and the adequate use of pesticides. A careful identification of pest/non-pest insects prevents the needless use of pesticides, while lowering input costs, protecting the environment and delaying the development of pesticide resistant strains. Another goal of sustainable agriculture is to implement the use of natural substances to fight pathogens. Several phytochemicals have been studied for their biological activity, including antioxidant, insecticidal and antibiotic properties. An increased concentration of phytochemicals in tissues may prevent the occurrence of disease and the spreading of harmful insects, and inhibit and/or reduce the spore germination of pathogenic fungi.

1.2.3. Biotechnology and Sustainability Is biotechnology compatible with sustainable agriculture? A survey of the literature indicates the presence of two opposite ways of answering this question. On the one side, biotechnology is seen as responsible for the increased commercialization of food production, in competition with food

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for human use. The enhanced market competitiveness caused by biotechnology is supposed to decrease world food security, to create a gap between rich and poor countries, to increase poverty, to decrease the ability to protect the environment and to generate a need for militarization to maintain order. On the other side, biotechnology is considered a useful tool to provide solutions to specific problems in sustainable agriculture. An important point is the correct understanding of biotechnology and its application. Owing to the peculiar characteristics of biotechnology, it can be of no help to sustainable agriculture in the short term. Its utility increases in the medium term and is of high usefulness in the long term, where it becomes a starting point for sound breeding programmes in sustainable agriculture. New technologies allow the identification of key plant genes to fight biotic and abiotic stress. In the past, geneticists have produced new varieties of plants with changes in developmental phases and plant architecture, and with higher levels of tolerance and resistance to environmental and biotic stress. This was performed by identifying the phenotypes among a large number of plants in a nesting population. Now the increase in our knowledge and gene-based diagnostics provide geneticists with more precise targets and assays to operate in crop improvement programmes. We can expect a potential increase in yield and a better quality of products that match the growing diversity of market needs. The new genetics will link agriculture with sectors beyond the food, feed and fibre industries; agri-business will contribute to public health and provide high-value products for the pharmaceutical industry and for those industries that still use petrol-based raw materials and chemical modification processes. With regards to plants producing phytochemicals, the biotechnological approach can be summarized in three points: x increasing crop productivity and phytochemicals yield; x increasing pest resistance; x increasing environmental stress tolerance. Increasing crop productivity is a matter of both agronomy and plant physiology. In terms of biotechnology, recent advances on the deciphering of several genes involved in the photosynthetic process have allowed the creation of mutants with improved photosynthetic capabilities. There is a direct relationship between the production of primary metabolites (above all sugars and lipids) and secondary metabolites, so any advantage gained

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with an increase of photosynthetic efficiency can be translated into a potential increase in the availability of basic structures to build compounds of practical interest. (See also the discussion on primary/secondary metabolism relationships in Chapter 10). Increasing phytochemical yield is a matter of both genetics and plant morphology. From a genetic point of view, increasing yield means overexpressing some genes involved in the secondary metabolism and the complete conversion of by-products whose accumulation lowers the content of quality compounds. From a morphological viewpoint, several phytochemicals accumulate in specialized tissues such as secretory structures (see Chapter 2). To produce new biological structures (i.e., more secretory tissues) and/or functions (enhanced enzyme activity), traditional plant breeders used crossing and backcrossing protocols. Now, genetic engineers manipulate DNA to increase the speed at which new biological structures and functions are produced, by short-cutting sexual reproduction and by-passing the limits of traditional breeding. Genetic engineering is approved by sustainable agriculture as long as its possible effects on the stability of the biosphere are under control. Many ecologists agree that gene flow is not an environmental problem unless it leads to undesirable consequences. In the short term, the spread of transgenic herbicide resistance via gene flow may create to growers logistical and/or economic problems. Over the long run, transgenes that either confer resistance to pests and environmental stress or lead to higher seed production possess the greatest likelihood of aiding weeds or harming non-target species. However, these outcomes seem unlikely for most currently grown transgenic crops. Many transgenic traits are likely to be innocuous from an environmental standpoint, and some could lead to more sustainable agricultural practices. It is crucial that molecular biologists, crop breeders and industry improve their understanding of ecological and evolutionary questions about the safety of new generations of transgenic crops. The major risk of applying resistance gene transfer is the development of pest resistance in weeds though gene flow via pollination. This is particularly important when herbicide-tolerant traits are transferred to crops. The case of sterile plants is quite particular, since these crops are reproduced vegetatively, but the risk is high in sexually reproducing plants. The presence of wild and weedy relatives varies among countries and regions. Table 1.3 shows examples of major crops grouped by their ability to disperse pollen and the occurrence of weedy relatives in the continental United States. This simple 2×2 matrix can be useful in identifying cases

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where gene flow from a transgenic crop to a wild relative is likely to occur. For crops where no wild or weedy relatives are grown nearby – as with soybean, cotton and maize – gene flow to the wild would not occur. Rice, sorghum and wheat have wild relatives in the United States and a relatively low tendency to outcross, which could allow transgenes to disperse into wild populations. The crops that have a high tendency to outcross and have wild relatives in the United States are sunflower, brassicas, carrot, squash, radish and poplar. There is a high potential for gene flow between these crops and their wild relatives, so care should be taken in growing transgenic varieties that might confer a competitive advantage on their hybrids.

LOW HIGH

Potential for outcrossing

Table 1.3. Major crops grouped by their ability to disperse pollen.

Soybean

Cotton Maize

Compatible weedy relatives nearby NO YES Rice Sorghum Wheat Sunflower Brassicas Carrot Squash Radish Poplar

The correct and wise use of biotechnology can improve our knowledge on gene responses to environmental changes, but we cannot ignore that any achievement has to be faced in normative terms. This norm relies again in the terms of a sustainable agriculture that evaluates the present aspirations for a continuous increase in standard of living and the right of future generations to live in a suitable environment. The increase in patents of new transgenic organisms tends to increase the viewing of organisms as tools (machines) used to produce goods rather than as members of the biosphere. This leads to an economic engagement towards the creation of new transgenic organisms and a depletion of funds devoted to the understanding of the many aspects we still ignore regarding natural life forms. If there is sustainability in biotechnology this will be the extremely powerful potential that this practice has to unlock understanding the secrets hidden in the genomes of already existing plants.

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1.2.4. Extraction of Phytochemicals Extraction requires energy, with varied resulting cost/benefit ratios depending on the country of cultivation. Developed countries utilize nonrenewable fossil-fuels by using high-input technologies, whereas developing countries use organic-fuel and lower-cost human power, with consequent deforestation, air pollution and environmental and human degradation. In developing and underdeveloped countries the cost/benefit ratio is usually kept low, often at the expense of product quality. In the case of essential oil-producing plants, a global overview on the distillation processes adopted by developed countries indicates the system used that is the most productive. On the other hand, the less efficient systems are those based on old family tradition technologies, which use technologies handed on from fathers to sons without consistent improvements. In some developed countries there is a consistent difference from grower to grower in the time of harvesting, managing the harvested material in the field (more or less dry material to be distilled) and distilling the plant material. Quite often, the distillers are old apparatuses with a low yield and a significant dispersal of thermal energy and essential oil into the environment. Eventually, the lack of subsides for extraction improvement leads many growers to give up cultivation of these plants in favour of staples like wheat and corn. In US peppermint oil production, the appropriate balance between mechanization of harvesting and distillation in tubs on one side, and multiple steam condenser systems on the other side, allow both the reduction of the cost/benefit ratio and the improvement (standardization) of distilled oil quality. The transportable tub allows cultivation of peppermint far away from the distillers and gives the opportunity of peppermint cultivation without the need of purchasing a distiller. This is the sustainable aspect of the US system, which reduces the dispersal of energy and concentrates the process in few distilling centres. With the adoption of improved distillers, able to utilize a reduced amount of fossil-fuel derived energy and to recover most of the hot water coming from the condensers (for example, to pre-heat the tubs), the use of alternative energy sources (most of the distillation is performed during the sunniest season) and environmentally-friendly cooling gases to improve condensation, there will be a chance for a sustainable future in the essential oil distillation process of peppermint and other aromatic plants. The last problem remains the plant material which is dispersed in the environment after extraction. Often, these plant residues still contain many phytochemicals and the leaching of these substances into the soil may

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perturb the natural soil equilibrium. The problem is particularly evident in those countries with restricted land availability. Adding fresh organic matter to the soil tends to boost plant growth less quickly than adding compost, which is organic matter partly decomposed by microorganisms and soil fauna. The spent material coming from the extraction of phytochemicals could be composted through processing with bacteria and/or fungi able to degrade phenolics and other soil interacting compounds, thus reducing soil contamination and pollution due to leaching. Many microorganisms degrade phenolic compounds by the synthesis of extracellular enzymes (i.e., lignin peroxidase), while other soil microorganisms convert other complex compounds into soluble products that can be taken up by plants and soil animals. The making of compost from spent material is one of the many goals for a sustainable agriculture in phytochemical production.

1.2.5. Toward what future? Only a sustainable practice in agriculture will allow the next generations to enjoy the world we are enjoying today. This requires a commitment to changing public policies, economic institutions and social values; phytochemicals production is part of the whole problem. There’s a link between restrictive organic farming and high-input agriculture, and it is sustainable agriculture. Many problems have still to be solved in sustainable agricultural practices, such as the lack of effectiveness, the lack of information, the management complexity, the scarce availability of biological agents, the high labour requirements, the lack of regulatory concerns and the high costs. Moreover, it is not known to what extent farmers will be willing to sacrifice predictable short-term profits for the unpredictable rewards of environmental stewardship. All these problems divert the race toward sustainability and the availability of funds for sustainable agriculture research in favour of the high-input agroindustry profitable in the shorter term. But this is a battle we must fight in terms of nutrient and energy dynamics and interaction among plants, animals and microorganisms, by balancing them with profit, community and consumer needs. The search for pest’s natural enemies, the adoption of alternative agricultural methods, and the deciphering and transfer of genetic information to improve phytochemical yields and resistance to biotic and abiotic stresses through biotechnology and in vitro systems, as well as the reduction of environmental pollution by the improvement of extraction

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systems, will allow a sustainable agriculture of phytochemicals for a sustainable future in developed, developing and underdeveloped countries.

1.3. Quantifying Biodiversity At the moment there is no available data that can precisely frame the distribution of all those species that make up the biological variability of our planet. We are also unable to objectively determine the role of biodiversity in maintaining ecosystem processes, to understand what could be the consequences of the depletion of biodiversity on atmospheric properties or to establish the global potential economic value of biodiversity. Although documented by centuries of taxonomic studies, our knowledge of the consistency of biological diversity is still in its infancy. We are still learning the vastness of the problem and at the same time we are destroying the knowledge that we still have to acquire. The simple observation of a physical map of our planet indicates how the distribution of species correlates to altitude gradients and their associated climatic variations. This does not mean that the biodiversity gradients we find are necessarily the consequence of altitudinal or latitudinal variations, but that many factors that affect biodiversity co-vary with altitude or latitude. Very often variations in biodiversity do not linearly follow latitude or altitude. For instance, the abundance of species in the tropical areas is certainly higher than that of temperate areas, but the wealth of species within the two environments is not directly related to latitude gradients. Comparing the hemispheres, the differences found among the floral species of rainforests have been mainly attributed to the higher homogeneity of the South American climate with respect to the higher heterogeneity of the North American continent. Sometimes it is difficult to explain why similar climatic zones show conflicting levels of biodiversity. For example, comparison between the temperate forests of central and eastern Asia, North America and Europe shows a 6:2:1 tree richness ratio. Therefore, the distribution of species and the meaning of their biodiversity cannot be studied only at low latitudes (such as in tropical zones) or at low altitudes (excluding premontane and mountainous altitude plains). Most modelling approaches developed for predicting plant species distributions have their roots in quantifying species–environment relationships. Three phases seem to have marked the history of species distribution models (SDMs):

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x non-spatial statistical quantification of species–environment relationship based on empirical data; x expert-based (non-statistical, non-empirical) spatial modelling of species distribution; x spatially explicit statistical and empirical modelling of species distribution. Species distribution models are empirical models relating field observations to environmental predictor variables, based on statistically or theoretically derived response surfaces. Species data can be simple presence, presence–absence or abundance observations based on random or stratified field sampling, or observations obtained opportunistically, such as those in natural history collections. Environmental predictors can exert direct or indirect effects on species, arranged along a gradient from proximal to distal predictors, and are optimally chosen to reflect the three main types of influences on the species: x limiting factors (or regulators), defined as factors controlling species eco-physiology (e.g. temperature, water, soil composition); x disturbances, defined as all types of perturbations affecting environmental systems (natural or human-induced); x resources, defined as all matter that can be assimilated by organisms (e.g. energy and water). These relationships between species and their overall environment can cause different spatial patterns to be observed at different scales, often in a hierarchical manner. For instance, a gradual distribution observed over a large extent and at coarse resolution is likely to be controlled by climatic regulators, whereas patchy distribution observed over a smaller area and at fine resolution is more likely to result from a patchy distribution of resources, driven by micro-topographic variation or habitat fragmentation. Although species richness is a natural measure of biodiversity, it is an elusive quantity to measure properly. For diverse taxa, as more individuals are sampled, more species will be recorded. The same, of course, is true for higher taxa, such as genera or families. The principles of species accumulation, rarefaction, species richness, and species density have been established for many decades. However, ecologists have only recently begun to incorporate these concepts into their measurements of species diversity patterns and evaluation of theory

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in community ecology and biogeography. These tasks are especially important as ecologists attempt to inventory species-rich communities and document the loss of species diversity from habitat destruction and global climate change. Ecologists may have avoided individual-based and sample-based rarefaction curves because they are computationally intensive, but public-domain software is now available for these calculations.

1.4. Classification and Characterization of Natural Compounds Classification is defined as the sorting into groups of organisms and molecules on the basis of similarities and/or differences. If we refer only to plants then we can add another term, taxonomy, which is part of the classification process. Systematics can study various aspects of taxonomy, including evolutionary processes, so the concept of systematics is seen as the merging of taxonomy, on the one hand, and short and long term evolutionary processes, on the other. The scientific basis for studying natural molecules rests without any doubt on the correct classification of the plants from which they are extracted. Many accurate analytical chemistry papers are unfortunately invalidated by the inaccurate, if not incorrect, classification of the starting plant material. For this reason, at the end of this introductory chapter, it is appropriate to discuss the botanical and chemical classification criteria. We can consider four important areas related to the concept of classification: taxonomy, evolution, character and data analysis.

1.4.1. Taxonomy Taxonomy is a sorting process where individuals are grouped to define a taxon, which in turn is ranked into categories. There are other aspects associated with the classification and these are the identification, where individuals refer to a taxon and the nomenclature that assigns a specific name to the taxon. Sometimes the two terms, nomenclature and identification are confused with classification and systematic, respectively. It should be remembered that nomenclature is the naming of groups and organisms and the rules governing the application of these names. Identification, on the other hand, involves referring an individual specimen to a previously classified and named group.

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1.4.2. Evolution Evolution is the change in heritable characteristics of biological populations over successive generations. Evolutionary processes give rise to the biodiversity at every level of biological organization, including the levels of species, individual organisms and molecules. In the study of evolutionary processes, there are sources of variability, organization of genetic variability in populations, population differentiation, reproductive isolation and species origin and hybridization. On the other hand, in the study of phylogenetics, there is divergence and/or development between all groups in time and space.

1.4.3. Character Characters are usually described in terms of their states, for example: hair present vs hair absent, where hair is the character, and present and absent are states. A taxonomic character is the characteristic of an organism that can be subdivided into at least two conditions (or states) and is used to build classifications and associated activities (especially identification). If the characteristics of an organism are used to determine the process of evolution, especially at the population level, then we refer to a systematic character. The character state is the particular form or value that is manifested by a variable character in a specific individual or taxon. The different characters’ types can be distinguished according to the organization of their states, the variability of characters and states, their specific utility and their general validity. There are various character types, for example quantitative characters, referring to metric (count or measure) estimates in which the states are expressed in numbers, and qualitative characters that describe the form and structure and where the states appear in descriptive words rather than in numbers. Filetic characters are mainly used in phylogenetic classifications, distinguishing the homologous characters from the analogous ones, while cladistic characters are the result of cladistic classification that seeks to determine bifurcations in evolutionary sequences in an attempt to use them as a classification criterion. Phenetic characters are not expected to reflect evolution. Another important point in phenetics is the use of unweighted characters.

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1.4.4. Data Analysis In order to classify organisms, we need comparative data. These can be obtained in various ways and a plant taxonomist must always be able to handle such data, no matter their origin or nature. There is an infinity of data that can be used for plant taxonomy, and we can broadly group them into seven main categories of data: morphological, anatomical, palynological, cytological, cytogenetic and genetic, chemical, and ecological. 1.4.4.1. Morphological Data Morphological data can be devised into two main types: macromorphological and micromorphological. The former are those from optical, stereoscopic microscope observations, obtained with a lens or simply observing the sample under consideration. Observing the shape of a plant is a clear example. Micromorphological data are obtained using sophisticated instrumentation such as scanning electron microscopy (SEM) and offer the possibility to add more accurate details, measurements and definitions of objects that are not otherwise visible. The surface of the leaves of an aromatic plant can reveal a world invisible to the human naked eye. Morphological data can be derived from vegetative morphological characters represented by leaf blades, epidermises, cuticles, shoots, stems and roots, but may also originate from reproductive morphological characters such as the corolla epidermis and fruit and seed surfaces. 1.4.4.2. Anatomical Data The anatomy, or the internal form, of an organism is another important source of taxonomic data. We can distinguish endomorphic data (as opposed to exomorphic or morphological data) that are derived by optical microscopy and ultrastructural data that are observable by the use of transmission electron microscopy (TEM). The internal analysis of leaves, stems and roots provides a much greater amount of data than can be obtained from the analysis of reproductive organs. The leaves provide many anatomical features, while shoots offer much taxonomic evidence. Roots provide fewer taxonomic data.

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1.4.4.3 Palynological Data A large amount of work has been carried out using pollen granules as taxonomic characters. In many studies, the number and shape of the openings, the shape of the granules and the structure of the external cell wall have been used at each level of classification hierarchy. External characteristics are evaluated by the use of optical and SEM microscopy, whereas internal ones (the study of the inner wall and protoplasm) are obtained with TEM. 1.4.4.4. Cytological Data Although cytology mainly describes the cell, the importance of cytology in taxonomy is centralized in the study of chromosomes. Different types of data derived from chromosomes have been used in taxonomy, data such as the number, amplitude, shape, behaviour during meiosis, and DNA content. The predominantly studied organs for mitosis are the meristematic cells of roots, while anthers are studied to obtain information about meiosis. Much used in taxonomy is the chromosome number, while karyotypes provide information on the amplitude and shape of the chromosomes, also indicating the position of the centromere. The chromosome banding technique also provides additional data to the karyotype, contributing considerably to taxonomic classification. 1.4.4.5. Cytogenetic and Genetic Data Cytogenetics studies chromosomes and their genetic implications, while genetics is the science of inheritance and variability. These two disciplines are often used together for taxonomic purposes as a source of comparative data. Observing chromosomes is extremely useful, but understanding the genetic content of a chromosome is definitely more important. There are currently few studies dedicated to the determination of the genetic basis of taxonomic characters used for classification purposes, while isozyme analysis has been used to provide a faster method for determining the genetic distances between taxa. Isozymes are allelic variants of genetically controlled enzymes, which carry various metabolic functions in plants. Cytogenetic data provide the ability to reveal degrees of relationship between taxa using homologies present in chromosomes, reproductive capabilities, and reproductive potential through natural and/or artificial hybridizations.

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1.4.4.6. Chemical Data In taxonomy, the analysis of chemical data allows the study of a variety of gene expressions and is an extremely powerful tool for a wide range of research. Taken to their extreme limit, chemical data should be able to go beyond cytology and genetics by allowing direct comparison with DNA sequences. It is possible to subdivide chemical data into micromolecular, macromolecular and other molecular types of data. Micromolecular data are the most commonly used, with phenolic compounds (see Chapter 6) belonging to this category and including phenylpropanes, benzoic acid derivatives, coumarins and furanocumarins, stilbenes, flavonoids, anthocyanidins, catechins, procyanidins, and polymeric forms represented by hydrolysable and condensed tannins. Terpenoids (see Chapter 7) also provide micromolecular data, especially monoterpenes and sesquiterpenes. Low molecular weight alkaloids, betaine and glucosinolates (see Chapter 9) correlate well with morphological and genetic data, providing ultimate and useful data for classification criteria. Among macromolecular compounds, there are molecules capable of solving taxonomic problems at higher hierarchical levels. Electrophoresis of seed proteins shows a number of bands that can be compared between different taxa and can be considered phenetically just like many other data. DNA-DNA hybridization has been successfully used in taxonomic studies. An extremely useful instrument is fragmentation by nucleic, mitochondrial and plastid DNA endonucleases, especially when applied to phylogenetic studies. Other molecules have been used for chemotaxonomic investigations as will be discussed in Chapter 5. 1.4.4.7. Ecological Data Ecological data represent a special type of comparative data when used in the study of taxonomy, with particular reference to plant–environment interactions. This is especially important in plants that produce secondary metabolites that, as we have already mentioned and will discuss in Chapter 3, are phenotypically responsive to environmental variations. One of the most commonly used ecological data in taxonomy is distribution. The correlation between maps and data obtained from vegetation zones provides a considerable amount of data usable to understand the similarities and the ecological differences between taxa. In the case of

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secondary metabolites, however, we must not forget that biotic and abiotic factors play a considerable role. Among abiotic factors are soil characteristics, geology, atmospheric conditions, humidity, climatic variations, altitude and quality and quantity of light. Among biotic factors, a significant influence on the distribution and reproduction of plants that produce secondary metabolites is exerted by predators, pathogens, weeds, and biotic associations (such as those that characterize plant–plant, plant– insect, plant–fungi and plant–bacteria interactions).

Suggested Reading Allahyari, M.S. (2009). Agricultural sustainability: implications for extension systems. Afr. J. Agricult. Res. 4, 781–786. Bajwa, A.A. (2014). Sustainable weed management in conservation agriculture. Crop Protection. 65, 105–113. Bradshaw, A.D. (1965). Evolutionary significance of phenotypic plasticity in plants. Adv. Genet. 13, 115–155. Brewer, M.J. and Goodell, P.B. (2012). Approaches and incentives to implement integrated pest management that addresses regional and environmental issues. Annu. Rev. Entomol. 57, 41–59. Brooks, T.M. et al. (2006). Global biodiversity conservation priorities. Science. 313, 58–61. Büchs, W. (2003). Biodiversity and agri-environmental indicators: general scopes and skills with special reference to the habitat level. Agric. Ecosyst. Environ. 98, 35–78. Convention on Biological Diversity (1992). United Nations Environment Programme (UNEP) Na.92-7807. 5 June. Conway, K.E. (1996). An overview of the influence of sustainable agricultural systems on plant disease. Crop Protection. 15, 223–228. Crouch, M.L. (1995). Biotechnology is not compatible with sustainable agriculture. J. Agric. Environm. Ethics, 8, 98–111. Dennis, E.S. et al. (2008). Genetic contributions to agricultural sustainability. Philos. Trans. Royal Soc. B-Biol. Sci. 363, 591–609. Duvick, D.N. (1995). Biotechnology is compatible with sustainable agriculture. J. Agric. Environm. Ethics. 8, 112–125. FAO (2003–2004). The state of food and agriculture, Chapter 5: health and environmental impacts of transgenic crops. Box 23. Goodland, R. (1995). The concept of environmental sustainability. Annu. Rev. Ecol. Syst. 26, 1–24.

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Gotelli, N.J. and Colwell, R.K. (2001). Quantifying biodiversity: procedures and pitfalls in the measurement and comparison of species richness. Ecol. Lett. 4, 379–391. Guisan, A. and Thuiller, W. (2005). Predicting species distribution: offering more than simple habitat models. Ecol. Lett. 8, 993–1009. Hoffman, C.A. and Carroll, C.R. (1995). Can we sustain the biological basis of agriculture? Annu. Rev. Ecol. Syst. 26, 69–92. Maffei, M. (1990). Plasticity and genotypic variation in some Mentha x verticillata hybrids. Biochem. Syst. Ecol. 18, 493–502. —. (1998). Sustainable Agriculture and Phytochemistry. Rec. Res. Devel. Phytochem. 2, 107–118. —. (1999). Sustainable methods for a sustainable production of peppermint (Mentha x piperita L.) essential oil. J. Essent. Oil Res. 11, 267–282. Mann, C.C. (1999). Crop scientists seek a new revolution. Science. 238, 310–316. Moir, W.H. and Mowrer, H.T. (1995). Unsustainability. Forest Ecol. Manag. 73, 239–248. Moran, K., King, S.R., and Carlson, T.J. (2001). Biodiversity prospecting: lessons and prospects. Annu. Rev. Anthropol. 30, 505–526. Pretty, J. (2008). Agricultural sustainability: concepts, principles and evidence. Phil. Trans. Royal Soc. B-Biol. Sci. 363, 447–465. Rozzi, R. et al. (2008). Changing lenses to assess biodiversity: patterns of species richness in sub-Antarctic plants and implications for global conservation. Front. Ecol. Environ. 6, 131–137.

CHAPTER TWO SITES OF SYNTHESIS AND STORAGE OF BIOACTIVE PLANT MOLECULES

Photosynthesis, the process by which plants build up organic molecules from carbon dioxide and water, provides the basic building blocks for the synthesis of many metabolites that are essential for plant life. Among these are secondary metabolites, which are synthesized using sugars, amino acids and numerous intermediates of primary metabolism. Some plants have evolved the ability to synthesize and accumulate secondary metabolites in significant quantities through specialized structures collectively defined as secretory tissues. This chapter will describe the main sites of synthesis and accumulation of the major secondary metabolites, with particular reference to secretory structures that produce lipophilic substances.

2.1. Secretion In general terms, secretion is the passage from the inside to the outside of the cell plasma membrane of molecules processed by the cell; such molecules (defined as secretion) have a specific function in the organism that produces them. The term secretion is often used together with the term excretion and in the literature there are numerous attempts to define the two terms. In animal biology, excretion is defined as the process by which metabolic waste substances and other non-useful materials are eliminated from the organism, while secretion is the process where the substance may have specific tasks after leaving the cells (e.g., may be involved in the organism’s growth and development processes). Secretion is a usual result of the metabolic activity of any cell, and is particularly evident in glandular cells. The process usually takes place by extrusion of material, but it may also involve the cell’s destruction. The definition of glandular cells depends on the degree of their cytological

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specialization, which is usually manifested by the presence of a dense cytoplasm with a high number of active organelles and membranes. It also involves a certain degree of physiological specialization, which depends on more or less specific processes for the elimination of substances. The concept of secretion contrasts with diffusion as it involves work to transport a substance through the plasma membrane against its concentration gradient, thus opposing the diffusion forces. Plant cells have the ability to extrude secreted substances out of the protoplasm or inside the vacuoles. Secretion occurs as: x intracellular storage, as is the case when several substances are encrusted inside the cell wall (lignins, cuticles, waxes, suberins, etc.), x intracellular secretion of secreted substances in compartments surrounded by membranes within the cell plasma membrane, and x extracellular secretion by releasing the secreted substances out of the plasma membrane, in which case, the intracellular accumulation can be either present or absent. The main mechanisms through which molecules are eliminated by the cell are illustrated in Figure 2.1 and can be classified into: x holocrine secretion, where substances are secreted after the disintegration of the cells that produced them; and x merocrine secretion, where substances are secreted from the cytoplasm of cells that remain intact; this latter process can be further subdivided into: o eccrine secretion, when the secreted substances pass through the plasma membrane or through the tonoplast along a concentration gradient or through an active transport process; and o granulocrine secretion, when the produced substances are accumulated in vesicles delimited by a membrane that may merge with the plasma membrane or with the tonoplast. Alternatively, the vesicles can be phagocytised by the plasma membrane surrounding them by eliminating their content out of the protoplast.

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We can also divide secretory cells into two large categories, depending on the nature of the secreted compound: (i) secretory cells that produce hydrophilic substances, and (ii) cells whose secretion is lipophilic. The first category encompasses secretory tissues that produce a mixture of various substances, mainly attributable to primary metabolism or related to basal metabolic processes. Mucilage glands are part of the hydrophilic secretion tissues; they secrete mucopolysaccharides and are typical of carnivorous plants such as the genera Drosophyllum, Drosera and Pinguicola. They are used to trap small insects that remain glued to the secretion.

Figure 2.1. The secretion process: cytological model. Elimination can be achieved by transmembrane transport of molecules using the eccrine secretion mode (1) or by transporting the molecules into vesicles using the granulocrine transport mode

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(2). The latter can occur as the secretion is (2a) or following modification of the secretion (2b) that can be accumulated in vesicles or cellular compartments. Storage in compartments such as the vacuole can be by molecular (3) or vesicular (4) transport. The cell lysis eventually leads to the loss of cellular integrity.

Much like mucilage glands are the digestive glands that, in addition to producing polysaccharides, secrete proteolytic enzymes. In some cases, as in Nepenthes, the modified leaves contain a fair amount of digestive fluids, while in others (as in the above mentioned Drosophyllum, Drosera and Pinguicola) secretion begins after insect contact and capture. Nectaries are secretory tissues that release a fluid with a high sugar content (the so-called nectar). They are mainly found in flowers, but there are also extrafloral nectaries. Other secretory tissues that produce hydrophilic substances are hydathodes that can be organized in glandular tissues and that extrude water and other substances into the outer environment. Very similar to hydathodes are the salt glands, which extract large amounts of mineral salts (particularly sodium chloride) by active transport and storage mechanisms. With regards to plant bioactive molecules, the major producers are secretory tissues that synthesize and store lipophilic substances. Their importance is morphophysiological, because of the differing nature of the forms and molecules produced, as well as economic, as the produced substances represent a non-negligible part of the world economy as functional compounds, due to their high bioactivity. Unlike hydrophilic secretions that are extruded predominantly by merocrine secretion, most lipophilic compounds are released by holocrine secretion. There are known cases where the compound is released in a first phase by a merocrine secretion to be finally expelled by holocrine secretion. Given the complexity and vastness of the structures we will discuss the major secretory structures involved in the mechanism of the extrusion of lipophilic molecules.

2.2. Glandular Trichomes Lipophilic molecules are secreted by a wide variety of anatomical structures, from simple cells such as epidermal idioblasts to aggregates. They are also secreted by structures with a large degree of specialization that are present on the plant surface (such as glandular trichomes) or inside roots, shoots, leaves and fruits (like resin ducts and lysigenous cavities). Among the secreted molecules, the major constituents are terpenoids (see

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Chapter 7), flavonoids (Chapter 6), fatty acids, waxes and aromatic amines. Volatile organic compounds (VOC) are emitted into the atmosphere and most of them are of biogenic origin. Several plant species store VOCs in specialized glandular trichomes, which release their contents in response to tissue damage, thus deterring herbivores or inhibiting microbial growth. Over 90% of VOC emissions are produced from natural forests around the world, the most important among them being the Amazonian rainforest. Up to 36% of the carbon taken up by plants is released as complex mixtures of VOCs. Unlike methane, VOCs produced by plants in the troposphere are extremely reactive, with lifetimes ranging from minutes to hours, contributing to the formation of an aerosol which diffuses light to produce the blue sky. VOCs are also released into the atmosphere from leaf and flower trichomes. The main features of VOCs released into the air are plant defence against herbivores and pathogens, attraction of pollinators and other animals and beneficial microorganisms, and seed dispersal as well as plant–plant communication signaling. In some plants, the release of VOCs can also seal wounds. Chemically, VOCs belong to the large group of terpenoids (homo-, mono-, di-, and sesquiterpenoids), fatty acids (that originate C6-volatile compounds and their derivatives, see Chapter 8) and aromatic phenylpropanoids (such as methyl salicylate and indole) as well as some alkanes, alkenes, alcohols, esters, aldehydes and ketones. To date more than 1800 volatile compounds have been isolated from more than 90 plant families, accounting for about 1% of all secondary plant metabolites. The composition of VOCs emitted by plants also depends on the plant damage, whether it is a single injury or continuous damage, such as that caused by herbivore attacks or the simple deposition of eggs (see Chapter 3). VOC production generally shows pronounced rhythmicity by emission of substances especially during the diurnal phase, in conjunction with the photosynthetic process. The pubescence is typical of the surfaces of many plant species and such term indicates the presence of structures emerging from the epidermis known as glandular trichomes or glandular hairs. The morphology of these structures is extremely variable and more than 300 morphological types have been described so far. These structures develop from protodermal cells formed because of anticlinal and periclinal divisions. Being

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protodermal extrusions, glandular trichomes are present on leaf blades, flowers and, in some cases, seed surfaces. In Arabidopsis thaliana mutants unable to produce leaf trichomes, the production of root hairs continues, clearly indicating that different genes control the formation of trichomes and root hairs. Regarding secretion, glandular trichome isolation techniques have allowed the demonstration that these structures are the only site of synthesis of certain substances, such as, for example, monoand sesquiterpenes. In some plant families, such as the Lamiaceae, the most common form of glandular trichomes is of the peltate type, characterized by a globular dome formed by the detachment of the cuticle from the cell wall. Other types of trichome include monoseriate and biseriate structures with one (capitate or sessile trichomes) or more (peltate trichomes) secretory cells. In any case, a basal cell always serves as the physical connection between the secreting cells and the rest of the leaf. Sometimes the metabolite flow passes through one or more connecting cells placed between the basal cell and the secretory cells; these cells are defined stem cells. As we will discuss in Chapter 7, the biochemistry of specialized metabolites produced by glandular trichomes is important because of the interaction that many VOCs have with the external environment, including herbivores, predators and pollinators. Understanding the mechanisms of formation and differentiation of trichomes and assessing the gene expression of the enzymes that produce bioactive molecules is of fundamental importance for sustainable use and biological control strategies. We will now discuss some types of glandular trichomes present in important plant families.

2.2.1. Glandular Trichomes of the Lamiaceae Family The Lamiaceae (Labiatae) family is particularly rich in species that produce flavours and fragrances used in popular medicine, mentioned in the Official Pharmacopoeias or used for various purposes by the food and cosmetics industries. In this family, the study of glandular trichomes has been extended to structure, histochemistry, ontogenesis, biochemistry and molecular biology, particularly in the major genera belonging to the Nepetoideae subfamily. The glandular trichomes present in this family are predominantly of two types: peltate and capitate (see above). The peltate trichomes consist of a

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secretory head formed of 4–14 secretory cells, depending on the species, supported by a stem cell linking the secretory head to the basal cell, which is placed next to the epidermal cells. In the capitate trichomes, the single secretory cell is thought to diffuse the secretion into the environment without accumulating it in the subcuticular space, as in peltate trichomes. In many Lamiaceae, there are up to three different types of trichomes, according to form and the mechanism of secretion. The ecological significance of peltate trichomes is related to their constitutive defence mechanism, because they accumulate the secreted substances. On the other hand, capitate trichomes show constitutive/ induced mechanisms, because some of them may emit the substances they produce directly into the environment. One of the most important genus in the family is Origanum. Glandular trichomes of Origanum dictamnus L. originate from a single protodermal cell. They are composed of a 12-celled secretory head, a unicellular stalk and a basal cell. During the early stages of trichome differentiation, the secretory cells possess a small number of plastids which contain globular inclusions. Similar inclusions are also observed in the plastids of the stalk and the basal cell. The lateral walls of the stalk cell progressively undergo cutinization. At the onset of secretion, the electron density of the plasmalemma region lining the apical walls of the head cells remarkably increases. These walls are impregnated with an osmiophilic substance identical in appearance to the content of the subcuticular space. In a following stage of the secretory process, osmiophilic droplets of various sizes arise in the cytoplasm of the secretory cells which undergoes simultaneously a reduction of its initial density. After secretion has been concluded, the protoplast of the head cells becomes gradually degenerated. Paradermal sections of glandular trichomes show the organization of twelve secretory cells in a central tetrad surrounded by eight peripheral cells. The lipophilic nature of the secretion can be demonstrated by histochemical staining of the glandular trichomes with the dye Sudan Black B (Figure 2.2). Only the secretory cells and the subcuticular space are stained, while the stalk cell, the basal cell and other mesophyll cells remain unstained.

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Figure 2.2. Ontogenesis of glandular trichomes in Origanum dictamnus. FC = basal cell; IGC = initial glandular trichome cell; MH = mother cell; SC = stem cell; SS = subcuticular space. The initial cell protrudes outwards (1–3) and then divides to give rise to MH and SC cells (4–6). The MH cell initiates anticlinal divisions that form the secretory head (6–8). Secretory cells produce VOCs that accumulate in the SS (9) and are released into the environment when the cuticle breaks down (10). Paradermal sections of the trichomes show twelve secretory cells (11), while staining with Sudan Black B shows the areas where secretion occurs (12). The same result is obtained by exposing leaves directly to osmium tetroxide vapors (13). From Bosabalidis and Tsekos, 1982.

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One of the most studied plants is peppermint (Mentha piperita). In this hybrid (as well in other species like spearmint, M. spicata), there are two types of glandular trichomes: peltate trichomes, containing 8 secretory cells, a stalk cell and a basal cell, and capitate trichomes, with a secretory cell, a stalk cell and a basal cell. Histochemical and biochemical studies have revealed that the secretory cells are the only site of synthesis of the lipophilic products (mainly monoterpenes and sesquiterpenes). During leaf development, a higher trichome number is produced on abaxial epidermises. Moreover, the trichome number is not fixed at the time of leaf emergence, and trichomes grow dynamically during the leaf ontogenetic development producing a VOC composition that is individually different, both from a quantitative and a qualitative point of view. With the use of morphometric techniques and microfluorimetric and electron microscopy (TEM and SEM) the dimension, the level of ploidy and the morphology of the nuclei of different cell types (basal, stem and secretory) have been investigated. The results showed a nuclear hypertrophy in the secretory cell of both trichome types, which was explained by polyploidization and by a variation in chromatin structure that may be related to increased transcriptional activity. In the genus Mentha, glandular trichomes are not restricted to leaves. The green parts of flowers, such as the calix, are particularly rich in glandular trichomes of the peltate and capitate type, while the density of trichomes is extremely low on the petals (Figure 2.3).

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Figure 2.3 Peltate and capitate trichomes of the genus Mentha. (a) Early division of secretory cells and distinction between basal and stem cells. Volatile terpenes accumulate in the subcuticular space (b) where the staining with pphenylenediamine shows the nature of both lipophilic (dark colour) and hydrophilic (light colour) substances inside the subcuticular space. In the stages of maturity (c), secretory cells degenerate and form large vacuoles, while the content of the subcuticular space is less electron-dense. By staining the DNA of the capitate trichomes, it is possible to quantify its nuclear content in the different cells. The fluorescence microscope image after dyeing with DAPI (d) shows a nucleus of the secretory cell with a higher ploidy (greater size) with respect to the nuclei of the other cells. (e) Mint flowers show many glandular trichomes, especially on the calix.

The leaves of Prostanthera ovatifolia possess glandular trichomes made of a basal cell sunken in the epidermis, a stem cell with a strongly cutinized

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cell wall and a secretory head formed of 16 cells (Figure 2.4). The subcuticular space contains a mixture of hydrophilic and lipophilic substances. The secretory cells possess numerous leucoplasts that are immersed in a dense cytoplasm. Volatile compounds are secreted into the subcuticular space by granulocrine secretion.

Figure 2.4 Peltate glandular trichomes of Prostanthera ovatifolia. (A) Epifluorescence microscopy shows peltate glandular trichomes (PGT) immersed in the epidermis (E). The content of trichomes causes a bright autofluorescence. A higher magnification (b) shows the typical shape of the trichomes and the subcuticular space filled with hydrophilic (Aq) and lipophilic (L) substances and delimited by the cuticle (EC). The intense fluorescence is due to the presence of cutin and suberin in the stem cell wall (St). From Gersbach, 2002.

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2.2.2. Glandular Trichomes of the Asteraceae Family The Asteraceae (Compositae) family is composed of numerous genera producing glandular trichomes that secrete lipophilic substances. In some cases, as in the genus Inula, mixtures of hydrophilic and lipophilic compounds are secreted. In this family, one of the most studied genera is Artemisia because of the economic importance of its terpenoids which are used both for flavouring and for the treatment of infectious diseases, such as malaria. In this genus, trichomes are of the biseriate type and are formed by two basal cells, two stem cells and three pairs of secretory cells. During the early phases of cell division, plastids are present as proplastids with a few thylakoids. When cellular divisions are completed, numerous chloroplasts differentiate in all trichome cell types. Figure 2.5 shows a cross section of a peltate glandular trichome of Artemisia annua.

Figure 2.5 Peltate glandular trichome of Artemisia annua; last stage of development of a peltate biseriate trichome. The two apical secretory cells and the four cells which contain chloroplasts are recognizable. Two stem cells and two basal cells are also evident. The arrows indicate a dense osmiophilic deposit in direct contact with the secretory cells. From Duke and Paul, 1993.

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2.2.3. Glandular Trichomes of the Geraniaceae Family The aromatic plant Geranium robertianum is covered by uniseriate glandular trichomes attributable to three types: type I trichomes are decumbent and possess an apical oval secretory cell, two stem cells and a basal cell; type II trichomes are erect and have a pear-shaped apical secretory cell, two stem cells and a basal cell; type III trichomes are much longer than the previous ones with an elongated apical cell, five long stem cells and a basal cell. Type I and II trichomes are mainly found on leaves, while type III trichomes are particularly abundant on flowers. While type I and II trichomes secrete terpenoids and phenols, type III trichomes accumulate anthocyanins and secrete flavonoids. Geranium (Pelargonium graveolens) also shows three morphologically distinct types of trichomes: non-glandular, peltate and capitate glandular trichomes. With regard to capitate trichomes of type I, there are three typologies. They are formed by a basal cell, a short stem (1–3 cells) and a globular head which can be round or elongated with the presence of secretion droplets. Type II, with a long stem and a pear shaped or spherical secretory head, have a very thin cuticle that forms a large subcuticular space filled with secreted material. The number of stalk cells ranges from 3 to 5. Type III capitate trichomes show a cup-shaped secretory head in which a cuticle fracture is often observed. The stem cell is short and this type of trichome is present on leaf blades, stems and sepals. The peltate glandular trichomes have a basal cell and a short stem, with cutinized side walls and a large flattened secretory head. In the subcuticular space, granular vesicles are involved in the secretory process. These trichomes are particularly present on the stems and calyxes (Figure 2.6).

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Figure 2.6 Glandular trichomes in the aerial parts of Pelargonium graveolens; (a), (a) and (f): SEM; (c–i): Light Microscopy (LM). (a) Capitate Glandular trichome Type I with one globular head cell on the abaxial side of leaf lamina (fixed material). Scale bar 50 μm. (b) Upper view of capitate glandular trichome Type I with one spherical head cell surrounded by stomata (ST) on the abaxial side of sepal. Scale bar 50 μm. (c) Cross section in leaf lamina examined under LM showing a capitate glandular trichome Type I with a unicellular spherical head. A few secretory droplets are observed. (N, neck cell; EO, essential oil; C, secretory cavity; D, droplet; T, stalk). G×100. (d) Capitate glandular trichome type I with unicellular elongated head and one stalk cell (T) observed on upper epidermis of stem. G×40. (e) Capitate glandular trichome type II showing a pear-like head with large subcuticular space nearly full of secreted material and elongated stalk (three or four cells) observed on upper epidermis of leaf lamina. G×100. (f) The crosssection of the abaxial side of the leaf lamina showing one capitate glandular trichome type III (Cg) with cup like head cell and elongated stalk (fixed material). Scale bar 50 μm. (g) Cross-section in sepal showing a capitate glandular trichome

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type III with basal cell (BC), stalk (T) and a cup-like head (H). G×100. (h) Upper view of peltate glandular trichome with one large flattered head (h) a torn cuticular sheath (Tc) with short stalk (T) and neck cell (N) obvious on upper epidermis of sepal. G×100. (i) Peltate glandular trichome exhibiting inner secretory vesicles (SV) in the secretory cavity with thick stalk (T) obvious on upper epidermis of stem. G×100. From Boukhris et al., 2013.

2.2.4. Glandular Trichomes of the Moraceae Family The cones of hops (Humulus lupulus) produce the compound xanthohumol and other hop terpenophenolics which accumulate primarily in peltate glandular trichomes. The latter are termed lupulin glands and are visible as yellow structures at the base of bracteoles in hop cones (Figures 2.7a and b). Lupulin glands are composed of a disk of biosynthetic secretory cells and a subcuticular cavity in which terpenophenolics and VOCs (i.e., monoterpenes and sesquiterpenes) are stored. Initially concave and cuplike, the trichomes develop a peaked appearance as the subcuticular cavity fills during ripening. Peltate trichomes are formed from a protodermal cell following two anticlinal divisions followed by two periclinal divisions, leading to the formation of the initial cells of the secretory head, stem and basal cell. Upon development, peltate glandular trichomes consist of a head made up of 30 to 72 cells, some stem cells and four basal cells. Bulbous trichomes are formed by a head with four (rarely eight) secretory cells, two stem cells and two basal cells (Figure 2.7).

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Figure 2.7 Morphology of Hop (Humulus lupulus) Cones and Lupulin Glands. (a) Cones of hop cultivar Taurus. Cones are about 5 cm in length. (b) Longitudinal section of a hop cone showing lupulin glands at the base of bracteoles. (c) A light microscopy image of ripe lupulin glands. Bar = 500 μm. (d) Scanning electron micrograph of a ripe lupulin gland showing the peaked appearance of the filled subcuticular sac. Bar = 100 μm. From Nagel et al., 2008.

2.2.5. Glandular Trichomes of the Cannabaceae Family In Cannabis sativa, bioactive cannabinoids are synthesized in glandular trichomes present mainly on female flowers. The main product of the secretion in C. sativa is ǻ9-tetrahydrocannabinol (THC). The formation of glandular trichomes begins with the enlargement of an epidermal cell that undergoes anticlinal divisions. A subsequent periclinal division forms a pair of upper cells that will give rise to the secretory cell and inferior cells

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that will form an auxiliary cell corona. The latter divide periclinally to give rise to stem and basal cells. Divisions occurring in the secretory cell layer produce a final number of 8–13 cells whose activity leads to the formation of a secretion that accumulates in the subcuticular space. The subcuticular space is also filled with fibrous material released from the surface of the secretory cells along with a large number of vesicles (Figure 2.8).

Figure 2.8 Trichomes of Cannabis sativa L.: (a) trichomes on the flower, (b) capitate-stalked trichome, (c) intact capitate-stalked trichome. T = head of the gland; S = stem. From Happyana et al., 2013.

Recent advances on the cellular localization of cannabinoids biosynthesis has focused on capitate trichomes as the main site of synthesis and storage. It was confirmed that the trichome secretory head is the main production site of cannabinoids demonstrating the presence of cDNAs encoding three polyketides, the MEP pathway and tetrahydrocannabinolate (TCHA) synthase.

2.2.6. Glandular Trichomes of the Solanaceae Family Glandular trichomes of the Solanaceae have been studied in detail, especially those of the Solanum species, because of their role in plant resistance. Typically, eight different types are distinguished of which four (types I, IV, VI and VII) are glandular capitate trichomes and four (types II, III, V and VIII) are non-glandular (Figure 2.9). Of the glandular trichomes, types I and IV are capitate, whereas types VI and VII are globular. The glandular trichome types differ in number of stalk and secretory cells, as well as in their chemical contents. In the cultivated tomato (Solanum lycopersicum), type I trichomes contain mostly acyl glucoses, while type VI trichomes contain terpenoids.

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Trichomes of type I and IV (that probably belong to the same type), look physically similar to non-glandular trichomes, but they differ by the presence of one or two glandular cells in the tip, which secrete acyl sugars. Type VI glandular trichomes are composed of four secretory cells on a two-celled stalk, and secrete metabolites that are stored under a waxy cuticle. In the cultivated tomato, type VI trichomes contain monoterpenes (mainly Į- and ȕ-pinene) and several sesquiterpenes.

Figure 2.9 Glandular trichomes in section Lycopersicon. Wild accessions have high densities of glandular trichomes that confer resistance to several pests. (a) Leaflet surface of Solanum habrochaites acc. LA 1777 with high densities of glandular trichome types IV and VI (b), and type I (c). (d) Surface of Solanum

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pennellii acc. LA 716 which is also covered by type IV trichomes (d, e), producing and secreting acyl sugars, and type VI trichomes, but in low density (f). (g) Surface of Solanum lycopersicum cv. Moneymaker with a low density of type VI trichomes (h) and type I trichomes. Sometimes, type IV-like trichomes (i) are observed on stems, veins, and on the leaflet edges. Metric bars = 500 ȝm (a, c, d and g) and 50 ȝm (b, e, f, h and i). From Glas et al., 2012.

2.3. Secretory Cavities and Resin Ducts Lipophilic secretion also occurs in secretory tissues that are located within the body of the plant. These tissues are capable of secreting their products in intercellular spaces that develop by either cell schizogeny (i.e., spreading apart of cells) or lysigeny (i.e., programmed cell death and dissolution). Sometimes these structures form by a mechanism that involves both stages of cellular development. In some cases, as we shall see later in the laticifers, the secreted substances accumulate within the cells. Secretory ducts that produce lipophilic substances are found in several plant families, especially in the Pinaceae, Anacardiaceae, Asteraceae, Hypericaceae, Leguminosae and the Apiaceae. In the conifers, secretory ducts produce a resin formed by mono-, sesquiand diterpenes, and for this reason they are also known as resin ducts. The development of the duct takes place via schizogeny, through the dissolution of the middle lamella between the duct initials and the formation of an intercellular space. The ducts are normally oriented parallel to the longitudinal axis of the organ containing them, but they may anastomose tangentially. In the conifers, resin ducts are found throughout the body of the plant and consist of elongated structures wrapped in epithelial cells surrounding an interior space. These cells are in turn surrounded by one or more layers of cells with relatively thick cell walls encrusted with pectic substances (Figure 2.10).

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Figure 2.10 Needle leaves of the gimnosperms contain many resin ducts. This picture shows the cross section of a pine tree needle leaf. The resin duct is surrounded by a cellular sheath with suberin thickening. The secretory cells are evident inside the duct.

Copaifera is a tropical genus of Leguminosae, subfamily Caesalpinioideae, which is characterized by the presence of internal secretory structures, such as canals and cavities comprising a lumen and a secretory epithelium. These structures are the sites of synthesis and storage of oils and oleoresins and have both ecological functions and commercial value. Internal secretory structures are present in the aerial vegetative organs of Copaifera langsdorffii, from the seedling to the adult stage. In cross sections, mature secretory tissues comprise a secretory epithelium delimiting a wide, round lumen where secretions accumulate. However, these secretory tissues have different shapes in longitudinal sections and can be classified as either spherical secretory cavities or elongated secretory canals. The cortex of epicotyl, hypocotyl, primary stem, pulvinus, petiole, rachis and midrib predominantly contain cavities, while canals are observed in the pith of these regions. Cavities are also observed in the mesophyll of eophylls, leaf primordia and expanded leaves (Figure 2.11).

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Figure 2.11 Cross sections of Copafiera langsdorffii vegetative organs. (a) Transverse section of pulvinus (bar = 500 μm). (b) Transverse section of the leaf blade (bar = 100 μm). (c) Transverse section of the rachis showing canals in the pith (bar = 50 μm). (d) Transverse section of the petiole showing a cavity in the cortex (bar = 50 μm). From Rodrigues et al., 2011.

2.4. Lysigenous Cavities Lysigenous cavities are present in many families, including the Myrtaceae, Rutaceae, Myoporaceae, Leguminosae and the Hypericaceae. These secreting structures are formed from a single epidermal cell. Some species show the formation of a meristemoid tissue characterized by cells with a dense cytoplasm and a large nucleus. In these structures, the first two divisions form two bicellular layers: an upper one and a lower one. From the latter, secretory cells are formed providing the epithelium surrounding the cavity inner space. In some families (such as the Myrtaceae), the formation of an inner cavity is the result of schizogeny of an initial group of cells; whereas in others (such as the Rutaceae), both schizogeny and lysigeny may occur. In any case, it cannot be excluded that also in the case of schizogeny the formation of the cavity may result from lysigeny. In legume leaves, secretory resin cavities are referred to as translucent glands; they are used in taxonomic descriptions as a diagnostic character

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in dichotomic identification keys. In Hymenaea stigonocarpa, the leaf resin of the epithelial cells is present in secretory cavities that are distributed randomly. These clearly defined cavities are covered with a single layer secretory epithelium. They are formed during the initial stages of the differentiation of leaf primordia. In the leaf blade, the secretory cavities are found predominantly near the adaxial surface. When observed in paradermal section, each cavity is located in the centre of an areole and equidistant in relation to the surrounding vascular tissues. Cells of palisade parenchyma form radial series that establish a connection between the vein and secretory cavity. However, there is no symplastic connection between the cells of the secretory epithelium and the mesophyll (Figure 2.12).

Figure 2.12 Secretory cavity ontogenesis in Hymenaea stigonocarpa leaf. (a) Detail showing initial cell of the secretory cavity (ic) between protodermal cells. In

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this stage IC move towards ground meristem (gm). (b) First IC division; note a space left between protodermal cells; a trichome initial cell (ti) is seen. (c) Cell cluster in the mesophyll, before lysis stage. (d and e) Final stage of secretory cavity differentiation; note a cell residue (d, arrow) and a large lumen (e). a–e metric Bars = 25 μm. (f) Young leaf transverse section showing the secretory cavity (sc) in the mesophyll; note the large secretory epithelium cells with distal convex pole. (g) Paradermal section of adult leaf showing the secretory cavity (sc) in the mesophyll; note the secretory cavity equidistance in relation to vascular tissues. From Paiva and Machado, 2006.

2.5. Oil-bearing Cells and Secretory Cells associated with Bacteria Other tissues able to produce lipophilic substances are represented by secretory cells that accumulate the secreted products inside their vacuoles. This is the case for VOCs produced by the odorous roots of the grass Vetiveria zizanioides Nash (Vetiver). Vetiver VOCs are produced in secretory cells localized in the first cortical layer outside the endodermis of mature roots. By using culture-based and culture-independent approaches to analyse the microbial community of the vetiver root, it was possible to demonstrate the presence of a broad phylogenetic spectrum of bacteria, including Į-, ȕ-, and Ȗ-proteobacteria, high-G+C-content grampositive bacteria, and microbes belonging to the Fibrobacteres/ Acidobacteria group. Isolated root-associated bacteria showed that most of them were able to grow by using Vetiver sesquiterpenes as a carbon source and to metabolize them by releasing into the medium a large number of compounds typically found in commercial Vetiver oils. Several of these bacteria were also able to induce gene expression of a Vetiver sesquiterpene synthase. Figure 2.13 shows a cross-section of a Vetiver root, where the VOC-producing cells are evidenced along with the associated bacteria. These results support the intriguing hypothesis that bacteria may play a major role in Vetiver VOC biosynthesis, opening the possibility to use them to manoeuvre the Vetiver oil molecular structure. These results are in accordance with the observation of Vetiver root ultrastructure using electron transmission microscopy where VOCs were detected in the inner cortical layer close to the endodermis. VOCs can be synthesized by a variety of other anatomical structures such as solitary cells and areas of epidermal cells. The typical fragrance of flowers results from VOCs occurring in the form of small droplets in the cytoplasm of the epidermal and neighbouring mesophyll cells of sepals. In flowers, the biosynthesis of VOCs usually occurs in epidermal cells,

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allowing an easy escape of VOCs into the atmosphere. Flowers usually produce their attractive fragrance in osmophores or in conical cells located on the petals. These cells do not store VOCs but release them into the air. In species belonging to the Orchidaceae and the Araceae, VOCs produced by osmophores also produce amines and ammonia.

Figure 2.13 Secretory cells and the presence of bacteria in the roots of Vetiveria zizanioides. (a) Presence of bacteria in the cell layer close to the endoderm. (b–g) V. zizanioides root electron micrographs showing the presence of endophyte bacteria. (b, c) Details of a cortical parenchymal cell and a lysigenous pocket with endogenous bacteria. (d, e) Lumen of a cortical parenchyma cell with bacteria adhering to the pectocellulosic wall. (f, g) Endophytic bacteria showing a substructure of the cell wall typical of gram-negative bacteria. (h–m) Laser scanning microscopy of V. zizanioides roots stained with SYTO 9, a specific fluorescence dye for bacteria. (h–l) The presence and location of live bacteria confirms their adhesion to the plant cell wall (arrows). (m) No bacteria were found in the same type of cells in axenic V. zizanioides plants cultivated in vitro. From Del Giudice et al., 2008.

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2.6. Laticifers Latex is a suspension or in some cases an emulsion of small particles dispersed in a liquid with a particular index of refraction. Although mainly of milky colour, this sap can also be yellow, orange, red, brown or even colourless. The chemical composition of the latex ranges from polyisoprenic hydrocarbons to triterpenols and from sterols to fatty acids or aromatic compounds. It can also contain carotenes, phospholipids, proteins and inorganic compounds, protein crystals, starch granules, tannins, alkaloids, proteolytic enzymes (papain), vitamins, calcium oxalate and malate. The tissues that contain latex are defined as laticifers and are present in about 12,500 species belonging to 900 genera and 20 families, especially dicotyledonous (e.g., Apocynaceae, Asclepiadaceae, Asteraceae, Euphorbiaceae, Papaveraceae and Sapotaceae) and less in monocots (Araceae, Liliacae and Musaceae). However, latex can accumulate in tissues that are not laticifers, as in Parthenium argentatum. Laticifers are divided into non-articulated and articulated. The former (also known as laticifer cells) are multinucleated and are derived from a single cell that extends enormously during plant development. In some species, laticifers grow in the form of elongated tubes, in other species they branch and are defined as not articulated branched laticifers. Articulated laticifers (also known as laticifer vessels) consist of series of usually elongated simple or branched cells. Laticifers are also divided into articulated not anastomized laticifers (not branched) and articulated anastomized laticifers (branched). Not articulated and unbranched laticifers are located in the stem outside of the primary phloem, being absent in the roots and secondary tissues. The species Camptotheca acuminata (Nyssaceae) is a tree native to southern China and Tibet. Interest in C. acuminata resides in the importance of certain bioactive molecules, in particular camptothecin (a pentacyclic quinoline alkaloid, see Chapter 9) and some of its derivatives. The distribution of laticifers and the ultrastructure of secretory cells were analysed in this species by light and electron microscopy and histochemical analysis to identify the major components of the latex. Histological analysis revealed that primary laticifers are already present in the leaf primordia. The vacuolar content of laticifers in the proximal area of the leaf is more intensely coloured with histochemical reagents. In some laticifers, the latex does not accumulate evenly and the histological

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analysis of the roots has not revealed the presence of laticifers in both primary and secondary tissues (Figure 2.14).

Figure 2.14 Laticifers of Camptotheca acuminata in transverse sections of young leaf (a and b) and stem (c, d, f, and h), and in longitudinal sections of stem (e and g). (a) Young leaf showing laticifers stained with Cresyl blue in the parenchyma delimited by vascular bundles (arrows) of the midrib and in the cortex just external to the phloem (arrowheads). (b) Laticifers stained with Hellram reagent (cherry red) are visible in the parenchyma delimited by vascular bundles and in the cortex just external to the phloem (arrowhead) of a young leaf midrib. (c) Stem with a laticifer stained with Hellram reagent in the primary cortex (arrow). (d) Stem showing laticifers stained with Nile blue in the pith and in the parenchyma near the phloem (arrow). (e) Stem with a nonarticulated laticifer stained with Hellram reagent. (f) A laticifer stained with Hellram reagent in the pith. (g) Thin section (1 μm) of stem showing a laticifer stained with toluidine blue; abundant masses of latex are visible. (h) A laticifer stained with toluidine blue in the cortex. Bars: a and d, 40 μm; b and g, 100 μm; c, 50 μm; e, f, and h, 30 μm. From Monacelli et al., 2005.

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Natural rubber is an elastic material mainly consisting of cis-polyisoprene (see Chapter 7) which is used to produce various industrial products, such as automotive tyres. Conversely, gum made from trans-polyisoprene shows less flexibility and reduced thermo-plasticity at low temperatures compared to cis-polyisoprene gum and is used as a raw material for golf balls, dental supplies and underwater cables. In nature, the production of cis-polyisoprene rubber is present in about 2500 plant species, among which Hevea brasiliensis is the most industrially used plant to produce natural rubber, but only a small number of plant species such as Eucommia ulmoides, Palaquium gutta, Manilkara Bidenta, Achras zapota, Garrya flavescens and Garrya wrightii are known to produce rubber with transpolyisoprene. In Eucommia ulmoides, a small native tree widely cultivated in China and belonging to the Eucommiaceae family, a microscopic technique has recently been developed that combines laser scanning confocal microscopy with a lipophilic fluorescent dye, the red Nile, able to emit a specific fluorescence for trans-polyisoprene, allowing the obtaining of localization images in situ of this compound. In this species, transpolyisoprene is initially synthesized in the form of granules in nonarticulated laticifers that change their shape into fibres during laticifer maturation. Non-articulated laticifers develop from single laticifer cells, which differ from the surrounding parenchymal cambial cells. Transpolyisoprene is present in the form of small dots in the secondary protofloema adjacent to the cambium cells and as fibrous structures in the adult phloem close to the bark. From these observations it can be deduced that the trans-polyisoprene accumulates first as granules and then undergoes changes that lead to the fibrous form. Based on observations in scanning electron microscopy, the accumulation of trans-polyisoprene occurs in the space enclosed by phloem cell walls.

Suggested Reading Appezzato-da-Gloria, B. et al. (2012). Glandular trichomes on aerial and underground organs in Chrysolaena species (Vernonieae – Asteraceae): Structure, ultrastructure and chemical composition. Flora. 207, 878–887. Baldwin, I.T., Halitschke, R., Paschold, A., von Dahl, C.C., and Preston, C.A. (2006). Volatile signaling in plant–plant interactions: “Talking trees” in the genomics era. Science. 311, 812–815.

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Berta, G., Dela Pierre, M. and Maffei, M. (1993). Nuclear morphology and DNA content in the glandular trichomes of peppermint (Mentha x piperita.L.). Protoplasma. 175, 85–92. Bicchi, C. and Maffei, M.E. (2012). The plant volatilome: methods of analysis, in High throughput phenotyping in plants: Methods and protocols., ed. J.Normanly (Totowa, NJ: Humana Press), pp. 289–310. Bosabalidis, A. and Tsekos, I. (1982). Glandular scale development and essential oil secretion in Origanum dictamnus L. Planta. 156, 496–504. Boukhris, M. et al. (2013). Trichomes morphology, structure and essential oils of Pelargonium graveolens L’Her. (Geraniaceae). Ind. Crops Prod. 50, 604–610. Brückner, K. et al. (2014). Characterization of two genes for the biosynthesis of abietane-type diterpenes in rosemary (Rosmarinus officinalis) glandular trichomes. Phytochemistry. 101, 52–64. Caissard, J.C. et al. (2012). Extracellular localization of the diterpene sclareol in clary sage (Salvia sclarea L., Lamiaceae). Plos One. 7, e48253. Chappell, J. (2008). Production platforms for the molecular pharming of alkaloid diversity. Proc. Natl. Acad. Sci. U.S.A. 105, 7897–7898. Cho, W.K. et al. (2010). Extended latex proteome analysis deciphers additional roles of the lettuce laticifer. Plant Biotechnol. Rep. 4, 311– 319. Choi, Y.E. et al. (2012). Tobacco NtLTP1, a glandular-specific lipid transfer protein, is required for lipid secretion from glandular trichomes. Plant J. 70, 480–491. Del Giudice, L. et al. (2008). The microbial community of Vetiver root and its involvement into essential oil biogenesis. Environ. Microbiol. 10, 2824–2841. Demarco, D., Castro, M.D., and Ascensao, L. (2013). Two laticifer systems in Sapium haematospermum: new records for Euphorbiaceae. Botany-Botanique. 91, 545–554. Dudareva, N., Negre, F., Nagegowda, D.A., and Orlova, I. (2006). Plant volatiles: Recent advances and future perspectives. Crit. Rev. Plant Sci. 25, 417–440. Duke, S.O. and Paul, R.N. (1993). Development and fine structure of the glandular trichomes of Artemisia annua. Int. J. Plant Sci. 154, 107– 118. Gersbach, P.V. (2002). The essential oil secretory structures of Prostanthera ovalifolia (Lamiaceae). Ann. Bot. 89, 255–260. Gershenzon J., Maffei M. and Croteau R. (1989). Biochemical and histochemical localization of monoterpene biosynthesis in the

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glandular trichomes of spearmint (Mentha spicata). Plant Physiol. 89, 1351–1357. Glas, J.J. et al. (2012). Plant glandular trichomes as targets for breeding or engineering of resistance to herbivores. Int. J. Mol. Sci. 13, 17077– 17103. Göpfert, J.C., Heil, N., Conrad, J., and Spring, O. (2005). Cytological development and sesquiterpene lactone secretion in capitate glandular trichomes of sunflower. Plant Biol. 7, 148–155. Göpfert, J.C., MacNevin, G., Ro, D.K., and Spring, O. (2009). Identification, functional characterization and developmental regulation of sesquiterpene synthases from sunflower capitate glandular trichomes. BMC Plant Biology. 9, e86. Gutierrez, D.G. and Luna, M.L. (2013). A comparative study of latexproducing tissues in genera of Liabeae (Asteraceae). Flora. 208, 33– 44. Happyana, N. et al. (2013). Analysis of cannabinoids in lasermicrodissected trichomes of medicinal Cannabis sativa using LCMS and cryogenic NMR. Phytochemistry. 87, 51–59. Harvest, T. et al. (2009). The latex capacity of opium poppy capsules is fixed early in capsule development and is not a major determinant in morphine yield. Ann. Appl. Biol. 154, 251–258. Heinrich, G., Pfeifhofer, H.W., Stabentheiner, E., and Sawidis, T. (2002). Glandular hairs of Sigesbeckia jorullensis Kunth (Asteraceae): Morphology, histochemistry and composition of essential oil. Ann. Bot. 89, 459–469. Hilker, M. and Meiners, T. (2002). Induction of plant responses to oviposition and feeding by herbivorous arthropods: a comparison. Entomol. Exper. Appl. 104, 181–192. Holopainen, J.K. (2004). Multiple functions of inducible plant volatiles. Trends Plant Sci. 9, 529–533. Kesselmeier, J. (2001). Exchange of short-chain oxygenated volatile organic compounds (VOCs) between plants and the atmosphere: A compilation of field and laboratory studies. J. Atm. Chem. 39, 219– 233. Kesselmeier, J. and Hubert, A. (2002). Exchange of reduced volatile sulfur compounds between leaf litter and the atmosphere. Atm. Environ. 36, 4679–4686. Maffei M., Chialva F. and Sacco T. (1989). Glandular trichomes and essential oils in developing peppermint leaves. I. Variation of peltate trichome number and terpene distribution within leaves. New Phytol. 111, 707–716.

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Maffei M., Gallino M. and Sacco T. (1986). Glandular trichomes and essential oils of developing leaves in Mentha viridis lavanduliodora. Planta Med. 52, 187–193. Maffei, M.E. (2010). Sites of synthesis, biochemistry and functional role of plant volatiles. South Afr. J. Bot. 76, 612–631. Maffei, M.E., Gertsch, J., and Appendino, G. (2011). Plant volatiles: Production, function and pharmacology. Nat. Prod. Rep. 28, 1359– 1380. Majdi, M. et al. (2011). Biosynthesis and localization of parthenolide in glandular trichomes of feverfew (Tanacetum parthenium L. Schulz Bip.). Phytochemistry. 72, 1739–1750. Marin, M., Koko, V., Duletic-Lausevic, S., Marin, P.D., Rancic, D., and Jic-Stevanovic, Z. (2006). Glandular trichomes on the leaves of Rosmarinus officinalis: Morphology, stereology and histochemistry. South Afr. J. Bot. 72, 378–382. Martins, F.M., Lima, J.F., Mascarenhas, A.A.S., and Macedo, T.P. (2012). Secretory structures of Ipomoea asarifolia: anatomy and histochemistry. Braz. J. Pharmacogn. 22, 13–20. Mithöfer, A., Wanner, G., and Boland, W. (2005). Effects of feeding Spodoptera littoralis on lima bean leaves. II. Continuous mechan-ical wounding resembling insect feeding is sufficient to elicit her-bivoryrelated volatile emission. Plant Physiol. 137, 1160–1168. Monacelli, B., Valletta, A., Rascio, N., Moro, I., and Pasqua, G. (2005). Laticifers in Camptotheca acuminata Decne: distribution and structure. Protoplasma. 226, 155–161. Nagel, J. et al. (2008). EST analysis of hop glandular trichomes identifies an O-methyltransferase that catalyzes the biosynthesis of xanthohumol. Plant Cell. 20, 186–200. Nakazawa, Y. et al. (2013). Histochemical study of trans-polyisoprene accumulation by spectral confocal laser scanning microscopy and a specific dye showing fluorescence solvato-chromism in the rubberproducing plant, Eucommia ulmoides Oliver. Planta. 238, 549–560. Olsson, M.E. et al. (2009). Localization of enzymes of artemisinin biosynthesis to the apical cells of glandular secretory trichomes of Artemisia annua L. Phytochemistry. 70, 1123–1128. Onoyovwe, A. et al. (2013). Morphine biosynthesis in opium poppy involves two cell types: Sieve elements and laticifers. Plant Cell. 25, 4110–4122. Paiva, E.A.S. and Machado, S.R. (2006). Structural and ultrastructural aspects of ontogenesis and differentiation of resin secretory cavities in

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Hymenaea stigonocarpa (Fabaceae-Caesalpinioideae) leaves. Nord. J. Bot. 24, 423–431. Pickard, W.F. (2008). Laticifers and secretory ducts: two other tube systems in plants. New Phytol. 177, 877–887. Rodrigues, T.M., Teixeira, S.D., and Machado, S.R. (2011). The oleoresin secretory system in seedlings and adult plants of copaiba (Copaifera langsdorffii Desf., Leguminosae-Caesalpinioideae). Flora. 206, 585– 594. Sallaud, C. et al. (2012). Characterization of two genes for the biosynthesis of the labdane diterpene Z-abienol in tobacco (Nicotiana tabacum) glandular trichomes. Plant J. 72, 1–17. Stout, J.M., Boubakir, Z., Ambrose, S.J., Purves, R.W., and Page, J.E. (2012). The hexanoyl-CoA precursor for cannabinoid biosynthesis is formed by an acyl-activating enzyme in Cannabis sativa trichomes. Plant J. 71, 353–365.

CHAPTER THREE FUNCTIONAL ROLE OF BIOACTIVE PLANT MOLECULES

In the previous chapters we discussed the distribution of secondary metabolites and we described how plants modify their tissues by creating special anatomical structures involved in the synthesis of bioactive molecules. The aim of this chapter is to provide information on the functional role of plant bioactive molecules, which are characterized by two common features: variability in chemical structure and high intraspecific variation. Traditionally, these two features were seen as the most obvious demonstration of the random origin of secondary metabolites, which were considered “ebbs and flows on the metabolism beach” or even waste and/or detoxification products. Today it is fully accepted that secondary metabolites are a special metabolic instrument that plants use to cope with the surrounding environment.

3.1. Primary and Secondary Metabolites We can make a general distinction between the metabolism involving metabolic pathways which are essential for growth and development (i.e., primary metabolism) and the metabolism that is unnecessary for such purposes (i.e., secondary metabolism). The focal point in framing secondary metabolites in a metabolic and functional context is to define their “dispensability” or “indispensability”. In the past, the dispensability of these molecules for growth and development processes limited the role of these molecules to waste or detoxification products. Today we know that many secondary metabolites are used in the ecosystem as chemical signals and metabolic defence tools. Secondary metabolites can therefore be considered as dispensable molecules for growth and development (except for the metabolic pathways involved in the synthesis of plant hormones) and indispensable molecules for the survival of species. The difference between primary and secondary metabolism is best expressed in

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functional rather than structural terms, since the same compound can exhibit qualities of both primary and secondary metabolites. The most evident quality of secondary metabolites is definitely their enormous structural diversity, as well as the restriction of their presence to certain families or genera and their high interspecific variability. The purpose of this chapter is to discuss the concept of chemical defences, developed by plants in relation to challenges of the biotic and abiotic environment that surrounds them. Generally, the strategies adopted by plants are three: x induction, that is, the formation of molecules for chemical defence as a response to attacks by pathogens or herbivores; x production of metabolically inactive pro-toxins that are activated by the action of an enzyme triggered by a biotic stress; x storage of defence compounds in constitutive sites of storage. There is no delimitation between these three strategies, and often constitutive defences can be boosted by inductive phenomena. However, constitutive defences (which accumulate in the secretory tissues as described in Chapter 2) represent the most characteristic example of secondary metabolite action. Plants evolved to adopt a constitutive strategy to anticipate the attack of predators, rather than defend themselves after attack. However, this does not diminish the high evolutionary significance of some induced defences such as the phytoalexins or the production of molecules capable of attracting predators of plants’ herbivores. Figure 3.1 illustrates the relationship between primary metabolism, secondary metabolism and their functional roles.

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Figure 3.1 Secondary metabolites originate from common precursors of primary metabolism. Their function covers all aspects of chemical interactions between plants and the surrounding environment. Notice how flavonoids and terpenoids derive from both carbon and fatty acids metabolism, and how the alkaloids derive from the fatty acids and nitrogen metabolism. The photosynthetic process is at the centre of the system and provides raw materials for the three major classes of metabolites. From Hartman, 2007, modified.

The vast array of secondary metabolites is not a random occurrence, but rather the result of a harmonious regulation of the various biogenic pathways that are perfectly integrated into the primary metabolism. According to many authors, plants use a few basic metabolic pathways, which diverge into infinite variants eventually leading to hundreds of thousands of molecules. According to some authors, one of the possible explanations for the origin of secondary metabolites can be found in the basal metabolism. Some intermediate compounds of a primary metabolic pathway may accumulate due to metabolic impairments or under environmental pressure. Accumulation of a metabolite can create serious metabolic problems in the “assembly” chain that leads to the production of the final compound. Sometimes the accumulation of an intermediate compound triggers a feedback reaction that can act upstream of a metabolic transformation; or simply its accumulation may be inhibitory

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with respect to the forming reaction. As a consequence, the metabolic process slows down, or stops, causing a number of impairments that may affect other metabolic pathways. However, if the excess of the primary metabolite is used by a parallel metabolic pathway to produce a secondary metabolite then its concentration decreases and the primary metabolism then resumes its normal activity. But the problem described for the primary metabolite may reoccur in the parallel secondary metabolic pathways, so there are several other parallel pathways that can “drain” undesired accumulation (Figure 3.2).

Figure 3.2 Secondary metabolites (S) are derived from the primary metabolic pathways of primary metabolism (P). Excess production of primary metabolites required the evolution of metabolic “vent valves” able to drain the excess (accumulation) of primary metabolites and prevent retroactive inhibitory reactions. The figure shows the metabolic interplay between primary and secondary metabolism as well as the need to produce more and more secondary metabolites to prevent primary and secondary metabolite accumulation. Eventually, secondary metabolites originating from different primary metabolites merge to produce new molecules.

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One of the peculiar characteristics of secondary metabolism is the high degree of freedom of its compounds, which may vary in quantity (in concentration) or in quality (chemical structure) without causing damage to the development and growth processes of the organ where they are produced. On the other hand, the primary metabolism used for plant growth and development must be maintained stably to ensure the structural and functional integrity of cells or organs. We will discuss again the relationship between primary and secondary metabolism in Chapter 11.

3.2. Phenotypic Plasticity Before discussing the functional role of bioactive molecules, we need to consider a general aspect: the capacity of plants to respond quantitatively to stress. A plant cannot escape changes in environmental conditions; that is, it must face environmental heterogeneity by adapting to a new or fluctuating environment. Plants can cope with the environment by changing the phenotypic expression of their genes, a property called phenotypic plasticity. For plants, phenotypic plasticity can be very important. Phenotypic plasticity allows phenotypic variations within a single generation and plays an important role in plant responses to environmental changes. Plasticity often involves altering gene expression and plant physiology in response to environmental cues. Moreover, it allows survival in non-optimal conditions, thus granting adaptation. Therefore, plasticity is likely to be very important for species that respond to global changes. Most of the phenotypic responses to climate change documented to date can be mostly attributed to plasticity, rather than to genetic variation. The plastic changes in the phenotype may be either adaptive or not. Given an appropriate genetic variability, adaptive phenotypic responses to the environment can evolve in populations that encounter predictable environmental change. Thus, adaptive phenotypic plasticity is the ability of a genotype to develop an appropriate phenotype for the local environment, allowing organisms to cope with environmental changes. Adaptive plasticity evolves easily in the presence of dispersion among populations of different ecological environments. This plasticity favours the colonization of new environments, but reduces the genetic divergence between them.

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It is well known that climatic changes are altering the availability of resources and conditions that are crucial to plant performance. Therefore, plants respond to these changes through phenotypic plasticity. Understanding the plasticity of responses is important in predicting and managing the effects of climate change on indigenous species and on cultivated plants. Plasticity can play a role both in the short-term response of plant populations to global change and in their long-term fate by maintaining genetic variability. In general, phenotypic plasticity can play a major role in guiding diversification and speciation. Finally, the trait of plasticity is not only capable of influencing the adaptive evolution of species in a significant way but is also the result of evolutionary processes. Therefore, phenotypic plasticity is a potentially important modulator of adaptation and evolution. With phenotypic plasticity studies it should become easier to determine what the effect is of the environment on the phenotype and to better understand the role of phenotypic plasticity. When phenotypic plasticity differs between genotypes, this event is described as the genotype– environment interaction or more simply G×E. The phenotypic response to the environment can be expressed in the reaction norms and G×E occurs when the slope of reaction norms differs for different genotypes. So the phenotype of the plant (P) is determined not only by the genetic composition of the plant (G) and by environmental factors (E), but also by their interaction (G×E), usually described by the linear model: P = G + E + G×E In a breeding programme, variety trials are usually conducted in several environments, to minimize the risk of discarding genotypes that potentially perform well in some, but not in all, environments. A plant phenotypic change is named a response. Since all changes in an individual that are not dependent on genetic variations are attributable to environmental (biotic and abiotic) responses, plasticity is a concept applicable to all intra-genotypic variability. Almost all characters exhibit a certain level of phenotypic plasticity, which can be grouped into four main categories: x Morphological plasticity, with the classic example of structural modifications of leaves in the same tree (shade leaves and sun leaves).

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x Physiological plasticity, which is very common. Examples are the differences in the photosynthetic rates of sun plants and shade plants. x Behavioural plasticity, which is typical of animals. Plants are static organisms, incapable of movement (excluding, of course, dissemination or pollination processes). Animals, on the other hand, can move toward less hostile areas. x Biochemical and chemical plasticity, related to changes in activity or conformation of proteins and to the consequent changes in the intracellular environment (pH, permeability, signal transduction pathway, etc.) and to the ability to produce special metabolites. In the plant kingdom, adaptation through plasticity occurs commonly and has evolved differently in different species. There is a considerable relationship between the plasticity of different characters, and the plasticity of one character can induce stability in another one. Among the various plasticities occurring in plants, many are typical of the secondary metabolism. Indeed, the synthesis of plant secondary metabolites is among the most plastic characters in the biosphere. Fluctuations in the quality and quantity of alkaloids, flavonoids, terpenoids and other molecules due to biotic and abiotic stress are clearly plastic responses. Recently a line of modern thinking on phenotypic plasticity and flexibility has suggested that multicellular organisms show a “mosaic physiology”. Mosaic physiology refers to groups of different phenotypes within individual organisms that perform related functions at the same time but that are distributed in space. Mosaic physiology stems from stochastic cell differentiation at the beginning of their diversification, which is then amplified by cell division and growth into the phenotypic macroscopic forms (cells, tissues, organs) that constitute the physiological steps of later life phases.

3.2. Chemical Defence from Biotic Stress 3.2.1. Chemical Defence in Prehistory Undoubtedly, the transition of plants from the sea to the land (terrestrialization) was one of the most significant evolutionary events in the history of life on Earth. Plants made their appearance on land ~400 million years ago, during the Silurian age. At that time there were already scorpions and centipedes, and scientists have not ruled out the possibility

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that during the Ordovician (Early Silurian) the first organisms appeared on land, including arthropods feeding on algae and members of the cyanophytes that had been predominant even before the Cambrian. That is to say, at that time, plant feeders (ancient herbivore arthropods) may have coexisted with plants. During the Permian, flora and fauna changed slowly, and gymnosperms and cycads took the place of Licopodales and tree ferns. At that time, many insects, probably 50% of the species, fed on plants. Butterflies appeared at the beginning of the Cretaceous period and contributed to pollination of flowering plants, which gradually evolved. Herbivorous insects were dominantly present in the Palaeozoic, as well as during the Mesozoic era. It is likely that the algae Charoficeae gave rise to the terrestrial plants that later evolved into woody plants. In fact, predation of leaves and seeds did not occur until the Carboniferous, given that the first documentation of feeding dates back to the Mississippian period. Fossil evidence of other feeding methods in the Palaeozoic, such as the formation of galls and root herbivory, appeared even later, from the middle to the end of the Pennsylvanian. Clearly, secondary metabolites helped ancient plants to withstand attacks from herbivores, as seen in contemporary ecosystems.

3.2.2. Chemical Ecology The origin of plant chemical ecology dates back to the late 1950s, when some evolutionary entomologists recognized the essential role of secondary plant metabolites in plant–insect interactions and suggested that the diversity of plant chemistry evolved under the selective pressure of herbivores. However, similar ideas were already present during the second half of the 19th century, but they were largely abandoned. Now, after more than 100 years, the molecular analysis of genes controlling the production of secondary metabolites underlines the perceptions of 19th century scientists such as von Marilaun, Errera and, above all, Stahl. Their ideas were probably lost as a result of the decisive rejection of all teleological thoughts by the physiologists who dominated biological research at that time. As we have discussed so far, plants, like animals, use chemicals for purposes that include defence, aggression, communication and reproduction. Chemical ecology deals with chemical interactions between organisms and between organisms and the environment, and the effect of chemical factors on population and ecosystem dynamics. The main purpose of this discipline is to describe the relationships existing in

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ecosystems and to predict the changes caused by both natural and anthropogenic compounds. Usually, the chemical compounds under study are of natural origin and of organic nature, and are sometimes produced also by microorganisms that live together with the plants. In order to better understand the ecological significance of the molecules produced by plants, we have to dwell on some basic considerations regarding the general interaction between plants and other organisms in the biosphere. Clarifying the chemical ecology of natural enemies, herbivores and host plants is important for the development of effective integrated pest management (IPM) programmes, where the abundance and distribution of natural enemies could be manipulated by semiochemicals for biological conservation control (BCC). Plants, as autotrophic organisms, provide the surrounding ecosystem the organic molecules that feed heterotrophic organisms, thus representing the main food source in the biosphere. Plants have evolved the ability to produce substances that can act as deterrents to their predators (herbivores, microorganisms and viruses). But as we will discuss later, the chemical struggle of plants is extended to their likes, in order to survive water, light and nutritional deficiencies. The food chain and the interactions with the surrounding world require an evolutionary calibration of plants’ processes in response to the pressures they receive, but this does not exclude that plant evolution in turn impels the evolution of their predators.

3.2.3. Coevolution Coevolution implies a tight ecological interaction between two or more species. Coevolution is an evolutionary process that prompts the genetic adaptation of a species in response to the natural selection imposed by another interacting species, and the effects might be reciprocal. Coevolution can occur between any interacting populations: prey and predator, pathogen, competitor or mutualists. The selective pressures that each individual can exert on another are expected to depend on the intimate nature and strength of the association. Since Darwin’s natural selection theory in The Origin of Species, coevolution has been recognized as a force that drives mutual adaptive evolution in interacting species. The dissection of coevolutionary relationships has shown the ecological ingredients necessary for plant–enemy coevolution. They can be summarized in three main points:

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x the natural enemy must have significant selective impacts on the host through a severe reduction of host-population fitness; x host resistance diversity must impact on the evolution of enemy virulence; x host and enemy populations must exhibit considerable genetic variability. In nature, however, the detection of coevolutionary processes is difficult. Therefore, most studies have focused on the detection of patterns of variation in genotypes or phenotypes that are consistent with either ongoing or past coevolution. In a natural environment, the selective pressure imposed by continuous interaction between host plant and enemy involves constant fluctuations in allele frequency. Co-evolution entails the rise of new alleles, by mutation or migration, and the fixing of those alleles in the population. Two models describe the dynamics of the coevolution process. The first, the Red Queen hypothesis, is synthesized as “running as fast as you can to stay in the same place”. It posits that for a given species adaptation increases its fitness against another interacting species, but at the same time such adaptation of the first species necessarily causes a decline in fitness of the second species. In interacting species, such coevolutionary interactions give rise to continuous natural selection for adaptation and counter-adaptation. The second evolutionary hypothesis of coevolution is known as the armsrace model. Coevolutionary dynamics are described as a continuous escalation of defences and counter-defences gained with new genetic traits that can be fixed in the population through a slow process. In such a model, genetic improvements are accumulated in both populations. Failure to recognize the dynamic nature of the interaction could result in misinterpretation of the genetic basis of coevolution. Theoretical models forecast many possible results in coevolution: stable polymorphisms, dynamic polymorphisms with cyclic or chaotic fluctuations in allelic frequencies of different amplitudes and selective screenings of favourable alleles. In nature, however, the detection of coevolutionary processes is difficult. Therefore, most studies have focused on the detection of variation patterns in genotypes or phenotypes that are consistent with both ongoing and past coevolution.

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There are some criteria to document the existence of a coevolutionary process between plants and herbivores. The presence and documentation of such evolutionary interactions depend on the existence of numerous conditions: x plants show intraspecific variations in proportion to the extent of the damage caused by herbivores; x the variability in the extent of the damage suffered is directly related to the presence or concentration of one or more secondary metabolites; x the damage suffered is directly related to fitness; x plants show additive genetic variance for the production of a given compound, depending on the damage suffered; x herbivores show intraspecific variability in the ability to tolerate secondary metabolites; x this capacity shows additive genetic variance; x this capacity is directly related to fitness. Further complicating the overall situation is the extreme variability occurring in the same plant depending on the organ, the stage of vegetative and reproductive development and the interaction of the plant with the surrounding environment. The distribution of secondary metabolites in a plant is not uniform and the attack of herbivores is usually directed towards those parts of the plant that are more defenceless. Recently, studies on chemical defence and herbivore resistance have been addressed to phenotypic responses as a result of environmental stress rather than genotype differences (see also the discussion above). Thus, a growing body of evidence indicates firstly that herbivores discriminate hosts based on both the amount and type of secondary metabolites, and secondly that secondary metabolism can be partially genetically determined. The important thing in evaluating the coevolutionary processes is to determine the cost of producing the secondary metabolites and compare it to the benefits obtained by being defended from herbivore attacks. In many cases, the ability to determine such a relationship is made difficult by the impossibility of assessing which kind of metabolic energy is spent by the plant. Progress in understanding the evolutionary dynamics in nature requires an in-depth characterization of processes occurring on space-time scales and requires greater genetic focus. One way to address these problems could be the development of new methods to provide experimental evidence of coevolution, beyond the traditional phenomenological approaches: that is, methods that focus more broadly on

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elements that can influence co-evolution by integrating genetic-molecular approaches. We will now discuss two out of the many aspects of plant coevolution. 3.2.3.1. Plant–herbivore Coevolution When it comes to herbivores, the first thought goes to ruminant quadrupeds populating the African savannahs, but the most noxious herbivores are very small and are more numerous than mammals both in overall population and in species. Many insects are herbivores and show an incredible ability to fight the chemical defences that plants produce to defend themselves from their attacks. Yet plants need insects for the pollination that allows many plant species to reproduce. In an environment where the availability and quality of host plants changes, phytophagous insects are under selective pressure to find quality hosts. They need to maximize their fitness by finding the suitable plants and avoiding the unsuitable ones. Thus, to detect signals coming from the host plants, insects have developed a finely tuned sensory system and a nervous system capable of integrating inputs from sensory neurons with a high space-time resolution. Insect responses to stimuli are not predetermined, but depend on the context in which they are perceived. While insects have developed the way they find their host, the selective pressure causes plants to do exactly the opposite, avoiding detection and increasing defence. Once on the plant, insect-associated molecules can activate or suppress defences depending on whether the plant or insect is more advanced in evolutionary terms. In nature, there is a complicated game of attraction and deterrence between plants and insects. Chemicals are synthesized by plants to attract insects (colours, scents and sugars) and at the same time the plants produce toxic, repellent and deterrent substances that sometimes are able to attract predators or parasites of plant feeding herbivores. Herbivorous insects are divided into two large groups. Polyphagous insects feed on a large number of plant species, while mono- or oligophagous insects specialize in one or few host plants that often belong to the same family or closely related families (sometimes grouped because of the production of a given class of secondary metabolites). For these specialist insects, plant’s chemical defences are no longer harmful. Herbivorous insects are able to perceive the presence of plant toxic substances and estimate the plant feed quality. Generalist phytophagous

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insects are deterred by plants that produce constitutively high amounts of secondary metabolites, even though they are still able to choose plants that produce high amounts of compounds with a lower level of toxicity. Other insects feed on small quantities of toxic molecules, thus avoiding lethal intoxication. Insects’ feeding on plants has prompted an antagonistic evolutionary interaction that has led to the development of a variety of plant defence strategies to avoid extinction. Plant defences can be classified into: x x x x

resistance to herbivores; herbivores’ tolerance; phenological escape from herbivores; overcompensation.

Tolerance reduces the negative effect of herbivory on plant fitness, but the genetic bases of such adaptive strategy are less clear than direct resistance traits. Moreover, the plant genotype and the environmental conditions can influence the tolerance capacity of plants under attack. Tolerance is associated to faster growth and higher photosynthetic capacity. The general assumption is that tolerance and resistance are genetic alternative defence strategies. The rationale of such statement comes from the observation that plants with effective resistance traits limit the area damaged by deterring enemies and do not need tolerance mechanisms to survive. Conversely, high-tolerance plants do not evolve strong resistance traits. However, on field experiments show evolution of mixed resistancetolerance strategies as in Datura stramonium in response to two chewing insects: the specialist Lema daturaphila and the generalist Epitrix parvula. These results confirm that generalist herbivores are more susceptible to plant secondary metabolites used as a resistance defence strategy, whereas specialists are less susceptible to resistance as a result of coevolution. In the latter case, tolerance is a more effective defence strategy for the plant. Moreover, in the case of contemporary attack from both enemies – a condition that occurs quite often in nature – the plant response lies between resistance and tolerance. In the case of resistance to herbivory, more information is available on molecular and genetic mechanisms. Plants have evolved the capacity to perceive elicitor molecules after herbivore attack. Cellular transduction mechanisms fine tune the activation and regulation at local and systemic

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levels of the genetic activation of biochemical pathways for the biosynthesis of defensive compounds. Unlike generalist herbivores that are usually more susceptible than specialists to plant secondary metabolites, specialist herbivores develop different biochemical mechanisms to disable the toxicity of such direct defence compounds. In many cases, selective pressure has evolved detoxification mechanisms through enzymatic inactivation or by sequestration of toxic compounds. Moreover, specialists can use the specific defensive chemicals as cues to locate the host plants (phagostimulant function). The extreme example of coevolution is those herbivores that sequester the defensive chemicals and use them as a protective compound against predators and parasites and to attract mates. The observed pattern of specialization reflects a consistent evolutionary relationship between host plant and specialists. Some herbivores, like the decorator crabs, seize chemical protection products by placing harmful plants outside their body and thereby reducing the risk of predation (Figure 3.3). Although these strategies allow herbivores to save on the costs associated with chemical defence synthesis, they often require specialized facilities to separate, selectively absorb and/or modify plant-derived bioactive molecules.

Figure 3.3 Decorator crabs attach pieces of seaweed, rocks and sedentary animals such as corals and sponges to their bodies as camouflage, to protect them from predators. And they can change their outfits to match their environment. (From http://bit.ly/2isata3)

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3.2.3.2. Plant–microbial Coevolution Plants are constantly interacting with the microbes present in their environment. For host plants, these interactions can range from beneficial (symbiosis) to detrimental (pathogenic). Beneficial associations assist the host plants in the assimilation of soil, water and nutrients – mainly nitrogen and phosphate – in exchange of carbon sources. The main plant symbioses are with mycorrhizal fungi and rhizobacteria. In both cases, the establishing of symbioses requires the recognition of specific chemical signals. Arbuscular mycorrhizal (AM) symbioses are the most ancient, over 400 million years, and have left an excellent fossil record of host plant–microbial interactions. The wide distribution of AM in all branches of plant phylogenetic trees suggests that symbioses might have been present in a common ancestor and perhaps were instrumental in the initial colonization of land. In symbioses with AM fungi, many genetic components have been conserved in eudicots, monocots and basal land plants. The Glomales are the only monophyletic mycorrhizal fungal lineage that has coevolved with land plants throughout their history; other mycorrhizal fungi have polyphyletic lineages that represent parallel or convergent evolution. The coevolutionary plant-AM model shows an increasing level of commitment and specialization by plants and fungi, according to the arms-race model. Moreover, the recent discovery of some genetic links between bacterial and fungal symbiosis has led to the hypothesis that rhizobia root–nodule symbiosis evolved from mycorrhizal functions. However, the main reported proof of co-evolution in plant symbioses comes from studies performed by evolutionary biologists and few data are reported on genetic selection of new alleles that improve fitness. Plants and pathogens are involved in an intimate detrimental physiological and ecological interaction. The strength of the selective pressure depends on the virulence of the pathogen, the driving force in the host–parasite coevolution. Virulence is a specific product of the plant–pathogen interaction; virulence does not depend on the parasite or the plant alone. The success of pathogen infection is determined by the combination of host and pathogen genotypes. A number of genetic models of infection have been proposed to investigate the reciprocal role in the infection.

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3.2.4. Constitutive Chemical Defence Plants, during their interaction and coevolution with herbivorous insects, developed a wide range of defence mechanisms to counteract insect attacks. These mechanisms can be generalized into two categories: preformed constitutive defences and inducible defences. Constitutive defences are physical and chemical barriers existing before the insect’s attack, while inducible defences include defence mechanisms that are activated by an external biotic attacking agent (viruses, bacteria, herbivores etc.). Plant parts that are either high in fitness or at a high risk of attack can be better protected by constitutive defence, while others may be better protected by induced defence. As we shall discuss, upon herbivore attacks constitutive defences may increase in quantity and vary in quality; therefore, they show inducible characters. Research over the last ten years has largely focused on induced defence because of the wide variety of elicitors available to trigger accumulation of defence compounds and the development of molecular instruments for the study of differential gene expression. On the other hand, studies on the roles and mechanisms of constitutive chemical defence are quite rare also because of the difficulty of manipulating constitutive compounds in the experimental context. A consistent number of studies indicates that the concentrations of constitutive bioactive metabolites are higher in younger, developing leaves than in older tissues. The growing list of bioactive molecules presenting this particular behaviour includes iridoid glycosides, phenolic compounds, alkaloids, furanocoumarins and volatile organic compounds. Recent studies have also found a similar distribution in below-ground tissues. Among the most effective constitutive defences, toxins occupy a dominant position. Plant toxins are compounds that exert negative effects on the growth, development or survival of another organism. The mechanisms of action of some plant toxins are well known. For example, saponins disrupt cell membranes, cyanide released by cyanogenic glycosides inhibits cellular respiration, while cardenolides are specific inhibitors of the Na+/K+-ATPase pump. However, the mode of action of many other toxins is still unknown. As we discussed in Chapter 2, the mode of toxins’ storage is often crucial to their effectiveness. Some plant species accumulate toxins in laticifers or in glandular trichomes. Constitutive toxins are released in large quantities

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as soon as these structures are broken down by the action of herbivores, by their movement on the surface of the plant or by the growth of pathogens. However, many defence compounds are also toxic to the plants that produce them; plants that rely on constitutive chemical defence must be able to synthesize and store these substances without self-poisoning. One strategy is to store toxins as inactive precursors, such as glycosides, in compartments separated from the enzymes that activate them. For example, it has long been known that glucosinolates present in plants of the Capparales order are stored separately from their activating enzyme, thioglucosidase myrosinase. Benzoxazinoids, which are mainly produced by members of the Gramineae family, are another class of glycosides that are activated at the time of tissue damage. The reversible hydrolysis of the inactive D-glucoside precursor leads to the generation of phytotoxic 2,4dihydroxy-1,4-benzoxazin-3-one acid (DIBOA) and its methoxy derivative. A common error is to consider constitutive defences as static and oppose them to induced defences that are certainly of a dynamic nature. A clear demonstration of the dynamism of constitutive defences has been shown in Mentha aquatica. The defence strategy of this species occurs through the production of terpenoids which accumulate in the glandular trichomes. These plants may have chemical barriers to potential herbivore colonists, and they appear to be accessible to a relatively few insect lineages which may be pre-adapted to chemically similar or related host plants. As some insects become adapted to these metabolites, interactions between the two groups of organisms occasionally lead to highly specific relationships, as in the case of M. aquatica and the herbivore Chrysolina herbacea. M. aquatica produces toxic terpenoids such as pulegone and menthofuran (see Chapter 7 for chemical formulae), while C. herbacea has the ability to produce deterrents for natural enemies using plant-based compounds. As a response to herbivore feeding, M. aquatica activates genes for terpenoid biosynthesis, diverting most of the terpene production toward the synthesis of menthofuran, which was found to repel C. herbacea in bioassay tests. Therefore, constitutive plant defence too can be modulated by interactions with herbivorous insects. The latter can trigger plant terpenoid gene expression and synthesis in a way that simple mechanical damage cannot. In conclusion, C. herbacea attacks undamaged M. aquatica, but it avoids herbivore-infested M. aquatica that will have responded to the herbivory by overproducing the deterrent compound menthofuran, thus reducing

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damage from further insect attacks. To sum up the behaviour of C. herbacea and M. aquatica, before and during the herbivory, we can note that: x unwounded plants emit pulegone, which acts as an attractant for C. herbacea; x herbivory induces gene expression and increases the content of the deterrent compound menthofuran, together with the release of the attractant compound pulegone; consequently, fewer insects are attracted to plants; x the intensive herbivory causes a reduction of pulegone content and a dramatic increase in repellent menthofuran content; x C. herbacea avoids feeding on plants with a consistent herbivore damage and moves towards undamaged plants (Figure 3.4).

Figure 3.4 The behaviour of Chrysolina herbacea and Mentha aquatica before and during herbivore feeding. (a) Undamaged plants emit pulegone, which acts as an

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attractant for C. herbacea. (b) Feeding activity induces gene expression and increases the content of the deterrent compound menthofuran, along with the emission of the attractant compound pulegone; as a result, fewer insects are attracted to the plants. (c) Intense feeding induces a reduction in pulegone content and a dramatic increase in the repellent compound menthofuran; C. herbacea avoids over-fed plants and moves towards undamaged plants. From Zebelo et al., 2011.

3.2.5. Induced Chemical Defence In induced processes, as opposed to the case of constitutive defences, the recognition of the attacking insect and the subsequent signaling is the prerequisite for a fast and efficient defence. Many of these strategies can be either constitutive or inducible or even both. Many forms of induced defence are not restricted to local responses at the wounding site, but can be detected systemically throughout the plant. In the case of chemical defences, the compounds used in both strategies are often the same. Thus, induced defences also involve the syntheses and accumulation of various secondary metabolites that influence insect attraction/deterrence and inhibit insect growth and development. In addition, proteins such as proteinase inhibitors, polyphenol oxidases and threonine deaminase, which inhibit insect digestive enzymes and/or decrease the nutritive value of the plant tissues, are employed in induced direct defences. In crop plants, protease inhibitors have been proposed as potential defence molecules to increase insect resistance. In tomato (Lycopersicum esculentum), proteinase inhibitors (PIs) were tested for their trypsin- and Helicoverpa armigera gut proteinases-inhibitory activity. Observation in the field revealed that H. armigera larvae infested leaves and fruits but not the flowers – a fact that has been correlated with the higher levels of PIs in the flower tissue. Phenolic compounds, such as tannins, are often correlated with resistance to herbivores, and in some trees their accumulation is induced by previous herbivory. Polyphenol oxidase (PPO) is an enzyme catalysing the oxidation of o-diphenolic compounds to oquinones, as well as the hydroxylation of monophenols to o-diphenols. Wounding of a hybrid poplar causes a strong induction of leaf PPO activity. In response to herbivore attack, many plants release volatile organic compounds (VOCs). Since these complex blends are fed by different biosynthetic pathways linked to a web of interacting signals, volatiles

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might be considered as a “volatilome” that is particularly sensitive to different external triggers. VOCs can carry various types of information: x for herbivores, to localize their host plants; x for indirect defence employing a third trophic level, by attracting natural enemies of the plant’s offender; x for neighbouring plants and for distant parts of the same plant, to adjust their defensive phenotype. Herbivore-induced plant volatiles represent phenotypically plastic responses of plants to herbivory which result in changes in the interaction between individuals in the insect–plant community. Induced plant responses often provide reliable information about the identity of the herbivores. At least in some systems, the amounts of VOCs emitted by the plant are correlated with the density of phytophagous insects feeding on that plant. These VOCs affect the flying behaviour of parasitoids, which are attracted towards the most profitable plants, those bearing the largest number of hosts. Animals usually require information about the current state of their habitat to optimize their behaviour. For this, they can use a learning process through which their estimate is continually updated according to the cues they perceive. The induction of VOC emissions occurs not only in response to herbivore feeding on leaves but also from the deposition of insect eggs on plant parts. The known indirect plant defence mechanisms induced by egg deposition act by supporting egg parasitoids in locating their hosts. For three tritrophic systems (host plants: elm, pine and bean), it has been shown that insect egg deposition induces a plant VOC pattern which attracts egg parasitoids, whereas in a further system consisting of Brassica (plant host), Pieris (herbivore) and Trichogramma (carnivore), egg deposition very likely induced a change of plant surface chemicals, thus arresting the egg parasitoids by contact cues in the vicinity of the eggs. Two types of inducible defences can be described in plants: direct defences and indirect defences. Direct defences include any character of the plant that alone can affect the susceptibility of host plants to insect attacks. Indirect defences, on the other hand, include characteristics of the plant which by themselves do not affect the susceptibility of the host plant, but which can serve as a bait for the natural enemy of the bug that attacks the plant. Natural enemies suppress the insect population and, consequently, reduce the damage caused by the insect on the plant.

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3.2.5.1. Signal Transduction Pathway and Early Events Plants respond to pathogen and herbivore attacks with systemic resistance. Much has been learned about the main signal cascades upon enemy perception, in particular about the systemic signals such as plant hormones and small RNAs and genes involved in the expression of local and systemic resistance. Induced resistance offers exciting prospects for the use of plant defences as an environmentally friendly means of protecting crops from parasites and pathogens. However, many issues still need to be resolved to understand the ecology of induced resistance in order to use artificially induced resistance as a reliable strategy for crop protection. Plants have evolved means to recognize and respond quickly to herbivory. These include the perception of molecular patterns and defence effectors, and the regulation of: x x x x x x x

cytosolic calcium ions; depolarization of the plasma transmembrane potential (Vm); control of ion efflux/influx; activation of mitogen-activated protein kinase (MAPK); protein phosphorylation; activation of NADPH oxidase; production of reactive oxygen (ROS) and nitrogen (RNS) species.

These events lead to a rise in: x production of the phytohormones jasmonic acid (JA), salicylic acid (SA) and ethylene; x expression of late defence response genes; x emission of VOCs; x production of toxic compounds. These events start locally at the feeding site but can spread systemically throughout the plant. Although the individual responses that comprise these pathways have been widely catalogued, the connections between them and their interdependence have received little research attention to date. In the sections that follow, we will analyse the role of early events in plant defence mechanisms against herbivores.

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3.2.5.2. The Sensitivity of the Plasma Membrane and the Role of Symplastic Signaling The plasma membrane, upon recognition of changes in the environment that surrounds the cell, starts a signaling cascade of events that eventually results in specific responses. Leaf damage by insect herbivores implies the direct delivery of elicitors or the indirect generation of plant cell wallderived elicitors that may bind specific receptors at the plant plasma membrane. Emerging evidence indicates that many high-affinity receptors for insect herbivores are located in the plant cell plasma membrane. The elicitor–receptor reaction produces variations in the Vm, which is defined as the difference in the electrochemical potential between the interior and exterior of the plant cell, that is, the electrochemical gradient across the plasma membrane. These variations can lead to either more positive (depolarization) or more negative (hyperpolarization) Vm values, and such events eventually lead to the generation of signaling cascades. In the Spodoptera littoralis–Phaseolus lunatus interaction, both direct herbivory and the insect’s oral secretions (OS) have been demonstrated to induce a rapid Vm depolarization. The same response has been shown in other plant species like Arabidopsis thaliana and Ginkgo biloba, as well as in lower plant species like the fern Pteris vittata. Interestingly, a significant Vm depolarization was observed in response to almost every stylet puncture during Myzus persicae phloem feeding. It is known that systemic signaling induced by biotic stressors is transduced by either chemical or electrical signals. OS from some insect herbivores contain effectors that overcome anti-herbivore defences. Herbivores possess diverse microbes in their digestive systems and salivary glands that can modify plant–insect interactions. For example, the Colorado potato beetle (Leptinotarsa decemlineata) larvae exploit the bacteria present in their OS to suppress the anti-herbivore defences of tomato plants. Furthermore, applying bacteria isolated from larval OS to wounded plants confirmed that microbial symbionts are responsible for this defence suppression. A further demonstration that salivary components are necessary to trigger plant responses to herbivore larvae has recently been provided. The ablation of the ventral eversible gland (VEG) of S. littoralis prompted a significant reduction in the Vm depolarization and significantly reduced both the cytosolic calcium concentration ([Ca2+]cyt) and the H2O2 burst. Moreover, VEG-ablated larvae induced a reduced defence-related enzyme expression and a reduced emission of plant volatiles.

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In general, three mechanisms are recognized for the transmission of electrical signals following herbivory: x action potentials (APs); x variation potentials (VPs); x system potentials (SPs). The OS of insect herbivores are known to cause both APs and VPs, but it is still unclear whether insect herbivory can cause SPs. SPs have been described as novel electrical long distance signals in plants that are induced by wounding, acting as the forerunners of slower chemical signals. SPs serve as backup APs and VPs, and can remain overlapped with APs and VPs in some instances. Although SPs have been demonstrated only in mechanically damaged tissues, it is difficult to exclude the occurrences of SPs following herbivore feeding. APs comprise a generic long distance signaling system that may act to potentiate a host response to subsequent signals delivered through alternative long distance information packages. An AP is a momentary change in the electrical potential of the plant cells that sense stimuli from environmental stressors, eventually leading to intercellular and intracellular communication. A number of substances strongly depolarize the plasma membrane and thus presumably activate voltage-gated ion channels. Although in principle it is possible that (anion) channels are directly activated by depolarization, the temporal sequence of the ion flux kinetics of barley leaves shows that Ca2+ is lost from the apoplast well before the apoplastic anion concentration (measured as Cl-) starts to increase. Therefore, channel activity is involved in APs. The more the channels are activated, the more rapid the depolarization will be, eventually leading to an accelerated depolarization that is measured as a membrane potential “break-through” typical of an AP. APs generated by herbivory propagate as fast electrical signals that travel through the entire plant from the point of origin of the perceived input at a speed of up to 40 cm sec-1. Herbivore-induced plant volatiles (HIPVs) trigger APs and VPs on nearby receiver plants. It is generally accepted that wounded leaves communicate their damaged status to other leaves through a long-distance process. Using non-invasive electrodes, the surface potential changes in A. thaliana were mapped after leaf wounding, and it was found that membrane depolarization is correlated with JA signaling domains in undamaged leaves. These results open new avenues for research in organ-to-organ wound signaling,

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demonstrating the existence of plant genes with functions similar to synaptic activity in animals. An open question remains: how are electrical signals propagated through the plant body? While animals have a nervous system that is specialized in the conduction of electrical signals, nothing similar is present in plants. Central to the success of these defences is the need for local and systemic communication between cells. For plant cells, which are surrounded by cell walls, symplastic continuity is achieved through the presence of plasmodesmata (PD). These plasma-membrane-lined channels, bridging the cell wall, provide symplastic continuity and provide soluble and membrane environments for the passage of small and large molecules, as well as the potential for electrical conduction. PD-located proteins (PDLPs) are type-I membrane proteins with receptor-like properties, although the nature of their potential ligands is not known. Using Arabidopsis plants mutated for pdlp genes, it was shown that the PDs role in the defence against herbivory and that some molecular responses to herbivory can be genetically distinguished from each other and from the overall defence response. However, although PDs have been correlated with gap junctions, no synaptic mechanisms of molecules are present to justify what has been demonstrated in the animal nervous system. Little is known about the electrophysiological responses of phloem sieve elements during wounding, and whether natural damaging stimuli induce propagating electrical signals in these tissues. Very recently, the use of living aphids and the direct current (DC) version of the electrical penetration graph (EPG) were used to detect changes in the membrane potential of Arabidopsis sieve elements (SEs) during caterpillar wounding. Feeding wounds in the lamina induced rapid depolarization waves in the affected leaf, increasing to the maximum amplitude (c. 60 mV) within 2 s. The distal electrical signals elicited by caterpillar cutting are indistinguishable from those elicited by a purely mechanical stimulus, demonstrating that the mechanical aspect of insect chewing is sufficient to induce the full electrophysiological response recorded in the phloem sieve elements of unwounded leaves. Therefore, it is conceivable that the depolarization waves produced by the sieve elements in response to remote wounding contribute to the global wound-activated surface potentials. Another interesting connection to electrical signaling could be the recent discovery that plants are able to discriminate between the vibrations caused by chewing and those caused by wind or insect song. It has been suggested that vibrations may represent a new long distance signaling

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mechanism in plant–insect interactions that might contribute to the systemic induction of chemical defences. The mechanisms used by plants to detect and respond to mechanical vibration have received experimental attention from several groups. 3.2.5.3. Calcium and other ions act as second messengers in plant–insect interactions An open question remains: what causes Vm variations? The candidate ion species responsible for Vm variations in plant cells following herbivory are calcium (Ca+2), protons (H+), potassium (K+) and chlorine (Cl-). Herbivore feeding causes a dramatic Ca2+cytosolic ion influx limited to a few cell layers lining the wounded zone. This response is limited to herbivory or biotrophic activity; neither single nor repeated mechanical wounding induces such significant changes in the cytosolic Ca2+ ion influx. The fact that single or repeated mechanical wounding alone is not sufficient to elicit significant [Ca2+]cyt variations points to oral factors (or herbivore-associated elicitors or herbivore-associated molecular patterns, HAMP) as triggers for a [Ca2+]cyt burst. Insect feeding and isolated insectderived elicitors are known to cause changes in the Ca2+ homeostasis resulting from the tight regulation of protein channels and transporters located in the plasma and organelle membranes. These events have been associated with Vm depolarization. In plants, [Ca2+]cyt is maintained in the nM range (100–200 nM), whereas in many organelles and in the apoplast, [Ca2+] reaches the mM range. The dynamics of spatial and temporal Ca2+ changes in the cytosol and/or in other compartments of the plant cell are now accepted to generate “calcium signatures”, which might be responsible for the initiation of specific downstream events that could eventually lead to the appropriate responses. Elicitor-dependent accumulation of second messengers such as Ca2+ and reactive oxygen/nitrogen species are central to many signaling and regulation processes in plants. However, the mechanisms that govern the reciprocal interrelation of Ca2+, reactive oxygen species (ROS) and reactive nitrogen species (RNS) signaling are only beginning to emerge. NADPH oxidases of the respiratory burst oxidase homolog (RBOH) family are critical components contributing to the generation of ROS, whereas Calcineurin B-like (CBL) Ca2+ sensor proteins together with their interacting kinases (CIPKs) have been shown to function in many Ca2+ signaling processes, including in the control of K+ channels. Figure 3.5 shows the role of Ca2+ signaling pathways following herbivory.

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Figure 3.5 Calcium signaling pathways after herbivory. Following herbivory, insect-originated elicitors bind on putative polysaccharide (ȕ-galactofuranose) or FACs receptors, leading to cytosolic Ca2+ homeostasis. This homeostasis is regulated by influx and efflux channels as well as by ATP-dependent Ca2+ pumps. The cytosolic Ca2+ increase induces the release of Ca2+ from cellular stores (mitochondria, vacuoles and the endoplasmic reticulum) via Ca2+ channels. The cytosolic Ca2+ increase triggers two cascades of signaling events: 1) the activation of inward K+ channels, which causes Vm depolarization, and 2) the activation of CBL-interacting protein kinases (CBL-CIPK), calmodulin-like protein (CML42 & CML43) and calcium dependent protein kinases (CPK3 & CPK13) signaling pathways, which are involved in the activation of transcription factors (e.g., HSFB2A) that lead to the expression of defence genes in the nucleus. The broken arrows indicate the calcium pathways contributing to the increased cytosolic calcium concentrations. From Zebelo and Maffei, 2015.

3.2.5.4. Oxidizing Chemical Defences: Reactive Oxygen (ROS) and Nitrogen (RNS) Species The unravelling of the mechanisms connected to changes in the Vm, [Ca2+]cyt, ROS and RNS production upon insect perception will undoubtedly shed critical light on the mechanisms used by plants to sense insect attacks. ROS and RNS constitute key features underpinning the

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dynamic nature of cell signaling systems in plants. Despite their importance in many aspects of cell biology, our understanding of oxidative and especially of nitrosative signaling and their regulation remains poor. Mechanically damaged lima bean leaves react rapidly and dramatically to H2O2 by inducing a strong Vm depolarization. However, leaves wounded by S. littoralis already show a reduced starting Vm, with the consequence of dramatically lower or even no responsiveness to H2O2 application. Thus, the calcium-induced potassium-dependent depolarization of the Vm following herbivory is linked to a reduction in the downstream responses of the attacked leaves to signaling molecules such as H2O2. The evidence of ROS involvement in plant–insect interactions is growing. The accumulation of reactive oxygen species seems to play a major role in determining the extent of tissue alterations during gall morphogenesis. The potential role of ROS in the defence systems of wheat (Triticum aestivum) and rice (Oryza sativa) against Hessian fly (Mayetiola destructor) larvae reveals a rapid and prolonged accumulation of H2O2 in wheat plants at the attack site during incompatible interactions. Moreover, insect infestation impacts the stress signaling network through the effects on the ROS and cellular redox metabolism, with particular emphasis on the roles of ROS in the plant responses to phloem-feeding insects. Pea aphid (Acyrthosiphon pisum) causes oxidative stress conditions in pea leaves through the enhanced production of H2O2 and O2-•. Another interesting question is how much insects can tolerate the increased ROS production in the attacked leaves. In the emerald ash borer (Agrilus planipennis), antioxidant genes quench ROS from both dietary and endogenous sources, whereas Lymantria dispar caterpillar larvae can tolerate elevated levels of ROS in their midguts without nutritionally significant changes in the compositions of susceptible essential amino acids in their food. The abundance of vitamin C (VitC) in plants influences their susceptibility to insect feeding as VitC is an essential dietary nutrient that serves as an antioxidant in the insect midgut and is also a substrate for plant-derived ascorbate oxidase, which can lead to the generation of toxic reactive oxygen species. Herbivores appear to influence both the de novo synthesis and redox cycling of VitC in their host plants, thereby potentially altering the nutritional value of crops and their susceptibility to pests. The recent development of genetically modified crops with enhanced VitC content provides both an impetus and a tool for further studies on the role of VitC in plant–insect interactions.

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Although nitric oxide (NO) was initially associated with plant defence responses against pathogens, reports indicate that NO is also involved in plant responses to insects. Nitric oxide-associated protein 1 (NOA1) is required for plant resistance in plant–herbivore interactions. NOA1silenced N. attenuata plants treated with mechanical wounding and M. sexta OS showed elevated levels of herbivory-induced JA but reduced concentrations of most carbon-based defensive compounds. These data suggest the involvement of NOA1 in N. attenuata’s defence against M. sexta attack, and highlight its role in photosynthesis and in the biosynthesis of jasmonates and secondary metabolites. In the interaction between S. littoralis and lima bean, both herbivory and robotic wounding induced marked accumulation of NO but with distinct temporal patterns. Herbivory caused a rapid and transient increase of NO levels, whereas the response to robotic wounding was of equal intensity but delayed. Interestingly, the time course of NO production parallels the induction of the volatile compound emission observed in lima beans, suggesting a possible involvement of NO in the signaling pathway leading to volatile emission. Figure 3.6 summarizes the changes in the ROS and RNS signaling pathways in response to herbivory.

Figure 3.6 The role of ROS and NRS in plant–insect interactions. Upon herbivory, insect originated elicitors bind putative polysaccharide (ȕ-galactofuranose) or

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FACs receptors, leading to the cytosolic Ca2+ homeostasis (see also Figure 3.5). The cytosolic Ca2+ increase triggers the CBL-CIPK signaling pathways, which activate the plasma membrane NADP oxidase (ROBH). This in turn generates the superoxide radical anion, which is eventually dismutated to hydrogen peroxide (H2O2) by the catalytic activity of superoxide dismutase (SOD). The activity of ROBH is also regulated by external ATP. H2O2 acts directly on herbivores or enters the cytosol through hydroperoxiporins, increasing the cytosolic H2O2 concentration. By the action of mitochondrial Fenton reaction and the activity of catalase (CAT) peroxidase (POD) and ascorbate peroxidase (APX), the levels of H2O2 are reduced. In contrast, the activity of peroxisomal xanthine oxidase, mitochondrial CIPI/CIPIII, Ubiquinone and chloroplastic PSI catalyse the conversion of O2 to superoxide radical anion (O2-•), which contributes to the elevation of the H2O2 cytosolic concentration by the action of SOD. Cytosolic Ca2+ increase may lead to nitric oxide synthase (NOS) activity, thus increasing the cytosolic NO level. The NO adduct S-nitrosoglutathione (GSNO) level is reduced by the activity of S-nitrosoglutathione reductase (GSNOR), producing the oxidized glutathione disulphide (GSSG) and NH3. Nitric oxide-associated protein 1 (NOA1) and N-nitroso species, as well as the activity of GSNOR have been associated with the phytohormone JA, triggering the transcriptional regulation of defence genes. From Zebelo and Maffei, 2015.

3.2.5.5. Priming A major disadvantage of induced defences is the time interval between the first attack and the activation of the defence, during which the plants remain vulnerable. One way to overcome this disadvantage is to use the information from the environment or from previous attacks to assess the future risk of being attacked and adjust the defence phenotype accordingly. VOCs provide information on the attack status of the emitting plant which can be used not only by higher trophic levels, but also by plants of the same or related/other species in the vicinity. Plants responding to VOCs change their defence-related gene transcription patterns and may increase the production of hormones involved in plant protection. Plants that perceive and interpret these signals with a response receive this so-called “priming” through triggering by volatile substances that are released by damaged plants or other environmental cues. By definition, priming is a mechanism which leads to a physiological state that enables plants to respond more rapidly and/or more robustly after exposure to biotic or abiotic stress. The primed state has been related to increased, more efficient activation of the defence response and enhanced resistance to challenging stress. In plants, priming plays a role in defence (defence priming) and seed germination (priming of seeds). So priming is the physiological state in which plants are able to activate defence

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responses better or faster. So far, priming has been interesting as a basic research tool rather than as applied science, but recent evidence that many pesticides act as priming agents for plants shows promise for the development of future practical applications. Moreover, it has recently been shown that priming also plays an important role in communication between plants (see below). In nature priming can also be the result of communication between plants. For example, volatile molecules emitted by Artemisia tridentata have a priming effect on nearby cultivated tobacco plants, resulting in the rapid production of trypsin protease inhibitors, with consequent and concomitant minor damage suffered from herbivores and a higher rate of mortality in young caterpillars of the herbivore Manduca sexta. Priming is therefore an important mechanism for plant induced resistance. Over the last few years, priming has emerged as a promising strategy in modern integrated pest management because it protects plants against pathogens and abiotic stress without significantly affecting fitness. Owing to their solely protective nature, pioneer compounds that exclusively induce the primed state in plants have not gained acceptance among farmers and growers. Successful modern plant protection agents at best combine both antimicrobial and priming–inducing activities, thus allowing reduced chemical input into the environment for effective and sustainable plant protection. 3.2.5.6. Plant–plant Communication: The Chemical Language In their communities, plants usually experience high plant density and grow along with a variety of neighbouring plants with which they may have an equally diverse range of interactions. Neighbours can be genetically related (e.g., offspring) or independent (e.g., other species) that can provide services (for example being the source of pollen) or pose a threat to fitness (competing or parasitic plants). As we have discussed, some volatile compounds emitted by plants fed by herbivores can act as airborne signals that can increase direct and indirect defences in remote parts of the same plants or in plants nearby. Field and laboratory studies have provided compelling evidence that plants receiving VOC signals are able to respond by activating signal pathways that ultimately lead to the gene expression and synthesis of defence metabolites.

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Airborne molecules that spread and transmit a chemical message are defined as infochemicals. An infochemical molecule is therefore a chemical that, in a natural context, conveys information in an interaction between two individuals, evoking in the receiver a behavioural or physiological response. The terms pheromone and allelochemical are subcategories of infochemical. A useful glossary of allelochemical terminology is as follows: x Allelochemical, an infochemical that mediates an interaction between two individuals that belong to different species. x Allomone, an allelochemical that is pertinent to the biology of an organism (organism 1) and that, when it contacts an individual of another organism (organism 2), evokes in the receiver a behavioural or physiological response that is adaptively favourable to organism 1, but not to organism 2. x Kairomone, an allelochemical that is pertinent to the biology of an organism (organism 1) and that, when it contacts an individual of another organism (organism 2), evokes in the receiver a behavioural or physiological response that is adaptively favourable to organism 2, but not to organism 1. x Synomone, an allelochemical that is pertinent to the biology of an organism (organism 1) and that, when it contacts an individual of another organism (organism 2), evokes in the receiver a behavioural or physiological response that is adaptively favourable to both organism 1 and organism 2. x Antimone, an allelochemical that is pertinent to the biology of an organism (organism 1) and that, when it contacts an individual of another organism (organism 2), evokes in the receiver a behavioural or physiological response that is adaptively favourable to neither organism 1 nor organism 2. It is conceivable to suppose that the same cell machinery involved in the perception of elicitors might also be involved in the perception of volatile cues. If plant–plant communication is proved to exist, the identification of such phenomenon would hold considerable promise for the agronomic improvement of staple food crops. In order to assess the contribution of herbivory to VOC emission, tomato (Solanum lycopersicum cv Microtom) plants were sampled from unwounded plants (controls), mechanically damaged plants (MD) and herbivore wounded (HW) plants. Within one hour from the onset of

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herbivore feeding, a dramatic increase was observed for several green leaf VOCs (GLVs), when compared to controls and MD plants. In order to assess whether VOCs emitted from HW tomato plants were able to induce a response in receiver tomato plants, rooted tomato plants were grown in a glass jar and offered to the herbivore Spodoptera littoralis. The jar was fluxed with clean air and the outgoing flux was directed towards leaves placed in a Faraday cage where Vm was measured (Figure 3.7). Upon perception of herbivore-induced plant VOC cues, tomato receiver plants respond with a strong Vm depolarization, which cannot be fully recovered by fluxing receiver leaves with clean air. Since changes in the Vm imply changes in the flux of ions across the plasma membrane, this observation suggests that some of the herbivore-induced VOCs open ion channels or form pores in the plasma membrane, as has been shown for several terpenoids and elicitors. These results indicate that plant perception of volatile cues from the surrounding environment is mediated by early events, occurring within seconds and involving the alteration of the plasma membrane potential and increases in the cytosolic calcium. GLVs are produced as long as herbivory is present; therefore the continuous emission of these molecules signals to neighbour plants the persistence of herbivore attack. Although the volatile language is still difficult to decipher, low molecular weight VOCs such as GLVs appear to prompt a faster and stronger Vm and calcium response when compared to higher molecular weight compounds such as monoterpenes and sesquiterpenes, which appear to act only on the Vm component of the plant cell response.

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Figure 3.7 Experimental design for evaluation of tomato (Solanum lycopersicum cv Microtom) plants’ responses to VOCs. Clean air (a) is fluxed to either a jar containing tomato plants fed by the herbivore Spodoptera littoralis (b) or an Erlenmeyer flask containing synthetic pure compounds (c), the choice being made through a three-way valve. A Gerstel glass liner is filled with Tenax and the VOCs emitted by herbivore-wounded plants are collected (d). VOCs coming from herbivore-wounded (b) plants are also directed to receiver tomato plants at the site where a probe is inserted in the leaf mesophyll cell (e), whereas the ground probe is inserted in the tomato stem. Both probes are connected to an oscilloscope for Vm measurement (f). Synthetic compounds (c) are either directly fluxed to tomato plants for Vm measurements (e) or to rooted tomato receiver plants, where a leaf is mounted on the CLSM stative (g) and calcium fluxes are analysed; even in this case the choice is made through a three-way valve. The insert (h) shows an enlargement of the CLSM stative were the tomato leaf is mounted. From Zebelo et al., 2012.

Plant volatiles can also drive a parasitic plant to its host. The parasitic plant Cuscuta pentagona exploits host plant volatiles during the selection and location of a suitable host. However, it remains to be clarified whether this parasitic plants’ localization of the host mediated by plant volatiles occurs also below-ground. However, it has been shown that this event can occur through non-volatile substances, mainly water soluble exuded radicals called strigolactones (see below). These substances that plants exudate in the rhizosphere stimulate interactions with arbuscular mycorrhizal fungi (AM) and are exploited by parasitic plants to stimulate

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the germination of their seeds, eventually leading to host plant parasitization. 3.2.5.7. Tritrophic and Multitrophic Interactions Among the numerous examples of plant–insect interaction, the most fascinating is the production of plant volatiles that attract predators of plant feeding insects, the so-called predators’ predators (or enemies of enemies). As we have discussed, the damage suffered by a plant as a result of herbivory causes the production of VOCs that inform natural enemies about the location at which they can find their prey. There are qualitative differences in the release of these substances that cause the predator to distinguish them from the background noise of all the other emitted volatiles. Such recognition is also species specific. It is evident that this type of interaction assumes the characteristic of a tritrophic interaction: plant–insect–carnivorous predator. Plant VOCs have the ability to spread in the environment faster and at greater distances than those produced by herbivores. Hence, carnivorous predators first perceive the plant airborne molecule and then, once the appropriate distance is reached, that produced by the herbivore. For example, females of the parasitic Cotesia marginiventris are not attracted by the smell emitted by the Spodoptera exigua larva that is feeding on corn leaves, but they recognize and are attracted by the volatiles emitted by the attacked plant. The plant produces GLVs, terpenoids and indole compounds that attract the parasite, and the duration of the herbivore feeding determines the type and amount of chemicals emitted and thus the potential capacity to attract Cotesia. After one hour of herbivory, emitted compounds include molecules derived from enzyme activity such as lipoxygenase that eventually generate aldehydes and low molecular weight alcohols. After six hours, terpenoids are emitted. Experimentally, it has been shown that simple abrasion or damage to the leaves causes the emission of volatile molecules typical of short time herbivore feeding (i.e., GLVs). These molecules do not exert any attractive effect on carnivorous predators, which are, rather, attracted to terpenoids, produced only after prolonged herbivory. The shift from mechanical damage induced volatiles (GLVs) to the production of terpenoids is due to the interaction of the herbivore’s OS with the damaged tissues. An interesting observation is that the predator-attracting molecules are produced only during the day and that they cease to be produced when the herbivore moves away from the plants.

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Learning the meaning of smells produced by the host plant is an extremely important aspect of the predator’s behaviour. This ability can help the predator to perceive the subtle differences that exist between signals coming from plant tissues attacked by herbivores and signals coming from mechanically damaged leaves. An important role in tritrophic interactions is played by the VOCs produced by bacteria. 2,3-Butanediol (2,3-BD) is produced by Enterobacter aerogenes, an endophytic bacterium that colonizes plants. The production of 2,3-BD by E. aerogenes makes maize plants more resistant to Setosphaeria turcica, the causal agent of northern corn leaf blight in maize. In contrast, plants inoculated with E. aerogenes are less resistant to the S. littoralis caterpillar. The effect of 2,3-BD on the attraction of the herbivore’s parasitoid is very variable; in fact the application of 2,3-BD to the plant head space has no effect on the parasitoids, but the application of the compound in the soil increases their attraction, suggesting that the effect of 2,3-BD on the parasitoid is indirect and depends on the composition of the microbial community. Therefore, tritrophic interactions are not limited to the events occurring above ground, but extend to those occurring below ground. The interactions between herbivores above and below ground level were first studied in ecological studies in which the researchers had experimentally manipulated the number of wild herbivores feeding on plants. The global pattern emerging from these studies is that belowground herbivores facilitate the feeding by above-ground herbivores, such as aphids. On the contrary, above-ground herbivores often reduce the performance of below-ground herbivores attacking the roots. Therefore, tritrophic interactions between plants, herbivores and carnivores/parasitoids can generally be influenced by plants as a defence strategy. The airborne plant volatiles induced by herbivory represent a true and proper “cry for help”, in full agreement with the concept of chemical language discussed in a previous section. Carnivorous mites were observed to use volatiles released from spider mite-infested lima bean (Phaseolus lunatus) plants to localize their prey. After this initial observation, the idea that herbivore-induced VOCs function as an indirect defence was rapidly confirmed. It is now widely accepted that VOCs can attract predatory arthropods and/or repel herbivores and thus serve as a means of plant resistance. Figure 3.8 depicts the biological effects and interactions of volatile organic compounds.

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Figure 3.8 Feeding by a herbivore (1) elicits the octadecanoid cascade that leads to the synthesis of jasmonic acid (JA) (2), which induces the release of VOCs and of extrafloral nectar (EFN) (3) from both the damaged and intact leaves. Several VOCs, such as Z-3-hexenyl acetate (4), induce indirect defences (EFN, VOCs etc.) in as yet undamaged leaves of the attacked plant. The VOCs then attract parasitic wasps (5) that parasitize herbivorous caterpillars (6) or beetles, and they attract predatory mites (7) that feed on smaller herbivores such as spider mites. Both wasps and mites also feed on EFN, as do ants (8), and both ants and mites may also be housed in domatia (domatia, plural of domatium, from the Latin “domus”). For the response of both con- and heterospecific herbivores, both attraction by VOCs (9) and repellent effects (10) have been reported. Volatile organic compounds can also be perceived by other plants belonging to the same or different species (11), which may be primed or directly induced depending on the concentration of VOCs in the headspace. These interactions do not stop above ground, since feeding on leaves can result in the transport of a systemic signal to roots where it elicits the synthesis of defensive compounds, such as nicotine, while feeding on roots by, for example, beetle larvae (12) can induce the release of VOCs, such as (E)-ȕ-Caryophyllene, from roots (13) and also elicit a systemic signal leading to the induced production of EFN or release of VOCs from aboveground parts (14). MeSA, Methyl salicylate; TMTT, 4,8,12,-Trimethyl-1,3,7,11,tridecatetraene. From Heil, 2007.

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3.2.6. Theories on Defence from Herbivores Numerous theories have been proposed to understand the meaning of plant protection from herbivore attack. Two hypotheses have been proposed: x the first considers the opposition between induced defences and constitutive defences; x the second considers the importance of competition as opposed to the intensity of competition. While the intensity of competition implies a reductionist reasoning, the importance of competition considers the evolutionary consequences, implying phenomenological considerations. It is the opinion of many authors that any individual will always try to make the greatest contribution to future generations rather than optimizing its individual growth. Below are described some theories on plant protection, developed by several authors in the last thirty years. The resource availability hypothesis (RAH), the most successful theory explaining plant defence patterns, predicts that defence investment is related to relative plant growth rate (RGR), which is associated with habitat quality. Thus, fast-growing species should show lower resistance than slow-growing species, which would lead fast growers to sustain higher herbivory rates, but the fitness consequences of herbivory would be greater for slow growers. Seedlings from tree species with higher RGR show a higher tolerance to herbivory. Among the three plant features included, only leaf lifespan showed a significant association with RGR, but RGR was the best predictor of tolerance. The production of defence compounds is only favoured when the cost of their production is less than the benefits obtained by increasing the defence against herbivores. The RAH hypothesizes that the amount and type of defences produced by plants depend on the availability of resources. So the plant has to decide whether to invest in growth and development processes or choose to produce defence molecules to survive. This choice becomes particularly important for plants that show slow growth, while those that grow rapidly will invest their metabolic capital in low-cost defensive molecules. In the seventies, Feeny and Rhoades & Cates formulated hypotheses explaining the evolution of plant defences based on plant apparency (Plant Apparency Hypothesis, PAH). Apparent plants are plants that are

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bound to be found or are susceptible to being discovered by herbivores. These plants are predicted to adaptively produce quantitative chemical defences (i.e., in high concentrations) as a consequence of the longevity of their leaf tissues. Secondary metabolites would be present in long-lived woody plants (highly apparent to herbivores) and mature tissue and would include phenolics and tannins. However, it is known that many of these compounds are not actually defensive in function and do not always act as deterrents in invertebrate herbivores. Conversely, unapparent plants are defined as being hard to find by their adapted herbivores and are characteristic of early successional communities. Although this hypothesis has provided an effective framework for developing new experiments, many inconsistencies have been discovered, and many relevant criticisms have been levied against the hypothesis, including that: x x x x

it ignores the role of upper trophic levels; it has not yielded easily testable hypotheses; it is plagued by many unrealistic assumptions; most plants include a complement of both qualitative and quantitative defences, so the assigning of chemical identities to plants or tissues is often an inaccurate generalization.

A recent meta-analysis tested the predictions of the RAH and some of the predictions of the PAH. They found that herbivory varied significantly across species; this variation could be attributed to the environmental resources of the species rather than species apparency, supporting the RAH. While the PAH paved the way for studies and subsequent hypotheses on the evolution of plant defences, it has in many ways lost its strength in explaining the occurrence and function of plant defences in their current form. The optimal defence theory (ODT) addresses intraspecific patterns of defence against herbivores. ODT predicts levels of defence in different plant tissues by considering the: x fitness value of an organ to a plant; x probability of attack to that organ; x costs of defence or resistance to attack. The ODT predicts that higher levels of defence or more constitutive levels of defence (vs induced defence) should be associated with plant tissues that are more valuable to plant fitness. The ODT also predicts that defence

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levels should be high and constitutive when plant tissues are attacked with a higher frequency than other tissue types. Finally, an important assumption of the ODT is that plants are able to regulate independently the levels of defence in different tissues. Some ecological studies suggest that independent regulation may occur. For example, different levels of constitutive and induced expression of furanocoumarins (see Chapter 6) were found in roots, leaves and reproductive structures in wild parsnip (Pastinaca sativa), and levels of glucosinolate (see Chapter 9) induction varied widely across plant tissues in wild radish. Moreover, molecular studies have identified high levels of gene and protein expression in floral tissues that are not found in other tissues. In other words, the ODT predicts high resistance to subsystems under attack (for example, in response to herbivores that attack roots) with no corresponding changes in different parts of the plant that are not at risk of attack (e.g., leaves in response to root herbivory). The growth rate hypothesis (GRH) predicts that plants that sustain rapid growth will dedicate less total resources to defence than plants that grow more slowly, in part because slow growers may not be able to tolerate damage. As with the ODT, the GRH assumes that plants have limited resource availability and that defences will have costs. The GRH can also be applied to specific plant tissues. For example, tissues that grow rapidly or have high rates of cell division (e.g., shoot apical meristems) should have fewer defences than slower growing tissues. The growth differentiation-balance hypothesis (GDBH) of plant resource allocation provides a framework for examining the impact of a resource gradient on the constant trade-off between growth and differentiation in the cells and tissues of plants, in particular with the consequences for plant defence. Thus nutrient-limited plants should accumulate carbon-based defences. The excess of carbohydrates, manifested by an increased foliar C:N ratio, can then be inexpensively converted to secondary metabolites. The GDBH provides a framework that predicts a trade-off between the costs of differentiation processes relative to the demand for photosynthates by growth (i.e., in the plant, growth is inversely related to differentiation). Differentiation includes all processes not involved in growth, including the synthesis of secondary metabolites. Furthermore, the hypothesis assumes that herbivory is a strong selective force for the production of secondary metabolites. The GDBH was developed using empirical evidence from plant species in northern boreal and temperate systems, leaving its applicability to species under different abiotic and biotic conditions questionable and problematic. In fact, the GDBH was unable to explain

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allocation to secondary metabolites in an African Savanna woody species (Combretum apiculatum), and suggests that mechanistic explanations of plant allocation should consider the integrative defensive effect of changing secondary metabolites. In the push–pull strategy (PPS), specifically chosen companion plants are grown in between and around the main crop. These companion plants release semiochemicals that repel insect pests from the main crop using an intercrop which is the “push” component, and attract insect pests away from the main crop using a trap crop which is the “pull” component. Such a system requires a complete understanding of the associated chemical ecology of plant–insect and plant–plant interactions on the different crops. Candidate plants need to be systematically evaluated in the laboratory and field trials. The science underpinning these interactions is vital in discovering and understanding the underlying mechanism of the companion plants.

3.2.7. Allelopathy The term allelopathy was introduced by the Austrian plant physiologist Molisch to refer to biochemical interactions between plants. Molisch referred to both the inhibitory and stimulatory biochemical interactions. Later, allelopathy was defined as any direct or indirect, beneficial or destructive effect by one plant on another through production of chemical compounds (allelochemicals/phytotoxins). These compounds are released into the environment from plant parts by leaching, stem flow, root exudation, volatilization, residue decomposition and other processes in both natural and agricultural systems. Allelopathy is the mechanism explaining inhibitory and/or stimulatory interactions in the soil–plant interface through bioactive products produced by biochemical pathways. An important point regarding allelopathy is that its effects depend upon chemical compounds released to the environment from living or decaying plants. Allelopathy reflects interdisciplinary, challenging and complex studies to unravel myths and facts and also invites the attention of global agronomists, botanists, soil scientists, microbiologists, phytochemists, foresters and ecologists. Allelochemicals can be classified into the following categories: x water-soluble organic acids; x straight-chain alcohols;

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x x x x x x x x x x

aliphatic aldehydes, and ketones; simple unsaturated lactones; long-chain fatty acids and polyacetylenes; quinones (benzoquinone, anthraquinone and complex quinones); phenolics; cinnamic acid and its derivatives; coumarins; flavonoids; tannins; steroids and terpenoids (sesquiterpene lactones, diterpenes, and triterpenoids).

Allelochemicals are released into the environment in various ways: exudation, percolation, leaching, secretion or simple decomposition. In general terms, allelopathy represents a particular aspect of what we have previously defined as the plant–plant interaction. In many cases, however, both directly and indirectly, allelopathy involves the presence of microbes, since the effectiveness of a chemical compound produced by a plant in exerting an effect on another plant depends on the speed with which the soil microorganisms are able to detoxify and further metabolize these compounds. In the soil, where most microorganisms reside, the efficacy of allelochemicals is affected by alterations in the root development and by the placement of a plant’s roots in relation to other plants’ rhizospheres. Although nearly a century has passed since Molisch’s observations, not all plant ecologists accept the concept of allelopathy as a significant factor in the mechanisms of competition between plant communities. Critics depend on the extreme difficulty of conducting studies that cover the many aspects of plant–plant interaction, but this does not exclude the existence, proven by hundreds of scientific publications, of the interaction between chemical molecules produced by certain plants and the physiological impairments suffered by others. However, chemicals with allelopathic functions have other ecological roles, such as plant protection, nutrition chelation and soil organism regulation by interfering with soil decomposition and fertility. On an ecosystem scale, the roles of allelochemicals can increase, mitigate or modify their functions in the community dimension. Allelopathy plays an important role in natural ecosystems, and has also the potential to become an important tool in cultivated ecosystems. If crop

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allelopathy is properly exploited for agricultural practices and production, agronomic applications could be addressed to: x biological control of insects, pests and diseases; x enhancement of soil quality by adding nutrients for crop plants during the decomposition of residues; x amelioration of the soil environment for microbes, x increase of crop diversity by rotation along with the reduction of weed and pest infestations; x crop rotation and the improvement of soil quality; x development of low-cost biopesticides with novel modes of action for sustainable and integrated pest management; x development of tolerance against abiotic stresses. An important issue emerging from studies on allelochemicals is their ability to interfere with biological membranes, causing signal transduction cascades and altering their permeability. To explain how such compounds alter the permeability two models have been proposed: x Allelopathic compounds (especially phenolics) solubilize in the cell membranes, thus causing the weakening of the membrane structure that causes the loss of ions outside the cell. The direct correlation between the inhibition of the electric potential and the logarithm of the partition coefficient of tested molecules supports this hypothesis. x Phenolic compounds cause the intake of anions and organic acids on the cell, which then leads to either a negative membrane potential or an increased membrane anionic strength. It cannot be excluded that the loss of ions from the cells treated with phenolic allelochemicals might be caused not only by the alteration of the membrane permeability but also, or alternatively, by the dissipation of the driving force that keeps the cell’s ion concentration at a high level. Recent studies show the existence of a complex network of interactions between allelochemicals and plant responses that involve many signal transduction pathways that lead to the expression of a vast number of stress response genes. Figure 3.9 summarizes early and late events in the interaction between plant cells and allelopathic substances.

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Figure 3.10 Interaction between allelopathic molecules and plant cells. Allelochemicals interact with the cell membrane causing Vm alterations, interact with membrane receptors, and affect the activity of ion and water transporters. These early events trigger signal pathways involving plant hormones and the transcriptional activity of the nucleus. The alteration of the enzymatic activity and protein synthesis affects respiration (mitochondria) and photosynthesis (chloroplasts), while the alteration of osmolarity and water status affect transpiration. All of these events ultimately reduce cell expansion and division by influencing growth and development processes. From Li et al., 2010, modified.

Allelopathy has been suggested as an innovative approach to tackle multifaceted issues of contemporary agriculture. A consistent body of knowledge suggests that secondary metabolites of living plants and dead residues are involved in many biochemical interactions in the biosphere. Many different types of useful products, such as low-cost biological pesticides and drugs, could arise from allelopathy studies. The search for these molecules is in full agreement with the sustainability principles that have been discussed in Chapter 1 and will serve, in addition to the understanding of the cellular and molecular mechanisms of the interaction of allelochemicals, to introduce natural, environmentally friendly and easily biodegradable compounds into agricultural practices. In addition,

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knowledge of potentially allelopathic plant species will be crucial in preventing previous crop residues from adversely affecting the germination or productivity of subsequent cultures. 3.2.7.1. Parasitic Plants and Allelochemicals A peculiar aspect of allelochemicals relates to parasitic plants. These plants are among the most problematic pests for farm crops around the world. Parasitic plants represent a taxonomically heterogeneous group of angiosperms that rely partly or completely on the host plant to obtain carbon, water and nutrients. Nutrients and water are acquired by attacking the roots or shoots of the host plant through specialized structures known as haustoria and penetrating the xylem and/or the phloem. The host attack site (root or shoot) classifies the type of the parasite, while the presence or absence of functional chloroplasts further defines the parasite as a hemiparasite or holoparasite respectively. A distinct functional and evolutionary difference exists between hemiparasites, which retain photosynthetic activity, and non-green holoparasites, which fully depend on their hosts for all essential resources. Parasitic plants are common to many natural and semi-natural ecosystems from tropical rainforests to the northern hemisphere and comprise approximately 4,500 species which account for 1% of angiosperm species within about 270 genera and more than 20 families. Orobanchaceae and Santalales are the largest monophyletic groups of parasitic plants, both of which also contain both hemi- and holoparasites. By contrast all the other lineages are small in terms of number of species and genera, and uniform in terms of the trophic strategy of their species. Some hemiparasitic species are non-green and thus completely dependent on their host during the initial period of their life, and this is typical of the closely related Striga and Alectra species, which require immediate contact with host roots after germination. Strigolactones were first identified as root-extruded host factors that stimulate the germination of parasitic plants of the genera Orobanche and Striga. More recently, strigolactones were also identified independently as a communication molecule between arbuscular mycorrhizal (AM) fungi and plant roots. Root extruded strigolactones stimulate AM fungi hyphal branching allowing them to grow towards the plant root. Purification of this plant-derived hyphal branching factor through fungal bioassays in combination with analytical chemistry has demonstrated that these chemicals were indeed the communication cues between fungi and the

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plant. As parasitic weeds have evolved the ability to exploit strigolactones as a means to recognize and infect their host plants, these molecules eventually behave as a double-edged blade, i.e., by bonding their friends (e.g., AM fungi) while hiding their foes (parasitic plants). Figure 3.11 depicts this dual aspect of the strigolactones.

Figure 3.10 The dual activity of strigolactones in the rhizosphere. To interact with the other soil organisms, plants secrete strigolactones in their rhizosphere through the strigolactones exporter PDR1. Diazotrophs and AM fungi respond to strigolactones, favouring their interaction with the plants (friend side of strigolactones). On the other hand, parasitic plants misuse the presence of

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strigolactones to recognize their hosts (foe side of strigolactones). From De Cuyper and Goormachtig, 2017.

The translocation and absorption of molecules and macromolecules from host plants to parasitic plants have been well documented. The first test of molecular translocation through a haustorian organ was the transmission of viral diseases between two host plants through the Cuscuta parasitic plant. In general, the molecular translocation between host and parasite ranges from the translocation of radiolabelled sugars, herbicides, plant viruses, siRNA and mRNA to protein transfer. Much less is known about the potential effects of translocation of non-nutritive bioactive solutes such as phytohormones, secondary metabolites, RNA and proteins on the development and physiology of parasitic plants and their successful interactions with other organisms such as herbivorous insects or microbial pathogens. An increasing number of recent studies have documented the transfer of these molecules from the host plant to the parasitic plant, implying that they can have a significant impact on the physiology and ecology of the parasite plant. It has recently been found that some of the defence responses induced by herbivores and pathogens, such as the increased synthesis of jasmonic acid and salicylic acid as well as the hypersensitive response, also occur in tomato plants attacked by the parasitic plant C. pentagona. There are therefore remarkable similarities between the defences induced in response to parasitic plants and those described above for herbivores and pathogens. Finally, a recent genomic study has shown that in the holoparasitic plant Rafflesia cantleyi (Rafflesiaceae), whose closest relatives have the largest flowers in the world, about 2.1% of the nuclear gene transcripts were probably acquired by its obligate host plant. But parasitic plants are not only deleterious, they also have medicinal properties. Important traditional uses are documented for the species Scurrula atropurpurea, Dendrophthoe pentandra and Helixanthera parasitica.

3.2.8. Chemical Defence from Microorganisms Microbial species are present in almost every natural habitat, accounting for about 60% of the total land biomass. This, together with their extraordinary genetic, metabolic and physiological diversity, poses a serious threat to plant health and growth, while on the other hand, it is an indispensable way to improve fitness and the ability to find nutrients. In this section we will not deal with the chemical mechanisms involved in

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symbiontic interactions, but we will limit our discussion to analysing the classes of bioactive molecules produced by plants to defend themselves from pathogenic microorganisms. Secondary plant metabolites can act on microbial cells in several ways. The main mechanisms include disruption of membrane function and structure (including the efflux system), disruption of DNA/RNA synthesis and function, intermediate-metabolism interference, induction of coagulation of cytoplasmic components and the breaking of cellular communication (so-called quorum sensing, QS). This antibacterial action usually includes the following sequence of events: x interaction of plant bioactive metabolites with the cell membrane; x their diffusion through the membrane (i.e., penetration within the cell); x the interaction of bioactive metabolites with the intracellular processes. Thymol, a p-menthane monoterpene alcohol, is produced in some aromatic plants such as Origanum vulgare and is one of the most active secondary metabolites. This compound provides a good example to illustrate the mechanism of the antimicrobial action of plant metabolites. This compound likely interacts with the outer and inner parts of the cytoplasmic cell membrane, by interacting with the polar heads of the lipid bilayer. This interaction alters the cell membrane and eventually leads to its disintegration or to increased permeability. Moreover, thymol also has a role in regulating the genes involved in the synthesis of the outer membrane proteins, inhibits the enzymes involved in thermal stress protection, and interferes with the synthesis of ATP and the citric acid pathway. Carvacrol, an isomer of thymol, interacts with the plasma membrane by inserting acyl chains between the phospholipids, thus affecting the membrane fluidity and permeability. This increased permeability leads to the efflux of ions and ATP, with the alteration of both the Vm and the pH gradient. By measuring the release of the lipopolysaccharides which are present in the outer membrane, it has been demonstrated that carvacrol also affects the outer membrane, the structure responsible for the resistance to Gram-negative bacteria. Trans-cinnamaldehyde, a phenolic compound, inhibits enzymes synthesizing the fungal cell wall by acting as a non-competitive inhibitor

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of ȕ- (1,3)-glucane synthase as well as being a mixed inhibitor of chitin synthase isoenzymes. A study on Saccharomyces cerevisiae has also shown that trans-cinnamaldehyde causes the partial destruction of the integrity of the cytoplasmic membrane, resulting in an excessive loss of the metabolites and enzymes from the fungal cell and the fungus death. During the antimicrobial action of trans-cinnamaldehyde, at least three processes occur: x at sub-inhibitory concentrations, enzymes involved in the cytokinesis are affected; x at higher concentrations, ATPase is inhibited; x at lethal concentrations, the cell membrane integrity is completely disrupted. Another phenylpropane, vanillin, also functions as an active membranedisrupting compound, but it also has intracellular targets. The literature data available on the antimicrobial action of many bioactive plant metabolites indicates that their primary target site is the cytoplasmic membrane. These substances can affect the structure and integrity, permeability or functionality of the membranes in many ways. For example, some antifungal plant compounds interact with ergosterol, which is the main sterol of the fungal membrane involved in important processes such as maintaining the membrane fluidity and integrity and regulating the fungal enzymes necessary for cell growth and division. Saponins (a family of triterpenes referred to in Chapter 7) have a potent antimicrobial activity generally attributed to their ability to form complexes with sterols of the fungal membranes, causing the formation of pores and the loss of membrane integrity. VOCs like linalool, linalyl acetate, menthol, limonene, eugenol and citral exert a proven effect by changing the cellular morphology and the fluidity of the membrane of many bacteria and fungi. In addition to the effect on cell membranes, the antimicrobial action of plant bioactive metabolites can be directed towards intracellular processes such as DNA/RNA/protein synthesis and cell-to-cell communication. This is the case with allicin, the main compound of garlic (Allium sativum), which is released when the bulb is crushed. Thiosulphinates readily react with the free SH groups of intracellular enzymes, leading to the inhibition of RNA synthesis. Overall, allyl isothiocyanate inhibits enzymes and causes protein alteration by the oxidative dissociation of disulphide bonds. One of the main groups of active plant compounds, the flavonoids, inhibits

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both cytoplasmic membrane function and DNA synthesis. Apigenin and quercetin, together with many other flavonoids, inhibit DNA gyrase.

3.3. Chemical Defence from Abiotic Stress Abiotic stress plays an important role in determining the productivity of crops and has a dominant effect on the distribution of plant species in different environments. Climate change exacerbates abiotic stress on a global scale, with uneven and unpredictable consequences. In addition, the interaction between abiotic and biotic stress has the power to damage agricultural production and natural ecosystems. One of the most interesting features in adapting to abiotic stress is the activation of multiple responses involving complex gene interactions. Knowing how these biological processes are modulated by abiotic stress is still an open challenge for both public and private research.

3.3.1. Plant Defence from Ultraviolet Radiation An inevitable stress on plants is exposure to Ultraviolet-B (UV-B, 280-320 nm) radiation. Although it represents only a small fraction of the electromagnetic spectrum, UV-B radiation is known to affect all living organisms, including higher plants. UV-B radiation levels depend on a number of factors such as weather, time, latitude, altitude, cloudiness and thickness of the ozone layer. Plants, during evolution, have developed multiple defence mechanisms against the stress imposed by exposure to UV-B radiation. Among the various effects induced by UV-B radiation, the most common is the modulation of secondary metabolism, including the increased concentration of phenolic compounds in leaf tissues and increased production and emission of isoprene and terpenoids. However, while the effect is clearly established for flavonoids, UV-B radiation does not always lead to an increase in the production of terpenoids. UV-A radiation was also found effective on the photomorphogenesis and essential oil chemical composition of Mentha piperita. The effect of UV-B radiation can also interfere with plant growth and development processes. Grape cuttings irradiated with supplemental UV-B applied both from aging to maturation and from the appearance of ripening at maturation show that UV-B radiation does not significantly alter the size of the berry, but increases the thickness of the skin. Moreover, UV-B radiation does not affect the time to reach maturation. In spite of this, the concentration of anthocyanins, colour intensity and skin flavonols were

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enhanced by excess UV-B radiation, especially in plants exposed during fruit setting. The quantitative and qualitative profile of flavonols in grape skin was also modified by UV-B radiation. For example, mono-substituted flavonols increase proportionally to the amount of UV-B radiation.

3.3.2. Plant Volatiles and Response to Extreme Climatic Conditions Another interesting aspect of the secondary metabolism response to adverse environmental conditions is the production of isoprene. Isoprene (or 2 methyl-1,3-butadiene) is a simple isoprenoid emitted into the atmosphere by many plant species. Once synthesized, this compound is not stored in the plant but is emitted into the atmosphere through the stomata as a result of its high volatility. Isoprene plays an important role in atmospheric chemistry because of its high reactivity. In the presence of high concentrations of nitrogen oxides, oxidation of isoprene by the hydroxide radical can increase ozone formation, thereby reducing the quality of air. In normal growth conditions, plants typically release into the atmosphere 0.5–2% carbon fixed by photosynthesis in the form of isoprene. Therefore, predicting how the emission of isoprene could change in the future will help to predict changes in atmospheric oxidants, greenhouse gases and organic aerosol concentrations. At the leaf level, the increase in the emission of isoprene with increasing temperature is offset by the reduction of its production rate caused by the increase in CO2. At the canopy level, the increase of the leaf area index in the presence of high CO2 concentrations can offset the reduction in the isoprene emission. On a global scale, the reduction in forest canopy may decrease the rate of emissions of isoprene, while the increased use of fertilizers can increase it. The impact of abiotic stress on volatile secondary metabolites is more controversial. In the case of volatile terpenes, the relationship between the uncoupling of photosynthesis and the emission of these substances is surprising, due to the high demand for photosynthetic carbon for terpene biosynthesis.

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Torres-Vera, R., Garcia, J.M., Pozo, M.J. and Lopez-Raez, J.A. (2014). Do strigolactones contribute to plant defence? Mol. Plant Pathol. 15, 211– 216. Tsuchiya, Y. and McCourt, P. (2012). Strigolactones as small molecule communicators. Mol. Biosyst. 8, 464–469. Turley, N.E., Godfrey, R.M. and Johnson, M.T. (2013). Evolution of mixed strategies of plant defense against herbivores. New Phytol. 197, 359–361. van Dam, N.M. and Heil, M. (2011). Multitrophic interactions below and above ground: en route to the next level. J. Ecol. 99, 77–88. Velikova, V.B. (2008). Isoprene as a tool for plant protection against abiotic stresses. J. Plant Interact. 3, 1–15. Volkov, A. (2012). Plant Electrophysiology: Methods and Cell Electrophysiology. Heildelberg: Springer. Weston, L.A. and Mathesius, U. (2013). Flavonoids: their structure, biosynthesis and role in the rhizosphere, including allelopathy. J. Chem. Ecol. 39, 283–297. Westwood, J.H., Yoder, J.I., Timko, M.P. and de Pamphilis, C.W. (2010). The evolution of parasitism in plants. Trends Plant Sci. 15, 227–235. Woods, H.A. (2014). Mosaic physiology from developmental noise: within-organism physiological diversity as an alternative to phenotypic plasticity and phenotypic flexibility. J. Exper. Biol. 217, 35–45. Yu, P., White, P., Hochholdinger, F. and Li, C.J. (2014). Phenotypic plasticity of the maize root system in response to heterogeneous nitrogen availability. Planta. 240, 667–678. Zebelo, A.S. and Maffei, M.E. (2015). Role of early signalling events in plant–insect interactions. J. Exp. Bot. 66, 435–448. Zebelo, S.A. et al. (2011). Chrysolina herbacea modulates terpenoid biosynthesis of Mentha aquatica L. PLoS One. 6, e17195. Zebelo, S.A., Matsui, K., Ozawa, R. and Maffei, M.E. (2012). Plasma membrane potential depolarization and cytosolic calcium flux are early events involved in tomato (Solanum lycopersicum) plant-to-plant communication. Plant Sci. 196, 93–100. Zimmermann, M.R., Maischak, H., Mithofer, A., Boland, W. and Felle, H.H. (2009). System potentials, a novel electrical long-distance apoplastic signal in plants, induced by wounding. Plant Physiology. 149, 1593–1600.

CHAPTER FOUR BIOACTIVE PLANT MOLECULES IN FOODS, DRUGS AND DIETARY SUPPLEMENTS

The ability of animals to use plant bioactive molecules to treat diseases is one of the most important heritages that humankind has acquired. Since prehistory, the evolution of humanity has been favoured by the presence of plants with medicinal properties, thus contributing to human survival ability. Even today, despite the radical change imposed by synthesis chemistry, a large amount of drugs used to cure many diseases directly or indirectly derives from plants. Recently, a number of new plant-derived molecules have entered the drug market, generating a wave of new studies and research both of a pharmacological and clinical nature. In particular, major advances have been made in the treatment of diseases such as cancer or pathologies due to infectious agents such as malaria. Alongside the pharmaceutical industry, which extracts and purifies bioactive substances from plants, there is a less standardized and controlled industry that uses medicinal plants in the form of herbs, extracts and powders as a support for a large amount of health disorders. In addition, research on natural products can be guided by knowledge of popular uses and traditions through ethnopharmacological and ethnopharmacognostic studies, contributing substantially to the innovation of medicinal products by providing new chemical structures and mechanisms of action. In this chapter, we will discuss the role of plant bioactive molecules as a source for drugs, but we will also analyse their role as dietary supplements. The aim of this chapter will not be the description of the numerous pharmaceutical applications of secondary plant metabolites, which are detailed in many phytochemical and pharmacognosy textbooks. Rather, we will focus on some aspects of the common use of medicinal plant extracts and how in the third millennium the issue of managing

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knowledge held in local cultures and the standardization of plant-based products are addressed. Depression, insomnia, immunodepression, senile dementia, cellular aging, oxidative stress, hypertension and inflammation are among the most common pathologies, with significant consequences for the world healthcare system, both economically and therapeutically. Some plants that show the potential to cure these diseases will be discussed in this chapter.

4.1. Dietary and Food Supplements During our lifetime we often have the opportunity to experience the effects of plant products, both consciously and unconsciously – from the cup of coffee or tea with stimulating effects, or chamomile and valerian with their sedative properties through to the flavours and fragrances of aromatic herbs and spices. In short, we all make use one way or another of bioactive principles. The conscious use of medicinal plants is extremely varied, ranging from the 500–600 species of western tradition to 3,000 in Indian traditional medicine and on to reach 7,000–8,000 species in Chinese Traditional Medicine. In many cases, the number of plant species used is proportional to the years of documented practice. There is evidence of the use of medicinal plants dating back to earlier periods of written documentation. An in-depth study of prehistoric rock records found on the plateau of Tassili-n-Ajjer in North-East Saharan Africa (Northeast of Haggar in Algeria) has shown that local populations, at a historical moment conventionally referred to as the “period of the Round Heads”, collected and used hallucinogenic plants. The hallucinogenic molecules were contained in seeds of a plant quite similar to a Convolvulaceae and not in the fungi, as is often generally indicated. Later on, Chinese writings dating to about 2,800 BC describe the use of 366 medicinal plants, while in the eighteenth century BC King Hammurabi of Babylon founded a public institution with the intent to list and collect information on the healing properties of some medicinal plants, transcribed on stone plates. In the fourth century BC Diocles of Carystus (also known by the Latin name Diocles Medicus), a student of Aristotle, listed some medicinal plants along with their use in a collection named Rhizotomika, but the idea of using medicinal plants for therapeutic purposes took on a character of science with the studies and descriptions of Hippocrates, Dioscorides and Galen.

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Medical botany experienced a major change at the beginning of the nineteenth century, with the development of chemistry, which made possible the isolation and purification of bioactive molecules. At the beginning of the twentieth century, Paul Ehrlich developed the theory that bioactive molecules acted as “magic bullets” capable of targeting their target selectively, without harming the body itself. Ehrlich’s laboratory discovered arsphenamine (Salvarsan), the first effective medicinal treatment for syphilis, thereby initiating and also naming the concept of chemotherapy. Individual molecules were judged more specific in their therapeutic action than botanical preparations and so the pharmaceutical industry was born. After decades of drug treatment with pharmaceuticals, the end of the last millennium has rediscovered the public interest in alternative medicines based on natural remedies. The pressure of this public interest has prompted the governments of many countries to consider more deeply the issue of regulating natural product extracts and has led medical and pharmaceutical associations to critically reconsider both the potential of these remedies and the use of these alternative therapies. In the United States, dietary supplements sales amounted to about $27 billion and increased by 4.5% from 2010 to 2011. The US is the most active market for nutraceuticals in the world, with a contribution of over $86 billion of the global revenues of nutraceuticals over the years. European countries spend about €6 billion annually on over-the-counter products based on medicinal plants. The UK, Germany, France and Italy are the major geographic markets in Europe. Asia-Pacific (including Japan) is expected to take the second largest market share after North America by 2018. In the United States, botanical products are regulated by the Food and Drug Administration. Usually, products used for dietary purposes or as dietary/food supplements are classified as foods, while products used to cure or prevent diseases are classified as drugs. Plant products sold as foods include vegetables, fruits, cereals and grain legumes, along with aromatic herbs such as spices and herbal teas (including tea). All these products are strictly controlled and subject to sanitary inspections by the health departments of the countries where they are marketed. In many cases, products are labelled by indicating the content of additives, expiry dates and specific claims, and therefore regulations are legally enforceable in the event of non-compliance or false declaration. Plant products can also be sold as supplements, these being substances sold with the intent of supplying the daily dietary requirements and including herbs or plant extracts, vitamins, minerals and amino acids.

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Unlike pharmaceuticals, that have the precise purpose of healing a particular disease or symptom and for which an informative prospectus about the action, contraindications, posology and side effects is provided, food and dietary supplements can only claim their possible nutritional support. Many dietary supplements present in the European market list statements that are inherent to: x benefits with respect to a disease caused by the lack of a nutrient element; x description of the role of a nutrient or dietary ingredient that affects the structure or function of the human body; x characterization of the mechanism through which a nutrient or dietary supplement acts to maintain that structure or function; x description of the general well-being obtained from the intake of a nutrient or dietary supplement.

4.1.1. Functional Foods Foods can be regarded as functional if they are satisfactorily demonstrated to affect beneficially one or more target functions in the body, beyond adequate nutritional effects, in a way that is relevant to either an improved state of health and wellbeing and/or a reduction of risk of disease. The term functional foods was first introduced in a report on the Systemic Analysis and Development of Food Functions sponsored by the Japanese Ministry of Education, Science and Culture during the period 1984–1986. Since any food is basically a supplier of nutrients to the body, its nutritional function is naturally understood to be of primary importance. This can probably be recognised as true in almost any part of the world in any period of history. Unlike functional ingredients, the target functions of functional foods are quite diversified, while reflecting regional trends of nutrition policy and research. Functional foods are different from nutraceuticals, which are defined as products produced from foods but sold in pills, powders and other medicinal forms not generally associated with food – demonstrated to have a physiologic benefit or reduce risk of chronic disease. The unique features of a functional food include the following: x it should consist of conventional ingredients or compositions and be consumed in the conventional form or method of food; x it should be consumed as part of the staple diet;

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x it is composed of naturally occurring (as opposed to synthetic) components, perhaps in unnatural concentration or present in foods that would not normally supply them; x it has a positive effect on target function(s) beyond nutritive value/basic nutrition; x it may enhance wellbeing and health and/or reduce the risk of disease or provide health benefits so as to improve the quality of life, including physical, psychological and behavioural performances; x it possesses authorized and scientifically based claims (i.e., it should be labelled as having a body control function). One of the major problems in standardization is definition. The definition of functional foods has been proposed by several authors and organizations, and can be summarized as follows by considering that: x functional foods should be distinguished from vitamins, minerals and other dietary supplements; x these foods should not be allowed to be accompanied by medical claims; x the altered functional effects of the foods must be substantiated and scientifically proven through laboratory and human studies. In addition, a functional food should satisfy the following criteria: x the food should be expected to contribute to the improvement of the diet and the maintenance/enhancement of health; x the health benefits of the food or its constituents should have a clear medical and nutritional basis; x based on medical and nutritional knowledge, an appropriate daily intake should be definable for the food or its constituents; x based on experience, the food or its constituents should be safe to eat; x the constituents of the food should be well defined in terms of physicochemical properties and qualitative/quantitative analytical determination; x there should be no significant loss in nutritive constituents of the food in comparison with those contained in similar types of food; x the food should be of a form normally consumed in daily dietary patterns, rather than consumed only occasionally;

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x the product should be in the form of a normal food, not in another form, such as pills or capsules; x the food and its constituents should not be those exclusively used as a medicine. It is also required that the functional foods: x remain as foods; x demonstrate their effects in amounts that can normally be expected to be consumed in the diet; x be consumed as part of a normal food pattern (i.e., not as pills or capsules). 4.1.1.1. Functional Foods or Phytopharmaceuticals? To distinguish between the regulatory approaches for food and for drugs, three criteria have been suggested: x Targeted effect: foods, unlike drugs, yield a future benefit rather than an immediate effect. x Target population: foods provide a broad-brush treatment approach to an entire population, whereas drugs are consumed by a targeted population of those who are ill or have medically accepted indicators of disease. x Safety: foods have a presumption of safety and are not allowed to have a benefit–risk equation applied to them, whereas the benefit– risk equation of drugs is an accepted part of their regulatory approval. The level of proof for drugs is well established internationally and does not include a presumption of safety. There are numerous studies that correlate diet with human health and the categories of compounds found in foods range from lipids to proteins, from simple sugars to polysaccharides, and from non-protein amino acids to vitamins. For example, hesperidin (1) a flavonoid common to many fruits and vegetables, has a potent chemopreventive action against cancer, while quercetin (2), another flavonoid present in foods, is able to inhibit both the binding activity of KB nuclear factor and oxidative damage to DNA in HepG2 cells. Although folic acid (3) is contained in many foods, its deficiency may be more frequent than is believed because of its sensitivity to various processing and cooking conditions.

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Recent studies have found that folic acid deficiency can increase the risk of cardiovascular disease through increased bloodstream intake of homocysteine. Of great importance is the discovery that diallyl sulphide (DAS, 4) and diallyl disulphide (DADS, 5) present in garlic are able to inhibit the activity of the enzyme arylamine N-acetyltransferase in human colon tumour (adenocarcinoma) cells. Both DAS and DADS decrease the Km and Vmax of the enzyme as a function of their concentration. Individual types of food cannot, however, overcome all nutritional deficiency. Because the amount of certain substances is lacking in some foods it is necessary to eat food in a varied and non-systematic way. A classic example is the amount of vitamins present in some foods that are served cooked. Table 4.1 illustrates the variability of the nutritional power between cooked and raw foods.

Table 4.1 Comparison of vitamin content in some cooked foods and in dietary supplements. Vitamin Vitamin A (UI) Vitamin C (mg) Thiamin (mg) Riboflavin(mg) Niacin (mg) Vitamin B12 (μg) Folate (μg)

Supplement 5,000.00 50.00 3.00 2.50 20.00 3.00 0.00

Broccoli 3,886.00 208.80 0.15 0.32 1.61 0.00 140.00

Carrots 11,294.00 1.06 0.02 0.03 0.23 0.00 6.39

Potatoes 0.00 9.90 0.13 0.03 1.77 0.00 12.02

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4.2. Plant Bioactive Molecules and the Treatment of Diseases Studies by a number of state and private bodies have found that between 30 and 50 percent of the adult population in industrialized countries uses Complementary and Alternative Medicine (CAM). CAM, as defined by the WHO and the FDA, covers a broad group of healthcare practices that are not integrated in the conventional healthcare system and includes use of herbs, use of dietary supplements (e.g., vitamins), traditional Chinese medicine, meditation, massage, chiropractic, praying, seeing a healer and more. An in-depth study on bibliographic data identified at least 25 highly scientific investigations that identified five major therapies: acupuncture, chiropractic therapy, homeopathy, herbal care and body massage. When these five therapies were evaluated by doctors, acupuncture obtained 43% consent, chiropractic 40% and the remaining part of consensus was for body massage. Further investigation found that younger physicians have a more optimistic approach towards CAM than older colleagues, although in general it is unclear whether doctors consider CAM a potent unspecific placebo or a specific effective remedy. Yet these five therapies are rapidly expanding and medical associations are aware that one of the most important tasks is to keep their patients informed of the potential risks or benefits they may have from using such therapies. At present the National Center for Complementary and Integrative Health (NCCIH) recognizes three types of CAM: x Natural Products. This category includes the use of herbs, vitamins and supplements. x Mind and Body Practices. This category includes yoga, chiropractic and osteopathic manipulation, meditation, massage therapy, acupuncture, relaxation techniques (such as breathing exercises, guided imagery and progressive muscle relaxation), Tai Chi, Qi Gong, healing touch, hypnotherapy and movement therapies (such as the Feldenkrais method, Alexander technique and Pilates). x Other Complementary Health Approaches. This category includes health approaches such as traditional healing, Ayurvedic medicine, Traditional Chinese Medicine (TCM), homeopathy and naturopathy (Figure 4.1).

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Figure 4.1 Different types of Whole-System Medicine (WSM) and Integrative Medicine (IM). The figure depicts prevalent IM practices that have been commonly used to improve health and ameliorate pathological conditions. Modified from Kanherkar et al., 2017.

In many countries, CAM practice is provided outside the national healthcare systems and practised by non-regulated personnel. CAM may therefore not be monitored by the safety mechanisms and reporting systems incorporated into mainstream regulatory and legislative frameworks. With regard to the use of medicinal herbs, plant extracts or derivatives from partial purification of plant extracts, the situation is complicated by the fact that, unlike the use of pharmaceuticals, it is difficult for the consumer to verify the state of purity, contamination, certification, authentication and standardization of the product they are purchasing. Therefore, although the use of medicinal plants is traditionally harmless, when these remedies are used for therapeutic purposes, they need a pharmacovigilance system capable of identifying their potential hazards.

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4.2.1. Interaction between Bioactive Plant Molecules and Drugs The term herbal products has become a common term which commonly refers to all types of preparations obtained from herbs, spices, roots, stems, leaves and other non-botanical materials of natural origin. The regional centres for pharmacovigilance of the various industrialized countries have the task of annotating the adverse effects caused by the intake of herbal products. In some cases, the occurrence of hepatitis has been reported (e.g., derived from the intake of wall germander extracts distributed in France), and examples of allergic, toxic, mutagenic and drug interactions are also known. When a therapeutic combination leads to an unexpected change in the patient’s conditions, this is described as an interaction with potential clinical significance. The effect of the herb–drug combination may: x x x x

be synergistic, or with an additive effect of one or more drugs; be antagonist, or with a negative effect of one or more drugs; lead to alteration of the effects of one or more drugs; lead to the production of idiosyncratic effects.

Interaction with drugs is among the major risks of the uncontrolled use of medicinal plant extracts. In many cases, it was found that patients treated with drugs were concurrently making use of natural products with medicinal action. There are clinical data provided by prestigious institutions that confirm the possibility of interaction between the bioactive molecules contained in medicinal herbs and certain drugs. For example, Echinacea purpurea, when used daily and for more than eight consecutive weeks, may have hepatotoxic effects and should therefore not be used with potentially hepatotoxic drugs such as anabolic steroids, amiodarone, methotrexate or ketoconazole. Non-Steroidal AntiInflammatory Drugs (NSAIDs) may cancel the effect of tansy when used to heal migraine. Ginseng may cause migraine, tremor and personality alteration episodes when taken together with phenazine sulphate. Valerian should not be taken with barbiturates as it may increase their sedative effects, while many plant extracts may interfere pharmacodynamically with the administration of digoxin or warfarin [3-(Į-acetonylbenzyl)-4hydroxycoumarin, 6]. Many plant extracts may eventually affect blood glucose concentration and should be avoided by people with diabetes mellitus.

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The risk of adverse effects due to interactions between herbal products and conventional drugs is often underestimated by consumers due to lack of information on the safety of herbal preparations. It has become very difficult to identify the possibility of the occurrence of an interaction due to: x the availability and easy accessibility of a large number of herbal products in the market, x the presence of multiple components of various pharmacological properties in the herbs, x a lack of data on the pharmacological action and the mechanisms of interactions of herbal products, x misinformation on the actual label content of herbal products, x the low potential of herb-induced adverse effects. Prior to reports of interactions between medicinal herbs and drugs such as digoxin, warfarin, protease inhibitors and oral contraceptives, few interactions between herbs and drugs had been documented. These studies are becoming more common, albeit rare in comparison to those on drug interactions. To date, the cardiovascular system, the central nervous system and the immune system are the most common therapeutic categories cited in the literature. Although many herbal medicines have good safety profiles, medicinal plant-based supplements are intended to be taken for a prolonged period, enabling enzyme induction and other interaction mechanisms. However, the increasing use of medicinal herbs in the community where people are being treated with prescription medications suggests that negative herb–drug interactions may have a significant impact on the public health. In the absence of further stringent studies to evaluate the clinical significance of herb–drug interactions, an assessment based on the evidence emerging from the scientific literature is essential to guide professionals involved in patient care. Herbal medicines may contain antagonistic components, which would reduce the drug efficacy and cause potential therapeutic failure. Acting or competition for the same drug target causes synergistic or antagonistic effects between herbal medicines and drugs. Warfarin (6) is the most commonly used of oral anticoagulants; however, despite its therapeutic usefulness, it can be unsafe because of its narrow therapeutic index, its interactions with other drugs and foods, and because its users often have

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special clinical conditions. Clinical findings regarding herb–warfarin interactions for 58 herbs highlight the clinical outcomes, severity of documented interactions and quality of clinical evidence. The clinical outcomes that may result from these interactions basically involve two possibilities: a risk of bleeding due to exacerbation of the anticoagulant effect of warfarin, and therapeutic ineffectiveness due to reduction of the effect of this drug because of blood clot stimulation. For example, garlic, ginger, ginkgo, ginseng, alfalfa (Medicago sativa), chamomile (Matricaria recutita) and danshen (Salvia miltiorrhiza) may enhance the anticoagulant activity of warfarin by targeting the same vitamin K epoxide reductase target or other critical components in the coagulation cascade. Thus all of these herbal products may increase the risk of bleeding in patients on chronic warfarin therapy. Aspilia africana (that contains alkaloids and tannins), when used along with antimalarial drugs like artemisinin (7) and chloroquine (8), was found to antagonize their effects. In general plants that potentialize the effect of warfarin act on platelet aggregation, inhibit platelet-activating factor (PAF), interact with the arachidonic acid cascade and increase warfarin metabolism. Table 4.2 lists the plant species involved in warfarin potentiation.

Table 4.2 Plants able to potentiate the effect of Warfarin (adapted from Leite et al., 2016) Description Plants that affect platelet aggregation

Plants with an inhibitor effect on

Plant species Allium cepa, Allim sativum, Arctium lappa, Arnica montana, Centipeda minima, Forsythia suspensa, Garcinia cambogia, Leonurus cardiaca, Mormordica charantia, Ocimum basilicum, Paeonia spp., Piper methysticum, Tanacetum parthenium, Trigonella foenum-graecum, Vaccinium uliginosum. Alpinia galangal, Boesenbergia pandurate, Calophyllum spp., Cinnamomum spp., Curcuma

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Description platelet-activating factor (PAF) Plants that interact with the arachidonic acid cascade Plants that increase warfarin metabolism

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Plant species spp., Ginkgo biloba, Goniothalamus malayanus, Hypericum patulum, Momordica charantia, Piper aduncum, Piper futokadsura, Thuja orientalis, Zingiber officinale. Crataegus monogyna, Piper methysticum, Serenoa repens. Allium sativum, Angelica sinensis, Matricaria recutita, Glycyrrhiza glabra, Harpagophytum procumbens, Lycium barbarum, Mangifera indica, Punica granatum, Serenoa repens, Trifolium pratense

The most common herbal medicines used for menopause are based on Cimicifuga racemosa, red clover (Trifolium pratense), Angelica sinensis and Oenothera biennis. Some of these medicinal herbs do not interact with conventional drugs, whereas others show significant interactions. Therefore interactions between prescribed herbs and prescribed drugs may occur and may lead to serious clinical consequences. Both pharmacokinetic and pharmacodynamic mechanisms have been considered to play an important role in these interactions, although the underlying mechanisms of simultaneous drug alteration effects and/or medicinal herb concentrations intake are still to be determined. The clinical importance of herb–drug interactions depends on many factors associated with the medicinal herb, the drug and the patient. However, because of the hepatotoxicity caused by some conventional anticancer drugs, herbal medicines including pomegranate (Punica granatum), Indian gooseberry (Phyllanthus emblica), mango (Mangifera indica), black cutch (Acacia catechu) and tea (Camellia sinensis) have been used for their hepatoprotective and antioxidant properties when hepatotoxic chemotherapeutic agents are utilized. Medicinal plants should be properly labelled to alert consumers of potential interactions when administered concomitantly with drugs and to advise on consultation with general practitioners. Table 4.3 lists some medicinal plants potentially interacting with drugs.

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Table 4.3. Some clinically relevant herb–drug interactions Herb Aloe

Latin name Aloe ferox

Astragalus

Astragalus membranaceus

Betel nut

Areca catechu

Bupleurum Ephedra

Bupleurum falcatum Ephedra sinica

Garlic

Allium sativum

Ginger

Zingiber officinale

Ginkgo

Ginkgo biloba

Ginseng

Panax ginseng

Significant or suspected interactions Enhances cardiac glycosides and antiarrhythmic drugs by reducing potassium due to the laxative effect Alters the immunosuppressive effects of cyclosporine, azathioprine and methotrexate. Causes rigidity, bradykinesia and jaw tremors when used with procyclidine. Enhances sedative effects. Causes hypertension with MAO inhibitors; reacts by producing cardiac arrhythmia with cardiac or alotane glycosides; increases the cardiovascular side effects of caffeine. Concomitant use of garlic and anticoagulants leads to increased risk of bleeding. Diallyl trisulfide has been shown to inhibit the activity and expression of P-glycoprotein. Increases the plasma concentration of chlorzoxazone and nevirapine. Induces changes in paracetamol pharmacokinetics. Decreases saquinavir and indinavir blood concentration. Concomitant use of ginger and anticoagulants can cause increased risk of bleeding. Concomitant use of ginkgo and nonsteroidal anti-inflammatory agents (NSAIDs) can cause an increased risk of bleeding and when used with warfarin it causes bleeding. Decreases omeprazole and tolbutamide blood concentration. Increases tanilolol blood concentration. Concomitant use of ginseng and antidiabetic agents can increase the risk of hypoglycaemia. Causes sleeplessness,

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Herb

Latin name

Goldenseal

Hydrastis canadensis

Grapefruit juice

Citrus paradisi

Green tea

Camellia sinensis

Hibiscus

Hibiscus sabdariffa

Impila

Callilepis laureola

Kava

Piper methysticum Glycyrrhiza uralensis

Liquorice Milk thistle

Silybum marianum

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Significant or suspected interactions tremor and headaches when used with phenelzine. Increases Cmax of nifedipine. Decreases debrisoquin urinary recovery ratio. When used with midazolam strongly inhibits CYP3A4/5 activity. Found to inhibit P-glycoprotein rhodamine-123 efflux in vitro in Caco-2 cells and in vivo in healthy volunteers. It was also found to increase the bioavailability of nifedipine in vivo in rats and talinolol in vitro in Caco-2 cells. Naringin, a furanocoumarinic phytochemical present in the juice, lowers the bioavailability of certain drugs. contains catechins that modulate Pglycoprotein transport in vitro and in vivo either through inhibition or activation by means of a heterotopic allosteric mechanism. Decreases folate blood concentration. Reduces the blood concentration of chloroquine and induces changes in paracetamol pharmacokinetics. Capable of interacting with renal functions and drug elimination and causes damage to the proximal convoluted tubules and the loop of Henle and was found to be hepatotoxic. Decreases 6-hydroxychlorzoxazone /chlorzoxazone serum ratio. Increases sensitivity to digoxin or other cardiac glycosides and enhances corticosteroids and thiazide diuretics. Flavonoids like apigenin, biochanin A, genistein and kaempferol enhance the accumulation of the BCRP substrate mitoxantrone by inhibition of BCRP in vitro and in vivo in MCF-7 M-X100 cells. Decreases metronidazole blood

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Herb

Latin name

Rhubarb

Rheum officinale

Sage

Salvia miltiorrhiza Hypericum perforatum

St John’s wort

Significant or suspected interactions concentration. Enhances cardiac glycosides and antiarrhythmic agents by reducing potassium through the laxative effect. Causes bleeding with warfarin. Concomitant use of digoxin and hypericin may reduce the efficacy of digoxin. It reduces the bioavailability of: warfarin (causes bleeding), serotonin reuptake inhibitors (causes a moderate serotoninergic syndrome), indinavir, digoxin, theophylline, cyclosporine, phenprocoumone and oral contraceptives. Decreases the blood concentration of the concomitantly used prescribed drugs. In the case of cyclosporine, changes in pharmacokinetics were associated with rejection episodes in transplant patients.

The Caco-2 model is used in pre-clinical investigations to predict the probable gastrointestinal permeability of drugs because it expresses the cytochrome P450 of microvilli and enterocytes, with characteristics identical to the small human intestine. The FDA recommends this model as an integral part of the biopharmaceutical classification system. Most specialized laboratories use the Caco-2 cell line for screening new chemicals by predicting its bioavailability, solubility and the possibility of drug–drug interactions or herb–drug interactions in the intestinal lumen. Another worthy consideration is the interaction between medicinal plants or alternative medicines and the treatment of diseases such as cancer. Since most alternative products are over-the-counter products, physicians, doctors, pharmacists and para-pharmacists have the joint responsibility to inform patients about product safety and potential interactions with other drugs, especially if patients are in anticancer therapy. However, a survey to assess what advice was given to breast cancer patients revealed that 68% of doctors did not ask whether patients had taken non-prescription medicines. On the other hand, the same survey found that 50–70% of these patients did not inform doctors or pharmacists as they believed that their physicians were not competent on medicinal plant extracts/herbs, fearing

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their doctor’s disagreement with CAM. Better knowledge of doctors is therefore needed to turn them into conscious partners who are aware of the potential benefits and risks of the joint use of medicinal plant extracts and drugs. Case reports coupled to pharmacokinetic trials constitute the highest level of evidence for herb–drug interactions. In the next paragraphs we will discuss some of the most widely used medicinal plant extracts and their potential interactions with drugs. 4.2.1.1. Interaction between Ginkgo Extracts and Drugs Geriatric patients over 50 years of age typically have a chronic disease occurring every decade. Each chronic disease generally requires long-term drug therapy, which means that most elderly patients require different medications to control their condition and/or maintain their health. The elderly often add medicinal herbs to drugs prescribed by their doctors, but as mentioned above they rarely inform their doctors about this. Ginkgo biloba has a long history of use in Traditional Chinese Medicine (TCM). Its products have been documented to include a wide range of pharmacological activities, such as neuroprotective, cardioprotective, antitumorigenic, antioxidant and stress alleviating effects, improvement of memory and protection against apoptosis. Many G. biloba products are available commercially, and the leaf extract is currently one of the bestselling herbal products worldwide. Ginkgo biloba extract is used as a herb drug or dietary supplement and as such is often administered in combination with other therapeutic drugs. Consequently, it is very important to identify the potential herb–drug interaction to guide the rational clinical use of drugs. Ginkgo extract was tested to study the ability to inhibit cytochrome P450 isoforms in human microsomes. CYP3A4 is considered to be the most important drug-metabolizing enzyme based on its high abundance in the liver and its participation in the metabolism of >60% of drugs. As such, the inhibition or induction of this enzyme is the source of numerous drug interactions, which has led to the effect of G. biloba extract on CYP450 becoming a major subject of research. An inhibitory effect of ginkgo extracts on cytochromes CYP1A2, CYP2C9, CYP2E1 and CYP3A4, but not CYP2D6, has been observed. Moreover, the incubation of human liver microsomes with a ginkgo based product significantly diminishes the activities of CYP2C8, CYP2C9 and CYP3A4, but not CYP2C19, CYP2D6 and CYP2E1.

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The effect of ginkgo in women with breast cancer treated with anastrozole (9) and letrozole (10) (both aromatase inhibitors) or with oestrogen antagonist tamoxifen (11) was assessed. Ginkgo did not alter the pharmacokinetics of these drugs and was considered safe when administered. Supplementation of ginkgo extracts to 12 healthy subjects for seven days did not alter the effect of S-warfarin and R-warfarin and did not affect clotting time and platelet aggregation. These data are in agreement with a meta-analysis that did not find an increased risk of bleeding during the concomitant intake of ginkgo and non-herbal medicines. Similarly, in a study on 11 healthy volunteers, ginkgo extracts did not alter the pharmacokinetics of flurbiprofen (tarenflurbil) (12), another substrate of CYP2C9.

Ginkgo terpene lactones meglumine injection (GMI) is a novel preparation of TCM containing 25 mg of ginkgo terpene lactones. The major ginkgo terpene lactones of GMI are ginkgolide A (13) (8.5 mg), ginkgolide B (14) (14 mg) and ginkgolide K (15) (1.0 mg). In comparison, the main components of EGb761, a typical Ginkgo biloba extract product, are flavonoids (24%) and terpenoides (6%: 3.1% ginkgolides and 2.9% bilobalide). The main effect of EGb761 is free radical scavenging, while that of GMI is for the treatment of cerebral apoplexy. GMI also possesses a variety of other pharmacological properties, such as neuroprotective properties and having a potential effect on cerebrovascular diseases. GMI was well tolerated during an open-label, placebo-controlled study. There was no systemic accumulation and were no significant effects on the pharmacokinetics of midazolam in healthy Chinese male subjects after repeated dosing of GMI.

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Intake of ginkgo alters the disposition of theophylline (16), nicardipine (17) and cyclosporine (18) and has been associated with seizures in patients treated with the antiepileptic drugs phenytoin (19) and sodium valproate (20). However, there was no effect of ginkgo on the pharmacokinetics of fexofenadine (21). The precise mechanisms behind the interactions are not fully understood, but the data suggest that the interaction at the pharmacokinetic level involve changes in the activity and/or expression of drug-metabolizing enzymes and/or transporters. An in vitro study using human hepatocytes found that ginkgo induces the expression of multiple drug-metabolizing enzymes and transporters, including P-glycoprotein, through selective activation of the pregnane X receptor, the constitutive androstane receptor and the aryl hydrocarbon receptor, possibly ascribed to the ginkgo terpenoids. Drug-induced nephrotoxicity is potentially lethal. When sodium aescinate (22) is given to patients to treat postoperative inflammation and oedema, adverse drug reactions and drug–drug interactions must be closely monitored. A 58-year-old man with phalangeal fractures suffered from acute kidney injury related to the interaction between sodium aescinate (22) and ginkgo extract. It is believed the interaction with the molecule is related to the downregulation of CYP2C9 and CYP3A4 by the presence of amentoflavone in ginkgo extracts. Ginkgo extracts were screened for agents active against xanthine oxidase (XOD). XOD, which can oxidize hypoxanthine or xanthine to uric acid and superoxide anion, was originally an important enzyme target for the treatment of gout. In recent years, many researchers have proposed XOD to be a promising target in the prevention of cardiovascular diseases because of its critical role in controlling reactive oxygen species. Extract of ginkgo inhibits XOD with an IC50 value of 56.23 ȝgௗmLí1. Ginkgo biloba flavones and coumarins exhibited the highest binding affinities, indicating that they might be the strongest XOD inhibitors.

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4.2.1.2. Interaction between Ginseng Extracts and Drugs Ginseng refers to any one of 11 species of perennial plants that belong to the genus Panax of the Araliaceae family. It is one of the most widely used herbs in the world. Its extracts are known to exert a wide range of pharmacological activities due to a diverse group of steroid saponins called ginsenosides. These are steroid glycosides with the hallmark steroid core, a carbohydrate chain and a tigloyl group. Based on their aglycones (non-sugar moiety), ginsenosides can be divided into two groups: the fourring carbon skeleton dammaranes and the five-ring carbon skeleton oleananes. The dammaranes are devised into two subgroups: the protopanaxadiols (PPDs) (Rb1, Rb2, Rc, Rd, Rg3, Rh2, Rh3 etc.) and the protopanaxatriols (PPTs) (Rg1, Re, Rf, Rg2, Rh1 etc.). The most abundant molecules are the ginsenosides Rb1, Rb, Rc, Rd, Re, Rf and Rg1. Depending on how it is processed, ginseng can be classified into 3 types: (i) fresh (unprocessed) ginseng, (ii) white ginseng (peeled and then dried), and (iii) red ginseng (peeled and then steamed). Red ginseng can be stored for extended periods of time because of the removal of humidity during the manufacturing process.

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When administered orally, ginseng is promoted as an adaptogen, which is defined as a natural substance that assists the body in coping with stress and normalizing bodily processes. The pharmacokinetics and the metabolism of ginseng have received limited attention. Understanding the pharmacokinetics of ginseng may reduce interaction in patients who use both ginseng and drugs. Ginseng is used to stimulate immune function and to improve cognitive function, physical stamina, concentration, work efficiency and memory. Additional purported uses of ginseng include depression, chronic fatigue syndrome, diabetes mellitus, various forms of cancer and numerous other diseases. Ginsenoside Rb1 (23) exhibits a significant antidepressant-like effect in mice behavioural tests, chronic animal models and drug interactions, and its mechanisms are mainly mediated by the central neurotransmitters of serotonergic, noradrenergic and dopaminergic systems. 20(S)-ginsenoside Rg3 (24) was generally well tolerated. In these studies, the molecule exhibited a pharmacokinetic profile suitable for once-every-2-days dosing. Data from a number of preclinical studies suggest that several ginsenosides and their deglycosylated metabolites are involved in the modulation of cytochrome P450 (CYP) enzymes, organic anion transporting polypeptide 1B1 (OATP1B1), P-glycoprotein and uridine diphosphate glucuronosyltransferases (UGTs). It is also possible that additional compounds present in ginseng may contribute to CYP modulation and that other metabolic enzymes and/or transporters such as breast cancer-related protein (BCRP), multidrug resistant proteins (MRPs), and organic anion transporters (OATs) may be altered by ginseng administration as well. The ability of ginseng to modulate the activity of uptake and efflux transporters, CYP and UGTs likely explains its involvement in drug interactions, although other unidentified mechanisms may also be involved.

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Warfarin patients are usually not advised to avoid taking ginseng products. In general, ginseng appears to have a low risk of cytochrome P450 involvement that appears to be only clinically relevant for those lowtherapeutic index drugs metabolized by CYP2C9. Ginseng administration produced a statistically significant 7% decrease (P = 0.003) in debrisoquine (25) urinary recovery ratios; however, the magnitude of this effect was not deemed to be clinically relevant. Conversely, no modulatory effects were identified for ginseng on CYP3A, CYP1A2 or CYP2E1. Perhaps the most clinically relevant effect of ginseng in healthy volunteers is its induction effects on CYP3A4, where it produces a 34% decrease in the AUC of the CYP3A4 substrate midazolam (26). After a two-week administration of the concentrated fermented red ginseng, no significant drug interactions were observed with some CYP probe substrates (caffeine [27], paraxanthine [28], losartan [29], omeprazole [30], 5-hydroxyomeprazole [31], dextromethorphan [32], dextrorphan [33], midazolam [26] and 1-hydroxymidazolam [34]). However, the inhibition of P-glycoprotein was significantly different between fermented red ginseng and the CYP probe substrates. The use of fermented red ginseng requires close attention due to the potential for increased systemic exposure when it is used in combination with Pglycoprotein substrate drugs. Because each of the ginseng components (primarily ginsenosides) is capable of exerting distinct pharmacological actions, it is difficult to consistently predict the drug interaction potential of such herbal product.

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4.2.1.3. Interaction between St John’s Wort Extracts and Drugs St John’s wort (SJW, Hypericum perforatum) is commonly used to relieve mild to moderate depression, anxiety and premenstrual syndrome. Other activities of the extract include sedative, nootropic, antischizophrenic, anticonvulsant, antibacterial and anti-inflammatory effects. Ten different kinds of bioactive molecules are present in SJW: flavonoids (including rutin [34], hyperoside [35], quercetin [2], quercitrin [36]), naphtodianthrones (including hypericin [37] and pseudohypericin [38]), acylphloroglucinols (including hyperforin [39] and adhyperforin [40]), proanthocyanidins, procyanidins, tannins, essential oil, amino acids, phenylpropanes, xantones and other hydrosoluble compounds (organic acids, peptides and polysaccharides).

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The daily dose recommended to induce an antidepressant effect is 900 mg, containing 0.3% hypericin or 5% hyperforin. These bioactive compounds act on the nervous system by inhibiting the synaptic reuptake of monoaminergic neurotransmitters. Hyperforin interacts pharmacologically by inducing CYP3A4 and the magnitude of this interaction is related to the hyperforin amount of the extract, which is highly variable. When healthy women using low concentrations of oral contraceptives were treated with SJW (containing 0.3% hypericin and 3.7% of hyperforin), the extract reduced systemic exposure to steroids and increased bleeding. Despite the safety of SJW as a monotherapy, its use in co-medication should be carefully considered. SJW is often taken in combination with other conventional medicines, creating the potential for herb–drug interactions. In mice, the effect of several SJW extracts on pentobarbital (41)-induced sleep time and pharmacokinetics resulted in impaired motor coordination caused by diazepam (42) and paracetamol (43). With respect to control, pre-treatment with SJW ethanolic extracts boosted the hypnotic effect of pentobarbital and compromised the motor coordination caused by diazepam; it also increased the plasma concentration of paracetamol. These results therefore show a remarkable interaction between SJW extracts and the pharmacodynamics of pentobarbital and diazepam and the pharmacokinetics of paracetamol.

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A schizophrenic patient, who was stable at a fixed dose with stable plasma levels of clozapine (44), got worse after initiating self-medication with SJW. It is possible that in addition to the induction of P450 enzymes, the induction of P-glycoprotein due to SJW was the cause of the aggravated psychiatric deterioration of the patient.

A recent European review of drug interactions with SJW revealed that it induces the metabolism of both S- and R-warfarin (6), which is thought to be mediated by induction of CYP1A2, CYP2C9 and CYP3A4. One case report of supratherapeutic international normalized ratios in a patient taking SJW and warfarin concurrently documented the patient’s development of a severe bleeding diathesis, manifested by hematemesis and melena. Since SJW co-administration with drugs can easily result in a severe decrease in plasma concentrations of CYP3A substrates, simultaneous intake should be avoided, at least for low-therapeutic drugs. SJW can lead to a significant CYP1A2 and CYP2D6 induction in WRL68, HepG2 and HepaRG cell lines. Moreover, hypericin seems to induce CYP1A2 in HepG2 cells and to inhibit its expression in HepaRG cells, while hyperforin induces CYP1A2 in HepG2 and in WRL-68 cells. Additionally, hypericin and hyperforin induce CYP2D6 in HepG2 cells but inhibit its expression in HepaRG and in WRL-68 cells. Since the observed effects in human hepatocytes suggest an expected in vivo effect, these results indicate that more studies are necessary to clarify the safety of SJW extract use either in terms of hepatotoxicity or herb–drug interactions, particularly with CYP1A2 and CYP2D6-metabolized drugs. Finally, the concomitant use of SJW with immunosuppressive (e.g., cyclosporine, [18]), antiretroviral (e.g., indinavir [45] and nevirapine [46]), cardiac (e.g., digoxin [47]) or antineoplastic (e.g., irinotecan [48] and

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imatinib [49]) drugs may result in a reduced plasma concentration of the prescribed drug and, hence, reduced efficacy.

4.2.1.4. Interaction between Echinacea Extracts and Drugs Echinacea purpurea, Echinacea angustifolia and Echinacea pallida are frequently used as medicinal plants for the prevention and treatment of the common cold, influenza and upper respiratory tract infection as well as for the unspecific enhancement of the immune system. Polysaccharides, glycoproteins, caffeic acid derivatives and alkamides have been considered as the most relevant constituents for activity. In healthy individuals, Echinacea increases by 29% the effect of caffeine (27) and by 14% that of tolbutamide (50). Multiple treatment of E. purpurea did not significantly alter the pharmacokinetics of docetaxel (51) chemotherapy, and therefore the extracts of this species can be safely combined with docetaxel in cancer patients. Etravirine (52) is an inhibitor of HIV non-nucleoside inverse transcriptase. Co-administration of E. purpurea with etravirine was found to be safe and well-tolerated in patients with HIV infection. In a library screening of commonly used nutraceuticals for their modulatory effects on the activity of cytarabine (53) and daunorubicin (54), two primary chemotherapeutics used to treat acute myeloid leukaemia, it was found that Echinacea extracts hindered acute myeloid

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leukaemia chemotherapy efficacy by significantly reducing the ability of cytarabine to induce cell death. Etoposide (55) is a cytotoxic, topoisomerase II inhibitor and a chemotherapeutic agent used in the treatment of lung cancer. In a clinical study, Echinacea contributed to the profound thrombocytopenia of a patient and therefore the administration of the extract of this plant should be avoided in patients receiving etoposide and, possibly, other chemotherapeutic drugs that are substrates of CYP3A4.

The pharmacodynamic and pharmacokinetic interactions in healthy subjects of either echinacea or policosanol (56) with warfarin (6) have been investigated. The apparent clearance of (S)-warfarin was significantly higher during a concomitant treatment with echinacea, but this did not lead to a clinically significant change in an International Normalized Ratio measurement. No evidence of any apparent effect of CYP2C9 interaction was found; neither echinacea nor policosanol significantly affected warfarin pharmacodynamics, platelet aggregation or baseline clotting status in healthy subjects. Therapeutic agents such as amitriptyline (57) (antidepressant), haloperidol (58) and olanzapine (59) (antipsychotic), theophylline (16), zileutin (60) (antiasthmatic), efavirenz (61) and nevirapine (46) are CYP1A2 and

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CYP3A4 substrates employed for the management of HIV/AIDS and associated disorders. Because of the high risk of E. purpurea preparations causing clinically significant herb–drug interactions via CYP3A4 and CYP1A2, it is recommended that clinicians should advise HIV/AIDS patients to avoid concurrent intake of E. purpurea and conventional drugs due to possible interactions even though the outcome could be delayed.

In conclusion, several studies have shown that Echinacea may induce the metabolism of drugs mediated by CYP1A2 and CYP3A4 and caution is advised when Echinacea is co-administered with substances with a limited therapeutic index or low oral bioavailability. One of the main factors that drives people to self-healing with natural products is the general belief that the bioactive ingredients of these herbs are safe. The European Union commissioned research through a programme called BIOMED (Biomedical and Health Research) to define European standards for the safe and correct use of phytochemicals for medicinal purposes. Likewise, many governmental bodies belonging to industrialized countries have defined standards enclosing them in summary monographs. This topic will be discussed in the next section.

4.2.2. Herbal Regulatory: Monographs The production of medicinal plants and food plants is subject to a set of rules that determine the procedures for processing and quality control, and the safety and identity of marketed products. The popular use of plantbased medicines throughout the world to heal, prevent and treat illness has prompted the development of monographs to define the identity and the qualitative and therapeutic criteria needed to provide basic information on the use of a given plant species. The term monograph is intended for only one subject, in this case only one species.

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In botany there are essentially three types of monographs: standard, therapeutic and combined. The first gives information on the identity of the plant (family, genus, species, subspecies, notomorph etc.) by using the classical taxonomic criteria already discussed in Chapter 1 to describe the morphological characteristics. Therapeutic monographs provide the definition of the drug (e.g., organ of the plant used, nomenclature etc.), a list of the major chemical compounds, uses and indications, contraindications, side effects, dosage, way of administration, posology, interactions with other substances and so on. Combined monographs associate standard information with therapeutic information, providing a greater amount of data than the other types. We will now discuss the features of the main monographs. 4.2.2.1. ESCOP Monographs The European phytochemicals market is characterized by a large number of products and one of the major problems in Europe is the harmonization of medicinal plant derivatives among the Member States. ESCOP was founded in June 1989 as an umbrella organisation representing national phytotherapy or herbal medicine associations across Europe. Since 1996, ESCOP has had the following non-profit aims and objectives: x to advance the scientific status of herbal medicinal products and to assist with the harmonization of their regulatory status at the European level; x to develop a coordinated scientific framework to assess herbal medicinal products; x to promote the acceptance of herbal medicinal products, especially within general medical practice; x to support and initiate clinical and experimental research in phytotherapy; x to improve and extend the international accumulation of scientific and practical knowledge in the field of phytotherapy; x to support all appropriate measures that will secure optimum protection for those who use herbal medicinal products; x to produce reference monographs on the therapeutic use of plant drugs; x to further cooperation among national associations of phytotherapy to advance these Aims and Objectives.

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More than 100 monographs have been published in different volumes and made available beginning in June 1997. ESCOP also publishes an app for Apple and Android devices summarizing ESCOP monograph conclusions on the uses, safety and quality standards of many European phytomedicines (http://escop.com/app/). Unlike the monographs typical of the Official Pharmacopoeia Member states, ESCOP monographs, such as those of the German Commission E, describe the therapeutic aspects of every phytomedicine including pharmacodynamic and pharmacokinetic properties along with preclinical and clinical safety data. The doses, side effects and much other information useful to assist healthcare professionals, industry, lawmakers and patients are indicated. Published monographs are more complete than those described by the German Commission E and also have a detailed bibliography. According to ESCOP, phytomedicines are defined as “medicinal products that contain as active ingredients only plants, parts of plants or plant material, or combinations thereof, whether in crude or processed state”. The term plant material includes juices, gums, fixed oils, essential oils and any other directly derived crude plant product. Phytomedicines do not include chemically defined isolated and purified chemical compounds either alone or in combination with plant materials. In addition, phytomedicines may contain excipients of plant or non-plant derivation. 4.2.2.2. WHO Monographs A series of volumes, the WHO monographs on selected medicinal plants aim to: x provide scientific information on the safety, efficacy and quality control of widely used medicinal plants; x provide models to assist Member States in developing their own monographs or formularies for these and other herbal medicines; and x facilitate information exchange among Member States. The WHO monographs, however, are not pharmacopoeial monographs; rather they are comprehensive scientific references for drug regulatory authorities, physicians, traditional health practitioners, pharmacists, manufacturers, research scientists and the general public. Each monograph follows a standard format with information presented in two parts followed by a reference list. The first part presents pharmacopoeial summaries for quality assurance. The second part includes sections on medicinal uses, pharmacology, safety issues and dosage forms. The

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descriptions under the medicinal uses section merely represent, for purposes of information exchange, the systematic collection of scientific information available at the time of each volume’s preparation and should not be taken as having WHO’s official endorsement or approval. Volume 1 contains 28 monographs published in 1999, Volume 2, published in 2003, includes 30 monographs, Volume 3 was published in 2007 and includes 31 monographs and Volume 4, which was published in 2009, includes 28 monographs. Each volume after Volume 1 has a general technical notice and two cumulative indexes to facilitate referencing; one lists the monographs in alphabetical order by plant name and the other according to the plant material of interest. 4.2.2.3. German Commission E German Commission E is the most widely accepted monograph system and since 1978 has published monographs on hundreds of medicinal herbs used in popular German medicine, approving many for consumption as non-prescriptive medicines. The commission gives scientific expertise for the approval of substances and products previously used in traditional, folk and herbal medicine. The monographs cover eighty percent of plant-based medicinal products on the German market. At the moment the number of monographs exceeds 450 units, widely revised, corrected and expanded. The main purpose of the German monographs is to inform on the therapeutic characteristics and on the use of a given plant by providing taxonomic nomenclature, botanical and chemical components, and pharmacological, pharmacokinetic and toxicological properties, along with clinical information, contraindications, side effects, and information for women during gestation and during lactation. There is also some information on interaction with other drugs, dosage and overdose. Commission E monographs are not always free from criticism. Toxic effects are less extensively documented than in the Lawrence Review of Natural Products, or Ellenhorn’s Medical Toxicology. Some monographs state or imply that certain herbs can kill, but others omit fatal reactions. Many of the monographs are too brief and all lack literature references, which reduces their scientific value. 4.2.2.4. USP The United States Pharmacopeia and the National Formulary (USP–NF) is a book of public pharmacopeia standards for chemical and biological drug substances, dosage forms, compounded preparations, excipients, medical devices and dietary supplements. If a drug ingredient or drug product has

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an applicable USP quality standard (in the form of a USP-NF monograph), it must conform in order to use the designation “USP” or “NF”. Drugs subject to USP standards include both human drugs (prescription, overthe-counter or otherwise) and animal drugs. Revisions are presented annually in the USP–NF, in twice-yearly Supplements, and as Accelerated Revisions on the USP website. The USP strengthens medicines quality assurance systems, increases the supply of quality-assured medicines and develops the capacity to detect and remove poor-quality medicines from the market. By sharing scientific expertise and providing technical support and leadership, the USP helps local regulators improve and sustain local health systems, and enables manufacturers to supply quality-assured essential medicines for years to come. The USP advances the quality, safety and benefit of medicines and foods across the globe through international policy and regulatory analysis, advocacy, and collaboration with governments, multilateral institutions, regional cooperation initiatives and other stakeholder organizations. The first USP was published in 1820. The Herbal Medicines Compendium (HMC), published by the USP Convention, is a freely available, online resource that provides standards for herbal ingredients used in herbal medicines. Standards are expressed primarily in monographs. The USP’s HMC employs validated analytical procedures for the tests specified in its monographs, using state-of-the-art analytical techniques and allied reference materials. Additional analytical methods and approaches may be referenced in general chapters, which are also available online. HMC standards may exist in one or more of the following stages: x Proposed for Development: these standards are in the initial developmental stage. Additional information will be required to complete the development. Interested parties are encouraged to submit their proposals to complete the monographs. x Proposed for Comment: these standards have undergone development and are being posted for a 90-day public comment period prior to their submission to the USP Expert Committee for inclusion in the HMC. x Final Authorized: the Expert Committee has authorized these standards for inclusion in the HMC.

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4.2.2.5. European Pharmacopoeia Since its foundation in 1964, the European Pharmacopoeia (Ph. Eur.) is Europe’s legal and scientific benchmark for pharmacopoeial standards which contribute to delivering high quality medicines in Europe and beyond. It is one of the multinational bodies that assure the quality of medicines and at the same time establish the relationships between pharmacopoeia, phytopharmaceuticals and industries. Ph. Eur’s task is to manually replace national pharmacopoeias, represent the Member States and refer to other states (e.g., Albania, Canada, Australia, China, Syria, Tunisia, Malaysia and Morocco). The aim of the Ph. Eur. is to promote public health through the provision of recognized common standards for the quality of medicines and their components. Such standards should be appropriate as the basis for the safe use of medications by patients. Moreover, their existence facilitates the free circulation of medicines in Europe and beyond. The 9th edition consists of 3 initial volumes and they will be complemented by 8 non-cumulative supplements. Furthermore, it contains 2366 monographs (including dosage forms), 361 general texts (including general monographs and methods of analysis) and around 2680 descriptions of reagents. It also includes a direct link to the knowledge database from each monograph. Free access is granted through its online archives.

4.2.3. Ethnofarmacognosy: The Root of Popular Culture Pharmacognosy is a branch of pharmacology and it treats, in some ways, simple drugs with pharmacological properties and provides the most varied cultural notions, specifying macro and microscopic morphological data and organoleptic characteristics capable of accurately identifying a drug. In ethnopharmacognosy, the prefix ethno points out the interest in popular knowledge, with the aim of re-evaluating, rediscovering, describing and making that knowledge accessible to the scientific community and the broader populace. As we pointed out at the beginning of the chapter, China and other eastern countries are the depositors of most popular cultures in the harvesting and use of medicinal plants. Aware of the importance of this popular cultural base, institutes such as the Chinese Institute of Information on Traditional Chinese Medicine and the Chinese Academy of Traditional Medicine Studies have produced a vast database that covers a broad spectrum of information on various therapies, including herbal medicines. Ethnopharmacognosy represents the link between herbs and people and its application helps not only in the

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obtaining of useful information from popular tradition, but also in the understanding of fluctuations in the variability of the pharmacological effects of plants of the same species present on different parts of the planet.

4.3. Mode and Action of Plant Bioactive Molecules As we have discussed and as we will see in depth in the following chapters, plants produce large amounts of secondary metabolites. Knowledge of their mechanism of action when interacting with other organisms (including humans) is important. In some cases, the modes of action of bioactive molecules are far less complex than they were previously supposed to be, but in others knowledge is still in its infancy. Medicinal plant-based medicines are becoming an issue for chemical therapies, especially those against cancer, and people are showing an increasing interest in natural remedies. Among the best known interactions are those with the cell cycle, the signal pathway through membranes, immunomodulation and toxic reactions.

4.3.1. Effect on Cell Division The reproductive cycle in cell life is characterized by four phases: G1, S, G2 and M. Cells that have temporarily or reversibly stopped dividing are said to have entered a state of quiescence called a G0 phase. In the G1 phase, or growth phase, nucleotides and enzymes are synthesized at a high rate and the duration of this phase is highly variable, even among different cells of the same species. In the S phase DNA synthesis occurs. The M phase (mitosis) consists of nuclear division (karyokinesis). In tissues where a fast cell replacement occurs, cell proliferation is particularly intense and is guaranteed by stem cells capable of rapid divisions. Cancer cells are basically proliferative elements and many studies have been carried out to identify cancer tumour oncogenes and tumour suppressors capable of controlling these cell proliferations. Although stem cells and tumour cells are potentially immortal, due to their ability to proliferate, cell death is not avoided. Vacuolarization and, ultimately, loss of membrane integrity cause a massive influx of calcium ions into the cells, leading to mitochondrial function inactivation and protein denaturation.

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4.3.1.1. Plant Bioactive Molecules Targeting Cell Cycle Protein synthesis is also involved in cell division processes and many alkaloids have the ability to inhibit this process, including the potent compound emetine, which is extracted from the species Psychotria ipecacuanha. Emetine (62) was found to arrest the cell cycle in the G2/M phase after 24 hours. Another alkaloid with the same properties is homoharringtonine (63) (currently in Phase II and III in human clinical tests for its anti-tumour capacity for some types of leukaemia), which has cytotoxic effects on the G1 and G2 phases of the cell cycle. Physalin A (64) is an active with an olide isolated from Physalis alkekengi var. franchetii, a traditional Chinese herbal medicine. Physalin A inhibited cell proliferation and induced G2/M cell cycle arrest in A549 cells suggesting that the molecule might be a promising therapeutic agent against non-small cell lung cancer. Extracts of Quisqualis indica exhibit strong cytotoxic activity against CCRF-CEM leukaemia cells. The most active compound isolated from the leaves and twigs of Q. indica is 25-O-acetyl-23,24-dihydro-cucurbitacin F (65). This compound reduced cell viability in a dose-dependent manner and arrested the cells at the G2/M interface. The accumulation of cells at the G2/M phase resulted in a significant decrease of the cell cycle checkpoint regulators cyclin B1, cyclin A, CDK1 and CDK2. The compound induced apoptosis in liposarcoma and rhabdomyosarcoma cells caspase-3 dependently. Therefore, 25-O-acetyl-23,24-dihydro-cucurbitacin F is a very interesting target for further investigation and for the development of novel therapeutics in sarcoma research. Cucurbitacins, a group of tetracyclic triterpenes extracted from the climbing stem of Cucumis melo, possess selective biological activities and functions against carcinogenesis. Cucurbitacin E (66) has the ability to disrupt cell actin and cell adhesion and shows inhibitory effects on cancer cell proliferation, actin polymerization and permeability. Cucurbitacin E is cytotoxic against malignant glioma GBM 8401 cells and induces cell cycle G2/M arrest. The molecule produced G2/M arrest as well as the upregulation of GADD45Ȗ and binding with CDC2. Both effects increased proportionally with the dose of cucurbitacin E, suggesting that the molecule-induced mitosis delay is regulated by GADD45Ȗ overexpression. These findings suggest that, in addition to the known effects on cancer prevention, cucurbitacin E may have antitumour activity in glioma

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therapy. Moreover, cucurbitacin E induces apoptosis of human prostate cancer cells via cofilin-1 and mTORC1. A mixture of some alkaloid components (e.g., 5,6-dihydrobicolorine [67] and 7-deoxy-trans-dihydronarciclasine [68]) from Hymenocallis littoralis was added to human liver hepatocellular cells HepG-2, human gastric cancer cell SGC-7901, human breast adenocarcinoma cell MCF-7 and human umbilical vein endothelial cell EVC-304. The mixture caused HepG-2 cycle arrest at the G2/M interface, induced apoptosis and interrupted polymerization of microtubules. In addition, expression of two cell cycle-regulated proteins, CyclinB1 and CDK1, was upregulated upon treatment. Upregulation of Fas, Fas ligand, Caspase-8 and Caspase-3 was observed as well, which might imply roles for the Fas/FsaL signaling pathway in the alkaloid-induced apoptosis of HepG-2 cells.

4.3.1.2. Plant Bioactive Molecules Targeting DNA Synthesis Among the enzymes that serve DNA synthesis the most sensitive to phytochemical compounds is topoisomerase, which has the task of selecting DNA filaments. DNA topoisomerase I (Topo I) is a crucial enzyme that works to relax supercoiled DNA during replication, transcription and mitosis. To date, a large number of Topo-directed agents (e.g., camptothecin [69], topotecan [70] and irinotecan [71]) are currently in clinical use. However, these compounds induce severe toxic side effects such as myelosuppression, nausea, hair loss, congestive heart failure and,

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in some cases, increased risk of secondary malignancies. Recently, epigallocatechin-3-gallate (72), a major polyphenolic constituent in green tea, has received much attention as a potential cancer chemopreventive agent with Topo I inhibitory activity. In particular, the isomers (í)epigallocatechin 3-O-(E)-p-coumaroate (73) and (í)-epigallocatechin 3-O(Z)-p-coumaroate (74) showed the most potent activities in the Topo I inhibition assay. Natural compounds such as genistein (75), an isoflavonoid present in soybeans, inhibit cell proliferation of certain types of cancer cells. According to some authors, genistein interacts with topoisomerase by stabilizing the enzyme–DNA complex in such a way as to alter gene expression and cell differentiation, and thus reduce cell proliferation. The proof of concept was obtained in genistein-resistant cells, where an alteration of the type II topoisomerase or a lower expression of the topoisomerase isoform IIb were evident.

Several labdane, halimane and clerodane diterpenoids isolated from the leaves of Vitex trifolia have been evaluated for DNA topoisomerases I inhibitory activity and cytotoxicity against HCT 116 cells. Compounds (76) and (77) exhibited equipotent Top1 inhibitory activity to the positive control, camptothecin (69), at 100 μM. Cryptolepine (78), a plant alkaloid isolated from the roots of the Central and West African shrub Cryptolepis sanguinolenta, was tested on the growth of human non-melanoma skin cancer cells. Cryptolepine increased

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the phosphorylation of ATM/ATR, BRCA1, Chk1/Chk2 and ȖH2AX; activated the p53 signaling cascade, including enhanced protein expressions of p16 and p21; downregulated the cyclin-dependent kinases, cyclin D1, cyclin A, cyclin E and proteins involved in cell division (e.g., Cdc25a and Cdc25b) leading to cell cycle arrest at the S-phase; and disrupted mitochondrial membrane potential. These changes in nonmelanoma skin cancer cells by cryptolepine resulted in a significant reduction in cell viability and colony formation and an increase in apoptotic cell death. Cuminaldehyde (79), an ingredient of the cortex of the plant Cinnamomum verum, led to lysosomal vacuolation with an upregulated volume of acidic compartment and cytotoxicity, together with inhibitions of both topoisomerase I and II activities in human colorectal adenocarcinoma COLO 205 cells. Two isoflavones, 5,7,4'-trihydroxy-6,8-diprenylisoflavone (80) and lupalbigenin (81), extracted from Derris scandens displayed cytotoxicity against three different cancer cell lines, KB, MCF-7 and NCI-H187, and acted as topoisomerase II agents. Telomeres, the protein–DNA structures found at the natural ends of eukaryotic chromosomes, are required to protect chromosomes from degradation and end-to-end fusion and to facilitate their complete replication. Recently, that the inhibitory effects of genistein (75) on telomerase-negative cells depend on the type II recombination pathway in yeast and the alternative lengthening of the telomere pathway in human tumours was demonstrated. Recently, the interference of several topoisomerase II-inhibiting polyphenolic compounds like delphinidin (82), resveratrol (83) or anthocyanin-rich berry extracts with clinically used topoisomerase II poisons were reported, regarding both their ability to stabilize cleavable complexes and to induce DNA strand breaks.

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4.3.1.3. Plant Bioactive Molecules Targeting Cytoskeleton and Mitosis Mitotic catastrophe is a type of cell death due to abnormal mitosis with spontaneous premature chromosome condensation. It causes a delayed mitosis-linked cell death and finally leads to apoptosis. Apples contain huge amounts of polyphenols that possess the potential to mediate cell cycle arrest, apoptosis and mitotic catastrophe in transitional cell carcinomas. Aurora kinases, which include aurora A (aurA), aurora B (aurB) and aurora C (aurC), are a family of serine/threonine kinases that play key roles in mitotic progression. Quercetagetin (84) is an aurora kinase inhibitor. Induction of mitosis-associated tumour cell death by quercetagetin is a promising strategy for developing novel chemotherapeutic anticancer agents. Pterocarpans are naturally occurring compounds that have a tetracyclic ring system derived from the basic isoflavonoid skeleton and an ether linkage between positions 4 and 2ƍ. These molecules induce increased frequencies of mitotic cells by inducing arrest in prometaphase. Immunofluorescence staining with an anti-Ȗ-tubulin antibody showed double-dot labeling in the spindle polar region of breast cancer cell lines, suggesting that pterocarpan treatment blocked centrosome segregation. The antiproliferative activity of pterocarpans arises with the inhibition of spindle pole separation during mitosis, leading to cell cycle arrest at prometaphase. These findings highlight the anticancer potential of these natural molecules as well as suggesting a new molecular tool for further investigations on mitosis.

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Curcumin (85), a natural polyphenol derived from the rhizome of Curcuma longa, shows anticancer properties both in vitro and in vivo. The induction of cell senescence upon curcumin treatment resulted from aberrant progression through the cell cycle. Moreover, the DNA damage acquired by cancer cells, due to mitotic disturbances, activates an important molecular mechanism that determines the potential anticancer activity of curcumin. However, curcumin may also be toxic and its toxicity was shown in proliferating bovine aortic endothelial cells, at concentrations relevant to the diet and below those previously reported in cancer models. Moreover, curcumin concentrations below the minimum 2 ȝM threshold required to induce hemeoxygenase-1 bound tubulin protein in vitro and triggered hallmark evidence of mitotic catastrophe in vivo. Concentrations as low as 0.1 ȝM of curcumin led to disproportionate DNA segregation, karyorrhexis and micronucleation in proliferating endothelial cells. While suggesting a mechanism by which physiological curcumin concentrations inhibit cell cycle progression, these findings describe heretofore unappreciated curcumin toxicity with potential implications for endothelial growth, development and tissue healing.

Mitosis causes a 20–100-fold increase in microtubule dynamics, providing an increased dependence on proper microtubule function during mitosis. Most microtubule targeting agents (MTAs) are frequently called “antimitotic drugs”. Defects in spindle assembly or spindle-kinetochore attachment activate the spindle assembly checkpoint, arresting cells prior to the metaphase–anaphase transition. This effect has been reported for microtubule inhibitors giving rise to the classic G2/M cell cycle arrest seen in cell culture experiments. Most MTAs bind one of four main sites/domains within microtubules impacting tubulin stability: the laulimalide site (stabilizing), taxane/epithilone site (stabilizing), colchicine site (destabilizing) or the Vinca alkaloid site. Vinca alkaloids bind with high affinity to one or a few tubulin molecules at the tip of microtubules but do not copolymerize with them. Extracts from the periwinkle plant Vinca rosea (now known as Catharanthus roseus) contain alkaloids

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including vinblastine (86) and vincristine (87), whereas semisynthetic derivatives of vinblastine are vinorelbine (88), vindesine (89) and vinflunine (90). Deoxypodophyllotoxin (91), a natural microtubule destabilizer, was isolated from Anthriscus sylvestris, and a few studies have reported its anti-cancer effect. This molecule inhibits cancer cell proliferation and induces G2/M cell cycle arrest accompanied by an increase in apoptotic cell death in SGC-7901 cancer cells. In addition, it causes cyclin B1, Cdc2 and Cdc25C to accumulate, it decreases the expression of Bcl-2 and activates caspase-3 and PARP, suggesting that caspase-mediated pathways were involved in deoxypodophyllotoxin-induced apoptosis.

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The alkaloid colchicine (92) was first extracted from the poisonous meadow saffron Colchicum autumnale. High affinity binding of colchicine to tubulin allows it to copolymerize into the structure; this process is slow and quasi-irreversible. Other agents have been discovered or designed to target the colchicine domain and are being developed as vascular disrupting agents against cancer. For instance, combretastatin A4 (93) isolated from the African bush willow Combretum caffrum is the most potent naturally occurring combretastatin in regards to tubulin binding ability and cellular toxicity. Tubulin has picked up great focus as a major target in drug discovery and, consequently, tubulin inhibitors have been pulling in considerable attention as anticancer agents. Paclitaxel (94, see also Chapter 9), a member of the taxane family, is one of the most useful and effective antineoplastic agents for the treatment of many forms of advanced and refractory cancers. It is a microtubule-stabilizing drug that selectively disrupts the microtubule dynamics, thus inducing mitotic arrest leading to cell death. This compound was originally obtained by extracting the peeled bark of the Taxus brevifolia. However, because of the very low content in the plant it has been produced semi-synthetically by the acylation of 10-deacetylbaccatin III (95), which is a precursor present in the needles of T. brevifolia as well as T. baccata. Paclitaxel acts as a chemotherapeutic agent by binding selectively to the subunit ȕ of tubulin proteins, promoting their polymerization and assembly, thereby stabilizing the formation of the microtubules. This effect leads to form a dysfunctional mitotic spindle, which causes profound mitotic arrest at the G2/M phase and eventually results in cell death through an apoptosis pathway. The combination of both anti-proliferative and cytotoxic properties contributes to the antitumour efficacy of paclitaxel.

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4.3.1.4. Plant Bioactive Molecules Targeting Apoptosis Apoptosis is a process of programmed cell death that occurs in multicellular organisms and that is caused by physiological signals (hormones and growth factors) that lead to rapid DNA damage, chromatin condensation and DNA fragmentation. The cell is also fragmented and is phagocytized by macrophages or neutrophils. There are three different mechanisms by which a cell undergoes apoptosis: extrinsic pathway, intrinsic pathway and perforin-granzyme apoptotic pathway. In the intrinsic pathway the cell kills itself because it senses cell stress, while in the extrinsic pathway the cell kills itself because of signals from other cells. The third type of mechanism, perforin/granzyme induced apoptosis, is mainly used by cytotoxic T lymphocytes. Apoptosis is a mechanism to eliminate precancerous and cancer cells and can be used as a novel target for cancer prevention studies. Recent studies have focused on the use of natural molecules as apoptotic agents that can be used in chemotherapy against cancer. Numerous pure constituents and crude extracts from plants have been studied for apoptosis induction. Apple polyphenols were shown to induce apoptosis by the activation of caspase-3, DNA fragmentation, the downregulation of VEGFR-2

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expression in adenoid cystic carcinoma, and the activation of caspase-3 expression and cleavage of poly(ADP ribose) polymerase. The induction of apoptosis and necrosis in drug-sensitive and multidrugresistant leukaemia cells was demonstrated by using Annona glabra (pond apple) seed extracts. Morusin (96) is a polyphenolic constituent of the root bark of Morus alba (mulberry) and other Morus species. In Annexin V-propidium iodide double staining assays, morusin significantly increased apoptosis in a dose-dependent manner in human breast cancer cells. Morusin induces apoptosis by suppressing survivin and inducing Bax proteins, suggesting that the molecule is a potentially effective therapeutic agent for the treatment of patients with breast cancer. Gallic acid (97) is an organic acid found in gallnuts, sumac, witch hazel, tea leaves, oak bark and other plants. Incubation of hypertrophic scar fibroblasts with 100–150 μM of gallic acid induced apoptosis through the Bcl2/Bax-mitochondrial-dependent pathway, thus indicating that the molecule might have the potential to be developed as a treatment for patients with hypertrophic scarring. Esculetin (98) belongs to the family of coumarins and is found in barley. Esculetin induces apoptosis in human hepatocellular carcinoma SMMC7721 cells, increases the activities of caspase-3 and caspase-9, promotes bax expression, decreases bcl-2 expression, triggers the collapse of mitochondrial membrane potential and increases cytochrome c release from mitochondria. Furthermore, pretreatment with IGF-1 before esculetin administration abrogated the pro-apoptotic effects of esculetin, while PI3K inhibitor increased the pro-apoptotic effects of esculetin. These results indicate that esculetin induces the apoptosis of SMMC-7721 cells through IGF-1/PI3K/Akt-regulated mitochondrial dysfunction. Licoricidin (99), a polyphenolic compound present in liquorice (Glycyrrhiza uralensis), inhibits SW480 human colorectal adenocarcinoma cells by inducing cycle arrest, apoptosis and autophagy, and is a potential chemopreventive or chemotherapeutic agent against colorectal cancer. Cardanol monoene (100) is the major phenolic component extracted from cashew (Anacardium occidentale) nut shell liquid. The molecule induces M14 human melanoma cell apoptosis via the ROS triggered

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mitochondrial-associated pathways, which supports the potential application of cardanol monoene for the therapy of melanoma cancer. Kaurenoic acid (101), obtained from copaiba (Copaifera langsdorfii) oil resin, induced an increase in cell DNA damage or micronucleus frequencies in gastric cancer cell lines in a dose-dependent manner. The gastric cancer and normal mucosa of stomach cell lines entering DNA synthesis and mitosis decreased significantly with kaurenoic acid treatment, and had an increased growth phase compared with non-treated cells. The treatment induced apoptosis (or necrosis) even at a concentration of 2.5 ȝg/mL in relation to non-treated cells. Moreover, this compound seems to be able to induce cell cycle arrest and apoptosis in gastric cancer cells. Homoharringtonine (63) and its semisynthetic formulation omacetaxine (102) have shown efficacy in chronic myeloid leukaemia. In myeloid leukaemia and myeloma cell lines, these agents inhibit ribosomal protein translation and consequently induce apoptosis via downregulation of oncoproteins (BCR-ABL1, cyclin D1, cMyc etc.), mitochondrial disruption, caspase activation and modulation of transforming growth factor-beta and tumour necrosis factor signaling pathways.

Some neolignans isolated from twigs of Nectandra leucantha showed a remarkable effect in vitro against cancer cell lines (SR BR-3, HCT, U87MG, A2058 and B16F10) during the induction of morphological, biochemical and enzymatic features of apoptosis, such as disruption of

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mitochondrial membrane potential, exposure of phosphatidylserine in the outer cell membrane, and-genomic DNA condensation and fragmentation. Naringenin (103) is a flavanone abundant in citrus plants, and was found to reduce apoptosis and oxidative stress in in vitro cortical neuron cells isolated from the brains of neonatal Sprague-Dawley rats and regulated the localization of the Nrf2 protein. Therefore, naringenin has healthpromoting properties because of its anti-apoptotic and anti-oxidant effects in cases of ischaemic stroke. Pterostilbene (104), a natural dimethylated analogue of resveratrol (83) from blueberries, alleviates sepsis-induced liver injury by reducing inflammatory response and inhibiting hepatic apoptosis, and the potential mechanism is associated with sirtuin-1 signalling activation. Punicalagin (105), a polyphenol extracted from pomegranate (Punica granatum) fruit, was shown to exert anti-proliferative activity in prostate cancer cells via induction of apoptosis and an anti-angiogenic effect. Tomatin (106) is a glycoalkaloid found in the stems, leaves and fruits of tomato plants. The apoptotic effect of tomatine was investigated in the MCF-7 human breast cancer cell line. Low concentrations of tomatine were very effective at the 48th hour and the compound showed antiproliferative effects in breast cancer cell lines. Silibinin (107) is an active component of silymarin (108), extracted from milk thistle and widely used as a hepatoprotective drug in liver injury, and it has been shown to suppress the growth of pancreatic cancer in vitro and in vivo without apparent toxicity. The combination of histone deactylase inhibitors with silibinin increased apoptosis through significant downregulation of survivin and activation of caspases, and promoted G2/M arrest via downregulation of cyclinB1/Cdk1 and cyclinA2. These results suggest that combination treatment with histone deactylase inhibitor and silibinin may be a novel and effective therapeutic approach for pancreatic cancer treatment.

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4.3.2. Effect of Plant Bioactive Molecules on Cell Membranes, Channels and Receptors In the cell, an accurate and continuous control of the homeostasis occurs through the presence of ion-specific ion channels (Na+, K+ and Ca2+ and Cl-) and active pumps for Na+, K+ and Ca2+ such as Na+/K+ ATPase and Ca2+-ATPase. Many plant bioactive molecules have a therapeutic value depending on their ability to modify the action of ion channels. It is known that different transient receptor potential (TRP) channels, which are molecular thermosensors that detect cold, warm and hot temperatures, may be involved in the action of some plant extracts. The capsaicin (109) receptor TRPV1 seems a good candidate to explain the skin irritancy of several essential oils, being expressed in keratinocytes from human epidermis and hair follicles. When 31 essential oils were investigated for the activation of hTRPV1 transfected in HEK293 cells, 4 of them gave positive results. In three cases, activation could be traced to a specific constituent (citronellol [110] for rose oil and geraniol [111] for palmarosa- and thyme oils). These channels are not only activated by certain natural products like capsaicin, menthol (112) and camphor (113), but also by various inflammatory signaling pathways. Menthol interacts

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not only with TRPM8, but also with related thermoTRPs, including TRPA1, making it difficult to dissect the various responses it evokes at the organ level. Camphor is noxious at high concentrations and activates various TRPs in a rather non-selective way. The TRPA1 channel is activated by a variety of noxious stimuli, including cold temperatures, pungent natural products and plant volatiles. It has been suggested that several of the compounds known to activate TRPA1, such as cinnamaldehyde (114) and mustard oil components, are also able to covalently bind certain cysteine residues in the ankyrin repeats of TRPA1. Menthol and camphor are local analgesics, probably acting via temperature-sensitive TRPV3 channels. The action of plant bioactive volatiles on different ion channels is probably one of the most important lines of research in this field. There is growing evidence that TRPs, and especially those sensitive to temperature (TRPV1-V4, TRPM8 and TRPA1), are primary candidates for mediating the sensory properties of plant volatiles. Menthol, thymol (115) and geraniol (111) perturb the dynamics of cellular membranes at different biophysical levels. Membrane toxic effects by these lipids may also be the reason why a large number of essential oils are powerful topical and gastrointestinal antimicrobials and nearly all of them are weakly to moderately antiseptic. Overall, the biological effects observed at micromolar concentrations may be related to the ability of these compounds to self-assemble and integrate into cellular membranes. Therefore, in vitro pharmacological investigations should be interpreted with great caution. However, it is important to stress that several plant volatiles also exert potent pharmacological effects in the nanomolar range, which implies specific interactions with proteins. Carvacrol (116) binds to and activates a number of effects on ion channels, but was recently shown to also activate peroxisome proliferator-activated receptor gamma (PPAR-Ȗ), causing inhibition (downregulation) of COX-2 expression and anti-inflammatory activity. This finding suggests that, apart from TRPV channels, also PPAR-Ȗ, a transcription factor sensitive to lipophilic ligands, should be investigated as a broadly tuned receptor for plant VOCs.

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Hinokiol (117) is a naturally occurring diterpenoid compound isolated from plants such as Taiwania cryptomerioides. This compound inhibits voltage-gated Na+ channels (VGSC) in a concentration-dependent and state-dependent manner in neuroblastoma N2A cells, differentiated neuronal NG108-15 cells and rat hippocampal CA1 neurons. Stimulation of the cell lines RIN14B or STC-1 with gingerol (118) increased intracellular calcium levels and serotonin or cholecystokinin secretion. The gingerol-induced intracellular calcium increase and secretion (serotonin or cholecystokinin) were completely blocked by ruthenium red, EGTA and TRPA1-specific siRNA. These results suggested that gingerol might improve the digestive function through secretion release from endocrine cells of the gut by inducing TRPA1mediated calcium influx. Gelsemine (119) is one of the principal alkaloids produced by the Gelsemium genus. Gelsemine directly modulates recombinant and native glycine receptors and exerts conformation-specific and subunit-selective effects. Gelsemine modulation is voltage-independent and is associated with differential changes in the apparent affinity for glycine. In addition, the alkaloid preferentially targets glycine receptors in spinal neurons and shows only minor effects on GABAA and AMPA receptors. Furthermore, gelsemine significantly diminishes the frequency of glycinergic and glutamatergic synaptic events without altering the amplitude. Myrsinane, premyrsinane and cyclomyrsinane diterpenes substituted with hydroxy, acetyl, propanoyl, isobutanoyl, 2-methylbutanoyl, n-hexanoyl, benzoyl and nicotinyl ester groups from Euphorbia falcata were bioassayed and several of the tested compounds were found to possess

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blocking activity on G protein-activated inwardly rectifying potassium ion channels. Falcatins A–C (120-122), which are potent for GIRK channels, exert low inhibitory effects on the human embryonic kidney cell channels.

Two modulators of Aquaporin-1 channels, bacopaside I (123) and bacopaside II (124), isolated from the medicinal plant Bacopa monnieri were tested in the Xenopus oocyte expression system. Bacopaside I blocked both the water and ion channel activities of Aquaporin-1 but did not alter Aquaporin-4 activity, whereas bacopaside II selectively blocked the Aquaporin-1 water channel without impairing the ionic conductance. The Ca2+ current through Cav1.2 channels is inhibited by three different classes of drugs, namely dihydropyridines (prototype nifedipine [125]; gating inhibitor), benzothiazepines and phenylalkylamines (diltiazem [126] and verapamil [127], respectively; channel blockers), which bind to distinct, but allosterically coupled, receptor sites close to the pore and to the proposed activation gate of the channel Į1-subunit. The natural plant product quercetin (2) increases the Cav1.2 current in clonal rat pituitary GH4C1 cells, possibly via cAMP-induced activation of serine/threonine kinase PKA, and seems to exert a direct effect on the channel protein without involving any of the main, endogenous modulators of the current. The analysis of the functional interaction between quercetin and either the stimulator myricetin (128) or the inhibitors resokaempferol (129), chrysin (130), genistein and 5,7,2ƍ-trihydroxyflavone (131) provided evidence of the highest apparent affinity of quercetin for the Cav1.2 channel and led to the hypothesis of a common recognition site for flavonoids.

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PTK specifically phosphorylates tyrosine residues on channel proteins. Among PTK inhibitors, the isoflavone genistein (75), a major bioactive constituent of soy, displays marked specificity by inhibiting PTK by competing for the ATP-binding site, while it has modest effects on serine/threonine kinases, PKA or PKC.

4.3.3. Immunomodulatory Effect of Plant Bioactive Molecules The immune system consists of cells and molecules that act by defending the organism from invasions of foreign molecules or microorganisms. As an intricate system composed of lymphoid organs, lymphocytes, macrophages, cytokines and receptors, the immune system constantly patrols the body and surveys the surroundings to eliminate any invaders. Immunomodulation involves immunostimulation or immunoinhibition of certain cellular and/or humoral immune responses. This suggests that the immune system functions as an open integrated system, rather than functioning in a strictly autarchic manner. One of the most promising recent alternatives to classical pharmacological treatment is the use of plant-derived immunomodulators for enhancing host defence responses.

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Indeed, the basic mechanism of the immunostimulatory, anti-tumour, bactericidal and other therapeutic effects of plant bioactive molecules is thought to occur via macrophage stimulation and modulation of the complement system. Although the immunomodulatory effects of some herbs have been extensively studied, research related to the possible immunomodulatory effects of many herbs and various spices is relatively scarce. Besides plant-derived polysaccharides, which are known to modulate innate immunity and, more specifically, macrophage function, several other secondary products have been demonstrated to exert immunomodulation. Among the signal molecules interleukin-1 (IL-1), tumour necrosis factor-Į (TNF-Į) and interferon-Ȗ (IF-Ȗ or an-IL-6) are the major players. Some plants produce bioactive molecules that stimulate macrophages to produce signal molecules such as TNF-Į, IL-1 and IL-6. These signals activate other components of the immune system and promote the migration of neutrophils from the bone marrow to the bloodstream. Eventually this leads to more intense antibody, phagocytic and oxidizing activity. Many herbs and food additives have been shown to exert immunomodulatory effects by stimulating various branches and components of the immune system. For example, black pepper (Piper nigrum) and cardamom (Elettaria cardamomum) have been shown to possess potent immunomodulatory effects. Some food products have been shown to trigger certain molecular and cellular mechanisms that lead to food allergies and oral tolerance including immunomodulation. Several experiments on humans – including a number of double-blind randomized trials – show health benefits. The most robust data come from tests on Echinacea purpurea extracts in the treatment of acute upper respiratory tract infections. Macrophages cultured in the presence of concentrations of echinacea extracts as low as 0.012 mg per litre produce significantly higher concentrations of IL-1, TNF-Į, IL-6 and IL-10 than non-stimulated cells. These results were correlated with an immunologically activated antiviral effect. Recent studies have shown that standardized preparations of echinacea able to induce a significant and selective antiviral and antimicrobial activity show multiple immunomodulatory activities, including stimulation of some immune functions such as phagocyte activity of macrophages and suppression of proinflammatory responses to epithelial cells to viruses and bacteria, as a

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consequence of alteration in the secretion of various cytokines and chemokines. This immunomodulation results from the up- or downregulation of relevant genes and their transcription factors. Overall, this bioactivity can be demonstrated at cytotoxic concentrations of the extract and appears to depend on multiple echinacea components rather than single ones. Nigella sativa, also known as black cumin, shows immunomodulatory and anti-inflammatory activities, which are associated with its major active ingredient, thymoquinone (132). Thymoquinone exerts anti-inflammatory effects by inhibiting COX and 5-LO biosynthesis in rat peritoneal leukocytes due to inhibited formation of thromboxane B2 and leukotriene B4 metabolites. In vivo anti-inflammatory properties of thymoquinone were extensively studied in two main inflammatory diseases, ulcerative colitis and experimental allergic encephalomyelitis. In the latter disease, the beneficial effects of thymoquinone are due to its ability to act as a glutathione inducer, allowing thymoquinone to serve as an anti-oxidant and anti-inflammatory agent. The monoterpene Į-pinene (133), produced by several aromatic plants, is effective against Leishmania amazonensis, a parasite responsible for the disease leishmaniasis. The effect is exerted through an immunomodulatory activity with increases in both phagocytic and lysosomal activity. Also, gallic acid (97) was able to reduce the number of L. amazonensis infected macrophages and the survival index and reduced parasitism with a concentration-dependent effect. Erucin (134), an isothiocyanate present in several species of the Brassicaceae plant family, inhibits the cell viability of human lymphocytes via the induction of apoptosis. The compound alters the expression of the interleukin (IL)-2 receptor and interferes with the function of human T lymphocytes by decreasing the activity of NK-cells. Carnosol (135) is a natural antioxidant derived from rosemary that significantly suppresses both the cellular and humoral activity of the immune system when experimented with on BALB/c mice. Interestingly, camosol is able to inhibit acquired immunity – more greatly than cyclophosphamide (136), which is a known potent immunosuppressant. Thymol (115) and carvacrol (116), the two major constituents of thyme, were tested on dendritic cell maturation and T cell activation. Thymol inhibited the proliferation of human lymphocytes as well as the Jurkat T

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cell line in a concentration-dependent manner. Furthermore, both thymol and carvacrol decreased mice lymphocyte proliferation, which indicated their immunosuppressive effects on lymphocytes. ǻ9-tetrahydrocannabinol (THC, 137) and cannabidiol (CBD, 138) are the most abundant phytocannabinoids in Cannabis plants and therapeutic applications for both compounds have been suggested. Results of animal studies show that these cannabinoids exert their immunomodulatory properties in four ways: induction of apoptosis, suppression of cell proliferation, inhibition of pro-inflammatory cytokine/chemokine production along with increase in anti-inflammatory cytokines, and induction of regulatory T cells. CBD has recently been emerging as a therapeutic agent in numerous pathological conditions since it is devoid of the psychoactive side effects exhibited by ǻ9-THC. CBD has been shown to exert immunosuppression through various other receptors in addition to the canonical CB1 and CB2. For instance, CBD, by enhancing endogenous adenosine signalling, mainly through the inhibition of its uptake, potently reduced the inflammatory lung response in an adenosine A2A receptordependent manner. CBD was also able to decrease total lung resistance and elastance, neutrophils’, macrophages’ and lymphocytes’ migration into the lungs, mieloperoxidase activity in tissue and the production of both pro-inflammatory cytokines tumour necrosis factor-Į (TNF-Į), interleukin-6 (IL-6), chemokines monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-2 (MIP-2) in the bronchoalveolar lavage supernatant. Cannabinol (CBN, 138) is a breakdown product of THC and acts as a weak agonist of CB1 and CB2 because of a lower affinity than THC toward receptors. Because neuroinflammation plays a significant role in essentially all neurodegenerative processes, CB2 stimulation became an attractive target for the development of neuroprotective therapies. CB2 is expressed in different types of leukocytes mediating cannabinoid anti-inflammatory effects and immunomodulation. In summary, cannabinoids can be neuroprotective via their immunomodulatory properties, which have been mainly attributed to CB2 receptors.

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4.3.4. Toxic Effect of Plant Bioactive Molecules Poisonous and toxic plants are among the most studied plants. The effect of plants on humans and animals can be healing, as we have just discussed, or fatal. The toxic effect may be skin-related, following contact, gastrointestinal, following ingestion, or lung-related, after inhalation. The toxicity can be extended to the liver, respiration and nervous system, and some compounds can cause cancer, by damaging DNA, which is eventually transmitted to unprotected daughter cells. Plant toxins can also generate teratogenic phenomena occurring when, in the developing foetus, cell proliferation, migration and differentiation are altered. 4.3.4.1. Kidney Injury The major risk factors for one of the most serious forms of kidney injury, chronic kidney disease, are diabetes mellitus, high blood pressure, heart disease and a family history of kidney failure. Clinical signs of kidney failure include polyuria, oliguria or anuria, dehydration, vomiting, diarrhoea and depression. Since the kidney is highly susceptible to toxic insults, drugs and plant toxins are a common source of acute kidney injury. As recently reported, in Africa, herbal medicine accounts for approximately 30–35% of all acute kidney injury cases. Several herbs have been related to kidney injury case reports. Among these herbs are Chinese yew (Taxus celbica) extract, impila (Callilepis laureola), morning cypress (Cupressus funebris), St John’s wort (Hypericum perforatum), thundergod vine (Tripterygium wilfordii),

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tribulus (Tribulus terrestris) and wormwood (Artemisia herba-alba). The top herbs are the aristolochic acid (140)-containing herbs such as Aristolochia spp. Other herbs affecting kidney health are those containing djenkolic acid (141), such as the Asian beans from Archidendron pauciflorum and Pithecellobium jeringa. Djenkolic acid is insoluble, precipitates into crystals and damages the kidneys, resulting in acute kidney injury. Swainsonine (142)-containing plants comprise a very important group of toxic plants, including several Ipomoea species. The main histologic lesions are vacuolations of kidney epithelial cells. Evidence of liver or kidney damage have been reported after the ingestion of extracts from Cycas, Lilium, Hemerocallis spp. and Ricinus communis 1–2 days after the exposure, whereas monocrotaline (143), a pyrrolizidine alkaloid present in Crotalaria species, shows nephrotoxic effects.

4.3.4.2. Liver Injury The liver is the main metabolizing organ involved in the biotransformation of xenobiotics and is responsible for the conversion of foreign chemical substances into metabolites that are readily excreted in the bile and urine. Despite the hepatoprotectant effect of many bioactive plant metabolites (including flavonoids), several plant extracts exert hepatotoxic activities. Liver injury can be classified as predictable (i.e., dose-related high incidence liver injury) or unpredictable (i.e., low incidence that may be dose-related or not). The hepatotoxicity of certain plant species has been associated with the presence of specific phytochemicals such as pyrrolizidine alkaloids, triterpene saponins, anthraquinones and ephedrine alkaloids. The primary polyphenolic compound of chaparral (Larrea tridentata) is nor-dihydroguaiaretic acid (144), which is the most likely compound to be

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responsible for the reported hepatotoxicity. This compound inhibits both lipoxygenase and cytochrome P-450 mono-oxygenase activity in rat epidermal and hepatic microsomes. The roots of Comfrey (Symphytum officinale) contain hepatotoxic pyrrolizidine alkaloids including among others intermedine (145), lycopsamine (146), lasiocarpine (147), symphytine (148) and uplandicine (149).

Greater celandine (Chelidonium majus) contains an alkaloid-rich orange coloured latex that causes acute cholestatic hepatitis. Isoquinoline alkaloids present in celandine that are known to be effective include sanguinarine (150) and berberine (151), whereas the main alkaloid present in the plants’ aerial parts is chelidonine (152). The hepatotoxicity of these compounds has been defined as a distinct form of herb-induced liver injury (HILI), due to an idiosyncratic reaction of the metabolic type. The fruit of Xanthium strumarium contains atractyloside (153) and carboxyatractyloside (154), that cause hepatotoxicity. Their hepatotoxic effects are associated with mitochondrial function inhibition and influence on fatty acid metabolism.

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The roots of several species belonging to the Polygonum genus contain emodin (155), that causes severe liver enzyme secretion. This compound and its analogues are also present in Cassia occidentalis, Rheum palmatum and Aloe vera. Saikosaponin D (156), isolated from Bupleurum falactum root, is a triterpene saponin with a steroid-like structure that may cause liver injury. In a mechanistic in vitro study on human LO2 hepatocytes, it was shown that this compound is capable of reducing cell viability, decreasing mitochondrial membrane potential, changing cell morphology and stimulating hepatocyte apoptosis. Wild germander (Teucrium chamaedrys)-induced hepatotoxicity is primarily mediated by the biotransformation of furanic neoclerodane diterpenes, like teucrin A (157), by CYP3A4 leading to the formation of electrophilic metabolites which deplete cellular thiol. Retrorsine (158) is a retronecine-type hepatotoxic pyrrolizidine alkaloid common in the genus Senecio. It is a mechanism-based inhibitor of CYP3A4. Retrorsine undergoes hepatic metabolic activation to generate the corresponding pyrrolic esters, which are chemically reactive. Evodiamine (159) and rutaecarpine (160) are the main active indoloquinazoline alkaloids of Evodia rutaecarpa. Rutaecarpine showed a dose related induction of the activities of hepatic cytochrome P450 1A, 2B and 2E, and the mechanisms may be associated with metabolic activation. Rutaecarpine and evodiamine can inhibit the activities of cytochrome P450 (CYP1A2/2B6/3A4) and exhibit a potential mechanism-based inhibition on CYP1A2 and CYP3A4 isoforms. L-Tetrahydropalmatin (161) is one of the main active ingredients isolated from Corydalis yanhusuo. This compound can induce apoptosis in the hepatocytes of BALB/c mice and human normal liver L-02 cells. Rhein (162) is an active ingredient in the root of rhubarb (Rheum rhabarbarum) and is a mechanism-based inhibitor of CYP2C19. This compound could cause rat hepatocytes and HL-60 cell apoptosis by generating ROS and eventually impairing mitochondrial functions. The roots of Angelica dahurica contain imperatorin (163) and isoimperatorin (164) which are mechanism-based inhibitors of CYP2B6;

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the formation of Ȗ-ketoenal intermediate may account for the enzyme inhibition. Fruits and seeds of the legume plant Psoralea corylifolia contain psoralen (165) and isopsoralen (166), two coumarin derivatives. These compounds have been characterized as mechanism-based inhibitors of CYP2B6. Moreover, psoralen and isopsoralen could suppress the activity and protein expression of CYP2E1 and increase the activity and protein expression of CYP3A11.

Menthofuran (167) is a p-menthane present in many Mentha species and hybrids (particularly in pennyroyal, Mentha pulegium). This compound is a mechanism-based inhibitor of CYP2A6 and can be oxidized by cytochrome P450 enzymes to a reactive electrophile (Ȗ-ketoenal, 168) that is capable of covalently binding to cellular proteins. The formation of Ȗketoenal may be associated with the pathogenesis of hepatotoxicity caused by both menthofuran and its precursor pulegone (169).

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Ephedrine (170), which is obtained from the plant Ephedra sinica and other members of the Ephedra genus, has been reported to cause acute hepatitis. In ephedrine-treated LX-2 cells, the compound triggers mitochondrial oxidative stress and depolarization, inhibits mitochondrial biogenesis and decreases the mitochondrial copy number. Several tumorigenic pyrrolizidine alkaloids, including riddelliine, (171) monocrotaline (143), lasiocarpine (147), retrorsine (158), heliotrine (172), clivorine (173) and senkirkine (174), are liver carcinogens. A set of exogenous DNA adducts commonly formed from these tumorigenic pyrrolizidine alkaloids has been recently characterized and are considered common biological biomarkers of pyrrolizidine alkaloid-induced liver tumour formation. The hydrolysis of dehydropyrrolizidine alkaloids generates dehydroretronecine (175), which is a reactive electrophilic metabolite. This compound can bind with glutathione or proteins to generate pyrroleglutathione conjugates or pyrrole-protein adducts respectively leading to detoxification or hepatotoxicity.

4.3.4.3. Cardiotoxicity Several plant toxins can affect the heart and cause cardiac manifestations. These plants contain alkaloids and other toxic agents that rapidly and significantly change heart rate and rhythm, eventually leading to death.

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Although many commonly occurring plants are toxic, only some plants are potentially lethal, and only when consumed in sufficient quantity. Plant species belonging to the genera Digitalis, Nerium, Thevetia, Convallaria, Strophanthus, Cheiranthus and Asclepias contain bioactive molecules acting as Na+/K+ ATPase blockers. The primary mechanism of action of Digitalis glycosides is their ability to inhibit membrane-bound Na+/K+-ATPase. As a consequence, Na+/Ca2+ exchange is promoted and more intracellular calcium becomes available for contractile proteins. This, in turn, increases myocardial contraction. Several species belonging to the genus Digitalis contain steroidal triterpenes called cardenolides, like digitoxin (176), digoxin (47), gitoxin (177), lanatoside C (178) and digitoxigenin (179). These compounds induce ventricular paroxysmal tachycardia. Accidental ingestion of foxglove (Digitalis purpurea) can cause significant cardiac toxicity. The toxic and ornamental plant Nerium oleander contains the bioactive molecule oleandrin (180). The basis for the physiological action of the oleander cardenolides is similar to that of the classic digitalis glycosides, i.e., inhibition of plasmalemma Na+/K+-ATPase. However, differences exist in the toxicity and extracardiac effects between the oleander and digitalis cardenolides. The toxicity of these compounds causes lifethreatening ventricular tachyarrhythmias, bradycardia and heart block. Cascabela thevetia or Thevetia peruviana poisoning are caused by the presence of thevetin A (181) and B (182), peruvoside (183), neriifolin (184) and ruvoside (185). The effect of these cardiac glycosides resembles that of digoxin (47). Convallaria majalis contains convallamarin (186) and convallatoxin (187) which act by inhibiting Na+/K+-ATPase revealing third-degree atrioventricular block. Ouabain (188) and strophanthin K (189) produced by Strophanthus gratus cause toxic arrhythmias with induction of ventricular fibrillation. In Cheiranthus cheiri, cardenolide cheirotoxin (190) and glucosinolate glucocheirolin (191) cause inverted T wave and bradycardia. The cardioactive steroid antiarin (192) produced by Antiaris toxicaria results in sinus tachycardia, T wave inversion and ST segment elevation.

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The milky wax exuding from the stem of Calotropis procera contains the cardiac glycosides calotropin (193) and uscharin (194) that cause bradycardia and a significant increase in the force of ventricular contraction. Cerberin (195), produced by Cerbera manghas, causes 1st, 2nd and 3rd degree heart block and sinus bradycardia.

The bufadienolide cardiac glycosides scillarenin (196), scilliglaucoside (197) and scilliphaeoside (198) are produced by Urginea maritima and Drimia maritima. Their toxicity depends on blockage of Na+/K+-ATPase causing bradycardia and complete atrio-ventricular heart block.

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The roots and seeds of Aconitum napellus contain the diterpene alkaloids aconitine (199) and mesaconitine (200) that show Na+ channel binding properties causing cardiac dysarhythmias, premature ventricular contraction and prolonged bidirectional ventricular tachycardia. Emerging evidence indicates that voltage-dependent Na+ channels have pivotal roles in the cardiotoxicity of aconitine. Moreover, aconitine significantly aggravates Ca2+ overload and causes arrhythmia and finally promotes apoptotic development via phosphorylation of P38 mitogen-activated protein kinase. Common yew (Taxus baccata) is a common decorative evergreen shrub with potentially fatal toxicity hallmarked by seizure, arrhythmia and cardiovascular collapse if ingested. The main cardiotoxic molecules of several Taxus species include taxol (paclitaxel, 94), taxine A (201) and, especially, taxine B (202). Another compound, 3,5-dimethoxyphenol (203), is used as a marker of Taxus ingestion. These molecules cause ventricular fibrillation and ventricular tachycardia. Adonitoxin, (204) produced by Adonis aestivalis, inhibits Na+/K+-ATPase and causes ventricular arrhythmias, bradyarrhythmias, atrioventricular block, ventricular premature beats, ventricular tachycardia and ventricular fibrillation.

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Veratrum album contains many alkaloids including protoveratrine A (205) and B (206), cyclopamine (207) and germerine (208), able to activate Na+ channels. By increasing Na+ permeability these molecules can cause significant bradycardia and hypotension on ingestion. Datura stramonium, Atropa belladonna and Hyoscamus niger contain belladonna alkaloids including atropine (209), hyoscyamine (210) and scopolamine (211), that exhibit anticholinergic toxicity.

Berberine (151) cardiotoxicity is dependent on the route and duration of administration of the drug extract.

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Species of the plant family Rubiaceae including Pachystigma latifolium, P. pygmaeum, P. thamnus, Pavetta harborii, Pavetta schumanniana and Fagodia homblei cause a syndrome characterized by heart failure and sudden death when ingested by ruminants. The bioactive molecule isolated from these species is pavettamine (212), that inhibits protein synthesis in the cardiac muscle and reduces systolic function. Cocaine (213), a naturally occurring alkaloid extracted from the leaves of Erythroxylum coca, besides its neurological effects (see below), is also a potent cardiotoxin. Its main effects are related to myocardial ischemia, infarction and scarring, bradycardia, acute hypertension as a result of an abrupt catecholamine surge and stroke. In contrast to other addictive drugs that exert their harmful effects through a limited mechanism, cocaine has a multitude of pathophysiological pathways by which it affects the cardiovascular system. The risk of myocardial infarction was found to increase up to 24-fold in the first hour after cocaine abuse. Cucurbitacin-I (214)-induced cardiotoxicity has been examined by investigating the role of MAPK-autophagy-dependent pathways in H9c2 cells. This compound increases the autophagy levels of H9c2 cells, most likely through the activation of an ERK-autophagy dependent pathway, which results in the hypertrophy and apoptosis of cardiomyocytes.

4.3.4.4. Neurotoxicity Neurotoxins are defined by their ability to cause structural damage or functional disturbance to nervous tissues upon the application of relatively small amounts. Almost all major recreational drugs, including caffeine (27), nicotine (215), THC (137), cocaine (213), amphetamines and heroin (216), are plant neurotoxins. These bioactive molecules interfere with neuronal signalling in the CNS, for example by binding to neurotransmitter receptors or interfering with neurotransmitter transport mechanisms. Polyacetylenes are widely distributed among the families Umbelliferae, Araliaceae and Asteraceae and are known to be neurotoxic in high

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concentrations. The genus Bupleurum comprises several species known for their content in polyacetylenes, with particular reference to B. longiradiatum. The neurotoxicity of polyacetylenes exhibits a relationship with the Ȗ-aminobutyric acid (GABA, 217) receptor pathway, and polyacetylenes have been shown to inhibit GABA-induced currents (IGABA) in a competitive manner. The major neurotoxic molecules of this species are bupleurotoxin (218), acetylbupleurotoxin (219) and oenanthotoxin (220). Other polyacetylenes such as falcarinol (221) and falcarindiol (222) are also potent neurotoxins. N,N-dimethyltryptamine (223) is an indole alkaloid widely found in plants. This molecule produces brief and intense psychedelic effects when ingested. Clivorine (224), an otonecine pyrrolizidine alkaloid from Ligularia species, causes neurotoxicity in PC12 cells by downregulating the nerve growth factor and its receptor signalling pathway. As many other pyrrolizidine alkaloids do, the molecule results in brain disorders, such as depression. Swainsonine (142) is an alkaloid that was initially discovered as a plant toxin in various Astragalus and Oxytropis species. The ingestion of the molecule also causes histological lesions to the CNS, including widespread neuronal vacuolation and axonal dystrophy. Gibberellic acid (GA3, 225) is an endogenous plant growth regulator used worldwide in agriculture; however, it also exerts physiological effects on mammals. Acetylcholinesterase activity in both the cerebellum and cerebrum of suckling rats during late pregnancy and early postnatal periods was inhibited after treatment with GA3. Moreover, GA3 caused an abnormal development of the external granular layer and a loss of Purkinje cells. Catha edulis is an evergreen plant that grows at high altitudes in East Africa and on the Arabian Peninsula. The plant produces the bioactive molecule cathinone (226), a potent natural amphetamine, along with cathine (227) and norephedrine (228). Ephedra species produce the amphetamines ephedrine (170) and pseudoephedrine (229). As many other amphetamines do, these molecules may cause microglial activation, oxidative stress and hyperthermia and act as substrate-type releasers. They bind to the plasma membrane monoamine

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transporters, being transported and translocated into the cytoplasm, stimulating neurotransmitter release through these transporters and promoting neurotoxic actions toward the CNS. The pyrrolizidine alkaloid monocrotaline (143) produced by Crotalaria retusa also exerts neurotoxic effects in primary cultures of astrocytes or neurons and in primary co-cultures of astrocytes/neurons obtained from the cerebral cortex of Wistar rats. Recent findings indicate that glutathione depletion is dependent on the metabolism and synthesis of active metabolites via the P450 system and that this phenomenon is involved in monocrotaline-induced neurotoxicity. Rotenone (230) is a crystalline isoflavone traditionally used as a broadspectrum pesticide, insecticide and piscicide. This molecule can trigger oxidative stress and low concentrations of rotenone which, during long times of exposure, can induce Parkinson-like features, with a selective dopaminergic neuronal cell death and the formation of Lewy body-like cytoplasmic inclusions. The existence of a novel mechanism of rotenone toxicity mediated by 70-kDa heat shock cognate chaperone protein (hsc70) indicates that dysfunction of both chaperone-mediated autophagy and macroautophagy can synergistically exacerbate alpha-synuclein toxicity, suggesting that hsc70 upregulation may represent a valuable therapeutic strategy for Parkinson’s disease. Endoplasmic reticulum stress in rotenone-induced neuronal death was also demonstrated and found to play a key role in neuronal death, rather than oxidative stress. Annonacin (231), isolated from Annona muricata fruit, bark and leaves, induces the death of cortical neurons at low micromolar concentrations.

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4.3.4.5. Genotoxicity When plant bioactive molecules react with cellular macromolecules, including DNA, they may cause cellular toxicity and/or genotoxicity. A genotoxin is a bioactive molecule that can cause DNA or chromosomal damage, and genotoxicity refers to potentially harmful effects on genetic material which are related to the induction of permanent transmissible changes in the amount or structure of the genetic material. Specific genotoxic events are considered hallmarks of cancer. The Aristolochiaceae family, notably in the genus Aristolochia and Asarum, contain genotoxic compounds known collectively as aristolochic acids. The major components of the plant extract are nitrophenanthrene carboxylic acids, which are genotoxic mutagens after metabolic activation.

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Tinctures of Aristolochia clematitis and Asarum europaeum inhibit DNA synthesis in human hepatoma HepG2 cells in a dose-dependent manner. One of the components of the plant extract, aristolochic acid I (232), is linked to the development of nephropathy and urothelial cancer in humans. Many studies have shown that 1,2-unsaturated pyrrolizidine alkaloids are hepatotoxic and exhibit a large variety of genotoxicities, inducing DNA binding, DNA cross-linking, DNA-protein cross-linking, sister chromatid exchange and chromosomal aberrations. As we have discussed, pyrrolizidine alkaloids are among the most potent natural toxins and 1,2-unsaturated pyrrolizidine alkaloids may act as genotoxic carcinogens in humans. After metabolic (oxidative) conversion of these molecules into dehydro-pyrrolizidine esters, adducts with proteins and DNA are formed. With the incubation of rat liver microsomes in the presence of calf thymus DNA, the amount of DNA adducts formed was found in the following rank order: retrorsine (158) > retrorsine-N-oxide (233) > heliotrine (172). Petasitenine (234), senkirkine (174), senecionine (235) and seneciphylline (236) were positive to the Ames test and in primary hepatocytes from Syrian golden hamsters and C3H/HeN mice. Primary DNA and chromosomal damage was also found by other pyrrolizidine alkaloids such as lasiocarpine (147), monocrotaline (143), riddelliine (171), heliosupine (237), echinatine (238), echimidine (239) and isatidine (240). The genotoxicity of some anthraquinones, such as emodin (155), has been confirmed in a variety of in vitro and in vivo assay systems. Potential genotoxicity has been reported for some volatile phytochemicals produced by aromatic plants, such as sassafras (Sassafras albidum), including safrole (241), eugenol (242), methyl eugenol (243), estragole (244) and ȕ-asarone (245). Other plant volatiles, such as anethole (246) pulegone (169), menthone (247) and camphor (113), were found positive in some genotoxicity tests, but their genotoxic potential remains to be confirmed.

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4.3.5. Plant Bioactive Molecules against Uropatogenic Escherichia Coli Urinary tract infections (UTIs) are widespread and affect a large proportion of the human population. Uropathogenic Escherichia coli (UPEC) is the main cause of community-acquired UTIs. Although antibiotics will continue to be an unavoidable source for the prevention of UTIs on a case-by-case basis, the excessive use of antibiotics and the longterm interference with intestinal microbiota, require alternative remedies to be sought. A plethora of molecules has been tested to reduce UPEC infections by exploiting their ability either to stimulate the immune system or to interfere with the UPEC ability to adhere and invade the urothelium. Phenolic compounds exert a strong antibiotic effect and can be generally subdivided into polymeric and not polymeric phenolics. Among polymeric phenolics, proanthocyanidins (PACs) represent an interesting class of compounds. These tannins are produced by different plants and those isolated from cranberry (Vaccinium macrocarpon) are particularly rich in A-type linkages (such as PAC-A2, 248), compared to the more common B-type linkages of other proanthocyanidins (PAC-B2, 249). Recently, both a standardized cranberry extract particularly rich in PAC-A and its purified

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PAC-A fractions were tested for their antiviral activity against Herpes 1 and Herpes 2. By using a combination of molecular and biochemical analyses, it was shown that PAC-A was able to specifically target viral glycoprotein gD and gB, thus causing the inability of viral particles to infect target cells. These new findings open the possibility that PAC-A may not only target the FimH lectins as D-mannose (250). Some non-polymeric flavonoids, such as quercetin (2), luteolin (251), scutellarein (252), phloretin (253) and genistein (75), are present in the diet and are excreted in the urinary system. These compounds inhibit the activity of cAMP and cyclic nucleotide phosphodiesterases and are considered as potential non-antibiotic therapeutic agents in the UTI setting. In silico studies revealed that the flavonoinds genistein and isorhamnetin (254) can be used as potential drug candidates against UPEC. Quercetin-3-galactoside (255), quercetin-3-glucuronide (256), protocatechuic acid (257), ferulic acid (258), p-coumaric acid (259) and dicaffeoylquinic acid (262) exert modifications in the bacterial surface structures responsible for binding to the occupied surface, whereas a correlation between antioxidant and antimicrobial activity has been demonstrated for ferulic acid, caffeic acid (260), quercetin and apigenin (261).

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4.3.6. Plant Bioactive Molecules for Brain and Mental Disorders Aging is caused by oxidative damage to DNA, proteins, lipids and other molecules. The resulting damage contributes significantly to the onset of degenerative diseases, including those affecting the brain, the sensory tissues and the cardiovascular system. Clinical data show that Ginkgo biloba extracts protect the brain by facilitating the absorption of neurotransmitters and at the same time reducing apoptosis. In sensory tissues, ginkgo extracts protect against retinal lipoperoxidation alterations. One of the most promising applications of ginkgo is in the treatment of Alzheimer’s. This psychiatric disease has received much attention in recent times. Although a cure for this disease has not yet been found,

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several drugs are currently used to reduce its progression. Psychometric tests have shown that a significant progress in patients treated with ginkgo extracts, and significant improvements in the psychopathological and dynamic functions have been noted, indicating also a positive effect in moderate dementia cases. The effects of ginkgo extract on brain pathology seem to be mainly due to the antioxidant properties attributable to the synergistic action of flavonoids, terpenoids and organic acids. Ginkgo extracts protect against damage caused by free radicals on biological systems. The extracts inhibit the production of nitrogen oxide in macrophages by inhibiting the activity of the forming enzyme and regulating the transcription of its gene. In addition, the extract inhibits the adhesion of lymphocytes to the endothelium, by acting in a beneficial way on the circulatory system. The active components of G.ࣟbiloba consist of flavonoids and terpenoids. Ginkgo flavonoid moiety has an inhibitory effect on monoamine oxidase (MAO) and mice studies have shown that the increase in age-dependent enzymatic activity is inverted by the presence of ginkgo flavonoids. Ginkgolide B (263) stimulates both the activity of the phospholipase D of polymorphonucleated human leukocytes and protein phosphorylation, suggesting its potential use in enhancing cellular activity. Ginkgo extracts have been shown to reduce the death of neurons in the hippocampus by reducing the concentration of non-esterified fatty acids, including arachidonic acid (264), which are released by the brain’s phospholipids during transient ischemia. Bilobalide (265), ginkgolide B and picrotoxinin (266) have been shown to negatively modulate the action of GABA at GABAA receptors. Moreover, animal studies have demonstrated that bilobalide has an anticonvulsant action and that ginkgolide A (267) reduces anxiety. In the presence of GABA (217), ginkgolide B was more potent than bilobalide in inhibiting the GABA-potentiating effect of propofol (268), equipotent against loreclezole (269) and allopregnanolone (270), and less potent against etomidate (271), diazepam (42) and thiopentone sodium (272). This indicates that in comparison to picrotoxinin, bilobalide and ginkgolide B differ in their effects on the different modulators. Hypericum perforatum extracts provide a variety of molecules, especially phenolic substances, all capable of passing the hematoencephalic barrier (or blood brain barrier). Leaves and flowers contain naphthodiantrones, xanthones, flavonoids, floroglucinols (e.g., hyperforin, 39) and hypericin (37). Low-hyperforin preparations are effective in treating depression. In

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addition, hypericin is also used to treat some forms of anxiety. In vitro studies indicate that hypericin is able to irreversibly inhibit monoamine oxidase (MAO) by actually increasing the amount of neurotransmitters in synapses between neurons and leading to a sense of psychological wellbeing. The in vitro inhibition of catecole-O-methyltransferase caused by H. perforatum flavonoids and xanthones appears to exert the same effect on neurotransmitters. In some cases, it has been shown that H. perforatum extracts are capable of inhibiting by 50% serotonin (273) absorption in synaptosomes, suggesting that one of the effects of the plant extract on depression may be the inhibition of serotonin absorption by postsynaptic receptors. While neither hypericin nor kaempferol (129) show inhibitory properties on the reuptake of serotonin, norephedrine (228) and dopamine (274), hyperforin has in many cases proved to be a potent non-specific inhibitor suggesting a basic role in biochemical models of antidepressant activity obtained with H. perforatum extracts. Rhodiola rosea is a botanical adaptogen with putative anti-stress and antidepressant properties. Its purified constituent, salidroside (275), and its aglycone, tyrosol (276), have been shown to produce a variety of mediator interactions with several molecular networks of neuroendocrine-immune and neurotransmitter receptor systems likely to be involved in the pathophysiology of depression. R. rosea demonstrates multi-target effects on various levels of the regulation of cell response to stress, by affecting various components of the neuroendocrine, neurotransmitter receptor and molecular networks associated with possible beneficial effects on mood. Therefore, it may be a useful strategy for treating the memory impairment induced by several neurodegenerative diseases.

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CHAPTER FIVE CHEMOTAXONOMIC SIGNIFICANCE OF PLANT BIOACTIVE MOLECULES

5.1. Overview on Chemotaxonomy Chemical data have often been used to overcome the difficulties encountered in the classification of species based solely on morphological data. Sometimes chemical data allow the going beyond where the morphological data stops and, in other cases, the results obtained are consistent with those obtained with classical taxonomy. Occasionally the extreme variability of the classes of compounds used for chemotaxonomic purposes creates contrasting results. For example, in the Leguminosae, phylogeny obtained by bio-molecular data agrees with that obtained by morphological data, but disagrees with that resulting from alkaloids or other natural substances analysis. One of the main reasons for these discrepancies is that secondary metabolites are molecules produced in response to external agents, both biotic and abiotic. In particular, the micromolecular features described by the data play an important role in plant adaptation to various stress conditions, especially to changing environmental conditions. By contrast, the features described by the data obtained from DNA analysis are insensitive to environmental changes or the attack of pathogens, although some authors speculate that the repeated fraction of the plant genome is susceptible to some environmental stresses that would cause DNA qualitative and quantitative changes. For the purposes of this chapter, we will focus on the main classes of compounds used for micromolecular analysis; however, we will also consider the correlation between micromolecular and macromolecular data. The most common micromolecular data include those of phenolic compounds such as phenylpropanoids, benzoic acids, coumarins and furanocoumarins, stilbenes, flavonoids, anthocyanidins, catechins, procyanidins and polymeric forms such as hydrolysable and condensed

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tannins. In addition, terpenoids may be listed as micromolecular compounds, especially monoterpenes and sesquiterpenes. Low-molecularweight alkaloids, betalains and glucosinolates correlate well with morphological and genetic data, providing additional useful information for classification criteria. Macromolecular compounds are often used to solve taxonomic doubts at higher hierarchical levels. For instance, electrophoresis of proteins contained in the seed storage tissues shows a number of bands that can be compared between different taxa and that can be considered phenetically as many other data can. In order to give an overview of the chemotaxonomic significance of specialized metabolites, we will present data on three major plant families, viz. the Leguminosae, Lamiaceae and Asteraceae, and we will add some information on other plant families.

5.2. Chemotaxonomy of Phenolic Compounds Phenolic compounds are among the most frequently used molecules in chemotaxonomic studies. They are divided into different classes, from simple phenols such as benzoic and cinnamic acids, stilbenes and coumarins to more complex structures such as flavonoids and anthocyanidins and their polymers. The extensive use of these compounds is principally due to the ease of their extraction and separation, together with their stability even at room temperature. Benzoic acids are the simplest molecules, made by a benzoic ring with an acid group in position 1 (see also Chapter 6). They may bear substituents of various natures, but the most common groups are -OH and -OCH3. Cinnamic acid derivatives, in particular trans-cinnamic acid (1), are basic building molecules for complex phenolic compounds and the constituents of one of the most widespread polymers in the plant kingdom, lignin. Flavonoids consist of three aromatic rings, two of which are found in all plants with the exception of algae, and are stored predominantly in the vacuole. By far the most used compounds in the chemotaxonomy of phenolic compounds are flavonoids and anthocyanidins. Among the main families used for chemotaxonomic surveys are the Asteraceae, Lamiaceae and Leguminosae. We will give detailed information about these families, while other families will be treated briefly.

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5.2.1. Asteraceae The effectiveness of flavonoids as molecular markers for phylogenetic and evolutionary studies in angiosperms has been widely accepted by the majority of botanists. In very large families, like the Asteraceae, the flavonoid distribution is not able to reflect the phylogenetic relationships among genera and even less explains the evolutionary steps of distinct tribes. However, flavonoids can be very useful, including in the case of the Asteraceae, in overcoming problems related to taxonomic revisions beyond the tribe level. From an evolutionary point of view, flavonoids can confirm the reticulate evolution of the tribes in this family. Flavonoids exist in plant cells in two main forms: aglycones and glycosides. The most detailed studies have been made on aglycones. Species showing high production of flavonoid exudates as well as those with the largest number of flavonoids among plants belong to the Asteroideae subfamily. Aglycones of flavones and flavonols, with substitutions in the 6 and 8 positions, which represent the basic structures of the Asteraceae flavonoids, are also widespread. While aglycones of this subfamily are useful taxonomic markers for species identification, they prove to be useless at the tribe or genus levels. In the Asteraceae, the majority of flavonoids are found in the leaves, stems and flowers as water-soluble glycosides. Nonetheless, the plant surfaces can also be rich in fat-soluble aglycone flavonoids either in the form of powdery deposits or mixed with resins and waxes. In the latter case they are usually produced by glandular trichomes (see Chapter 2), secreted outwards with other substances and laid on the epicuticular layers. A comprehensive study on anthocyanidins of the Asteraceae has shown that most of these molecules are acetylated with organic dicarboxylic acids. Succinic acid is exclusively found in the Cynareae tribe while in other families the acetylating acid is malonic acid. The family comprises other rare anthocyanidins which are acetylated by (di)malonylate derivatives and glycosylated by glucuronic acid. In the Asteraceae there are three predominant basic structures of anthocyanidins, i.e., cyanidin (2), delphinidin (3) and pelargonidin (4). Unlike other flavonoids, anthocyanidins are useful chemotaxonomic markers for tribe identification. For example, in the Astereae, 3malonylglucoside derivatives are found, while in the Heliantheae there is a second acyl group. In the Senecioneae substituents of malonic acid and

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caffeic acid (5) exist, while in the Anthemideae there are molecules with two units of malonic acid linked to glucose in position 3. Finally, the acetylating compound in the Cynareae is succinic acid.

5.2.2. Lamiaceae Several phenolic compounds are present in the Lamiaceae family, and in this case as well, the most studied structures are flavonoids and anthocyanidins. There is a general correlation between the chemistry of flavonoids and the systematics of Lamiaceae, both within the family and at genus level. One of the family features is to accumulate 5,6-dihydroxy-7,8dimetoxyflavones, except for in the Lamioideae subfamily, which is particularly rich in 8-hydroxyflavone-7-allosyl glucosides or p-coumaryl glycosides, and in the Nepetoideae, which accumulate 6-hydroxy flavones. 5,7-Dihydroxy-6-methoxyflavone and 5,6-dihydroxy-7-methoxyflavones with a substituted B-ring and 5,6-dihydroxy-7,8-dimethoxyflavones are characteristic flavonoid constituents of the subfamily Nepetoideae and of the tribe Saturejeae. One of the most studied genera is Salvia. The chemotaxonomic diversity of Salvia species shows a consistent intraspecific diversification based on differing quantities and qualities of flavones, flavonols, flavanones, isoflavones, dihydroflavonols and chalcones. Only about 40% of Plectranthus species were found to produce exudate flavonoids, which were mainly flavones. Flavanones were restricted to five species of Plectranthus, whereas flavonols were only found in two species of Coleus. The most common flavones, occurring in both genera, were cirsimaritin (6) and salvigenin (7), which are methoxylated at the 6 and 7 positions. 6Hydroxylated flavones such as scutellarein (8) and ladanein (9) were restricted to Plectranthus species. A significant intraspecific variation also occurs in the genus Origanum. Taxa in subgeneric populations accumulated flavonoids with methoxyl groups at both C-6 and C-4'; however, taxa in other subgeneric groups did not accumulate 4'- or 6methoxylated compounds. The widespread genus Stachys comprises about 300 species and is considered to be one of the largest genera of the Lamiaceae. Several flavonoids, among others apigenin (10), chrysoeriol (11), penduletin (12), luteolin-7-O-ȕ-D-glucoside (13) and stachyspinoside (14), are characteristic of this genus and have been used for chemotaxonomic purposes.

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As regards the anthocyanidins, a direct comparison with other families indicates that a distinctive feature of the Lamiaceae is the higher frequency of aromatic and aliphatic acylations. A cyanidin derivative present in several members of the Lamiaceae has been characterized as the related 3p-coumarylglucoside-5-malonylglucoside (15). In the blue-purple flowers of Triteleia species one of the main anthocyanidins is delphinidin 3-transp-coumaroylglucoside-5-malonylglucosides (16). A good correlation was also found between flower colour and anthocyanidin type in several Lamiaceae species.

5.2.3. Leguminosae Anthocyanins and flavonoids have also been used as chemotaxonomic markers in the large family of the Leguminosae. Despite the massive amount of information that has accumulated, much remains to be done to obtain good quality comparative data that allows a chemosystematic interpretation at higher taxonomic levels. In the Cesalpinioideae subfamily, numerous anthocyanidins are in the form of 3-glucosides and 6-alkylated-3-soforosides of cyanidin. In the Lotoideae subfamily, the tribe of the Genisteae is characterized by 3,5-diglucosides of pelargonidin, the Trifolieae by 3-glucosides of delphinidin, the Galegeae by 3,5diglucosides of delphinidin, the Hedysareae by 3,5-diglucosides of malvidin and the Phaseoleae by 3-soforosids of pelargonidin and cyanidin. Flavonoids bearing 6,7-(dimethylpyran) and 8-(Ȗ,Ȗ-dimethyl allyl) substituents such as mundulin (17) and minimiflorin (18) are characteristic for some Lonchocarpus species, whereas in the genus Sophora the presence or absence of oligostilbenes and prenylated flavonoids has been found to represent an important chemotaxonomic character. Common to this family are flavonols and flavones. Among flavonols, those with the highest chemotaxonomic value are kaempferol (19) and quercetin (20). Quercetagetin (21), gossypetin (22) and their derivatives are primarily found in the subfamilies Mimosoideae, Caesalpinioideae and Loteae, whereas chalcones are distributed mainly in the Lotoideae. A survey of foliar flavonoids in the swartzioid legume genus Cordyla s.l. revealed that three species, C. haraka, C. pinnata and C. richardii, were rich in the flavonol pentaglycosides 3-O-Į-L-rhamnopyranosyl(1Æ3)-ĮL-rhamnopyranosyl (1Æ2)[Į-L-rhamnopyranosyl(1Æ6)]-ȕ-Dgalactopyranoside-7-O-Į-L-rhamnopyranosides of quercetin and kaempferol (cordylasins A [23] and B [24], respectively). Flavone

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glycosides have also been demonstrated to possess a chemotaxonomic character. Esterification of anthocyanins appears to be a unique apomorphy for the tribe Podalyrieae (including Liparieae). When present, these compounds are esterified with coumaric or acetic acid, while species from other tribes have glycosides only. In the Podalyrieae and in the related Crotalarieae, only hydroxylated anthocyanins (derivatives of cyanidin [2] and peonidin [25]) were found. Isoflavonoids are the characterizing flavonoids of the Leguminosae and, in general, the unique occurrence of isoflavonoids in the subfamily Papilionoideae and perhaps the frequent absence of a hydroxyl group in position five are interesting features. Isoflavonoids are similar to flavonoids in the structural formula, but the B aromatic ring is linked in position 3 rather than in position 2. Isoflavones are peculiar to Lotoideae and are uncommon in other subfamilies. In addition, they are also rare in other plant families, and for this reason they possess a remarkable chemotaxonomic value. Their structures range from simple molecules, such as genistein (26), to very complex molecules such as toxicarol isoflavone (27). There are many classes of isoflavones: the derivatives of genistein and of orobol (28), 6-hydroxylated isoflavones and isoprenoid isoflavones. Two isoflavone glycosides, wistin (29) and ononin (30), were isolated as major constituents of Glycyrrhiza (the liquorice genus) species and found to be good chemotaxonomic markers. Interesting compounds related to isoflavonoids are rotenones, pterocarpans, coumestans, 2-arylbenzofurans and coumaronochromones.

5.2.4. Other Plant Families Flavonoids have been used for the chemotaxonomic characterization of many families. In some cases, as for the Anarthriaceae, results were perfectly in line with the phylogenetic assumptions gathered by biomolecular analyses. In the Cuscutaceae, some species accumulate derivatives of cinnamic acid, others accumulate flavonoids and others still have a cinnamates/flavonoids ratio of 1:1. More than thirty structures belonging to flavanones and chalcones are in the resins produced by the Xanthorrhoea genus (Xanthorroeaceae) and have been used to solve taxonomic problems.

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Significant taxonomic markers of the Asphodelaceae family are the root anthraquinones. Due to the presence of peculiar anthraquinones such as aloesaponarin II (31), the genus Lomatophyllum was included in the genus Aloe. Over 90 flavonoid constituents have been discovered and characterized for their chemotaxonomic significance, including 38 new compounds from 15 species of the genus Iris (Iridaceae). The pattern of phenolic compounds has been successfully used to solve controversial classifications in several plant genera, including Drosera, Anthurium, Aletris, Crataegus and Bagassa. Wood and bark from many species of both hardwood and softwood trees contain many types of flavonoid compounds. Most chemotaxonomic studies have been conducted on flavonoids in the extracts from softwoods such as Podocarpus, Pinus, Pseudotsuga, Larix, Taxus, Libocedrus, Tsuja, Taxodium, Sequoia, Cedrus, Tsuga, Abies and Picea, as recently reviewed.

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5.3. Chemotaxonomy of Terpenoids The terpenoids make up another important class of specialized metabolites which has been widely used for chemotaxonomic studies. The number of repetitions of a five-carbon-atom isoprenoid building block defines several chemical groups within this class (see Chapter 7). For this reason, terpenoids are also known as isoprenoids. Instead of analysing the chemotaxonomic significance of terpenoids in the different plant families, we will dissect these compounds into their different classes.

5.3.1 Monoterpenes Monoterpenes are, along with sesquiterpenes (see below), the main constituents of essential oils extracted by steam distillation from many plant families, but especially in the Lamiaceae, Rutaceae, Apiaceae, Asteraceae and in several gymnosperms. The power of monoterpenes in determining and, sometimes, correcting the taxonomic status of plants is proven by several scientific reports. For instance, the family Pittosporaceae was classified in the Rosanae super-order, but a large number of species belonging to this family encompasses volatile compounds stored in resin ducts and lysigenous pockets (see also Chapter 2). However, this does not occur in other families ascribed to Rosanae, while it is common in those belonging to the superorder Aralianae, which includes the Apiaceae and the Araliaceae families. In gymnosperms, the most well studied genera for chemotaxonomic purposes are Pinus and Abies (Pinaceae), Juniperus, Thuja and Cupressus (Cupressaceae), and Taxus (Taxaceae). The most representative chemotaxonomic monoterpene markers are Į-pinene (32), ȕ-pinene (33), bornyl acetate (34), sabinene (35), Į- thujone (36), ȕ-thujone (37), fenchone (38) and p-cymene (39). In angiosperms, Lamiaceae is one of the most investigated families. This family, which includes more than 3500 species, can be easily divided into two categories: species that produce and store volatile compounds (Nepetoideae) and species where these compounds are less abundant (Lamioideae). However, the major distinction between the two families resides in the presence of iridoids, glycosylated monoterpenes like 5,9-epipenstemoside (40), which are generally absent in the Nepetoideae. Among the Nepetoideae the most studied genera for the chemotaxonomic significance of their essential oils are Lavandula, Ocimum, Origanum, Rosmarinus, Salvia, Teucrium and Thymus. In the genus Mentha, chemical

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varieties were identified in virtually every species studied. The chemotaxonomic survey based on monoterpenes has proven to be useful in understanding the intraspecific variation patterns, in defining some species and in establishing the degree of hybridization of some natural populations. The chemical distribution of the essential oil and the chemotaxonomic significance of these compounds have also been analysed in the genera Wiedemannia, Nepeta, Stachys and Micromeria. In terms of chemotaxonomic significance, the most representative monoterpenes in the Lamiaceae family are isopinocamphone (41), 1,8cineole (42), borneol (43), camphor (44), linalool (45), linalyl acetate (46), methyl chavicol (47), eugenol (48), methyl eugenol (49), menthol (50), pulegone (51), thymol (52), p-cymene (39), carvacrol (53), Ȗ-terpinene (54), Į-terpineol (55), isoiridomyrmecin (56) and menthofuran (57). In the Asteraceae family, monoterpenes are useful for species discrimination. In the genus Achillea the presence of oxygenated monoterpenes such as camphor (44), 1,8-cineole (42), artemisia alcohol (58), santolina alcohol (59) and terpinen-4-ol (60) is used for discerning different species. In the same genus, ȕ-pinene (33), camphor (44) and 1,8cineole (42) have proved to separate species according to their geographical origin. Monoterpenes are taxonomic markers in several other Asteraceae, such as in the genus Chiliadenus, in which camphor (44) is an excellent chemotaxonomic marker, or in the genus Artemisia, where the presence of ȕ-thujone (37) has allowed the discrimination of spontaneous populations, hardly distinguishable by morphological data. Moreover, monoterpenes are also chemotaxonomic markers in the genus Chrysanthemum, with camphor (44), chrysanthenone (61), safranal (62) and myrcene (63); in Santolina, that contains borneol (43), santolina triene (64) and the irregular monoterpenes iso-lyratol (65) and lyratyl butyrate (66); in Tagetes, containing (Z)-ȕ-ocimene (67); and in Eriocephalus, with high contents of camphor (44) and Ȗ-terpinene (54). Although monoterpenes are not found in Leguminosae leaves, they are quite frequent in flowers through the action of scent glands, or osmophores, which are predominantly floral secretory structures that secrete volatile substances during anthesis (see also Chapter 2). A larger quantity of monoterpenes is located in Acacia flowers, characterized by the presence of (Z)-ȕ-ocimene (67), while a phenolic monoterpene, bakuchiol (68), is exclusively present in Psoralea corylifolia. Linalool (45) and its derivative cis-linalool pyran oxide (69) are characteristic floral scents of Ceratonia siliqua and Cyathostegia mathewsii, whereas the chemical profile obtained from the floral scent of some Parkia species

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shows the presence of (Z)-ȕ-ocimene (67). This last compound is also typical of Browneopsis disepala’s floral scent, along with Į-pinene (32) and ȕ-pinene (33). Iridoids are a group of monoterpenes which show a particular importance for chemotaxonomic studies. These compounds are typical of major families including the Valerianaceae, Dipsacaceae, Rubiaceae, Gentianaceae, Apocynaceae, Oleaceae, Lamiaceae and Ericaceae. However, they are only found in the Rutanae, Rosanae, Cornanae, Loasanae, Gentiananae, Lamianae and Ericanae super-orders, indicating that the ability to synthesize these compounds occurred a few times in the plants phylogeny.

5.3.2. Sesquiterpenes As are monoterpenes, sesquiterpenes too are components of essential oils and for this reason have been widely used as chemotaxonomic markers.

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Several of the Lamiaceae store sesquiterpenes in their secretory tissues. Germacrene D (70), (E)-ȕ-caryophyllene (71), į-cadinene (72), (E)-ȕfarnesene (73), spathulenol (74) and Ȗ-muurolene (75) are the major constituents in the genus Stachys, whereas Pogostemon cablin (patchouli) essential oil is unique because it consists of over 24 different sesquiterpenes, including patchouli alcohol (76). The sesquiterpene 14hydroxy-Į-humulene (77) is the main constituent of the essential oil of Salvia argentea, whereas in the genus Teucrium, (E)-ȕ-caryophyllene (71), germacrene D (70) and caryophyllene oxide (78) are the major chemotaxonomic markers. Į-Humulene (79) is characteristic of the genus Hyptis, whereas E-E-Į-farnesene (80) and oplopanone (81) are frequently present in the genus Origanum. Į-Bisabolol oxide B (82) is a chemotaxonomic marker of Anisomeles indica, whereas aromadendrene (83), viridiflorene (84), ȕ-selinene (85) and valencene (86) are typical of Lepechinia paniculata, and bicyclogermacrene (87) is a chemotaxonomic marker of the genus Hypenia. The genus Salvia is characterized by the presence of sesquiterpene lactones (see Chapter 7), which represent powerful chemotaxonomic markers. Germacrane sesquiterpenoids, with an unusual ǻ3-15,6-lactone moiety, are typical of Salvia scapiformis, whereas eudesmane-type sesquiterpenes, such as plebeiolide A (88) and plebeiafuran (89), are characteristic of Salvia plebeia. In the Leguminosae, 4-Į-copaenol (90), kaurene (91), cyclosativene (92) and cis-Į-bergamotene (93) are characteristic compounds of the genus Copaifera. Five typical sesquiterpenes are present in the heartwood of Dalbergia odorifrea, whereas in Pterodon pubescens the sesquiterpene oplopanone (81), which bears a modified cadinane skeleton, is being reported for the first time in this genus. (E)-Nerolidol (94) is present in the flowering shoots of Lupinus varius, whereas the guaiane sesquiterpene, (1ȕ,6Į,10Į)-guai-4(15)-ene-6,7,10-triol (95), and the lignan (+)lariciresinol 9ƍ-stearate (96) are typical of the aerial parts of Tephrosia vogelii. Finally, (E)-ȕ-farnesene (73) is present in the floral scent of Cyathostegia mathewsii. Sesquiterpene lactones are a particular feature of the Asteraceae, where they may have concentrations of up to 2% of the dry weight of the plant. One of the advantages in using sesquiterpene lactones as chemotaxonomic markers instead of monoterpenes and other sesquiterpenes is their lower volatility, which allows a more efficient extraction. These compounds are

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particularly useful at low levels of the taxonomic hierarchy, as shown in the Ambrosia psilostachya. In the Asteraceae, more than 4000 structures of sesquiterpene lactones have been isolated from leaves and accumulated in the glandular trichomes. In the Heliantheae (Asteraceae) tribe, sesquiterpene lactones were used for the taxonomic discrimination of subtribes. The Coreopsidinae and the Milleriinae have only germacranolides, which in contrast are absent in the Ecliptinae and the Engelmaniinae. Melampolides and ambrosanolides are missing in the Helianthinae, Neurolaeninae, Verbesinae and Ziniinae. In the Astereae tribe, and especially in the subtribe Asterinae, guaianolides are widespread. In particular, in the Gonosperminae (Anthemideae, Asteraceae) the three genera Gonospermum, Lugoa and Inulanthera are characterized by the presence of several eudesmane sesquiterpene lactones, whereas the germacranolide taraxinic acid ȕ-glucopyranosyl ester (97), ainslioside (98), and crepidiaside B (99) are common constituents of Taraxacum species. The guaianolide leucodin (100) and the eudesmanolide tanacetin (101) are chemotaxonomic markers of Cichorium spinosum, whereas several guaianolides and germacranolides characterize the genus Achillea. In Viguiera radula (Heliantheae), the co-occurrence of germacranolides and heliangolides, stored in glandular trichomes, shows the same general sesquiterpene lactone pattern as in many other members of the section Paradosa. Several other plant families contain sesquiterpene lactones. For instance, costunolide (102), parthenolide (103) and lipiferolide (104) were isolated from the leaves of several Magnolia species, which belong to the Magnoliaceae family, whereas the seco-prezizaane-type sesquiterpene 3,4dehydroneomajucin (105) is present in the fruits of Illicium jiadifengpi (Illiciaceae family).

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5.3.3. Diterpenes Several diterpenes have been used for chemotaxonomic purposes using acyclic diterpenoids and the bi-, tri- and tetracyclic diterpenoids, including labdanes, halimanes, clerodanes, abietanes, pimaranes, kauranes, cembranes, tiglianes, salviatrienes and many other compounds. In the Lamiaceae, the genus Salvia is one the most studied. About 800 compounds have been isolated from 130 Salvia species and more than 500 are diterpenes. Salvia species are a rich source of tanshinones and royleanones, as well as their analogues. For example, the roots of Salvia hypoleuca contain the diterpenes manool (106) and 7Į-acetoxyroyleanone (107), Salvia gilliessi is characterized by the presence of icetexone (108) and 12-hydroxy-11,14-diketo-6,8,12-abietatrien-19,20-olide (109), Salvia divinorum contains salvinorin A (110), Salvia cuspidata contains the

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abietane 12-hydroxy-11,14-diketo-6,8,12-abietatrien-19,20-olide (111), Salvia pomifera shows the presence of the diterpene carnosol (112) and salviol (113), whereas the reddish root and rhizome of Salvia miltiorrhiza consist of abietane quinone diterpenoids including tanshinone I (114) and cryptotanshinone (115). In the genus Hyptis (Lamiaceae) diterpenoids are extracted from a limited number of species and the main compounds are hyptol (116) and 15-ȕmethoxyfasciculatin (117). The neo-clerodane diterpene teufruintin A (118) is characteristic of Teucrium fruticans, whereas the labdane-type diterpenoid 5-ethoxy-3-(2-((R)-4-hydroxy-2,5,5,8a-tetramethy1-3-oxo3,5,6,7,8,8a-hexahydronaphthalen-1-y1)ethyl)furan-2(5H)-one (119) is typical of Leonurus japonicus. Several diterpenes have been used as chemotaxonomic markers in the genus Sideritis, including 2-hydroxyisophytol (120), whereas the labdane diterpenoid leoleorin (121) is typical of Leonotis leonurus. Most of the diterpenes found in the Leguminosae have been isolated from the Cesalpinioideae subfamily. The majority of them show a labdanic skeleton. As for mono and sesquiterpenes, quantitative changes in diterpenoids may have considerable chemotaxonomic significance, especially at genus and species levels. In the Detarieae (Cesalpinioideae) the distinguishing characteristic is the presence of bicyclic labdanes, while in the Caesalpinieae the major compounds are the alcaloidic diterpenes. For instance, Caesalpinia sappan, which is distributed in Southeast Asia, contains the diterpene phanginin (122), C. echinata produces cassane-type diterpenoids designated as echinalide H (123), C. furfuracea twigs contain the isopimarane diterpene caesalfurfuric acid A (124), whereas C. pulcherrima is characterized by the cassane-type diterpene isovouacapenol E (125).

5.3.4. Triterpenes Triterpenes include several classes of compounds, many of which are powerful deterrents towards herbivores. Phytosterols, pentacyclic triterpenes, saponins and sapogenins, cardiac glycosides and limonoids (see also Chapter 7) are among the most represented categories. Some classes of triterpenoids (such as the sitosterols) are widespread while others (e.g., pentacyclic triterpenes) show taxonomic value only at higher hierarchical levels.

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In the Lamiaceae, oleanolic acids and ursolic acids are common constituents. Derivatives of ursolic acid are characteristics of the Nepetoideae subfamily, principally in the genera Teucrium, Lavandula and Rosmarinus, while in Ajuga spp. steroidal compounds such as ecdysone (126) and ajugasterone (127) are found. Species with high oleanolic acid levels belong to the genera Salvia and Nepeta, whereas low oleanolic acid contents are found in Marrubium alysson and M. thessalum. In general, the subfamily Lamioideae appears to be poorer in both oleanolic and ursolic acids than the subfamily Nepetoideae. The Macaronesian Sideritis normally contains pentacyclic triterpenes including rhoiptelenol (128), Įamyrin (129) and obtusifoliol (130). In the Asteraceae, the roots of the genus Nannoglottis contain cycloartanetype, ursane-type, oleanane-type and A-frideooleanane-type triterpenes. Triterpenes from Saussurea spp. comprise ca. 26 compounds of four types – lanostane, oleanane, ursane and lupane – whereas in Tanacetum vulgare monohydroxy triterpene alcohols and sterols are present. Steroidal sapogenins with atypical structures such as pogosterol (131) are typical of some species of the Vernonia genus, whereas triterpenoid sapogenin lactones are typical of the Grindelia species. In the Leguminosae, most saponins are of the triterpene type, with steroidal saponins being rare. Cardenolides are restricted to a few (but not all) members of the genera Coronilla and Securigera. Triterpenes are clearly the main chemotaxonomic markers for most of the Ericales. The triterpene skeletal types, including oleanane, ursane and lupane, are present in the Marcgraviaceae, Lecythidaceae, Sapotaceae and Theaceae. One of the most important classes of metabolites represented in the Ericales is that of saponins, especially of the triterpene type, and their occurrence is very common in many families of this order. Flower buds of some Camellia sinensis varieties produce typical oleananetype triterpene oligoglycosides, e.g., floraassamsaponin I (132), whereas in the Rubiaceae family the leaf pentacyclic triterpenes ursolic acids and the stem bark acyl lupeols have been reported from many species. The chemosystematic significance of triterpenoid accumulation in the Meliaceae shows a wide distribution of seco-dammarane derivatives in the Aglaia species. In addition, various biogenetic trends towards cycloartane, tirucallane, apotirucallane and lupane basic skeletons have been described for the genus.

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Triterpenes are also present in the epicuticular layers, mixed with waxes and alkanes. In the Palmae, there are several triterpenes of lupanic skeleton, which are characteristics of the Butia and Orbignya genera, whereas triterpenes were detected in the waxes of Sedum species, the major triterpene being ȕ-amyrenyl acetate (133).

5.3.5. Tetraterpenes The most common compounds in this class of terpenoids are carotenoids. In general, orange-red flower colour is determined by carotenoids; however, since they have a presence in virtually all plant parts they are not usually involved in chemotaxonomic investigations. Nevertheless, their distribution in flower petals is not universal and thus can be used to solve some taxonomic problems. Excellent results were obtained by analyzing the carotenoids of Rosaceae and Asteraceae petals. The compounds used for these studies are Į- and ȕ-carotene, lutein, flavoxantine and violaxanthin. In the Leguminosae, species belonging to the Acacia genus have only ȕ-carotene (134) and lutein (135), while in the morphologically similar genus Ulex, taraxanthin (136) is also found. Genera such as Genista, Cytisus and Laburnum are difficult to be discerned merely on a morphological basis, but possess clear-cut patterns of variation in carotenoids which can be helpful for taxonomic investigations of the Genisteae. Finally, studies on Astragalus designate the rubixanthin (137) as an outstanding chemotaxonomic marker, because it is only found in this genus.

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5.4. Chemotaxonomy of Secondary Products Containing Nitrogen Numerous specialized metabolites contain in their carbon skeletons one or more nitrogen atoms. Among the most important classes are alkaloids, glucosinolates, cyanogenic glycosides and several non-protein amino acids (see Chapter 9). Alkaloids are nitrogen heterocyclic compounds with a powerful pharmacological action, whereas betalains are red or yellow alkaloids only found in the Caryophyllales and absent in plants producing anthocyanins. Glucosinolates contain a sulfur atom in addition to a nitrogen atom. These compounds are only produced by the Capparales. The cyanogenic glycosides elicit a toxic compound (HCN) and are found in several food plants such as cassava. The non-protein amino acids are widespread in the plant kingdom and play an ecologically crucial role in chemical defence.

5.4.1. Alkaloids The taxonomic criterion applied to the study of the alkaloids is based on the origin of the amino acid precursor. The first category of alkaloids to be considered are the derivatives of tyrosine (and phenylalanine), formed by the decarboxylation of dihydroxyphenylalanine. This group includes 1benzylisoquinoline alkaloids, alkaloids of the Amaryllidaceae, alkaloids of the Erythrina spp. and betalains. In the Rutaceae, benzylisoquinoline alkaloids have been used at the genus level to outline a group of species within the family. Unfortunately, other attempts to use these compounds in chemotaxonomic analysis as distinctive traits of related groups of plant families have failed. For instance, Rutaceae, Papaveraceae and Berberidineae are related families containing benzylisoquinoline alkaloids, but these compounds are also found in the Euphorbiaceae, Buxaceae and Rhamnaceae, which are phylogenetically separated from the previous families. However, some classes of benzylisoquinoline alkaloids are specific to certain plant families, such as the dibenzazonine alkaloids of the Menispermaceae and Leguminosae or the spiroisoquinoline alkaloids of the Fumariaceae and Papaverinae. In the Amaryllidaceae, a family of monocot angiosperms, there are alkaloids that share the same precursors of benzylisoquinolinie alkaloids. Groups of plants producing these alkaloids, e.g. norpluvine (138), separate from those that produce benzylisoquinoline alkaloids, thus confirming the drift of monocotyledons from ancestors similar to

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Polycarpicae. Recently, several studies have dealt with alkaloids extracted from the Leguminosae (Erythrina), thus broadening the knowledge about the quali-quantitative variations in species evolved from geographically distant areas. Erythroidine alkaloids, such as Į-erythroidine (139), are present in the stem bark of Erythrina poeppigiana, whereas the dimeric Erythrina alkaloid spirocyclic (6/5/6/6) erythrivarine A (140) has been isolated from Erythrina variegata. As already mentioned, the betalains are typical of the Centrospermae (Caryophyllales) and are commonly found in the Aizoaceae, Amaranthaceae, Basellaceae, Cactaceae, Chemopodiaceae, Didiereaceae, Nyctaginaceae, Phytolaccaceae and Portulacaceae, but are absent in the Caryophyllaceae and Molluginaceae. The chemotaxonomic value of betalains proved to be particularly useful in the classification of some Caryophyllales species of uncertain taxonomic position. However, there are still concerns in separating the Caryophyllales into two groups according to the presence/absence of betalains. The second alkaloids category consists of indole-seco-loganine derivatives. Secologanin (141) is an iridoid monoterpene able to form alkaloids. The indole-secologanin alkaloids are found in the Loganiaceae, Apocynaceae and Rubiaceae families and in some taxa of the Cornales order. Three types of basic structures characterize these alkaloids: I, II and III, whose most important representatives are ajmalicine (142), vincadifformine (143) and eglandine (144) respectively. Type III alkaloids are only found in the Apocynaceae, making these molecules useful for chemotaxonomic classifications at the family or superior level. In many cases, the ability to accumulate these alkaloids depends on the biosynthetic pathway for the formation of seco- and iridoids precursors. The Asclepiadaceae are unable to synthetize iridoids and indole-loganine alkaloids; instead they produce steroidal cardenolides, a class of compounds detectable only in a few genera belonging to the Apocinaceae. Derivatives of anthranilic acid make the third alkaloids category. These alkaloids are limited to a large family, the Rutaceae, where furoquinolines are broadly represented. Unfortunately, this latter class of compounds also occurs occasionally in other families such as the Solanaceae, Asclepiadaceae and Apocinaceae, while acridones are typical of the Rutaceae only. Alkaloids deriving from the amino acids lysine and ornithine make the fourth class of compounds. Tropane alkaloids are typical of the

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Solanaceae, Erythroxylaceae, Proteaceae, Euphorbiaceae, Rhizophoraceae, Convolvulaceae and Cruciferae, but are also found, though rarely, in other plant families. Tropane alkaloids are characteristic of the genera Datura, Brugmansia (tree datura) and Duboisia of the Solanaceae. Chemotaxonomic studies have demonstrated that the tropanic nucleus has evolved several times during the angiosperms phylogeny. However, in distinct plant families these alkaloids differ for substitutions and stereochemical arrangements on the structure of the tropanic core. Nevertheless, the use of these molecules is primarily at the lower levels of the taxonomic hierarchy. The pyrrolizidine alkaloids also belong to the fourth category. These are a group of molecules formed by approximately 360 structures with a distribution restricted to certain taxa. On the basis of taxonomical and biogenetic implications, we can distinguish five main classes of pyrrolizidine alkaloids: type I (or senecionine type) is typical of the tribe Senecioneae (Asteraceae) and is formed of a group of more than 100 compounds in which a necine base and necic acid form macrocyclic diesters; type II (or triangularine type) consists of more than 50 different structures, is typical of the Asteraceae and Boraginaceae families and is represented by open-chain diesters; type III (structurally similar to type I, monocrotaline type) comprises approximately 30 structures and is characteristic of the Fabaceae; type IV (or lycopsamine type) is made up of monoesters scattered in more than 100 molecules of the Eupatorieae (Asteraceae) tribe; finally, type V (or phalaenopsine type) is typical of the Orchidaceae. According to many authors, the chemotaxonomic significance of quinolizidine alkaloids is high because they are primarily found in the Sophoreae, Padalyrieae (sensu lato) and Genisteae (Papilionoideae, Leguminosae) tribes. In addition, chemotaxonomic studies allow the separation of basal shrub species that accumulate alkaloids, such as racemic (+/-) sparteine (145) and/or matrine (146), from more recent species that produce (-)-sparteine. The tribe Sophoreae contains the most diverse forms of quinolizidine alkaloids, and the variation in this compound production has also been used to separate species of the genus Lupinus. Other families able to produce these alkaloids are the Berberidaceae, Ranunculaceae, Solanaceae, Chenopodiaceae and Rubiaceae. For instance, the alkaloid pattern of Leontice leontopetalum (Berberidaceae) is characterized by quinolizidine alkaloids of the lupanine-type with lupanine (147) as the main compound, whereas Leontice ewersmannii accumulates quinolizidine alkaloids of the matrine-

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type and Į-pyridone-type as the major compounds. A chemical dichotomy was demonstrated for the species of the section Spartioides of the genus Genista (Fabaceae: Genisteae): one group of species contained the Įpyridone alkaloids cytisine (148), N-methylcytisine and anagyrine (149), while the other group contained lupanine (147), 13-hydroxylupanine (150) and its esters as major alkaloids. Finally, the quinolizidine alkaloids retamine (151), 17-oxoretamine (152) and 12-Į-hydroxylupanine (153) were detected in the aerial parts of Genista ephedroides, whereas jussiaeiine A (154) is recognized as a marker of the genus Ulex.

5.4.2. Glucosinolates As we have already mentioned, this category of nitrogen compounds is restricted to the Capparales order and shows a fully dissimilar structure in the Brassicaceae family. These compounds are extremely useful at the low levels of the classification hierarchy. In the genus Aurinia (Brassicaceae), glucoalyssin (155), glucobrassicanapin (156) and glucoberteroin (157) are the major compounds, whereas gluconasturtiin (158) and glucobrassicin (159) are typical of Barbarea (Brassicaceae) species. Gluconapin (160) and progoitrin (161) are characteristic of the seeds of oilseed turnip (Brassica rapa var oleifera) collected from several different geographic regions, whereas glucotropaeolin (162) is present in some Lepidium (Brassicaceae) species.

5.4.3. Cyanogenic Glycosides Cyanogenesis (i.e., the production of HCN from damaged plant tissue) requires the presence of two biochemical pathways, one that controls the synthesis of the cyanogen glycoside and the other that controls the production of a specific, hydrolyzing ȕ-glucosidase (see Chapter 9). The cyanogenic glycoside of Eucalyptus nobilis is prunasin (163) (Dmandelonitrile ȕ-D-glucoside), whereas a cyanogenic glucoside, 6'-Ogalloyl sambunigrin (164), is present in the tropical tree Elaeocarpus sericopetalus (Elaeocarpaceae). This was the first formal characterization of a cyanogenic constituent in the Elaeocarpaceae family and the second for the Malvales order. Moreover, E. sericopetalus contains the highest foliar concentrations of cyanogenic glycosides known for leaves of any tree. The cyanogenic glycosides lucumin (165) and prunasin (163) have been reported for the first time in the Lamiaceae family, from foliage of the rare Australian endemic rainforest tree Clerodendrum grayi, whereas passibiflorin (166), a bisglycoside containing the 6-deoxy-ȕ-D-

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gulopyranosyl residue, (Passifloraceae) species.

was

isolated

from

several

Passiflora

5.4.4 Non-protein Amino Acids While twenty amino acids are involved directly in protein structure, there are thousands of other non-protein amino acids (NPAAs) that do not play such a role. However, non-protein amino acids play various roles in plant ecology and survival strategies and are powerful chemotaxonomic markers. Some NPAAs are only found in a few plant families, while others are widespread among living organisms. For example, Ȗ-methyleneglutamic acid (167) is present in the Leguminosae, Liliaceae and Cannabinaceae while canavanine (168) has a very restricted distribution in the Lotoideae (Leguminosae) subfamily. For instance, lathyrine (169) is only present in the genus Lathyrus. Among NPAAs, the pipecolic acid hydroxylated derivatives are exclusively found in the Mimosoideae and are restricted to a few genera, with particular reference to the Inga genus. In the Leguminosae, there is a perfect match between the classification based on morphological cues and those using NPAAs as chemotaxonomic markers. This means that the mutations that occurred in various biosynthetic pathways for the production of amino acids contributed to the evolution of the Leguminosae just like those mutations that have changed the morphology. In the seeds of several Australian Acacia species, the major NPAAs are djenkolic acid (170), mimosine (171) and lanthionine (172), whereas azetidine-2-carboxylic acid (173) is a toxic and teratogenic NPAA found in the roots of sugar beets and garden beets (Beta vulgaris). The relationship between several Bocoa species with Ateleia and Cyathostegia species is supported by the presence of the rare Ateleia-type NPAA 2,4methanoproline (174), whereas O-oxalylhomoserine (175) has been isolated from the aerial parts of Lathyrus latifolius.

5.5. Chemotaxonomic Significance of Fatty Acids The efficacy of seed fatty acids for taxonomical surveys has been proved at the genus and species level; however, these compounds lose their chemotaxonomic significance at the family or higher hierarchical level. The most abundant fatty acids are those with 16 and 18 carbon atoms, i.e.,

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palmitic acid (and its unsaturated derivatives, palmitoleic and palmitolenic acids) and stearic acid (and its unsaturated derivatives oleic, linoleic and linolenic acids). In the Leguminosae, the linoleic acid (176) percentage varies between 40 and 67%, while fatty acids with chains of less than 14 carbon atoms are rare; in species belonging to the Taxaceae, Pinaceae, Taxodiaceae and Cupressaceae, Į-linolenic acid has been used as a chemotaxonomic marker to distinguish between groups of families. Moreover, in conifer seeds, six '5-olefinic acids may occur in variable proportions depending on the family. These are taxoleic (177), pinolenic (178) and sciadonic (179) acids (all double bonds in cis configuration). In the Pinaceae and Cupressaceae, the seed fatty acids are rich in ǻ5-olefinic acids, including 20-carbon-atoms acids with two, three and four unsaturations. Chemotaxonomic grouping of the Pinatae families (Pinaceae, Taxodiaceae, Taxaceae and Cupressaceae) was based on data obtained from these molecules, using multivariate statistical analysis. With this procedure, it was possible to further discriminate many Pinaceae genera (Pinus, Abies, Cedrus, Piceae and Larix). The study of “unusual” fatty acids becomes a powerful tool for chemotaxonomic investigations, as demonstrated in the Ranunculaceae, where some ǻ6 fatty acids were only found in one genus or in a few closely related genera. Free fatty acids and fatty acids esterified with mono-, di- and triglycerides are present in the non-polar fraction of some Lamiaceae leaves. In the genus Mentha, fatty acids show several degrees of interspecific variability depending on the class of lipids analysed (mono-, di- or triglycerides) and the organ from which they are extracted (leaves, stems, flowers). Hence these compounds proved to be very useful in chemotaxonomic studies at a lower level of hierarchical classification. An analysis carried out on Lavandula hybrids highlighted the existence of distinct chemotypes within a large population of wild plants scattered on alpine altitudinal gradients. In contrast, at higher hierarchical levels leaf and flower fatty acids show no taxonomic significance.

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5.6. Chemotaxonomic Significance of Surface Alkanes In the early sixties, Eglinton published a series of works on the taxonomic significance of alkanes deposited as epicuticular layers on plant surfaces. The power of these substances is mainly due to their presence not only in plants, but also in the bacteria, fungi and animal kingdoms. Recently a survey of more than 550 species belonging to several plant families supported the hypothesis of Eglinton at the family, subfamily and tribe levels. Epicuticular alkanes have a lower discriminating power at the species level, because they vary considerably depending on the plant developmental stage as well as on environmental factors. A study on nine major families of angiosperms reported that, in general, the chemical composition of epicuticular alkanes consists of linear and branched chain molecules consisting of 21–36 carbon atoms. In the Leguminosae and Apiaceae, 21–25 carbon atom alkanes are the prevailing compounds, whereas n-alkanes and iso-alkanes with a higher number of carbon atoms (34–36) are characteristic of the Gramineae. The Boraginaceae and Solanaceae mostly produce alkanes with 29 carbon atoms, the Lamiaceae and Verbenaceae are characterized by alkanes of both 31 and 33 carbon atoms, whereas in the Compositae and Scrophulariaceae alkanes with 31 carbon atoms prevail.

5.7. Correlation between Micromolecular and Macromolecular Data As we have discussed, the identification of plants that contain bioactive molecules depends on morphological, anatomical and chemical analytical factors. However, such factors are influenced by the surrounding environment and/or depend on the degree of plant growth, or by the sample extraction method. This means that, according to environmental conditions, the same genotype can express different chemical patterns or, conversely, several genotypes can respond to the same environmental pressure with the same phenotypic expression (see also Chapter 1 for a discussion on phenotypic plasticity). Chemotypes (or chemical phenotypes) are generally considered to be the phenotypic expressions of genotypes, although different chemotypes can derive from the same genotype. Very often, the identification of plant specimens in a mixture is difficult to achieve and this problem is particularly evident when plant blends are in

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powder form. DNA analysis of these powders is relatively fast and the presence of more stable molecules makes quantification easier, provided that specific primers are used for target genes. In this context, molecular genetic methods have proven to be very effective in genotypic discrimination. Molecular genetic methods have several advantages over classical morphological and chemical analyses. For instance, the genetic method requires genotype instead of phenotype, and therefore DNA-based experiments have become widely employed techniques for the rapid identification of herbal medicines. Using PCR approaches, nanogram quantities of DNA are required to amplify and yield sufficient amounts of template DNA for molecular genetic analysis. Recently, the phylogenetic relationships of some higher plant species have been evaluated using sequences of a 5S-rRNA gene spacer region. The 5SrRNA region is a component of all ribosomes, except in the mitochondria of certain species. In all higher eukaryotes, 5S-rRNA is transcribed from hundreds to thousands of genes. Genes encoding 5S-rRNA are located separately from the 18S-26S rRNA gene clusters and organized into tandem repeats, with alternative arrays of sequences coding 5S-rRNA and non-transcribed spacers (NTSs) in one or more sites in the genome. The two gene clusters can be localized either in a linked state, and therefore on the same chromosome, or independently on different chromosomes in the genome.

5.7.1. Using the 5S-Rrna Gene for the DNA Fingerprinting of Plants Producing Bioactive Molecules Relevant results obtained with RFLP analysis of nuclear DNA have been obtained from several medicinal and aromatic plants using the 5S-rRNANTS gene. Successful application of NTS comparison has been obtained at both the interspecific and the intraspecific level. Below are some examples from gymnosperms and angiosperms. 5.7.1.1. Molecular and Chemical Correlation in the Gymnosperms In contrast to angiosperms, the structure and organization of the 5S-rDNA locus has only been characterized in a few gymnosperm species. In Douglas fir (Pseudotsuga spp.), plants known to produce an essential oil characterized by monoterpenes such as bornyl acetate, camphene and Įpinene, sequencing and Southern hybridization of the 5S-rRNA gene

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revealed repeat units of 888 and 871 bp in length, the latter with a 17 bp deletion in the NTS. In the silver fir (Abies alba Mill.), one of the most important conifers in many eastern European mountain forests, which is characterized by the presence of an essential oil containing terpenoids such as limonene, ȕ-phellandrene (182), Į-pinene (32), ȕ-pinene (33) and camphene, PCR amplification of the gene and NTS region, sequence analysis and Southern hybridization using a homologous probe detected DNA sequences of approximately 550 and 700 bp. In Picea glauca, a plant producing an essential oil containing į-3-carene (180), sabinene (181), ȕ-pinene (33), borneol (43), linalool (45), ȕphellandrene (182), ȕ-caryophyllene (71) and camphor (44), and in Pseudotsuga menziesii, the 5S-rDNA repeats have a conserved 120 bp transcribed region and an NTS that varies not only in size (from 101 bp in P. glauca to 880 bp in P. menziesii) but also in the number of different size classes, whereas in Asian pines, the length of the NTS varies (382– 401 bp in Pinus bungeana and 538– 608 bp in four diploxylon pines). In the conifer Pinus radiata, a species with an essential oil particularly rich in Į-pinene (32) and ȕ-pinene (33), the 5S-rRNA gene (5S-DNA) has been cloned and characterized at the nucleotide, genomic and chromosomal levels. Sequencing revealed a repeat unit of 524 bp which is present in approximately 3000 copies per diploid genome. In the genus Larix, known to produce essential oils, divergent size classes of 5S-rDNA were identified in L. decidua and L. kaempferi using either selective amplification of gene and spacer, sequence analysis or homologous probe hybridization. Two highly divergent unit size classes of approximately 650 and 870 bp were detected in both species. 5.7.1.2. Molecular and Chemical Correlation in the Angiosperms Many more data are available from angiosperms. Australia is unique in having a single genus of tree, Eucalyptus (family Myrtaceae), dominating its forests and woodlands. Sequences of the 5S-rDNA repeat have been determined from two Angophora species and nineteen Eucalyptus species; the tandemly repeated 5S-rRNA genes were highly conserved, while the non-coding intergenic spacers were variable. A 50 bp repeating element, which has undergone duplication and modification in certain taxa, was identifiable within the spacer and accounted for much of the variability. Based on the modifications of the 50 bp element, it is apparent that the spacer from bloodwood (informal subgenera Blakella and Corymbia)

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species of Eucalyptus was more similar to that of Angophora than to nonbloodwood species of Eucalyptus. An important medicinal and essential oil plant belonging to the family Solanaceae is Capsicum. In some species of this genus, the compounds that are primarily responsible for the pungency are capsaicin (8-methyl-nvanillyl-6-nonenamide) (183) and a group of similar substances called capsaicinoids, which includes dihydrocapsaicin (184) and nordihydrocapsaicin (185). In sequence analysis, the repeating units of the 5S-rRNA genes in the Capsicum species were variable in size (278–300 bp). In sequence comparison with other members of the Solanaceae, the coding region was highly conserved but the spacer regions varied in size and sequence. The genus Brassica is known for its medicinal properties. Sinapine (Osinapoyl choline) (186), a choline ester of sinapic acid, is one of the major phenolic choline esters in oil-extracted rapeseed meal and a component of Semen Sinapis Albae (white mustard seed), a traditional Chinese medicine. The 5S-rRNA gene from Brassica campestris has been cloned, sequenced and characterized; the 5S-rDNA repeat unit is 495 bp in length and consists of a highly conserved 119 bp coding region and a variable noncoding spacer region, which separates it from the coding region of the next repeat unit. The phylogenetic relationship of Acorus gramineus and three types of A. calamus was analysed by comparing the 700 bp sequences of 5S-rRNA gene spacer regions. Although there was no intraspecific variation in the essential oil profile of A. gramineus, A. calamus was classified into two chemotypes: chemotype A, in which ȕ-asarone is a major essential oil constituent, and chemotype B, which contained mainly sesquiterpenoids. The 5S-rRNA spacer region of both diploid (ȕ-asarone-free) and triploid (ȕ-asarone-rich) A. calamus were amplified by PCR, using a pair of primers located at the 3ƍ and 5ƍ ends of the coding sequence of the 5SrRNA gene. The PCR products were digested using EcoRI and the restriction profile of the spacer domain was shown to be different for the two cytotypes. Along with chemical analysis of alcoholic extracts, sequence analysis coupled to restriction mapping was demonstrated to represent a powerful tool to distinguish the A. calamus diploid cytotype from the others (Figure 5.1, lanes 1 and 2). Salvia divinorum Epling & Játiva-M. is a perennial herb belonging to the family Lamiaceae and is most recognized for its hallucinogenic properties.

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The active ingredient of S. divinorum is the neoclerodane diterpene salvinorin A (187), a psychotropic molecule that produces hallucinations. For this reason, S. divinorum is a frequently used hallucinogen, with a potency in producing hallucinations similar to that of LSD. Molecular fingerprinting using the 5S-rRNA-NTS region allowed the rapid and precise identification of S. divinorum. By aligning the isolated nucleotide sequences, great diversities were found in the spacer regions of S. divinorum when compared to those of S. officinalis. Specific S. divinorum primers were designed on the sequence of the 5S-rRNA gene spacer region. In addition, a PCR–restriction fragment length polymorphism (PCR–RFLP) method was applied using NdeI and TaqI restriction enzymes. An NdeI site, absent in S. officinalis, was found in the S. divinorum NTS region at 428–433 bp. For TaqI, multiple sites (161–164, 170–173 and 217–220 bp) were found in S. officinalis, whereas a unique site was found in S. divinorum (235–238 bp) (Figure 5.1, lanes 3–6). Thus, even in this case, molecular analysis of the 5S-rRNA-NTS led to the precise and unequivocal identification of a species. S. divinorum is often sold, in legal or illegal markets, as a powder that can be easily adulterated by adding dried leaves of other species, thus making it hard to establish the purity of samples. Recently a new mathematical model for the quantitative analysis of S. divinorum in a biological mixture was developed by quantifying DNA by means of SYBR Green I fluorescence dye quantitative real-time PCR (qRT–PCR). This model is based on relative quantification of DNA extracted from a mixture vs a reference DNA extracted from a known amount of the pure species. The results of this work showed an almost perfect correspondence between qRT–PCR calculated weight and the weight estimated by an analytical weighted method, proving the effectiveness of this method for the quantitative analysis of a given species in a plant mixture. Į-Thujone (36) and ȕ-thujone (37) are natural terpenoids also associated with common wormwood (Artemisia absinthium L.) and Roman wormwood (Artemisia pontica L.), absinthe’s most widely used ingredients. There is currently a heated debate on the toxicity of absinthe and thujones, but European Union legislation has imposed a limit of 35 ppm on the total amount of these compounds in alcoholic beverages. To overcome this issue, thujone-free chemotypes of A. umbelliformis have been selected by horticultural techniques. Two chemotypes of A. umbelliformis (with and without thujone) used to prepare a local liqueur, genepi, were studied and specific A. umbelliformis primers were designed on the sequence of the 5S-rRNA gene spacer region. When a PCR–RFLP

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method was applied, using RsaI and TaqI restriction enzymes, the two chemotypes were clearly distinguished (Figure 5.1, lanes 7–10).

Figure 5.1 Some examples of DNA fingerprinting by the use of PCR– RFLP of the 5S-rRNA-NTS region. Acorus calamus: PCR–RFLP analysis using EcoRI; digested products from triploid (lane 1) and diploid (lane 2) 5S-rRNA gene spacer regions. Salvia divinorum and Salvia officinalis: PCR–RFLP analysis using TaqI; S. divinorum undigested PCR products (lane 3), S. officinalis undigested PCR products (lane 4), S. divinorum TaqI PCR-digested products (lane 5), S. officinalis TaqI-digested PCR products (lane 6). Artemisia umbelliformis: PCR product of the NTS spacer of chemotypes of A. umbelliformis containing thujone (Au1, lane 7) and without thujone (Au2, lane 8), Au1 RsaI PCR-digested products (lane 9), Au2 TaqI PCR-digested products (lane 10). L, bp ladder. From Gnavi et al., 2010.

Hybridization of Mentha species is very common, but identification of these hybrids may be difficult. Since the clarification of the relationships between species and hybrids is hard to achieve, over the past years many attempts have been made for classification purposes. New approaches have been proposed to analyse the genus Mentha, using biomolecular data as support for taxonomical identification. Recently, amplification of the NTS of the 5S-rRNA gene has been successfully used to characterize some Mentha species, revealing high specific variability. Cloning and sequencing of all amplified NTS fragments enabled the discrimination of almost all Mentha species studied. In silico and experimental analyses identified specific restriction sites on the amplified 5S-NTS regions, facilitating the rapid and unambiguous discrimination of all the different

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Mentha species by PCR-RFLP. Moreover, a direct comparison between essential oil composition and DNA fingerprinting confirmed a relationship between chemical and molecular data. By using the same molecular strategy that allowed the unequivocal identification of some Mentha species, the NTS of the 5S-rRNA gene was amplified to characterise the industrial crop peppermint, M. x piperita, and some important Mentha interspecific hybrids: M. x dalmatica, M. x dumetorum, M. x rotundifolia, M. x maximilianea, M. x smithiana, M. x verticillata, M. x villosa. DNA amplification, sequence and cluster analysis revealed differences in the 5SrRNA NTS region of Mentha hybrids. Peppermint and all other hybrids were unequivocally discriminated by RFLP analysis by using TaqI restriction enzyme, while a further discrimination between M. x dumetorum and M. x verticillata was obtained by XhoI restriction enzyme. Essential oil composition showed clustering patterns similar to DNA fingerprint, with a clear discrimination between plants producing menthofuran (57) (e.g., M. aquatica and its related hybrids, including peppermint) and those containing piperitenone oxide (188) (M. longifolia and its related hybrids) (Figure 5.2).

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Figure 5.2 Correlation between molecular data (DNA fingerprinting) and chemical data. (a) Cladistic analysis on the DNA sequences of the spacer region of the 5S-rRNA gene clearly separates M. cervina and M. gattefossei that are classified in the Eriodontes section by other species of Mentha. Nodes indicate the bootstrap values. (b) Schematic representation of sexual species of the genus Mentha and of some of the resulting hybrids. (c) Cluster analysis performed on DNA sequence data of the spacer region of the 5S-rRNA gene with root obtained using Salvia divinorum as an outgroup. The analysis clearly separates M. longifolia and M. aquatica and their relative hybrids. (d) Cluster analysis of the main components of the essential oil. A cluster gathers the parental species M. aquatica together with all the hybrids that produce mentofuran. All other hybrids are linked in the second cluster along with M. longifolia for their piperitone oxide content. From Capuzzo and Maffei, 2014 and 2015, modified.

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UNIT II BIOCHEMISTRY OF BIOACTIVE PLANT MOLECULES

CHAPTER SIX THE SHIKIMATE PATHWAY: AROMATIC AMINO ACIDS AND PHENOLIC COMPOUNDS

The purpose of this unit is to study the main biosynthetic pathways for the production of the most important plant bioactive molecules. Having discussed the distribution, function and chemotaxonomic significance of these substances in the plant kingdom we will now turn our attention to the biosynthetic mechanisms at the base of their production. This chapter will focus on phenolic compounds by describing the shikimate pathway.

6.1. The Biosynthesis of Simple Phenolics As a general distinction, we can divide the large family of phenolic compounds into simple (i.e., C6-C3 compounds) and complex (i.e., C6-C3C6 compounds) molecules. Although being widely covered in many biochemistry books, it is important to recall at least the main steps of the biosynthetic pathway for the production of the aromatic building block C6C3. The basic molecule for the production of the various phenolic structures is an aromatic acid, trans-cinnamic acid, that derives from the deamination of the amino acid phenylalanine by the action of phenylalanine ammonia lyase (PAL). A very similar reaction transforms tyrosine into p-hydroxycinnamic acid and is catalysed by tyrosine ammonia lyase (TAL).

6.1.1. The Shikimate Pathway and the Biosynthesis of Chorismate The shikimate pathway leads to the synthesis of the aromatic amino acid precursor chorismate. The reactions begin with the condensation of erythrose 4-phostate (1), from the pentose phosphate pathway, with

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phosphoenolpyruvate (PEP, 2), from glycolysis. This reaction is catalysed by 3-deoxy-D-arabino-eptulosonate 7-phosphate (DAHP, 3) synthase (SHKA, SHKB), which is activated by the presence of tryptophan and Mn2+.

The heterocyclic ring of DAHP is transformed into cyclohexane by 3dehydroquinic acid (4) synthase (SHKC), an enzyme that requires Co2+ and NAD+ as cofactors. This reaction occurs by elimination of the phosphoryl group. In the next step, the 3-dehydroquinate dehydratase (SHKD) removes a water molecule from 3-dehydrochinic acid to form 3dehydroshikimic acid (5), which is finally transformed into the intermediate shikimic acid (6) by shikimate:NADP oxidoreductase (shikimate dehydrogenase, SHKE), that uses NADPH2 as a cofactor.

Shikimic acid is phosphorylated at the 3-hydroxyl group to 3phosphoshikimic acid (7) by the chloroplastic enzyme shikimate kinase (SHKF). 3-Phosphoshikimic acid is then transformed into 5enolpyruvylshikimate 3-phosphate (EPSP, 8) by EPSP synthase (SHKG). This enzyme catalyses the reversible production of EPSP and phosphate by binding a PEP (2) molecule to 3-phosphoshikimic acid. This enzyme is the target of one of the most used commercial herbicides in agricultural practices: glyphosate (9) (N-[Phosphonomethyl]glycine). The use of this herbicide inhibits the synthesis of EPSP by competitive inhibition with respect to phosphoenolpyruvate. Glyphosate induces the plant’s inability to synthesize aromatic amino acids, eventually causing death. The pathway ends with the 1,4-trans cleavage of phosphate from EPSP catalysed by chorismate synthase (SHKH) that requires a FMNH2 to form chorismic acid (10).

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The term “corismic” refers to the metabolic bifurcation that leads to the synthesis of prephenic acid (and hence the amino acids phenylalanine and tyrosine) and anthranilic acid (the precursor of tryptophan).

6.1.2. Aromatic Amino Acid Biosynthesis Aromatic amino acids are important precursors of several bioactive molecules. In general, their biosynthesis takes place in the chloroplast. Phenylalanine and tyrosine share common precursors and their biosynthesis starts from the intramolecular rearrangement of chorismate to yield prephenic acid (11), a reaction catalysed by two isoforms of chorismate mutase (CM1, which is localized in the plastid, and CM2, which is cytosolic). Another common enzyme is prephenate aminotransferase, that utilizes glutamate (12) as an amino group donor to transfer NH2 to prephenate, thus generating arogenate (14). The reaction generates Į-ketoglutarate (13) from glutamate. The biosynthesis of phenylalanine (15) is catalysed by arogenate dehydratase, that catalyses the removal of a water molecule and the decarboxylation of arogenate. Tyrosine (16) biosynthesis is catalysed by arogenate dehydrogenase, that decarboxylates arogenate through an NADP+-dependent reaction.

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The aromatic amino acid tryptophan is important not only for the biosynthesis of proteins, but also for many other important plant metabolites, including the phytohormone auxin and several bioactive metabolites, such as indole alkaloids, acridone alkaloids and indole glucosinolates (see Chapter 9). The first step in the tryptophan biosynthesis is catalysed by anthranilate synthase (AnS), an enzyme consisting of two subunits (Į and ȕ). The two subunits act in concert: the Į-subunit removes the enolpyruvyl side chain and catalyses the amination of chorismate in concert with the aminotransferase activity of the ȕsubunit to form anthranilic acid (17). The next step is the transfer of a phosphoribosyl moiety (from 5-phosphoribosyl-1-pyrophosphate) to anthranilate catalysed by phosphoribosylanthranilate transferase (PAT) to yield 5-phosphoribosylanthranilic acid (18). Mutation of the gene coding for this enzyme generates dwarf plants, probably because of impaired auxin biosynthesis. The enzyme phosphoribosylanthranilate isomerase (PAI) catalyses the conversion of 5-phosphoribosylanthranilate to 1-(ocarboxyphenylamino)-1-deoxyribulose 5-phosphate (CDRP, 19). The next step in tryptophan biosynthesis is catalysed by indole-3-glycerolphosphate synthase (IGPS), that is responsible for the ring closure and decarboxylation of CDRP with the formation of indole-3-glycerol phosphate (20). This enzyme generates the indole (21) structure, which is important for the synthesis of many bioactive metabolites, including indole alkaloids. The final step in the tryptophan synthesis is catalysed by tryptophan synthase (TS), which is present in plants in two independent

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subunits (Į and ȕ) that may also function as a stable Įȕ heterodimer, by forming a hydrophobic tunnel between them. As observed in Salmonella thyphimurium, the Į-subunit catalyses the transformation of indole-3glycerol phosphate into indole and glyceraldehyde 3-phosphate (22), whereas the ȕ-subunit catalyses the binding of L-serine (23) to indole, thus generating tryptophan (24) and a water molecule.

6.1.3. Phenylpropanoid and Lignin Biosynthesis Plant phenolic compounds originate from the catabolic reactions of the shikimate pathway. Some simple phenolics, such as the benzoic acid derivatives, are obtained from the shikimate pathway whereas phenylpropoanoids derive from the deamination of phenylalanine and tyrosine. As mentioned, PAL and TAL are the most important enzymes in the phenylpropoanoid pathway. These enzymes convert phenylalanine and tyrosine to trans-cinnamic acid (25) and p-coumaric acid (26) respectively. Phenylalanine is the preferred substrate in the majority of vascular plants, although some monocotyledons can utilize both tyrosine and phenylalanine. A rapid increase of PAL activity levels represents an early response to attempted penetration by pathogens, and a partial suppression of PAL gene expression may lead to increased pathogen susceptibility.

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Variously substituted phenylpropanes which are found in plants originate from this metabolic pathway and are present both as vacuole-stored compounds and cell wall components. Among the most commonly found phenylpropanes are p-coumaric acid (26), caffeic acid (27), ferulic acid (28) and sinapic acid (30). The transformation of these acids leads to the biosynthesis of the monoliglols p-coumaryl alcohol (30), coniferyl alcohol (31) and sinapyl alcohol (32) respectively, the main constituents of the plant polymer lignin. The key steps in the conversion of phenylpropane acids to monolignols are characterized by four types of reactions including hydroxylations (CYP), O-methylations (COMT), CoA ligations and oxidoreductive reactions involving NADPH. The conversion of pcoumaric acid (26) to caffeic acid (27) requires a hydroxylation step that is catalysed by a microsomal NADPH-dependent cytochrome P450 enzyme. An O-methyltransferase introduces a methyl group in a regiospecific manner by using S-adenosylmethionine as a cofactor and methylates caffeic acid to ferulic acid (28). The latter compound is hydroxylated by a P450 monoxygenase to yield 5-hydroxyferulic acid (29) which is then methoxylated to sinapic acid (30). A two-step ATP-dependent/reduced coenzyme-A ligation first generates the AMP derivatives which are then converted to the corresponding CoA esters, including p-coumaroyl-CoA (31), caffeoyl-CoA (32), 5-hydroxyferuloyl-CoA (33), feruloyl-CoA (34) and sinapoyl-CoA (35). The next step is catalysed by two sequential NADPH-dependent reductions: the first, catalysed by cinnamoyl-CoA reductase, is responsible for the formation of p-coumaraldehyde (36), coniferaldehyde (37) and possibly 5-hydroxyconiferaldehyde (38) and sinapaldehyde (39). The second step is catalysed by the reductase cynnamoyl alcohol dehydrogenase that yields the monolignols p-coumaryl alcohol (40), coniferyl alcohol (41) and sinapyl alcohol (42). These monolignols are then transformed into two classes of plant metabolites: the lignans and the lignins.

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There is increasing evidence that lignin is made up of many other monomer derivatives in addition to the above mentioned monolignols. Many plants substantially contain lignin from other monomers and all

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lignins contain traces of apparently recurring intermediates of the monolignols or products resulting from incomplete reactions. Peroxidases and laccases are the enzymes responsible for the polymerization of the monolignols in the complex lignin structure. There is increasing evidence that the macromolecular lignin assembly is not based on a “random coupling” of monolignols. Instead, there seems to be a strong biological control over the result of phenoxy radical coupling driven by a class of proteins involved in lignin biosynthesis. In the lignin polymer, there are several terminal groups including alcohols, aldehydes, esters and diols. Direct polymerization of hydroxycinnamoyl, caffeoyl and 5hydroxyconiferoyl alcohols has recently been demonstrated to produce lignin with catechoyl and 5-hydroxyguaiacoyls in a process considered unconventional. The following figure (modified from Karkonen and Koutanieni, 2010) illustrates a hypothetical lignin structure and its precursor monolignols.

6.1.4. Other Chorismate Derivatives Other important chorismate-derived compounds include 4hydroxybenzoate (4HBA), phenol, gastrodin and the phytohormone

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salicylic acid. 4HBA (43) is a building block molecule for several compounds, including the antibacterial parabens. Chorismate (10) is the precusrsor of 4HBA, which is formed through catalysis of chorismate pyruvate lyase (CPL). The decarboxylation of 4HBA by 4HBA decarboxylase (4HBAD) yields phenol (44), whereas catalysis by aromatic carboxylic acid reductase (ACAR) produces 4-hydroxybenzaldehyde (45). The latter compound is reduced by alcohol dehydrogenase (ADH) to 4hydroxybenzyl alcohol (46), which is then glusosylated by UDPglucosyltransferase (UGT) to gastrodin (47), an important bioactive component of Gastrodia elata. The biosynthesis of the phytohormone salicylic acid has been demonstrated to start from chorismate (10) which is then transformed to isochorismate (48) by isochorismate synthase (EntC). Next, isochorismate lyase (PchB) catalyses the reaction that yields salicylic acid (49).

6.1.5. Benzoic Acid Derivatives Benzoic acid derivatives are quite common in the plant kingdom. The biosynthetic pathway for the production of these molecules derives from either the PAL and TAL enzymatic transamination products, or by

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oxidation of shikimic acid. Benzoic acid is formed from trans-cinnamic acid (25) by a series of reactions where the first step is catalysed by a hydratase that binds a hydroxyl group to carbon 7 of phenylpropane to form ȕ-hydroxyphenylpropionate (50). The deacetylation of this compound results in the formation of benzaldehyde (51), which is subsequently oxidized by benzaldehyde dehydrogenase (BD) to benzoic acid (52).

Several dehydroshikimate-derived compounds, such as gallic acid, protocatechuic acid, catechol, cis,cis-muconic acid (ccMA) and vanillin, have been produced in recombinant microbial systems, thus helping their biosynthetic pathway elucidation in plants. Gallic acid (53) is an important trihydroxylated benzoic acid. Its biosynthesis starts from 3-dehydroshikimic acid (5). There are two pathways leading to the synthesis of gallic acid: the first uses shikimate dehydrogenase (SDH), which leads to direct synthesis of gallic acid via an enolization process; the second uses dehydroshikimate dehydratase (AroZ), that forms protocatechuic acid (54) after dehydration. The latter compound is then converted into gallic acid by a still uncharacterized hydroxylase. Protocatechuic acid is decarboxylated by protocatechuic acid decarboxylase (AroY) to catechol (55), which is then cleaved by catechol 1,2-dioxyganase (CatA) to yield cis,cis-muconic acid (56).

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Vanillin is one of the major plant flavouring agents in terms of economic importance. The compound is produced by cured beans of Vanilla planifolia. The biosynthesis of this compound starts from protocatechuic acid (54) which is transformed to protocatechuic aldehyde (57) by the action of an aromatic carboxylic acid reductase (ACAR). A methyl transferase that uses SAM as a methyl donor (COMT) methylates protochatecuic aldehyde to yield vanillin (58), which can be transformed to vanillyl alcohol (59) by alcohol dehydrogenase (ADH).

6.1.6. Coumarins and Furanocoumarins Some important trans-cinnamic acid derivatives are coumarins. These are physiologically bioactive molecules present in various plant families, but above all in the Apiaceae and the Rutaceae, although the Leguminosae and the Moraceae possess several genera able to accumulate coumarins in substantial quantities (see also Chapter 5). Their functional role spans from chemical defence to the sequestration of metals (e.g., iron) in the soil. Based on their structural and biosynthetic properties, we can categorize plant coumarins into: simple coumarins, furanocoumarins and pyranocoumarins. The first step in the synthesis of simple coumarins is the o-hydroxylation of various phenylpropanes. In Arabidopsis, this catalysis is operated by a 2-oxoglutarate-dependent dioxygenase (2OGD) that acts specifically on feruloyl-CoA and CoA thioesters of cinnamate. In Ruta graveolens, 2ODG catalyses the hydroxylation not only of feruloyl-CoA (33) but also of pcoumaroyl-CoA (31). Hydroxylation of cinnamic acid (25) and caffeic acid (27) is followed by a still unknown reaction that generates coumarin (61) and esculetin (62) respectively. Upon hydroxylation, CoA thioesters of coumarate (31) and ferulate (33) undergo a spontaneous rearrangement to yield umbelliferone (63) and scopoletin (64) respectively.

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Important coumarin derivatives are furanocoumarins. Furanocoumarins are natural plant metabolites characterized by a furan ring attached to carbon 6 and 7 (linear type) or 7 and 8 (angular type) of a benzo-Į-pyrone (coumarin). Therefore, the substitution position in the furan distinguishes two large groups of compounds: linear furanocoumarins (defined as psoralenes) and angular furanocoumarins (defined as angelicins). The former group of furanocoumarins is distributed in plant families including the Rutaceae, Moraceae, Leguminosae and Apiaceae, whereas the latter group of furanocoumarins are primarily confined to the Apiaceae and Leguminosae. The psoralene-type furanocoumarins are known for their photosensitizing and phototoxic properties and comprise marmesin, psoralen and bergapten. The coumarin umbelliferone is the building block for the biosynthesis of both linear and angular furanocoumarins.

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In parsnip (Pastinaca sativa) microcosmes, an umbelliferone dimethylallyltransferase (UDT) catalyzes the reaction between umbelliferone and dimethylallyl pyrophosphate and umbelliferone in the presence of Mg2+ as a cofactor to yield demethylsuberosin (65). The latter compound is transformed to (+)-marmesin (66) and further converted to psoralen (67) by two consecutive cytochrome P450-dependent monooxygenases, marmesin synthase and psoralen synthase. The hydroxylation of psoralen most likely occurs at either the 5- and/or 8position of bergaptol (5-hydroxypsoralen, 68), xanthotoxol (8hydroxypsoralen, 69), and 5,8-dihydroxypsoralen (70). The formation of bergaptol, catalysed by psoralen 5-monooxygenase in the presence of oxygen and nicotinamide adenine dinucleotide phosphate, has been characterized as inducible cytochrome P450 monooxygenase from microsomes of cultured Ammi majus cells. Bergaptol O-methyltransferase catalyses the methylation of the 5-hydroxy group of bergaptol to yield bergapten (71). Finally, a prenyltransferase attaches a geranyl group to bergapten to produce bergamottin (72).

The biosynthesis of the angular furanocoumarins isobergapten and sophondin starts from the catalysis operated by umbelliferone 6prenyltransferase (U6DT), a prenyltransferase that yields osthenol (73) from umbelliferone and dimethylallyl pyrophosphate. In the next step (+)columbianetin synthase, with a reaction mechanism analogous to that of psoralen synthase, uses a cytochrome P450 monooxygenase to hydroxylate osthenol to (+)-columbianetin (74). Angelicin synthase

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cleaves the hydroxylated prenyl chain of (+)-columbianetin to yield angelicin (75). The latter compound is the substrate of two specific hydroxylases: angelicin 5-hydroxylase converts angelicin to 5-hydroxy angelicin (76), which is then methoxylated to sophondin (77); however, angelicin 6-hydroxylase converts angelicin to 6-hydroxy angelicin (78), which is finally methoxylated to isobergapten (79).

6.1.7. Biosynthesis of Stilbenes Stilbenes are a family of phenolic compounds composed by two benzene rings separated by either an ethane or ethene bridge. These molecules are particularly widespread in the mosses, but are also present in the Vitaceae, Dipterocarpaceae, Gnetaceae, Pinaceae, Fabaceae, Poaceae, Leguminoseae and Cyperaceae. Their physiological role is mainly related to their function as plant growth regulators and as pathogen-induced molecules. Because of their proven bioactivity, resveratrol and its derivatives, including pterostilbene, piceid and viniferins, are the most studied stilbenes. However, many other stilbenes including piceatannol, pinosylvin, combretastatins, polydatin (piceid), mulberroside and various oligostilbenes are also known to display valuable biological activities and beneficial effects. The biosynthesis of stilbenes starts from the catalytic activity of stilbene synthase (STS), that uses as substrates one CoA-ester of a cinnamic acid derivative and three malonyl-CoA units. STS activity on trans-cinnamoylCoA (80) yields the stilbene trans-pinosylvin (81), whereas when pcoumaroyl-CoA (31) and caffeoyl-CoA (32) are used as substrates the products of the catalysis are trans-resveratrol (82) and trans-piceatannol (83), respectively. The activity of different S-adenosyl-L-methionine (SAM)-dependent O-methyltransferases (OMT) leads to the production of

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methoxylated stilbenes. Methoxylation of trans-pinosylvin generates pinosylvin monomethyl ether (84), whereas in grapevine the methylation of trans-resveratrol yields pterostilbene (85). Methylation of transpiceatannol yields rhapontigenin (86). Glycosyltransferases (UGT) catalyse the glycosylation of stilbenes, with the formation of piceid (87) from tans-resveratrol and astringin (88) from trans-piceatannol. Finally, stilbenoid-specific prenyltransferases catalyse the prenylation of transresveratrol to yield arachidins (e.g., arachidin-3, 89).

6.2. The Biosynthesis of Complex Phenolics Phenylpropanoid building blocks like trans-cinnamate may function as starting molecules for the formation of a higher level of phenolic compound complexity. By far, the most well-known phenolic compounds are flavonoids, which are molecules characterized by the presence of two aromatic rings bound by a heterocyclic ring formed following the cyclization of a propane residue. Flavonoids are among the most studied secondary metabolites and modern molecular biology techniques make the biosynthetic pathway of flavonoids one of the best available systems in nature by which to study the mechanisms of gene expression regulation.

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6.2.1. The Biosynthesis of Flavonoids Flavonoids are a class of bioactive molecules common to all plants from which thousands of structures have been characterized. Depending on the degree of oxidation, flavonoids are classified into subclasses: aurones, flavones, flavanones, flavonols, flavanols, isoflavones and anthocyanins. The carbon atoms of the aromatic rings may be variedly substituted with hydroxyl, methoxyl and acyl groups. Moreover, hydroxyl groups can be further glycosylated. The biosynthesis of flavonoids starts from a reaction catalysed by chalcone synthase (CHS), a dimeric polyketide synthase that condensates three malonyl-CoA (90)-derived acetyl-CoA (91) molecules and one molecule of p-coumaroyl-CoA (31) to generate tetrahydroxychalcone (92). In some species, the combined action of CHS with NADPH reductase generates isoliquiritigenin (93), a 6’-deoxychalcone. The activity of aurone synthase (AUS), a polyphenol oxidase, generates the yellow aurone pigments found in several plant petals of some Asteraceae species. These compounds are distinguished on the basis of the presence of a hydroxyl group at position 4. AUS activity on tetrahydroxychalcone generates 4,4’,6’-trihydroxyaurone (94), whereas the cyclization of isoliquiritigenin catalysed by AUS generates the aurone hispidol (95). Further hydroxylation of hispidol generates sulfuretin (96).

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Tetrahydroxychalcone is also cycled through a Claisen condensation to form naringenin (97), a flavanone, by the catalytic action of chalcone isomerase (CHI). Flavanones are reduced to flavones, like apigenin (98), through the activity of flavone synthase (FNS), whereas the cytochrome P450 flavanone 3-hydroxylase (FHT), catalyses the hydroxylation of the molecule at position 3 to yield the dihydroflavonol dihydrokaempferol (99), which is then reduced by flavonol synthase (FLS) to kaempferol (100), a flavonol. The cytochrome P450 flavonoid 3’-hydroxylase (F3H) catalyses the hydroxylation of apigenin (98) to kaempferol (100). The enzyme isoflavone synthase (IFS) catalyzes the C-2 to C-3 migration of the aryl group of naringenin and the NADPH-dependent cytochrome P450 hydroxylation to yield 3-hydroxyisoflavanone (101), the precursor of isoflavonoids such as genistein (102), which is formed by the action of 2hydroxyisoflavanone dehydratase (IFD) and biochanin A (103), through the action of isoflavanone O-methyltransferase (IOMT). The NADPH-dependent dihydroflavonol 4-reductase (DFR) catalyses the oxidation of dihydrokaempferol to leucopelargonidin (104), which is then dehydrated to the anthocyanidin pelargonidin (105) by the catalytic action

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of anthocyanidin synthase (ANS). Leucoanthocyanidin reductase (LAR) catalyses the reduction of leucopelargonidin to the leucaoanthocyanidin afzelechin (106). Finally, the UDP-glucose:flavonoid 3-Oglucosyltransferase (FGT) generates the anthocyanin pelargonidin 3glucoside (107).

6.3. Polymeric phenolic compounds Both simple and complex phenolic compounds can polymerize in high molecular weight structures known as tannins. These polymers have the ability to complex with carbohydrates and proteins and for this reason they have long been used in the tanning industry. According to Freudenberg’s classic definition, plant tannins are divided into two classes: hydrolysable tannins, which can be described as gallic acid esters with a central polyol, typically ȕ-D-glucopyranose; and condensed tannins (also known as proanthocyanidins), which originate

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from flavonoids. To this subdivision another group was added: the florotannins, made by floroglucinol units.

6.3.1. The Biosynthesis of Hydrolysable Tannins As mentioned, hydrolysable tannins are metabolites of a polyol to which one or more gallic acid (53) molecules are bound through an ester bond, which in turn can oxidatively bind to other gallic acid units and produce a large variety of metabolites. The biosynthesis of these compounds implies the esterification of gallic acid with UDP-glucose to produce 1,2,3,4,6-pentagallylglucose (108), which represents an important branching point of the biosynthetic pathway. The addition to this molecule of further galloyl residues leads to the subclass of gallotannins, which are characterized by meta-digalloyl groups, while the oxidation of penta-gallylglucose leads to ellagitannins (109), a subclass that is characterized by hexahydroxydiphenyl residues. Mono- and penta-substituted gallylglucoses are often referred to as “simple gallylglucoses” to distinguish them from “complex gallylglucoses”, the real gallotannins. Unlike the limited distribution of gallotannins in nature, ellagitannins are typical constituents of many plant families where they are found in a large structural variety, a feature that is due to their tendency to form dimeric (110) and oligomeric derivatives. The formation of these compounds is catalysed by laccase-like soluble enzymes that use oxygen to oxidize pentagallylglucose to ellagitannin. Further oxidative catalysis leads to the dimerization and polymerization of ellagitannins.

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6.3.2. The Biosynthesis of Condensed Tannins Condensed tannins, or proanthocyanidins, are polyphenolys formed by repetitive flavan-3-ol units. The biosynthesis of proanthocyanidins starts from phenylalanine, then through the flavonoid pathway is channeled into the synthesis of anthocyanidins, which are then converted into monomers (catechin) through a reaction catalysed by leucoanthocyanidin reductase (LAR). Afterwards, with anthocyanidins as the substrate, anthocyanidin reductase (ANR) provides another pathway for the synthesis of proanthocyanidin monomers (epicatechin). Thus, LAR and ANR are specific genes in proanthocyanidin synthesis. Starting from leucocyanidin (111), the catalytic action of ANS yields cyanidin (112), which is then converted into epicatechin (113) by the action of ANR. Catalysis by LAR converts laucocyanidin into catechin (114). Catechin and epicatechin are the building blocks of most proanthocyanidins. The linkage between successive monomeric units in proanthocyanidins occurs between the 4-position of the “upper” unit and the 8-position of the “lower” unit and the stereochemistry can be either Į or ȕ. For instance, procyanidin B1 (115) is formed by condensation of catechin and

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epicatechin. A-type proanthocyanidins present an unusual second ether linkage between an A-ring hydroxyl function of the bottom unit to C2 of the upper-unit. Procyanidin A2 (116) represents a typical dimeric A-type compound. Cranberry extract contains proanthocyanidins consisting primarily of epicatechin dimers to heptamers, with at least one A-type linkage. Fractionation of cranberry extracts yields a complex mixture of epicatechin, catechin, dimers of gallocatechin and epigallocatechin and a series of proantocyanidin oligomers. A typical A-type proanthocyanidin from cranberry is depicted in compound 117.

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Suggested Reading Akagi, T., Katayama-Ikegami, A. and Yonemori, K. (2011). Proanthocyanidin biosynthesis of persimmon (Diospyros kaki Thunb.) fruit. Sci. Hortic. 130, 373–380. Boerjan, W., Ralph, J. and Baucher, M. (2003). Lignin biosynthesis. Annu. Rev. Plant Biol. 54, 519–546. Dixon, R.A., Xie, D.Y. and Sharma, S.B. (2005). Proanthocyanidins – a final frontier in flavonoid research? New Phytol. 165, 9–28. Dubrovina, A.S. and Kiselev, K.V. (2017). Regulation of stilbene biosynthesis in plants. Planta. 246, 597–623. Gross, G.G. (2008). From lignins to tannins: forty years of enzyme studies on the biosynthesis of phenolic compounds. Phytochemistry. 69, 3018– 3031. Hao, Z.Y. and Mohnen, D. (2014). A review of xylan and lignin biosynthesis: foundation for studying Arabidopsis irregular xylem mutants with pleiotropic phenotypes. Crit. Rev. Biochem. Mol. Biol. 49, 212–241. Hung, W.L., Suh, J.H. and Wang, Y. (2017). Chemistry and health effects of furanocoumarins in grapefruit. J. Food Drug Anal. 25, 71–83. Jeandet, P. et al. (2010). Biosynthesis, metabolism, molecular engineering and biological functions of stilbene phytoalexins in plants. Biofactors. 36, 331–341. Karkonen, A. and Koutaniemi, S. (2010). Lignin biosynthesis studies in plant tissue cultures. J. Integr. Plant Biol. 52, 176–185. Krueger, C.G., Reed, J.D., Feliciano, R.P. and Howell, A.B. (2013). Quantifying and characterizing proanthocyanidins in cranberries in relation to urinary tract health. Anal. Bioanal. Chem. 405, 4385–4395. Larbat, R. et al. (2009). Isolation and functional characterization of CYP71AJ4 encoding for the first P450 monooxygenase of angular furanocoumarin biosynthesis. J. Biol. Chem. 284, 4776–4785. Larbat, R. et al. (2007). Molecular cloning and functional characterization of psoralen synthase, the first committed monooxygenase of furanocoumarin biosynthesis. J. Biol. Chem. 282, 542–554. Lee, J.-H. and Wendisch, V.F. (2017). Biotechnological production of aromatic compounds of the extended shikimate pathway from renewable biomass. J. Biotechnol. 257, 211–221. Molitor, C. et al. (2015). Latent and active aurone synthase from petals of C. grandiflora: a polyphenol oxidase with unique characteristics. Planta. 242, 519–537.

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Muir, R.M. et al. (2011). Mechanism of gallic acid biosynthesis in bacteria (Escherichia coli) and walnut (Juglans regia). Plant Mol. Biol. 75, 555–565. Saini, S.S. et al. (2017). Benzaldehyde dehydrogenase-driven phytoalexin biosynthesis in elicitor-treated Pyrus pyrifolia cell cultures. J. Plant Physiol. 215, 154–162. Shimizu, B.-I. (2014) 2-Oxoglutarate-dependent dioxygenases in the biosynthesis of simple coumarins. Front. Plant Sci. 5, e549. Stanjek, V. and Boland, W. (1998). Biosynthesis of angular furanocoumarins: mechanism and stereochemistry of the oxidative dealkyla-tion of columbianetin to angelicin in Heracleum mantegazzianum (Apiaceae). Helv. Chim. Acta. 81, 1596–1607. Stanjek, V., Miksch, M., Lueer, P., Matern, U. and Boland, W. (1999). Biosynthesis of psoralen: mechanism of a cytochrome p450 catalyzed oxidative bond cleavage. Ang. Chem. Int. Ed. 38, 400–402. Vogt, T. (2010). Phenylpropanoid biosynthesis. Mol. Plant. 3, 2–20. Weng, J.K. and Chapple, C. (2010). The origin and evolution of lignin biosynthesis. New Phytol. 187, 273–285. Xie, D.Y. and Dixon, R.A. (2005). Proanthocyanidin biosynthesis – still more questions than answers? Phytochemistry. 66, 2127–2144. Yan, J., Cai, Z., Shen, Z., Ma, R. and Yu, M. (2017). Accumulation of proanthocyanidin monomers in two genotypes of blood-flesh peach. J. Horticult. Sci. Biotechnol. 92, 513–520. Yáñez, J.A. et al. (2013). Polyphenols and flavonoids: an overview. In Flavonoid Pharmacokinetics: Methods of Analysis, Preclinical and Clinical Pharmacokinetics, Safety, and Toxicology, eds. K.M. Davies and J.A. Yanez (Whiley), 1–69.

CHAPTER SEVEN THE BIOSYNTHESIS OF TERPENOIDS

Plants produce a large amount of lipids that are used in a variety of metabolic and structural functions. The main lipid constituents of biological membranes are fatty acids. However, plants produce other lipids and the most important and varied are the terpenoids. Terpenoids, with over 30,000 molecules identified so far, are present in all living organisms and are particularly diverse in plants, where they originate from two main biosynthetic pathways: the mevalonic acid pathway (MVA) and the methylerythritol phosphate pathway (MEP). Both pathways produce the 5-carbon base precursor isopentenyl diphosphate (or isopentenyl pyrophosphate, IPP). Terpenoids are also referred to as isoprenoids, because some terpenoids decompose to form the volatile compound isoprene. The condensation of two isoprenoids gives rise to monoterpenes, composed of 10 carbon atoms, while the addition of another isoprenoid unit to a monoterpene originates the sesquiterpenes, composed of 15 carbon atoms. The subsequent condensation of an IPP molecule with a sesquiterpene forms the diterpenes, composed of 20 carbon atoms, while the condensation of two sesquiterpenes gives rise to the triterpenes (30 carbon atoms). Finally, two diterpene molecules condense to form the tetraterpenes, containing 40 carbon atoms. Intermediate categories of terpenoids are the emiterpenes, with 5 carbon atoms, the sesterterpenes, made up of 25 carbon atoms, and the sesquarterpenes, containing 35 carbon atoms. Finally, the last category of terpenoids is represented by isoprenoid polymers; with a variable number of some hundreds of residues, these compounds are defined as polyterpenes.

7.1. Two Biosynthetic Pathways produce all Plant Terpenoids As we have mentioned, terpenes originate from the condensation of isoprenoid units. IPP and its isomer, dimethylallyl diphosphate (or

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dimethylallyl pyrophosphate, DMAPP) are the starter compounds for the synthesis of all terpenoids. We will analyse separately the two metabolic pathways that lead to the formation of IPP.

7.1.1. The Mevalonic Acid Pathway In the MVA, a residue of acetyl CoA (1), originating from glycolysis or lipid degradation, is condensed with another acetyl CoA residue by the enzyme acetyl CoA acetyltransferase (AACT) which produces acetoacetyl CoA (2), with the release of reduced CoA. The enzyme hydroxymethylglutaryl CoA synthase (HMGS) condenses a third acetyl CoA molecule with acetoacetyl CoA to form the six-carbon compound 3hydroxy-3-methylglutaryl-CoA (HMG-CoA, 3). This compound is reduced by HMG-CoA reductase (HMGR) in two coupled NADPHrequiring reactions to form mevalonic acid (4). This reaction is a ratedetermining step in the entire MVA pathway. Mevalonate is activated by two phosphorylated steps catalysed by the enzyme mevalonate 5-kinase (MVK), which produces mevalonate-5phosphate (5) and phosphomevalonate 5-kinase (PMK) which forms mevalonate-5-diphosphate (6). These reactions are followed by an ATPdependent decarboxylation catalysed by mevalonate diphosphate decarboxylase (MDD), which forms the unsaturated hydrocarbon compound isopentenyl diphosphate (IPP, 7). Two structurally unrelated IPP isomerases (IDI-1 and IDI-2) are responsible for the IPP crossconversion into dimethylallyl diphosphate (DMAPP, 8). Recently, an enzyme (isopentenyl phosphate kinase, IPK) has been identified to transform isopentenyl phosphate, formed as a result of decarboxylation of mevalonate-5-phosphate to IPP, via an enzyme that has not been characterized so far.

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7.1.2. The Methylerythritol 4-Phosphate Pathway Between 1990 and 2000, several researchers contributed to the discovery of the second metabolic pathway for the production of IPP, which is present in bacteria, algae and plants. The name of this pathway comes from one of the key metabolic intermediates and the pathway starts from the condensation of pyruvic acid (10) and glyceraldehyde 3-phosphate (11) to yield 1-deoxy-D-xylulose-5-phosphate (DXP, 12), a reaction catalysed by 1-deoxy-D-xylulose-5-phosphate synthase (DXS), a thiamine pyrophosphate utilizing enzyme. DXP is then isomerized in a reductive manner by the DXP reductoisomerase (DXR, also known as IspC) to yield 2C-methyl-D-erythritol-4-phosphate (MEP, 13). Cytidine triphosphate is then transferred by the catalysis of MEP cytidyl transferase (IspD) to give 4-(Cytidine 5’-diphospho)-2C-methyl-D-erythritol (CDP-ME, 14). This reaction is followed by an additional phosphate group from ATP by cytidyl MEP kinase (IspE ) that produces 4-(Cytidine 5’-diphospho)-2Cmethyl-D-erythritol 2-phosphate (CDP-MEP, 15). The linking of a phosphate group to the phosphate moiety of the nucleotide leads to the formation of a cyclic diphosphate and the loss of the cytidyl monophosphate to give 2C-methyl-D-erythritol-2,4-cyclodiphosphate (MEcPP, 16), a reaction catalysed by MEP-2,4-cyclodiphosphate synthase (IspF). The last two steps are catalysed by two iron-sulfur reductases: (E)-4hydroxy-3-methylbut-2-enyl diphosphate synthase (IspG) and (E)-4-

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hydroxy-3-methylbut-2-enyl diphosphate reductase (IspH). IspG catalyses the opening of the ring and the reductive dehydration of the MEcPP to form (E)-4-hydroxy-3-methylbut-2-enyl diphosphate (HMBPP, 17), whereas IspH catalyses the reductive dehydration of HMBPP to form IPP (7) and DMAPP (8). It is therefore clear that in organisms using the MEP pathway the activity of IDI is not essential for survival, even though it plays an important role in modulating the regulation of cellular concentrations of both IPP and DMAPP.

7.1.3. Comparing the Two Pathways Most organisms retain only one route for the biosynthesis of isoprenoid precursors. For example, many, but not all, eubacteria, as well as cyanobacteria, make exclusive use of the MEP pathway. In contrast, archaebacteria, staphylococci, streptococci and enterococci mostly use the MVA pathway (Fig. 7.1). Based on genome sequencing, eukaryotes, with the exception of the photosynthetic ones, generally use only the MVA pathway. Higher plants and some algae maintain both routes with different localization; the MVA pathway occurs in the cytoplasm while the MEP pathway is localized in the plastids. Some organisms, after acquisition of the MEP pathway as a consequence of endosymbiosis, completely lost the MVA pathway.

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Figure 7.1. Distribution of the MVA and MEP pathways within different kingdoms. MVA: mevalonate pathway. MEP: 2C-methyl-d-eryhtritol 4-phosphate pathway. Archaebacteria, fungi and animals exclusively use the MVA pathway, whereas eubacteria, red algae and higher plants use both MVA or MEP pathways to synthesize isoprenoids. Cyanobacteria and unicellular algae use exclusively the MEP pathway for isoprenoid biosynthesis. From Hammerlin et al., 2012, modified.

7.2. Hemiterpenes The simplest terpene produced by plants is isoprene (2-methyl 1,3butadiene, 18), a five carbon atom molecule, which is a hemiterpene. Despite the assonance of the term, isoprenoids are not made up of isoprene, but originate from IPP and DMAPP as described in the above biosynthetic pathways. Isoprene is synthesized from DMAPP (8), which is produced by the catalytic activity of IspH on HMBPP (17). The action of isoprene synthase (IspS) catalyses the conversion of DMAPP to isoprene.

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7.3. Monoterpenes The production of DMAPP polymers originates with the “head-to-tail” condensation of IPP (7) and DMAPP (8) deriving from the MEP pathway, a reaction catalysed by a prenyltransferase (GPP synthase) that generates the precursor of all monoterpenes, geranyl diphosphate (GPP, 19).

GPP undergoes numerous transformations leading to the formation of more than 1000 monoterpene structures. The main feature of monoterpenes is that they are extremely volatile compounds. They represent the majority of the molecules contained in essential oils and are responsible for the scent of numerous aromatic plants, such as those belonging to the genera Rosmarinus, Salvia, Mentha, Ocimum, Satureja, Thymus, Achillea, Artemisia etc. The cyclization of GPP occurs via enzyme defined cyclases that lead to the formation of various monoterpene skeletons. Among these the most common are: menthanes (20), pinanes (21), camphanes (22), caranes (23), fenchanes (24), thujanes (25), iridanes (26), pyrethranes (27) and cannabinols (28).

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Menthol biosynthesis is one of the most defined monoterpene pathways and represents a good example. This monoterpene alcohol accumulates along with other similar compounds in the glandular trichomes of the genus Mentha (see Chapter 2), but is also present in other species, such as in some varieties of fragrant geraniums (Pelargonium spp.). The menthol biosynthesis takes place in eight steps and begins with the cyclization of GPP (19) to the hydrocarbon limonene (29), a reaction catalysed by the plastidial enzyme limonene synthase (LS). The overall reaction involves ionization, isomerization, cyclization and deprotonation steps, leading to the formation of the limonene enantiomer as a stereochemical consequence of the initial helical folding of the GPP at the active site of the enzyme. An OH group in position 3 is inserted on limonene by the action of a cytochrome P450-dependent monoterpene hydroxylase (limonene 3-hydroxylase, L3OH) which uses oxygen and NAPDH to form trans-isopiperitenol (30). This process leads to the hydroxylation of the pmentane in position 3, an essential catalysis for menthol formation. A similar reaction in Mentha spicata mediates a regiospecific hydroxylation in position 6 (L6OH), transforming limonene (29) into trans-carveol (31), in the biosynthetic pathway that leads to the monoterpene carvone (32). The oxidation of trans-isopiperitenol (30) catalysed by an NAD-dependent isopiperitenol dehydrogenase (iPD) produces isopiperitenone (33) which is reduced by an NADPH-dependent isopiperitenone reductase (iPR) to cisisopulegone (34). Isopulegone isomerase (iPI) catalyses the reaction that transforms cis-isopulegone to pulegone (35). The latter compound plays a central role in the entire metabolic pathway as it is the substrate of different reactions that lead to the synthesis of important monoterpenes.

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Pulegone reductase (PR), an NADPH-dependent enzyme, catalyses the reduction of pulegone to menthone (36) and isomenthone (37), whereas menthofuran synthase (MFS) is a microsomal enzyme that uses pulegone as a substrate to perform a P450-dependent oxidation that leads to the synthesis of menthofuran (38). The last step in the biosynthetic path is the reduction of menthone to menthol (39). This reaction is catalysed by menthone reductase (MR), a cytosolic enzyme. In the genus Mentha there are other compounds that accumulate in the glandular trichomes. Piperitenone (40) originates from isopiperitenone (33) and can be further reduced to piperitone (41) or oxidized to piperitenone oxide (42). Piperitone can also be oxidized to piperitone oxide (43). Another noteworthy compound is carvone (32) that, as we have mentioned, is formed by hydroxylation of limonene. Trans-carveol (31) is subsequently oxidized to carvone by carvone dehydrogenase (CDH).

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7.4. Sesquiterpenes The condensation of IPP (7) with a molecule of GPP (19) forms a molecule consisting of 15 carbon atoms. Fifteen being equivalent to 1.5 times 10, these terpenes are termed sesqui-(one and a half)-terpenes. The precursor of all sesquiterpenes is farnesyl pyrophosphate (FPP, 43). The superior complexity of these molecules, when compared to that of monoterpenes, generates a higher number of molecular structures, with more than 7,000 known compounds.

The cyclization of FPP allows the formation of structures with one, two and three cycles. Sesquiterpenes can be classified as: farnesanes (44), bisabolanes (45), humbertianes (46), sesquicanphanes (47), cuparanes (48), carotanes (49), longifolanes (50), caryophyllanes (51), humulanes (52), germacranes (53), elemanes (54), vetisperanes (55), eremophylanes (56), patchoulanes (57), ishwaranes (58), pinguisanes (59) and many other classes (including lauranes, tricotecanes, camigranes, thujopsanes, cycloperphoranes, acoranes, cedranes, cadinanes, cubebanes, oplopananes, picrotoxanes, hymacalanes, longipinanes, longicamphanes, clovanes, illudalanes, protoillidanes, illudanes, hirsutanes, lactaranes, sterpurianes, marasmanes, pentalenanes, africananes, eudesmanes, oppositanes, guaianes, pseudoguaianes, cyperanes, trixanes, aromadendranes and aristolanes).

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Sesquiterpene lactones are important derivatives of sesquiterpenes. These molecules are very common in the plant kingdom and are particularly present in some families, among which the Asteraceae. Sesquiterpene lactones are characterized by the presence of a four-carbon and one oxygen heterocyclic ring and by the presence of a ketone group. The most important sesquiterpene lactone classes are: germacranolides (60), eudesmanolides (61), eremophylanolides (62), guaianolides (63), xanthanolides (64) and ambrosanolides (65). Certainly the sesquiterpene lactone on which the hopes of many researchers are based is artemisinin (73), a seco-cadinane. This molecule is produced by Artemisia annua, known in Chinese Traditional Medicine as “qinghaosu”, and is used in the treatment of flu, fever and malaria. Artemisinin is particularly active against Plasmodium falciparum strains resistant to chloroquine therapy.

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The first step of artemisinin (73) biosynthesis is the least controversial, and the majority of the enzymes involved in the conversion of IPP (7) and its isomer DMAPP (8) to form amorpha-4,11-diene (68) have been isolated and characterized in A. annua. It is believed that the IPP used is derived from both the MVA and MEP pathways, but it has recently been demonstrated that the central isoprenoid unit of the artemisinin precursor FPP (43) is primarily biosynthesized from the MEP pathway. An intermediate compound in the biosynthesis of artemisinin is the sesquiterpene bicyclic compound amorpha-4,11-diene (68), which is formed from FPP by the action of a sesquiterpene cyclase, amorpha-4,11diene synthase (ADS). Most possibly, FPP is first isomerized to nerolidyl diphosphate (66); then the ionization of nerolidyl diphosphate is followed by the closure of the ring to generate the bisabolyl cation (67) which undergoes a 1,3-hydride shift that allows a second closure of the ring between position 1 and 10 to generate the amorphane skeleton. Finally, the deprotonation of C-12 or C-13 leads to the formation of amorpha-4,11diene (68). The second phase in the biosynthesis involves the modification of the isopropylidene group (C-11, C-12 and C-13) of amorpha-4,11-diene, to produce biosynthetic intermediates such as dihydroartemisinic acid and artemisinic acid. Historically, it was hypothesized that artemisinic acid was the starting point for the last step of artemisinin biosynthesis. However, recent evidence suggests that dihydroartemisinic acid (71) is the true arsenisinin precursor. This compound is produced after two oxidations: the first yielding dihydroartemisinic alcohol (69) and the second generates dihydroartemisinic aldehyde (70). These steps are catalysed by CYP71AV1 and artemisinic aldehyde double bond reductase (DBR2) respectively. Further, the oxidation of dihydroartemisinic aldehyde to dihydroartemisinic acid occurs via an aldehyde

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dehydrogenase, ALDH1. The subsequent steps in the biosynthetic pathway require the presence of dihydroartemisinic acid as a precursor. According to some authors, the conversion of this acid into artemisinin could take place not enzymatically, with a four-step spontaneous oxidation: 1) a photo-sensitization reaction of the double bond in 4,5 of dihydroartemisinic acid with a molecular oxygen singlet (through an “ene” mechanism); 2) Hock cleavage of the resulting tertiary allyl hydroperoxide (72); 3) oxygenation of the enolic product of the Hock cleavage; and 4) cyclization of the hydroperoxyl aldehyde proximal to the 1,2,4-trioxanic artemisinin (73) structure. These reactions have been demonstrated predominantly in vitro and many other compounds are produced in addition to artemisinin. Therefore, there is an open possibility that other biosynthetic reactions might be involved in the biosynthesis of this important compound.

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7.5. Diterpenes Diterpenes are terpenoid containing 20 carbon atoms. The precursor of all diterpenes is geranylgeranyl diphosphate (GGPP, 74) which is formed by the “head-to-tail” condensation of FPP (43) with a unit of IPP (7). After production of IPP and DMAPP in plastids, GGPP synthase represents a crucial point responsible for the biosynthesis of all diterpenes. GGPP synthases are a small family of genes that encode 11 homologues in Arabidopsis thaliana.

There are different classes of diterpenes, divided into bicyclic (labdanes, [75]; and clerodanes, [76]), tricyclic (pimaranes, [77]; and abietanes, [78]), tetracyclic (kauranes, [79]; beyeranes, [80]; gibberellanes, [81]; stemaranes, [82]; stemodanes, [83]; and aphidicolanes, [84]) and macrocyclic (cembranes, [85]; daphnanes [86]; and taxanes [87]) structures.

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As discussed in Chapter 4, one of the most studied diterpenes is paclitaxel (taxol), which is present at extremely low concentrations in the barks of Taxus brevifolia. Paclitaxel is a tetracyclic diterpene biosynthesized in nineteen steps starting from two large structural building blocks, a diterpenoid (baccatin III) and a phenylpropanoid side chain (phenylisoserine). In the paclitaxel biosynthesis, the origin of the precursors IPP and DMAPP has been shown to belong to both MVA and MEP pathways. The first step in the pathway is the cyclization of GGPP (74) to taxadiene (88), a reaction catalysed by taxadiene synthase (TASY). The produced tricyclic structure subsequently undergoes numerous regio- and stereospecific oxygenations and acylations mediated by a variety of cytochrome P450 oxygenases and acyltransferases. Taxadiene is then hydroxylated at the 5 position to produce taxadien-5Į-ol (89) by the cP450 taxadiene-5Įhydroxylase (T5ĮH). Taxadiene-13Į-hydroxylase (T13ĮH) catalyses the conversion of taxadien-5Į-ol to taxadien-5Į-13Į-diol (90), which in turn undergoes four further P450-dependent hydroxylations at the C1, C2, C4 and C7 positions, an oxidation at C9 and an epoxidation between C4 and C5 to yield the hypothetical compound 2-debenzoyltaxane (91). A catalysis operated by taxane 2Į-O-benzoyltransferse (TBT) produces 10deacetylbaccatin III (92), which is subsequently acetylated at the C10 position by 10-deacetylbaccatin III-10-O-acetyltransferase (DBAT) to yield baccatin III (93). The enzyme baccatin III-13-O-phenylpropanoyl transferase (BAPT) attaches a ȕ-phenylalanine-CoA (94) moiety to the taxane core to produce 30-N-debenzoyl-20-deoxytaxol (95), which is then hydroxylated by a P450 to N-debenzoyltaxol (96). The latter compound is converted into the final compound paclitaxel (97) by the enzyme 30-Ndebenzoyl-20-deoxytaxol-N-benzoyl transferase (DBTNBT). The latex of some plants belonging to the genus Euphorbia contains some toxic substances that can cause poisoning to both animals and humans. These substances belong to a class of tetracyclic diterpenes, the tiglianes. The most progressed compound in this class of natural products is phorbol 12-myristate 13-acetate, whose biosynthetic pathway originates from the cyclization of GGPP (74) into casbene (98) by casbene synthase (CS). The cyclization of casbene occurs through the formation of the tricyclic latirane (99) and the subsequent formation of the tetracyclic tigliane (100) structures. Subsequent oxygenations form the molecular structure of phorbol (101), which is first esterified with a residue of acetate (102) and then with a residue of myristic acid to form phorbol 12-myristate 13acetate (103).

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7.6. Sesterterpenes The condensation of a molecule of GGPP (74) with one of IPP (7) generates geranylfarnesyl diphosphate (GFPP, 104) a compound made with 25 carbon atoms and the precursor of all sesterterpenes.

Sesterterpenes are natural substances that are not widespread in the plant kingdom but that can be found especially in fungi and in marine organisms. Sesterterpenes and sesterterpene lactones have been reported from several Salvia species, including S. lachnocalyx, S. sahendica, S. dominicana, S. yosgadensis, S. syriaca and S. mirzayanii. Lachnocalyxolide A (105) is a sesterterpene lactone isolated from S. lachnocalyx, whereas salvileucolide methylester (106) has been isolated from S. sahendica. Yosgadensolide A (107) is a sesterterpene lactone isolated from S. yosgadensis.

7.7. Triterpenes Triterpenes are terpenoids synthesized from intermediates of the MVA pathway. They are one of the largest families of compounds in the class of terpenoids, with over 20,000 molecules characterized. Triterpenes are produced by many species of the plant kingdom, but other organisms are also able to biosynthesise these molecules. The first product in the

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biosynthetic pathway of triterpenes is presqualene diphosphate (PSPP, 108), which is not formed by condensation of IPP with GFPP, but by the “head-to-head” condensation of two FPP (43) molecules to form a structure with 30 carbon atoms. In plants, the reaction is catalysed by the enzyme squalene synthase (SQS). PSPP undergoes an Mg2+-dependent ionization which leads to the loss of the diphosphate and the subsequent reduction (through NADPH) to form squalene (109), the precursor of all triterpenes.

Squalene is then oxidized to position 2,3 by squalene epoxidase (SQE), which leads to the synthesis of 2,3-oxidosqualene (110). In plants there are more than 100 basic triterpenes, the result of over 80 cyclases that use 2,3oxidosqualene as a substrate. The cyclization of 2,3-oxidosqualene is catalysed by enzymes known as oxidosqualene cyclase (OSC), which generate sterols and other triterpene structures in a metabolic pathway that includes: (a) substrate bonding and pre-organization (bending); (b) initiation of the reaction through protonation of the epoxide; (c) cyclization and rearrangement of carbocationic species; and (d) synthesis by means of deprotonation or water capture to obtain the final triterpene. The first structural folding step is crucial for the biosynthesis of triterpenes. For example, the chair-boat-chair conformation (CBC) organizes the cyclization for the formation of the protosteryl cation which gives rise to sterols; whereas the chair-chair-chair (CCC) conformation directs the cyclization towards the dammarenyl cation that generates the different triterpene structures. Following folding, OSCs initiate the cyclization reaction by protonating the epoxide group of 2,3oxidosqualene. Cycloartenol synthase (CAS) and cucurbitadienol synthase (CPQ) use CBC conformations to form cycloartenol (111) (and all plant sterols, including brassinosteroids) and cucurbitadienol (112) (and cucurbitacins) respectively. ȕ-amyrin synthase (BAS) instead uses the CCC conformations and catalyses the synthesis of ȕ-amyrin (113) (and all glycosidic triterpenes, including boswellic acids).

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Triterpenes account for many bioactive molecules, including phytosterols, triterpene saponins and the modified triterpenoids such as limonoids and quassinoids. Among the most widespread phytosterols, ȕ-sitosterol and stigmasterol along with brassinosteroids (like brassinolide) represent about 70% of all steroidal compounds contained in plants.

7.7.1. Ecdysteroids Ecdysteroids are steroid hormones that control the development and reproduction of arthropods, but they are also bioactive molecules produced by a wide range of vascular plants and defined as phytoecdysteroids. In plants there are more than 300 molecules, often present as mixtures; however, the main component is usually 20-hydroxyecdysone (116), a moulting hormone of most arthropods. In plants, cholesterol (114) is transformed into 7-dehydrocholesterol (115) and then into ecdysone. Plants are able to produce Į-ecdysone (117) and ȕ-ecdysone (118) and other modified phytoecdysones that are used as defence molecules against insects.

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7.7.2. Saponins Saponins are glucosylated derivatives of pentacyclic tritepenes such as lupeol and owe their name to the ability to generate foam, just like soap, when mixed in aqueous solutions. They are present in the family Caryophyllaceae (genus Saponaria) and Rosaceae (genus Quillaja), produced by roots and stems and with powerful hemolytic action if injected into the bloodstream. In liquorice (Glycyrrhiza glabra) glycyrrhizin is one of the main saponins. In this plant there are three OSCs: ȕ-amyrin synthase (BAS), lupeol synthase (LUS) and cycloartenol synthase (CAS). These three enzymes are involved in the cyclization of 2,3-oxidosqualene to generate oleananic triterpene saponins (glycyrrhizin and soysaponin), litercanic triterpenes (e.g., betulinic acid) and phytosterols respectively. The biosynthesis of glycyrrhizin involves a series of oxidative reactions of the precursor ȕ-amyrin (113) in positions C-11 (two-phase oxidation) and C-30 (three-phase oxidation), followed by the transfer of a glycosylic moiety to the C-3 hydroxyl group. Oxidation in C11 by CYP88D6 leads to the synthesis of 11-keto-ȕ-amyrin (119), which is then hydroxylated at position C30 by CYP72A154 to form 30-hydroxy11-keto-ȕ-amyrin (120). This compound is oxidized to glycyrretaldehyde (121) and subsequently oxidized to glycyrrhetinic acid (122). The catalysis of UDP-glucuronosyl transferase (UGAT) leads to the bonding of two residues of glucuronic acid on the hydroxyl group at position C3 to form glycyrrhizin (123).

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In addition to the saponins formed by glucosylation of triterpenes, there are also steroidal saponins, which are present mainly in the Dioscoreaceae, Agavaceae and Liliaceae families. An example is given by dioscin, formed by diosgenin (a spimstanol saponin) linked to rhamnosylic and glucorhamnosylic residues. Diosgenin is found in the species of the genus Dioscorea, but also in fenugreek (Trigonella foenum-graecum). The biosynthesis of diosgenin starts from the cyclization of 2,3-oxidosqualene (110) to cycloartenol (111), a reaction catalysed by cycloartenol synthase (CAS). Cycloartenol can be either methylated to 24-methylenecycloartanol (124) by sterol C-24 methyltransferase (SMT) or transformed into cholesterol (114) by sterol side chain reductase (SSR). 24Methylenecycloartanol is then transformed into sitosterol (125) and diosgenin (126) by the catalysis of sterol-3-ȕ-glucosyl-transferase (S3ȕGT) and 26-O-ȕ-glucosidase (26ȕG), whereas cytochrome P450s play major roles in catalysing the transformation of cholesterol into diosgenin (126). Finally, a UDP-glycosyltransferase (e.g., 3-O-sterol glycosyltransferase) converts diosgenin into dioscin (127).

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7.7.3. Limonoids Tetranortriterpenoids, also known as limonoids, including azadirachtin, nimbin and salannin, are modified triterpenoids present in many plant species, but particularly in the families Meliaceae and Simaroubaceae. The seeds of Azadirachta indica produce one of the most complex limonoids, azadirachtin, which is of particular interest as an agrochemical due to its active role as an insect feeding deterrent. There are limited biosynthetic studies on this molecule with many proposed speculations. According to a biosynthetic hypothesis, tirucallol (128) appears to be the most probable precursor. This molecule is further transformed into azadirone (129), which is acetylated to nimbolide (130), whereas subsequent oxidations form salannin (131), marangine (132) and azadiractol (133). The latter compound would eventually lead to azadyractin (134); however, the exact sequence of reactions is still unknown.

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7.7.4. Quassinoids Quassinoids have a skeleton with 20 carbon atoms and can easily be confused with diterpenes. Quassinoids are molecules produced by species belonging to the Simaroubaceae family with particular reference to the genus Quassia. Quassin (138) is extracted from the wood of this species and it has been hypothesized that a possible precursor of quassin may be euphol (135), which would undergo an allyic isomerization to yield butyrospermol (136) that is subsequently transformed into apoeufol (137) and finally transformed into quassin (138).

7.7.5. Cardenolides and Bufadienolides Another category of triterpenes is that of cardiac glycosides. Based on their chemical structure, cardiac glycosides are divided into cardenolides and bufadienolides; both groups have a common structural base consisting of a steroidal nucleus, a glycosidic portion and a lactone portion. The

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glycosidic portion may consist of a large variety of sugars, among which the most common are glucose, galactose, mannose and rhamnose. The lactone portion instead allows division of the cardiac glycosides into two groups: the cardenolides, possessing an unsaturated butyrolactone ring with five atoms and the bufadienolides that contain an unsaturated pyranic ring with six atoms. Bufadienolides are less represented in the plant kingdom (they are limited to some species of the genera Helleborus, Drimia and Urginea) and have been found in some animals such as the toads of the Bufonidae family, to whom they owe their name. Bufadienolide glycosides such as hellebrin (139) are present in the genus Helleborus, whereas the bufadienolides rubellin (140) and riparianin (141) have been isolated from the bulbs of Drimia macrocentra and Urginea riparia respectively.

Digitoxigenin, extracted from the species Digitalis purpurea, belongs to the group of cardenolides. The biosynthesis of this compound starts from the transformation of cholesterol (114) into pregnenolone (142), catalysed by the sterol side chain cleaving enzyme (SCCE). NAD:3ȕ-hydroxysteroid dehydrogenase (3ȕ-HSD) transforms the pregnenolone into isoprogesterone (143), which is then transformed into progesterone (144) by ǻ4,5-3-ketosteroid isomerase (3-KSI). Progesterone-5ȕ-reductase (P5ȕR) reduces progesterone to 5ȕ-pregnane-3,20-dione (145), whereas the activity of NADH:3ȕ-hydroxysteroid dehydrogenase (3ȕ-HSD) causes the dehydrogenation of 5ȕ-pregnane-3,20-dione into 5ȕ-pregnane-3ȕ-ol20-dione (146). The latter compound is then transformed by an unknown steroid, 14ȕ-hydroxylase to 5ȕ-pregnane-3ȕ-14ȕ-diol-20-one (147), which is further hydroxylated to 5ȕ-pregnane-3ȕ-14ȕ,21-triol-20-one (148) by another still unknown steroid, 21ȕ-hydroxylase. Malonyl coenzyme A:21hydroxypregnane 21-O-malonyltransferase (21MaT) catalyses the transfer of a malonyl moiety that generates 21-O-malonyl-5ȕ-pregnane-3ȕ-14ȕdiol-20-one (149), the direct precursor of digitoxigenin (150).

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7.8. Sesquarterpenes Sesquarterpenes are a rare class of terpenes with 35 carbon atoms identified only in a few species of bacteria (Bacillus and Mycobacterium). The biosynthetic pathway uses one molecule of FPP (43) to which four IPP (7) molecules are added through catalysis of hexaprenyl diphosphate synthase (HepPPs), which forms the precursor hexaprenyl diphosphate (HepPP, 151).

7.9. Tetraterpenes The “head-to-head” condensation of two molecules of GGPP (74) catalysed by phytoene synthase (PS) leads to the formation of 15-cisphytoene (152), the precursor of all tetraterpenes (C40 terpenoids).

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15-Cis-phytoene undergoes a series of transformations that lead to the formation of increasingly unsaturated metabolic intermediates.

7.9.1. Carotenoids Carotenoids are essential hydrophobic compounds, usually coloured in yellow, orange or red, and play an important role in photosynthesis, photoprotection and the production of phytohormones. Some carotenoids, such as Į-and ȕ-carotene, are precursors of vitamin A. The hundreds of carotenoid structures known to date can be divided into two main groups: carotenes (non-oxygenated molecules) and xanthophylls (oxygenated carotenoids). In the biosynthetic pathway of carotenoids, the colourless 15-cis-phytoene (152) is desaturated and isomerized to form the reddish lycopene (155) (all trans) through a series of reactions comprising the catalysis of the enzymes phytoene desaturase (PDS) and 15-cis-ȗ-carotene isomerase (ZISO), with the production of the intermediate compound 9,9'-cis-ȗcarotene (153). Catalysis operated by ȗ-carotene desaturase (ZDS) produces the intermediate prolycopene (154) which is transformed by carotenoid (pro-lycopene) isomerase (CrtISO) to lycopene (155). Next, type İ and type ȕ lycopene cyclases (LCYE and LCYB, respectively) produce two types of orange carotenes from lycopene: Įcarotene (with a ȕ ring at one end and one İ ring at the other), and ȕcarotene (from the cyclization of the two ends of lycopene to form two ȕrings). The two branches of the biosynthetic pathway that perform these cyclization reactions are defined ȕ,İ and ȕ,ȕ pathways. In the first pathway, lycopene (155) is cyclized by lycopene İ-cyclase (LCYE) leading to the formation of į-carotene (156), which is then transformed to Į-carotene (157) by the catalytic activity of lycopene ȕ-cyclase (LCYB). The hydroxylation of Į-carotene catalysed by İ hydroxylase (CHYE) leads to the formation of zeinoxanthin (158), while the hydroxylation catalysed by ȕ-hydroxylase (CHYB) is responsible for the production of a yellowish

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xanthophyll, lutein (159). Both CHYE and CHYB are cytochromes P450 (CYP97). In the ȕ,ȕ pathway, two lycopene ȕ-cyclases (LCYB) catalyse the formation of Ȗ-carotene (160) and subsequently of ȕ-carotene (161). Two hydroxylations catalysed by carotenoid ȕ-hydroxylase (CHYB) produce first ȕ-cryptoxanthin (162) and then zeaxanthin (163). Next, violaxanthin (164) is produced from zeaxanthin by the catalytic activity of zeaxanthin epoxidase (ZEP), whereas neoxanthin (165) is produced from violaxanthin by neoxanthin synthase (NSY). Violaxanthin deepoxidase (VDE) can transform violaxanthin back into zeaxanthin, in the so-called xanthophyll cycle. Xanthophylls such as violaxanthin and zeaxanthin represent a category of extremely important compounds involved in light energy transduction mechanisms of photosynthetic organisms. Important carotenoid derivatives are the group A vitamins. Vitamin A1 (or retinol) is a diterpene, but derives from the oxidative metabolism of ȕ-carotene that mammals ingest from plant foods. Another high-value xanthophyll used as a food supplement is astaxanthin (or 3,3'-dihydroxy-ȕ,ȕ-carotene-4,4'dione), a xanthophyll synthesized by many bacteria and marine algae and a small group of plants and mushrooms. The compound is used both as a dietary antioxidant and as a food supplement in the fishing industry to give colour to the fish. Astaxanthin biosynthesis has recently been elucidated in the flowers of Adonis aestivalis. The biosynthetic pathway of astaxanthin begins with the activation of the carbon 4 of a ȕ ring of ȕ-carotene (161) in a reaction catalysed by carotenoid ring-ȕ 4-dehydrogenase (CBFD) that leads to the formation of a 4-hydroxy derivative (166). The pathway continues with further dehydrogenation of this carbon to produce a carbonyl in a reaction catalysed by the carotenoid 4-hydroxy-ring-ȕ 4-dehydrogenase (HBFD) which forms the keto group (167). The biosynthetic pathway ends with the addition of a hydroxyl group to carbon 3 in a reaction catalysed by CBFD that generates astaxanthin (168).

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7.9.1.1. Abscisic Acid In higher plants, the sesquiterpenoid hormone abscisic acid does not originate from FPP; rather it derives from the oxidation of carotenoids. The pathway occurs in two compartments: plastid and cytosol. In the plastid, trans-neoxanthin (169) is isomerized by a still unknown isomerase to 9’-cis-neoxanthin (170) which is exported to the cytosol. Here the compound is cleaved by 9-cis-epoxycarotenoid dioxygenase (NCED) to form the C15 compound xanthoxin (171). The latter compound undergoes the reduction of the epoxy group and the oxidation of the secondary alcohol group to form ABA-aldehyde (172) which is subsequently oxidized to abscisic acid (ABA, 173).

7.9.1.2. Strigolactones Strigolactones are plant hormones that regulate signals in parasitic and symbiotic interactions. Strigolactones, like ABA (173), are synthesized from carotenoids. An important biosynthetic intermediate in strigolactone biosynthesis is carlactone. This compound has a carbon skeleton similar to that of strigolactones and has been identified as the product of sequential reactions catalysed by three biosynthetic enzymes: D27, an iron-binding,

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plastid-localized ȕ-carotene isomerase; CCD7, a carbon 7 carotenoidcleavage dioxygenase; and CCD8, a carbon 8 carotenoid cleavage dioxygenase. These enzymes use ȕ-carotene (all trans) (161) as a substrate. In these sequential reactions, D27 catalyses the reversible isomerization in C-9 of ȕ-carotene (all trans) to produce 9-cis-ȕ-carotene (174). CCD7 preferentially splits 9-cis-ȕ-carotene to produce ȕ-ionone (175) and 9-cis-ȕ-apo-10'-carotenal (176), which is then cleaved by CCD8 to produce (R)-carlactone (177), which is an intermediated compound in the strigolactone pathway containing only A and D rings with an enol ether bridge. Carlactone acts as an endogenous precursor for more specific strigolactones that possess the same stereochemistry in position C-20 (configuration R). For instance, the natural compound 5-deoxystigol has a (+)-5DS (178) or (-)-2’-epi-5DS (179) configuration. In rice, the stable labelled isotope of carlactone is transformed into strigolactones such as orobancol (180) and sorgolactone (181). Only 11-R-carlattone acts as a precursor for strigolactones.

7.10. Polyterpenes The last terpenes class is that of the polyterpenes, which are large polymers formed by the repetition of a consistent number of isoprene units. The most well-known molecules belonging to this class are gum (182) and gutta-percha (183). The former is a linear polymer with double

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bonds predominantly in cis conformation extracted from the rubber tree (Hevea brasiliensis), whereas gutta-percha is extracted from species of the genus Palaquium and is characterized by double bonds that give conformations predominantly in trans. Trans-1,4-polyisoprene (TPI) is a non-petroleum-based material that possesses more elasticity with resistance to biological degradation than cis-polyisoprene (natural rubber). TPI is synthesized using IPP as a substrate deriving from the two terpene pathways: MVA and MEP. A putative trans-isoprenyl diphosphate synthase (TIDS) has been identified and TIDS2 and TIDS4 from Eucommia ulmoides were confirmed as having farnesyl diphosphate synthase (FDPS) activity. Other putative enzymes, TIDS1 and TIDS3, might be long-chain trans-polyprenyl diphosphate synthases. Rubber elongation factor (REF) is the most abundant protein found on the rubber particles or latex from Hevea brasiliensis and is considered to play important roles in natural rubber (cis-polyisoprene) biosynthesis.

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Seki, H. et al. (2008). Licorice beta-amyrin 11-oxidase, a cytochrome P450 with a key role in the biosynthesis of the triterpene sweetener glycyrrhizin. Proc. Natl. Acad. Sci. USA. 105, 14204–14209. Seki, H. et al. (2011). Triterpene functional genomics in licorice for identification of CYP72A154 involved in the biosynthesis of glycyrrhizin. Plant Cell. 23, 4112–4123. Seto, Y. and Yamaguchi, S. (2014). Strigolactone biosynthesis and perception. Curr. Opin. Plant Biol. 21, 1–6. Seto, Y., Kameoka, H., Yamaguchi, S. and Kyozuka, J. (2012). Recent advances in strigolactone research: chemical and biological as-pects. Plant Cell Physiol. 53, 1843–1853. Sharkey, T.D. and Monson, R.K. (2014). The future of isoprene emission from leaves, canopies and landscapes. Plant Cell Environ. 37, 1727– 1740. Sharkey, T.D. and Monson, R.K. (2017). Isoprene research – 60 years later, the biology is still enigmatic. Plant, Cell Environ. 40, 1671–1678. Shumskaya, M. and Wurtzel, E.T. (2013). The carotenoid biosynthetic pathway: thinking in all dimensions. Plant Sci. 208, 58–63. Soliman, S. and Tang, Y. (2015). Natural and engineered production of taxadiene with taxadiene synthase. Biotechnol. Bioengin. 112, 229– 235. Thimmappa, R., Geisler, K., Louveau, T., O'Maille, P. and Osbourn, A. (2014). Triterpene biosynthesis in plants. Ann. Rev. Plant Biol. 65, 225–257. Vaidya, A.D.B. and Devasagayam, T.P.A. (2007). Current status of herbal drugs in India: an overview. J. Clin. Biochem. Nutr. 41, 1–11. Weathers, P.J., Elkholy, S., and Wobbe, K.K. (2006). Artemisinin: the biosynthetic pathway and its regulation in Artemisia annua, a terpenoid-rich species. In Vitro Cell. Devel. Biol. Plant. 42, 309–317. Yamane, H. (2013). Biosynthesis of phytoalexins and regulatory mechanisms of it in rice. Biosci. Biotechnol. Biochem. 77, 1141–1148. Yang, F.-Y. et al. (2010). Bufadienolides and phytoecdystones from the rhizomes of Helleborus thibetanus (Ranunculaceae). Biochem. Syst. Ecol. 38, 759–763. Zerbe, P. et al. (2014). Diterpene synthases of the biosynthetic system of medicinally active diterpenoids in Marrubium vulgare. Plant J. 79, 914–927.

CHAPTER EIGHT OXYLIPIN BIOSYNTHETIC PATHWAY

Fatty acids, the main lipid components, are common to all living cells and are the key components of membranes and storage lipids. Beside these central functions, fatty acids and their derivatives also play an important role as signal molecules and as precursors of volatile molecules. In addition to fatty acid turnover within lipids, oxidation of unsaturated lipids is one of the major reactions of lipid metabolism. Plant oxylipins are a distinct class of lipid metabolites that derive from the oxidation of unsaturated fatty acids. Plant oxylipins include hydroperoxide hydrocarbons, hydroxyl-, oxo- or keto-fatty acids, divinyl ethers, volatile aldehydes and the plant hormone jasmonic acid and its precursor (oxophytodienoic acid).

8.1. Biosynthesis of Oxylipins In most cases, the first step in oxylipin biosynthesis is the formation of fatty acid hydroperoxides which are catalysed by either enzymatic processes or by chemical (auto)oxidation. The main substrates are linoleic acid (1) [18: 2 (Ÿ6): where x: y (Ÿ) is a fatty acid containing x carbon atoms and y double bonds in Ÿ position from the methyl terminal], Įlinolenic acid (2) [18: 3 (Ÿ3)] and ruganic acid (3) [16: 3 (Ÿ3)]. Hydroperoxides of these fatty acids are predominantly formed by lipoxygenase (LOX). Plants possess a variety of different LOX isoforms that can be divided into two groups: Type 1, containing all 9-LOX isoenzymes found exclusively outside the plastids, and Type 2, which includes all isoenzymes located in the plastid, such as the 13-LOX. Hydroperoxides (4) can also be generated by Į-dioxygenases (Į-DOX). Further hydroperoxide conversions can take place through several alternative routes, including those catalysed by divinyl ether synthase (DES), with the formation of divinyl ether (5), LOX, with the formation of ketooctadecadi(tri)enoic acids (6), allene oxide synthase (AOS), that forms epoxy-octadecatrienoic acids (7), hydroperoxide lyase (HPL), that

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generates (3-Z)-aldehydes (8) and Ȧ-oxo fatty acids (9), peroxigenase (PXG), that generates hydroxyl- (10) and epoxyl- (11) derivatives, or epoxy alcohol synthase (EAS), that catalyses the formation of epoxyalcohols (12, 13). Along with the enzymatic conversion, which leads to the formation of pure oxylipin enantiomers, oxidative stress and the formation of reactive oxygen species can lead to chemical peroxidation of membrane lipids. The most abundant fatty acids, linoleic acid (1) and linolenic acid (2), are particularly susceptible to oxidation by free radicals, providing racemic mixtures of peroxy fatty acid radicals. Oxylipin degradation products include compounds known as green leaf volatiles, which will be discussed in the next section.

8.2. Biosynthesis of Green Leaf Volatiles (Glvs) Green leaf volatile (GLV) is a generic name used to refer to a volatile sixcarbon compound produced and/or emitted by plants. (E)-2-Hexenal (14) was the first member of the GLVs to be isolated, in 1912. Thereafter, more than ten GLV compounds have been isolated. Because they have peculiar green leaf-like odour properties, we can smell them during mowing or the chewing of green vegetables. As such, the most prominent feature of GLVs is that they form rapidly after disrupting green leaves. Because intact leaves homogenized with care to suppress enzyme reactions (e.g.,

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extraction with organic solvents or disruption under anaerobic conditions) form only small quantities of GLVs, their rapid formation in nature seems to be achieved through de novo synthesis. This is in contrast to the synthesis of terpenoids. Rapid emission of volatile terpenoids after tissue disruption is typical of herbal plants that constitutively store terpenoids in glandular trichomes (see Chapter 2).

8.2.2. Site of Synthesis of GLVS GLVs are quickly released from wounded tissues. This happens during both abiotic and biotic stress. Mechanical wounding is the major cause of GLV release; it can be caused either by natural events or herbivory. However, emission of GLVs is influenced by environmental conditions that do not necessarily require wounding, such as temperature or soil conditions. Rising temperature substantially increases VOC emissions and light is another important abiotic factor that influences GLV emission. The emission of GLVs in conditions where the leaf tissues are intact requires their release through leaf stomata. However, recent findings indicate that in stress conditions where stomata close, GLVs and other volatiles are still emitted.

8.2.3. Biochemical Pathway to GLV Production Generally, free fatty acids are considered the commencing substances of the GLV pathway. Experiments have demonstrated that following labeling with 13CO2, the alcohol moiety of one of the main GLVs, (Z)-3-hexenyl acetate (15), was unlabelled, while the acetate moiety showed strong labelling. Therefore, Į-linolenic acid (1), the precursor of the alcohol moiety, is obtained from storage products. This view has been accepted because plant lipoxygenases (LOX) usually prefer free fatty acids rather than fatty acyl groups in lipids, at least in vitro. The lipoxygenase pathway presents the most widely accepted enzymatic pathway for conversion of unsaturated fatty acids, especially linolenic acid (1), to GLVs. Lipoxygenase (LOX) (linoleate:oxygen oxidoreductase) constitutes a large gene family of non-heme iron containing fatty acid dioxygenases, which are ubiquitous in plants and animals. LOX enzymes are referred to as 9and 13-LOXs and act on polyunsaturated fatty acids at the C9 and C13 positions respectively, yielding two groups of hydroperoxides. 9-LOXs comprise a subfamily of proteins that share high amino-acid sequence identity (~60%) to one another, but 13-LOXs are more diverse, sharing only ~35% sequence identity among themselves.

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During GLV biosynthesis, the C18 acids (e.g., linolenic acid, 1) contained in membrane lipids are removed from the lipid moiety by lipase, oxidized by LOX to the corresponding 13-hydroperoxy derivative (e.g., 13hydropreoxy linolenic acid, 16) and then cleaved to C12 and C6 compounds by hydroperoxide lyases (HPL), a P450 protein that catalyses the scission of hydroperoxides at the level of the oxygenated function, releasing volatile C6 or C9 carbonyl fragments. With (13S)hydroperoxyoctadecatrienoic acid (13-HPOT, 16) as the precursor, the first C6 GLV compound synthesized by the LOX/lyase pathway is (Z)-3hexenal (17), which is then converted to other GLVs such as (E)-2-hexenal (14) (leaf aldehyde) that leads to (E)-2-hexen-1-ol (18), or to (Z)-2hexenol (19) (leaf alcohol) and 3-(Z)-hexenyl acetate (15) (leaf acetate). The latter is formed by the reaction between (Z)-3-hexenol (20) and acetyl-CoA, a reaction catalysed by an acyltransferase. The C9 compound (e.g., 12-oxo-(Z)-9-dodecenoic acid, 21) is then hydroxylated to 9hydroxy-12-oxo-(Z)-9-dodecenoic acid (22). (Z)-3-Hexenal (17) produced by HPL is quite unstable and is converted to (Z)-2-hexenal (14) non-enzymatically or through the activity of (Z)-3:(E)2-enal isomerase. Some plants, such as soybean, possess two pathways that metabolize (Z)-3-alkenals. One is a soluble (Z)-3:(E)-2-enal isomerase that transforms (Z)-3-hexenal and (Z)-3-nonenal (23) into the corresponding (E)-2-alkenals. The other is a membrane-bound system that converts (Z)-3-hexenal and (Z)-3-nonenal into the corresponding hydroxyl derivatives. The aldehydes produced in plant tissues are usually reduced to alcohols by the catalysis of alcohol dehydrogenase (ADH) or cinammic aldehyde dehydrogenase, which are soluble enzymes whose activity depends on the supply of reducing equivalents in the form of NADPH. ADH1 converts hexanal into hexanol and (Z)-3-hexenal into (Z)-3-hexenol. Volatile alcohols are substrates for ester formation catalysed by alcoholacyl transferase (AAT), the enzyme responsible for most of the sweet and fruity sensory notes found in plant food products, which esterifies a volatile alcohol with acyl-CoA derivatives to produce volatile esters. In Arabidopsis, AAT has been characterized as a member of the BAHD acyltransferase family, called CoA:3-(Z)-hexenol acetyltransferase (CHAT) and predicted to be localized in the cytoplasm. The concentration of free fatty acids in intact tissues is generally low, thus rapid and massive formation of GLVs seems to be supported by the

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liberation of free fatty acids from membrane lipids. If this is the case, a lipase that liberates free fatty acids serving as substrates of LOX should be essential to the control of the rapid formation of GLVs after the disruption of leaves. When plants are fed with enriched 13CO2, not all GLVs exhibit similarly strong labelling patterns (hexenyl acetate vs hexyl acetate), indicating the existence of different precursors. Alcohols are strongly labelled but the acetate moiety, acetyl-CoA, is likely to derive from a different cellular pool to that used in chloroplastic fatty acid synthesis, or is rapidly synthesized after the end of labelling.

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When a targeted lipidome analysis was carried out with disrupted leaf tissues of an Arabidopsis thaliana mutant lacking active HPL, accumulation of free forms of fatty acid hydroperoxides that should be cleaved by HPL in wild type Arabidopsis lines was hardly observed. In addition, in wild type Arabidopsis that had an active HPL, the amount of C12 oxo-acid (traumatin; 12-oxo-(Z)-dodec-9-enoic acid, 22) that should be formed in a stoichiometric manner with its counterpart, i.e., C6 volatiles, was significantly lower than was expected. Instead of a free form of traumatin, an accumulation of traumatin that was esterified as galactolipids was evident. These lines of evidence indicate that the acyl groups of galactolipids are converted into GLVs through LOX and HPL reaction without being liberated from the glycerol backbone by a lipase. The resultant galactolipid hydroperoxides are also catalysed by HPL. The esterified traumatin is found in cabbage (Brassica oleracea), tobacco (Nicotiana tabacum), tomato (Solanum lycopersicum) and common bean (Phaseolus vulgaris); thus it was expected that GLV formation without releasing free fatty acid is rather common, showing a distinct contrast to the esterified oxophytodienoic acid (see below) found only within the Brassicaceae. While GLVs are usually defined as saturated and unsaturated C6 alcohols, aldehydes and esters, it has recently been shown that C5 compounds (2pentenyl acetate, 23; and 2-penten-1-ol, 24) can be constituents of the GLVs as well. C5 volatiles such as 1-penten-3-one (25), (E)-2-pentenal (26), 3-pentanone (27), 1-pentanol (28), and 1-penten-3-ol (29) are the most important volatiles contributing to tomato flavour; however, compared to the well-known C6 volatile biosynthetic pathway, the synthesis of C5 compounds is less established. The formation of 1-penten3-one (25) from soybean (Glycine max) is dependent on 13-HPOT. In a proposed pathway, LOX oxygenates 13-HPOT, followed by a ȕelimination, which generates the C5 compounds. C5 compounds such as 1penten-3-ol (29) and (Z)-2-pentenal (30) were also observed to accumulate at higher levels in HPL-depleted transgenic potato and Arabidopsis plants, but the mechanism was not identified. One of the LOX isozymes in tomato (TomloxC) was essential to the formation of C5 volatiles in tomato fruits. Also in this case, HPL or HPL-like enzymes are not needed, but the LOX catalysed oxygenation of fatty acid, and the hydroperoxide thus formed, subsequently cleaved in a hemolytic way to yield C5 compounds.

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8.3. Biochemical Pathway to Jasmonates Jasmonates are important regulators of plant response to biotic and abiotic stresses, as well as fundamental regulators of development. Jasmonic acid (JA, 31), the main compound of this class, is synthesized from lipid components and is then converted into different metabolites, including its isoleucine conjugate. The biosynthesis of JA and (+)-7-iso-JA-isoleucine (32) occurs in three different compartments of the plant cell. In the chloroplast, Į-linolenic acid is released from the galactolipids of the thylakoid membranes by phospholipase A1 (PLA1). Į-Linolenic acid is then oxygenated by a 13lipoxygenase (13-LOX) to give rise to the 13-HPOT (16), which is converted into an unstable epoxide by the 13-oxene-synthase (13-AOS) and then cyclized by an oxide cyclase (AOC) to cis-(+)-12-oxophytodienoic acid (OPDA, 33). OPDA is then transported to the peroxisomes where the cyclopentenone ring is reduced by the OPDA reductase 3 (OPR3), which forms 3-oxo-2-(2-pentenyl)-cyclopentane-1octanoic acid (OPC-8, 34). Subsequently, the ȕ-oxidation mechanism of fatty acids catalyses the shortening of the carboxylic acid side chain to form (+)-7-iso-JA (35), which is released into the cytosol and epimerizes to (-)-JA (31).

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Conjugation with amino acids, such as isoleucine, is catalysed by jasmonic acid-amino acid synthase (JAR1), which gives rise to conjugates such as (+)-7-iso-JA-isoleucine (32). An important modification of JA is the catalysis operated by the JA carboxyl methyl transferase (JMT) which produces the volatile compound methyl jasmonate (36). This compound is involved in plant communication and is a powerful inducer of plant responses to biotic stimuli.

Another compound that derives from JA is cis-jasmone, a molecule highly valued in perfumery that controls pollination, attracts the parasitoids of herbivores and serves as a regulator of gene expression. Two pathways lead to the formation of cis-jasmone: in the first pathway (Pathway A), the key step is the conversion of cis-OPDA (33) into iso-OPDA (37). This compound is then degraded by ȕ-oxidation into 3,7-didehydrojasmonic acid (38). The second pathway (Pathway B) involves reduction of cisOPDA by OPDA reductase 3 (OPR3) and subsequent ȕ-oxidation to yield (+)-7-iso-JA (35). The latter compound is then transformed to 3,7didehydrojasmonic acid (38), which produces, by spontaneous decarboxylation, cis-jasmone (39).

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Suggested Reading Andreou, A., Brodhun, F. and Feussner, I. (2009). Biosynthesis of oxylipins in non-mammals. Progr. Lipid Res. 48, 148–170. Bottcher, C. and Pollmann, S. (2009). Plant oxylipins: plant responses to 12-oxo-phytodienoic acid are governed by its specific structural and functional properties. Febs J. 276, 4693–4704. Christensen, S.A. et al. (2013). The maize lipoxygenase, ZmLOX10, mediates green leaf volatile, jasmonate and herbivore-induced plant volatile production for defense against insect attack. Plant J. 74, 59–73. Connor, E.C., Rott, A.S., Zeder, M., Juttner, F. and Dorn, S. (2008). 13Clabelling patterns of green leaf volatiles indicating different dynamics of precursors in Brassica leaves. Phytochemistry. 69, 1304–1312. D'Auria, J.C., Pichersky, E., Schaub, A., Hansel, A. and Gershenzon, J. (2007). Characterization of a BAHD acyltransferase responsible for producing the green leaf volatile (Z)-3-hexen-1-yl acetate in Arabidopsis thaliana. Plant J. 49, 194–207. Dabrowska, P. and Boland, W. (2007). iso-OPDA: an early precursor of cis-jasmone in plants? Chembiochem. 8, 2281–2285. Engelberth, J., Alborn, H.T., Schmelz, E.A. and Tumlinson, J.H. (2004). Airborne signals prime plants against insect herbivore attack. Proc. Natl. Acad. Sci. U.S.A. 101, 1781–1785. Farmer, E.E., Almeras, E. and Krishnamurthy, V. (2003). Jasmonates and related oxylipins in plant responses to pathogenesis and herbivory. Curr. Opin. Plant Biol. 6, 372–378. Howe, G.A. (2004). Jasmonates as signals in the wound response. J. Plant Growth Regul. 23, 223–237. Joo, Y.C. and Oh, D.K. (2012). Lipoxygenases: potential starting biocatalysts for the synthesis of signaling compounds. Biotechnol. Adv. 30, 1524–1532.

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Krieg, P. and Furstenberger, G. (2014). The role of lipoxygenases in epidermis. Biochim. Biophys. Acta Mol. Cell Biol. Lipids. 1841, 390– 400. Lange, B. and Turner, G.W. (2013). Terpenoid biosynthesis in trichomes – current status and future opportunities. Plant Biotechnol. J. 11, 2–22. Maffei, M.E. (2010). Sites of synthesis, biochemistry and functional role of plant volatiles. South Afr. J. Bot. 76, 612–631. Maffei, M.E., Gertsch, J., and Appendino, G. (2011). Plant volatiles: production, function and pharmacology. Nat. Prod. Rep. 28, 1359– 1380. Matsui, R. et al. (2017). Elucidation of the biosynthetic pathway of cisjasmone in Lasiodiplodia theobromae. Scientific Rep. 7, 6688. Mosblech, A., Feussner, I. and Heilmann, I. (2009). Oxylipins: structurally diverse metabolites from fatty acid oxidation. Plant Physiol. Biochem. 47, 511–517. Salas, J.J., Garcia-Gonzalez, D.L. and Aparicio, R. (2006). Volatile compound biosynthesis by green leaves from an Arabidopsis thaliana hydroperoxide lyase knockout mutant. J. Agric. Food Chem. 54, 8199– 8205. Schaller, F. (2001). Enzymes of the biosynthesis of octadecanoid-derived signalling molecules. J. Exper. Bot. 52, 11–23. Schaller, F., Schaller, A. and Stintzi, A. (2004). Biosynthesis and metabolism of jasmonates. J. Plant Growth Regul. 23, 179–199. Shen, J. et al. (2014). A 13-lipoxygenase, Tom-loxC, is essential for synthesis of C5 flavour volatiles in tomato. J. Exper. Bot. 65, 419–428. Shiojiri, K. et al. (2006). Changing green leaf volatile biosynthesis in plants: An approach for improving plant resistance against both herbivores and pathogens. Proc. Natl. Acad. Sci. USA. 103, 16672– 16676. Sugimoto, K. et al. (2014). Intake and transformation to a glycoside of (Z)3-hexenol from infested neighbors reveals a mode of plant odor reception and defense. Proc. Natl. Acad. Sci. USA. 111, 7144–7149. Wasternack, C. (2007). Jasmonates: an update on biosynthesis, signal transduction and action in plant stress response, growth and development. Ann. Botany. 100, 681–697. Wasternack, C. and Hause, B. (2013). Jasmonates: biosynthesis, perception, signal transduction and action in plant stress response, growth and development. An update to the 2007 review in Annals of Botany. Ann. Botany. 111, 1021–1058. Zebelo, S.A. et al. (2011). Chrysolina herbacea modulates terpenoid biosyn-thesis of Mentha aquatica L. PLoS One. 6, e17195.

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Zebelo, S.A., Matsui, K., Ozawa, R. and Maffei, M.E. (2012). Plasma membrane potential depolarization and cytosolic calcium flux are early events involved in tomato (Solanum lycopersicum) plant-to-plant communication. Plant Sci. 196, 93–100.

CHAPTER NINE BIOSYNTHESIS OF BIOACTIVE NITROGEN-CONTAINING MOLECULES

In addition to proteins, nitrogen bases and some hormonal classes such as cytokinins and polyamines, many other plant molecules not directly involved in the primary metabolism contain nitrogen. Among the most important nitrogen-containing bioactive compounds we will consider glucosinolates, cyanogenic glycosides and alkaloids. The degree of specialization of some species in producing bioactive molecules containing nitrogen is so advanced that even under nitrogen deficiency conditions these species continue to synthesize secondary metabolites to the detriment of primary metabolism. We discussed the importance of these molecules as defence compounds in Chapter 3; in this chapter we will explore some representative biosynthetic pathways involved in the production of the main classes of nitrogen compounds produced as bioactive molecules. We will begin by describing the toxic compounds containing a cyano group, the cyanogenic glycosides, and we will continue with the so-called “mustard oil” compounds, better known as glucosinolates. We will then discuss the widest and most important group of bioactive molecules containing nitrogen, the alkaloids. We will end the chapter with the biosynthesis of betalains.

9.1. Biosynthesis and Catabolism of Cyanogenic Glycosides Cyanide is a toxic compound for most living organisms and this is due to its ability to bind metals of functional groups or ligands of numerous enzymes. Examples of cyanide inhibition are the inhibition of cytochrome c oxidase in the respiratory chain, the inhibition of plastocyanin reduction in photosynthesis and the inhibition of catalase activity in photorespiration. Plants are able to metabolize cyanide; they possess an

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alternative oxidase and release this substance during the synthesis of the phytohormone ethylene. In some plants, the production of cyanide and cyanogenic compounds is so high that it can be used as a metabolic source of nitrogen or as a chemical weapon to defend the plant against herbivore attacks. Cyanogenic glycosides are synthesized from specific amino acids in a series of reactions catalysed by two membrane-related multi-functional P450 cytochromes (P450aa and P450ox) and a soluble UDPglucosyltransferase, with an oxime and an Į-hydroxynitrile (cyanohydrin) as key intermediates. Cyanogenesis occurs when the ȕ-glucosidic bond is hydrolyzed by a specific ȕ-glycosidase to form an unstable Įhydroxynitrile, which dissociates into hydrogen cyanide (HCN) and a ketone in a reaction that either occurs spontaneously at high pH or is catalysed by an Į-hydroxynitrilase. The general scheme for the production and catabolism of the cyanogenic glycoside dhurrin involves the formation of the oxime (E)-phydroxyphenyl acetaldehydeoxime (2) starting from the amino acid Ltyrosine (1) by catalysis of CYP79A1 and the subsequent CYP71E1dependent oxidation of the oxime to the cyanohydrin phydroxymandelonitrile (3). This last compound is glucosylated by the glucosyltransferase UGT85B1 to form the cyanogenic glycoside dhurrin (4).

The catabolism of dhurrin (4) starts with the hydrolysis of the molecule catalysed by dhurrinase, which releases glucose and generates the cyanhydrin p-hydroxymandelonitrile (3). The catalytic action of Įhydroxyinitrile lyase releases hydrogen cyanide (5) and produces the ketone p-hydroxybenzaldehyde (6).

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Another cyanogenic glycoside is amygdalin, produced in the seeds of almonds (Prunus amygdalus var. amara) and in many other species of the same genus (peaches, apricots, plums and cherries). Amygdalin (7) is a cyanogenic diglucoside that generates prunasin (8) after hydrolysis of the first glucose. In turn, this compound can be further hydrolysed to the cyanohydrin benzaldehyde mandelonitrile (9), which is cleaved into benzaldehyde (10) and hydrogen cyanide (5).

9.2. Biosynthesis and Catabolism of Glucosinolates Closely related to cyanogenic glycosides are glucosinolates, sulphurcontaining glucosidic compounds. These molecules are not toxic per se, but their enzymatic degradation gives rise to a complex mixture of toxic compounds including isothiocyanates and their derivatives. In general, the site of synthesis of these molecules is located in the outer parts of the fruit and in the leaves. Unlike the main classes of natural plant metabolites, glucosinolates include a relatively small but variegated group of nitrogen- and sulfur-containing compounds, which are largely limited to species of the Brassicales, which include species of the genus Brassica as well as the model plant Arabidopsis thaliana. The basic structure of glucosinolates (the glucone) derives from specific protein amino acids and includes a ȕ-thioglucosyl residue bound to an Į carbon to form a sulphated ketoxime. The extensive modification of the glucosinolate lateral side chain and the chain elongation of the amino acids are responsible for the chemical diversity of more than 120 identified

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structures. These stable and hydrophilic compounds are normally sequestered in the vacuoles of many plant tissues. Loss of cell integrity initiates the degradation of glucosinolates by hydrolysis of the glucosidic bond, a reaction catalysed by the enzyme myrosinase. Myrosinases are specific ȕ-thioglucosidases located in the idioblasts (myrosine cells) that are scattered in tissues of plants producing glucosinolates. The biosynthesis of glucosinolates proceeds in three phases: (i) amino acid side chain elongation; (ii) formation of a core glucosinolate structure; and (iii) secondary modifications of the side chain. The first process of chain elongation is the deamination of amino acids (11) such as methionine to the corresponding 2-oxo acids by a branchedchain amino acid aminotransferase (BCAT). The 2-oxo acids are precursors of the elongation reaction through a methylene group. The process of continuous elongation continues through the catalysis of methylthioalkylmalate synthase (MAM), isopropylmalate isomerase (IPMI) and isopropyl malate dehydrogenase (IPMDH). Finally, the 2-oxo elongated acids are transformed into the corresponding amino acids by BCAT. This chain elongation also occurs in the biosynthesis of aromatic glucosinolates, but does not occur in the formation of indole glucosinolates. Amino acids, including those with an elongated shape, are then used for the formation of the basic structure of glucosinolates. Cytochromes P450 (CYP79s) convert amino acids to aldoximes (12), which are then oxidized into the active forms by CYP83s. The active forms are transformed into thiohydroxymates (13) by conjugation and the C-S lyase (SUR1) reaction. The thiohydroxymates are finally converted first into desulphoglucosinolates (14) by the S-glucosyltransferases of the UGT74 family and then to glucosinolates (15) by catalysis of the sulfotransferases (SOTs). After formation of the glucosinolate structure, the side chains are modified by oxygenation, hydroxylation, alkenylation, benzoylation and methoxylation processes. The S-oxygenation of aliphatic glucosinolates is a common variant catalysed by Flavin monooxygenases (FMOGS-OXS). The S-oxygenated aliphatic glucosinolates, such as glucoraphanin (16), are found in many Brassicaceae. Alkenyl glucosinolates such as sinigrin (17) are produced from 2-ketoglutarate-dependent dioxigenases (AOPs) from S-oxygenated glucosinolates. Glucobrassicin (18) is an indolic glucosinolate which in Arabidopsis is hydroxylated by CYP81F2.

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The catabolism of glucosinolates occurs when plant tissues are damaged because of the disruption of plant tissues. Glucosinolates (19) are hydrolysed quickly with inherent myrosinase (ȕ-thioglucoside glucohydrolase, thioglucosidase, EC3.2.1.147), with the release of glucose and the production of an array of bioactive molecules including isothiocyanates (20), thiocyanates (21), nitriles (22), goitrin (23) and epithionitriles (24), depending upon pH and other conditions. As the glucosinolates and myrosinases are stored in different compartments, tissue rupture is necessary to bring them into contact. Glucosinolate hydrolysis then forms different breakdown products that have variable bioactivities.

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9.3. Biosynthesis of Alkaloids Alkaloids are widespread in the biosphere and are present in plants, animals (insects and marine invertebrates), fungi and microorganisms. Among the several known chemical structures, many have medical applications and for millennia have been known and used for the treatment of diseases. Other alkaloids have long been known for their ability to act on the nervous system by blocking nerve communications or altering them, causing moderate, harmful or lethal effects. Some alkaloids are powerful antibiotics. Because of their powerful biological activity, many of the roughly 12,000 known alkaloids have been exploited as pharmaceuticals, stimulants, narcotics and poisons. Unlike many other types of secondary metabolites, the many classes of alkaloids have unique biosynthetic origins. By definition, alkaloids are compounds that contain nitrogen in heterocyclic rings or in extracycles, and they can be divided into at least four groups:

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x The classic type of alkaloid in which there are more or less protonated secondary or tertiary amines which are hydrophilic at pH < 7.0 or non-protonated secondary or tertiary amines which are lipophilic at pH > 8.0. x Very polar quaternary amino compounds which are charged at all pH values. x Neutral amino compounds, including amide alkaloids. x N-oxides, generally soluble in water, present in many classes of alkaloids (e.g., pyrrolizidine alkaloids). As we discussed in Chapter 5, alkaloids can be subdivided from a biosynthetic point of view on the basis of their biogenetic origin. Although the major source of nitrogen and carbon for building alkaloids derives from amino acids, not all alkaloids derive from these molecules. Based on these considerations we can further subdivide the alkaloids into four groups: x Alkaloids deriving from amino acids (ornithine, arginine, lysine, histidine, tryptophan, phenylalanine and tyrosine). x Alkaloids deriving from purine. x Amino terpenes. x Polyketidic alkaloids, where the nitrogen atom is introduced into a polyketide carbon skeleton. The last two categories of alkaloids are certainly the most studied due to the large number of new molecules isolated and identified in insects and marine animals. For convenience, we will divide the various classes of alkaloids by grouping them according to the universally adopted nomenclature.

9.3.1. Biosynthesis of Piperidine Alkaloids Plant piperidine alkaloids are formed from acetic acid (25) or lysine, depending on the species. Hemlock (Conium maculatum) is known for its alkaloids which have been considered powerful poisons since the time of Plato. These simple piperidine derivatives possess a side chain consisting of three carbon atoms, and were once considered lysine derivatives. The metabolic pathway for the synthesis of these molecules takes place in the aerial parts of the plant and the biosynthesis begins with the amination of 5-ketooctanal (26), catalysed by L-alanine transaminase, to form 5ketooctylamine (27). The condensation of this compound gives rise to Ȗ-

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coniceine (28) (an extremely toxic alkaloid) that is reduced to coniine (29). Methylation of coniine by a methyltransferase that uses S-adenosyl methionine as a methyl donor leads to the synthesis of N-methylconiine (30).

Some species, such as Nicotiana glauca, accumulate anabasine, a pyridine-piperidine alkaloid similar to nicotine (see below) in both structure and effects, but much more toxic to human health. The piperidine ring of anabasin derives from cadaverine, which is formed by decarboxylation of lysine (31) by lysine decarboxylase (LDC). Cadaverine (32) is then oxidized by amine oxidase (AO) to 5-aminopentanal (33), which spontaneously cyclizes to form ǻ1-piperidein (34). The further coupling of a ǻ1-piperidein ring with a pyridine ring along with a series of reductions generates anabasine (35).

9.3.2. Biosynthesis of Tropane Alkaloids Tropane alkaloids, such as hyoscyamine and scopolamine, are an important class of plant anticholinergic bioactive metabolites present in different plant species including the genera Hyoscyamus, Atropa and Datura. In some species of Duboisia the biosynthesis of the tropane alkaloids hyoscyamine and scopolamine occurs through a biosynthetic pathway that starts from the amino acids ornithine and/or arginine, which are transformed into putrescine by ornithine decarboxylase. The methylation of putrescine (36) by putrescine:SAM N-methyltransferase (PMT) generates N-methylputrescine (37) which is subsequently deaminated oxidatively to 4-methylaminobutanal (38) by catalysis of methylputrescine oxidase (MPO). The spontaneous cyclization of 4-methylaminobutanal

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gives rise to N-methyl-ǻ1-pyrrolinium cation (39), which is also a precursor of other alkaloids such as nicotine and cocaine (see below).

Although not fully understood, scopolamine biosynthesis proceeds with the possible condensation of a molecule of the N-methyl-ǻ1-pyrrolinium cation (39) with acetoacetic acid (40) giving rise to hygrine (41), whose cyclization generates tropinone (42). This last molecule is subsequently reduced to tropine (43) by tropinone reductase I (TR-I). Tropine then condenses with the phenylalanine-derived phenyllactate (44) to generate littorine (45), which is transformed by littorine mutase/monooxygenase (CYP80F1) to hyoscyamine aldehyde (46) in a two-step reaction catalysed by Cyp80F1, probably with an alcohol dehydrogenase involved as a second enzyme. Hyoscyamine aldehyde is then reduced to hyoscyamine (47) and then two hyoscyamine 6ȕ-hydroxylases (H6H) convert hyoscyamine via 6ȕ–hydroxy–hyoscyamine (48) into scopolamine (49). As described above, the diamine putrescine generates an N-methyl-ǻ1pyrrolinium cation, which is an important metabolic intermediate for the formation of two other important molecules belonging to the pyrrolidine alkaloids: nicotine and cocaine. The pathway that generates nicotine starts from L-aspartate (50), which is oxidized by L-aspartate oxidase (AO) to Įiminosuccinic acid (51). The latter compound condenses with glyceraldehyde 3-phosphate (52) to form quinolinic acid (53), a reaction catalysed by the enzyme quinolinate synthetase (QS). Quinolinate phosphoribosyltransferase (QPT) converts quinolinic acid to nicotinic acid mononucleotide (54), which is then transformed to nicotinic acid (55) either directly by a nicotinic acid mononucleotide glycohydrolase (NAMN-GH), or through a multi-step process involving the synthesis and degradation of the ubiquitous coenzyme NAD.

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Nicotinic acid is then reduced to 3,6-dihydronicotinic acid (56) which is subsequently decarboxylated and then condensed with the N-methyl-ǻ1pyrrolinium cation (39) by catalysis of nicotine synthase (NS) to produce nicotine (57). How this metabolite becomes decarboxylated and coupled to the pyrrolidine ring, and whether additional intermediates are involved, remains a mystery.

One of the best known tropane alkaloids is cocaine (65), the benzoic ester of 2-carbomethoxy-3ȕ-tropine (methylecgonine, 63). The phenolic component of cocaine is formed from phenylalanine (58) which is

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successively deaminated to trans-cinnamic acid (59) by phenylalanine ammonia lyase (PAL), hydroxylated to 3-hydroxy-phenylpropanoate (60) and decarboxylated to form benzoyl-CoA (61). The transformation of the N-methyl-ǻ1-pyrrolinium cation (39) through a series of not yet demonstrated reactions leads to the formation of the oxobutanoic acid intermediate (62). The reduction of this compound generates methylecgonone (63) which is reduced to methylecgonine (64) by the action of methylecgonone reductase (MecgoR). The esterification of benzoyl-CoA (61) with methylecgonine (64) is catalysed by cocaine synthase (CS), a member of the BAHD (tyramine Nhydroxycinnamoyltransferase/serotonin N-hydroxycinnamoyltransferase) family of plant acyltransferases, and leads to cocaine (65) formation.

9.3.3. Biosynthesis of Benzylisoquinoline Alkaloids Benzylisoquinoline alkaloids are derivatives of L-tyrosine and are diversified due to an intricate biochemical network of intramolecular bonds, reductions, methylations, hydroxylations and other reactions that generate about 2,500 known structures. Many of these alkaloids are used as pharmaceuticals or as precursors of semi-synthetic drugs. Examples include the analgesic morphine, the cough suppressor codeine, the muscle relaxants papaverine and tubocurarine and the antimicrobial agent sanguinarine. The biosynthesis of these alkaloids can be dissected into three components: (i) formation of the benzylisoquinoline structure; (ii) rearrangement of the benzylisoquinoline skeleton in various ring configurations through carbon-carbon and carbon-oxygen phenol coupling; and (iii) addition of functional groups thus increasing the number of compounds in each subgroup.

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The six main natural sources of benzylisoquinoline alkaloids are: opium poppy (Papaver somniferum), Californian poppy (Eschscholzia californica), Mexican prickly poppy (Argemone mexicana), Coptis japonica, common meadow-rue (Thalictrum flavum) and a barberry (Berberis wilsoniae). The decarboxylation of the amino acid precursor tyrosine (66) generates tyramine (67) whose hydroxylation leads to the formation of dopamine (68). Alternatively, tyrosine can be hydroxylated to form dihydroxyphenylalanine (69) which subsequently is transformed into dopamine (68) by a decarboxylating enzyme. The transamination of tyrosine forms 4-hydroxyphenylpyruvic acid (70) which undergoes decarboxylation to form 4-hydroxyphenylacetaldehyde (71). The combination of the latter compound with dopamine occurs by catalysis operated by norcoclaurine synthase (NCS) and gives rise to (S)norcoclaurine (72), the precursor of all benzylisoquinoline alkaloids.

The incision of the immature capsules of Papaver somniferum produces a morpho-physiological reaction that leads to the formation of a whitish latex rich in alkaloids. Exposure to the air of this latex gives rise to opium, a resinoid in which the content of alkaloids can exceed 25%. The compound used as it is has analgesic and narcotic properties, but is also used to calm coughs. The tincture obtained from opium, laudanum, is included in the lists of numerous official pharmacopoeias and was once used as a narcotic. A benzylisoquinoline alkaloid present in opium is papaverine, a compound with a spasmolytic and vasodilatory action. Two routes for papaverine biosynthesis have been proposed: (i) an N-methyl pathway involving (S)reticuline and the N-demethylation of an unspecified intermediate by a hypothetical enzyme; and (ii) an N-desmethyl pathway involving (S)norreticuline and precluding the requirement for N-demethylation. Recent

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studies have demonstrated that the major route to papaverine is likely the N-desmethyl pathway. The biosynthesis of papaverine starts with the methylation of (S)-norcoclaurine (72) to give rise to (S)-coclaurine (73), a reaction catalysed by S-adenosyl-L-methionine:norcoclaurine 6-Omethyltransferase (6OMT). Hydroxylation by an uncharacterized enzyme (3ƍOHase) and methylation operated by a still unidentified 3ƍ-Omethyltransferase (3ƍOMT) transform (S)-coclaurine into (S)-norreticuline (74). Catalysis of norreticuline 7-O-methyltransferase (N7OMT) and (S)norreticuline 4ƍ-O-methyltransferase (4ƍOMT) generates (S)tetrahydropapaverine (75). The final step of papaverine (76) biosynthesis involves oxidation of the fully O-methylated and N-desmethyl compound (S)-tetrahydropapaverine by the catalytic activity of dihydrobenzophenanthridine oxidase (DBOX).

Thebaine is a morphine (see below) antagonist and the biosynthesis of this compound begins with the methylation of (S)-coclaurine (73) catalysed by coclaurine N-methyltransferase (CNMT) giving rise to (S)-Nmethylcoclaurine (77). This compound is hydroxylated by (S)-Nmethylcoclaurine 3ƍ-hydroxylase (NMCH, CYP80B3) to (S)-3ƍ-hydroxyN-methylcoclaurine (78), which is converted by 3ƍ-hydroxy-Nmethylcoclaurine 4ƍ-O-methyltransferase (4ƍOMT) into (S)-reticuline (79). The latter is epimerized to (R)-reticuline (80) via the dehydrogenation of (S)-reticuline to a 1,2-dehydroreticulinium ion, which is subsequently reduced to (R)-reticuline. (R)-reticuline is converted by the CYP salutaridine synthase (SalSyn, CYP719B1) into salutaridine (81). The reduction of the ketonic group of this compound generates salutaridinol (82), a reaction catalysed by the short-chain dehydrogenase/reductase salutaridine reductase (SalR). Subsequently, salutaridinol is O-acetylated by salutaridinol 7-Oacetyltransferase (SalAT) to form salutaridinol-7-O-acetate (83), which undergoes spontaneous cyclization to generate thebaine (84). The demethylation of thebaine by thebaine 6-O-demethylase (T6ODM) generates neopinone (85) which spontaneously rearranges into codeinone (86), whose reduction by the aldo–keto reductase codeinone reductase

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(COR) gives rise to codeine (87). This alkaloid is the most used among all those present in opium. It is an analgesic with about a tenth of the power of morphine and is used in the treatment of coughs. The demethylation of codeine by codeine O-demethylase (CODM) generates morphine (88), a powerful alkaloid with analgesic and narcotic actions, used in the treatment of pain and still irreplaceable in this use. A synthetic product derived from morphine is diamorphine, better known as heroin (89). The synthesis is obtained by acetylation of the two hydroxyl groups of morphine. A compound able to mimic the conformation of piperidines is methadone (90), which is used for medical purposes to treat people addicted to morphine and its derivatives.

9.3.4. Biosynthesis of Indole Alkaloids The decarboxylation of tryptophan to triptamine is the first step for the formation of indole alkaloids. One of the plant species known for the presence of indole alkaloids is Madagascar periwinkle (Catharanthus roseus). The alkaloid fraction (more than 150 different structures) of the plant extract contains highly bioactive indole alkaloids of terpenoid nature including vinblastine and vincristine. Vinblastine is used in the treatment of Hodking syndrome, whereas vincristine has superior antitumour activity, but is also more neurotoxic. The biosynthesis of the indole alkaloids vinblastine and vincristine involves two metabolic pathways described in Chapters 6 and 7, namely

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the shikimic acid and the MEP pathways. The first pathway gives rise to the amino acid tryptophan (91) which is decarboxylated to tryptamine (92) by tryptophan decarboxylase (TDC). The second pathway gives rise to geranyl diphosphate (GPP), which is transformed into geraniol (93) by geraniol synthase (GES), a member of the terpene synthase family. The enzyme cytochrome P450 geraniol 10-hydroxylase (G10H), also known as geraniol 8-oxidase (G8O, the name we will use here), is responsible for the hydroxylation of geraniol into 8-hydroxygeraniol (94). The next enzyme, 8-hydroxygeraniol oxidoreductase (8-HGO), oxidises the hydroxy group to an aldehyde in the substrates 8-hydroxygeraniol and 8-oxogeraniol in the presence of NAD+ to yield 8-hydroxygeranial (95), and 8-oxogeranial (96), respectively. An iridoid synthase (IS) functions for the cyclization of 8-oxogeranial into iridodial (97). The CYP enzyme iridoid oxidase (IO) catalyses the conversion of iridodial into 7-deoxyloganetic acid (98), which is glucosylated by the enzyme 7-deoxyloganetic acid glucosyltransferase (7-DLGT) forming 7-deoxyloganic acid (99) using UDP-glucose as the sugar donor. 7-deoxyloganic acid hydroxylase (7DLH), a P450 hydroxylase, catalyses the formation of loganic acid (100). The enzyme loganic acid methyltransferase (LAMT) catalyses the methylation of loganic acid to form loganin (101), whereas secologanin synthase (SLS) catalyses the conversion of loganin to secologanin (102). Finally, the condensation of tryptamine and secologanin, catalysed by strictosidine synthase (STR), generates strictosidine (103).

Strictosidine is then deglucosylated by strictosidine-ȕ-D-glucosidase (SGD) to yield an unstable aglycon, cathenamine (104), which is reversibly converted to 4,21-dehydrogeissoschizine (105) and then to

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stemmadenine (106); the latter is partly transformed into catharanthine (107) and partly transformed into tabersonine (108) by still unknown enzymes. The hydroxylation of tabersonine by tabersonine 16-hydroxylase (T16H) generates 16-hydroxytabersonine (109), which is methylated by 16-hydroxytabersonine O-methyltransferase (16OMT) to 16methoxytabersonine (110). Hydration of the 2,3-double bond of 16methoxytabersonine by an unidentified hydroxylase generates 16methoxy-2,3-dihydro-3-hydroxytabersonine (111), which is transformed to desacetoxyvindoline (112) by 16-methoxy-2,3-dihydro-3hydroxytabersonine N-methyltransferase (NMT). Hydroxylation at position 4 of desacetoxyvindoline by desacetoxyvindoline-4-hydroxylase (D4H) generates deacetylvindoline (113) which is acetylated to vindoline (114) by deacetylvindoline-4-O-acetyltransferase (DAT). The coupling of catharanthine (107) and vindoline (114) is catalysed by Į-3’,4’anhydrovinbastine synthase (AVLBS or PRX1), a basic peroxidase that produces anhydrovinblastine (115), the direct precursor of the two bisindole alkaloids vinblastine (116) and vincristine (117).

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9.3.4.1. Biosynthesis of Quinoline Alkaloids One of the most interesting modifications in the structure of indolic terpene alkaloids is the formation of the quinolinic structure. A genus known for the presence of such structures is Cinchona, belonging to the Rubiaceae family. In this genus, the bark accumulates a discrete amount of quinine, quinidine, cinchonine and cinchonidine alkaloids, known for their antimalarial properties. In the biosynthesis of quinine, strictosidine (103) is deglucosylated and the opening of the pyranic ring leads to the synthesis of corynantheal (118), which undergoes a structural rearrangement to form cinchonaminal (119). The opening of the pyrrole ring poses the molecular premises for the formation of an intermediate molecule (120) that gives rise to cinchonidinone (121) (which equilibrates with its epimer cinchoninone, 122). Cinchonidinone is methoxylated to quinine (123),

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whereas the NADPH-dependent reduction of cinchoninone leads to the synthesis of cinchonidine (124) and cinchonine (125).

9.3.4.2. Biosynthesis of Pyrroloindole Alkaloids The seeds of the Calabar bean (Physostigma venenosum) contain a powerful poison that causes paralysis and cardiac arrest. Among the alkaloids present, the most abundant is physostigmine (or eserine), used in ophthalmology as an antagonist of atropine. Due to its properties this alkaloid is used as a cure against anticholinergic poisons and seems to be particularly effective in the treatment of Parkinson’s and Alzheimer’s diseases. The biosynthesis of this compound has been indirectly elucidated in the bacterium Streptomyces griseofuscus. In physostigmine biosynthesis, tryptophan (91) is hydroxylated by tryptophan 5-hydroxylase to generate 5-hydroxytryptophan (126), which is decarboxylated by a pyridoxal 5’-phosphate (PLP)-dependent decarboxylase (PsmH) to 5hydroxytryptamine (127). The latter compound undergoes N-acetylation catalysed by 5-hydroxytryptamine N-acetyltransferase (PsmF) generating N-acetyl-5-hydroxytryptamine (128). The next steps are the carbamylation of N-acetyl-5-hydroxytryptamine catalysed by normelatonin Ocarbamoyltransferase (PsmE) followed by a methylation catalysed by a carbamoyl N-methyltransferase (PsmA) to yield an intermediate compound (129), which is methylated by a C3-methyltransferase (PsmD) and then undergoes a spontaneous cyclization to form the pyrroloindole skeleton (130). The latter compound is then methylated by an Nmethyltransferase (PsmC) to yield a product (131) which is then deacetylated by a deacetylase (PsmB) that generates a compound (132). Finally, this compound is N-methylated to physostigmine (133) by PsmC.

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9.3.4.3. Biosynthesis of Ergot Alkaloids Ergotism is a disease suffered due to the contamination of foodstuffs (especially rye flour) by fungi belonging to the genus Claviceps. The cause of the disease (also known as rye horn) is the presence of some alkaloids produced by the fungus with the participation of metabolites provided by the plant. Ergot alkaloids of the same kind are also produced by plants belonging to the Convolvulaceae family, such as Ipomea and Rivea. The structures of ergot alkaloids contain the ergoline tetracyclic ring. Ergot alkaloids can be divided into classes based on substituents bound to the ergoline skeleton; the main classes include clavines, simple derivatives of lysergic acid and ergopeptides. The biosynthetic pathway of ergot alkaloids begins with the prenylation of tryptophan (91) with a molecule of DMAPP, a reaction catalysed by dimethylallyltryptophan synthase (dmaW) that generates dimethylallyl tryptophan (134). The methylation of this compound by dimethylallyltryptophan N-methyltransferase (easF) leads to the formation of 4-dimethylallyl-L-abrine (135). Subsequently, a series of oxidative steps catalyse the intramolecular cyclization of the prenyl and indole portions to form the tricyclic ring of chanoclavine-I (136), a reaction catalysed by chanoclavine-I synthase (easE). Chanoclavine-I is then oxidized by chanoclavine I dehydrogenase (EasD) to form chanoclavine-Ialdehyde (137), a precursor common to all classes of ergot alkaloids.

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The reduction of the chanoclavine-I-aldehyde catalysed by agroclavine dehydrogenase (easG) leads to the production of agroclavine (138) which is hydroxylated by agroclavine monooxygenase (CloA) to form elymoclavine (139). Another catalysis by CloA hydroxylates elymoclavine to either paspalic acid (140) or D-lysergic acid (141).

9.3.5. Biosynthesis of Purine Alkaloids The purine bases adenine and guanine, fundamental components for the synthesis of nucleic acids, are transformed by some plants into alkaloids, the purine alkaloids. Some important molecules belong to this category, including caffeine, theobromine and theophylline. Caffeine is a central nervous system stimulant, while theobromine has a diuretic action as well as having a relaxing effect on smooth muscles. Theophylline has a smaller action on the central nervous system than the previous alkaloids, but has a greater relaxing effect on smooth muscles. Caffeine is produced, along with traces of theobromine and theophylline, by Coffea Arabica, Coffea canephora (Rubiaceae) and Camellia sinensis (Theaceae), whereas a higher content in theobromine is present in Theobroma cacao (Sterculiaceae).

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The biosynthesis of these alkaloids is a four-step sequence consisting of three methylation and one nucleosidase reactions. The starting molecule is a xanthine backbone deriving from purine nucleotides. The first step is the N-methylation of xanthosine (142), a reaction catalysed by a SAMdependent xantosine 7-N-methyl transferase (XMT) to form 7methylxanthosine (143). This compound is then hydrolysed by Nmethylnucleosidase (NS) generating 7-methylxanthine (144). Further methylation catalysed by a different SAM-dependent N-methyltransferase (theobromine synthase, TS) leads to the synthesis of theobromine (145) which is further methylated by caffeine synthase (CS) to produce caffeine (146).

Theophylline biosynthesis starts from xanthosine (142), which is first hydrolysed to xanthine (147) by NS and then methylated to position 3 to 3-methylxanthine (148) by N3-methyltransferase (N3MT). This compound is finally methylated to theophylline (149) by the anzymatic activity of N1methyltransferase (N1MT).

9.3.6. Biosynthesis of Other Alkaloids Some alkaloids are derivatives of the shikimic acid pathway, ephedrine (160) being one. This alkaloid, together with pseudoephedrine (161), is produced by species belonging to the genus Ephedra (Ephedraceae) and is used in Chinese Traditional Medicine for its bronchodilating and decongestant properties.

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The biosynthesis of these compounds starts from L-phenylalanine (58), which is deaminated by phenylalanine ammonia lyase (PAL) to transcinnamic acid (59). This compound undergoes a side-chain shortening by at least two possible pathways: ȕ-oxidative and non-ȕ-oxidative routes. In the first route, trans-cinnamic acid is transformed to cinnamoyl-CoA (150) by cinnamate:CoA ligase (CNL). Cinnamoyl-CoA hydratasedehydrogenase (CHD) first converts cinnamoyl-CoA to 3-hydroxy-3phenylpropanoyl-CoA (151) and then forms 3-oxo-3-phenylpropanoylCoA (152), whereas 3-ketoacyl-CoA thiolase (KAT) catalyses the formation of benzoyl-CoA (61). In the second route, trans-cinnamic acid is hydrated to 3-hydroxy-3-phenylpropanoic acid (153), which is transformed by retro-aldol cleavage into benzaldehyde (10), which is oxidized to benzoic acid (154) by either benzaldehyde dehydrogenase (BALDH) or aldehyde oxidases 4 (AO4). When benzoic acid is formed it is condensed with pyruvic acid (155) to form 1-phenylpropane-1,2-dione (156), a reaction catalysed by a not yet identified enzyme (either a ThDPdependent pyruvate decarboxylase [ThPDC] or an acetolactate synthase [AHAS]). Transamination of 1-phenylpropane-1,2-dione generates (S)cathinone (157) which is then reduced to (1R,2S)-norephedrine (158) and (1S,2S)-pseudonorephedrine (cathine) (159). These compounds are then methylated by a N-methyltransferase to (1R,2S)-ephedrine (160) and (1S,2S)-pseudoephedrine (161).

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Capsaicin is the alkaloid responsible for the pungent taste of chilli pepper (Capsicum sp.) fruits. The biosynthesis of this alkaloid requires both phenylpropanoid and the branched-fatty-acid pathways. In the first pathway, as for ephedrine, phenylalanine (58) is transformed by PAL to trans-cinnamic acid (59) and the catalytic action of cinnamic acid 4-hydroxylase (C4H) generates coumaric acid (162). This compound is transformed into 4-coumaroyl-CoA (163) by 4-coumarate-CoA ligase (4CL), whereas hydroxycinnamoyl transferase (HCT) generates caffeoylCoA (164). Caffeic acid O-methyltransferase (COMT) methylates caffeoyl-CoA to yield feruoyl-CoA (165). This last compound is then used as a substrate for the synthesis of vanillin (166) by a still not characterized acyltransferase (HCLH) (note that in this case protocatechuic aldehyde is not the direct precursor of vanillin as described in Chapter 6). Finally, an aminotransferase (pAMT) aminates vanillin to vanillylamine (167). In the second pathway, valine (168) is transformed to Į-ketoisovalerate (169) by branched-chain amino acid transferase (BCAT), whereas isovalerate dehydrogenase generates isobutyryl-CoA (170). This compound enters the fatty acid synthesis (with the activity of ketoacylACP synthase [KAS] and acyl-CoA synthetase [ACL] followed by acylACP thioesterase [FAT]) and is then transformed into 8-methyl-6nonenoic acid (171), which is transformed into 8-methyl-6-nonenyl-CoA (172) by acyl-CoA synthetase (ACS). Finally, capsaicinoid synthase (CS) (along with an acyl transferase, AT3), catalyses the condensation of vanillylamine (167) and 8-methyl-6-nonenyl-CoA (172) to yield capsaicin (173).

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9.4. Biosynthesis of Betalains Betalains are vacuolar nitrogen containing pigments having a core structure known as betalamic acid. Condensation of betalamic acid with cyclo-DOPA and its glucosyl derivatives, and with amino acids and their derivatives, leads to the formation of two respective categories of betalains: violet betacyanins and yellow betaxanthins. Because of the presence of the 1,7-diaza motif, some researchers have claimed these compounds as chromoalkaloids; however, their stability in slightly acidic pH seems to negate this claim. All betalains exhibit absorption maxima in both UV and visible regions owing, respectively, to the phenolic nature of the betalamic acid and the conjugated dienes of the 1,7-diazaheptamethin substructure. In the betalain biosynthetic pathway, the formation of L-DOPA (175) from tyrosine (174) is catalysed by CYP76AD6, which uniquely exhibits only tyrosine hydroxylase activity. L-DOPA is then converted to cyclo-DOPA (177) via dopaquinone (176), a reaction catalysed by a cytochrome P450 (CYP76AD1). A key enzyme in betalain biosynthesis, DOPA 4,5dioxygenase (DOD), catalyses the extradiol aromatic ring cleavage by converting DOPA (175) to 4,5-seco-DOPA (178), that then nonenzymatically rearranges to betalamic acid (179), which constitutes the basic backbone of all betalains.

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Betalamic acid is subjected to spontaneous Schiff base condensation with amino acids and amino derivatives to form betaxanthins (180) through aldimine bonding. In the biosynthesis of betalains, cyclo-DOPA (177) condenses with betalamic acid (179) to form betanidin (181), which is glucosylated by a glucosyltransferase (B5GT) to produce betanin (182).

Suggested Reading Agerbirk, N. and Olsen, C.E. (2012). Glucosinolate structures in evolution. Phytochemistry. 77, 16–45. Arce-Rodriguez, M. and Ochoa-Alejo, N. (2017). An R2R3-MYB transcription factor regulates capsaicinoid biosynthesis. Plant Physiol. 174, 1359–1370. Ashihara, H., Sano, H. and Crozier, A. (2008). Caffeine and related purine alkaloids: biosynthesis, catabolism, function and genetic engineering. Phytochemistry. 69, 841–856. Beaudoin, G.A.W. and Facchini, P.J. (2014). Benzylisoquinoline alkaloid biosynthesis in opium poppy. Planta. 240, 19–32. Bunsupa, S., Komastsu, K., Nakabayashi, R., Saito, K., and Yamazaki, M. (2014). Revisiting anabasine biosynthesis in tobacco hairy roots expressing plant lysine decarboxylase gene by using N-15-labeled lysine. Plant Biotechnol. 31, 511–518.

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De Luca, V., Salim, V., Levac, D., Atsumi, S.M. and Yu, F. (2012). Discovery and Functional Analysis of Monoterpenoid Indole Alkaloid Pathways in Plants. San Diego: Elsevier Academic Press Inc. Facchini, P.J. (2001). Alkaloid biosynthesis in plants: biochemistry, cell biology, molecular regulation, and metabolic engineering applications. Annu. Rev. Plant Physiol. Plant Mol. Biol. 52, 29–66. Facchini, P.J. and De Luca, V. (2008). Opium poppy and Madagascar periwinkle: model non-model systems to investigate alkaloid biosynthesis in plants. Plant J. 54, 763–784. Florea, S., Panaccione, D.G. and Schardl, C.L. (2017). Ergot alkaloids of the family Clavicipitaceae. Phytopathology. 107, 504–518. Gandia-Herrero, F. and Garcia-Carmona, F. (2013) Biosynthesis of betalains: yellow and violet plant pigments. Trends Plant Sci. 6, 334– 343. Gleadow, R.M. and Moller, B.L. (2014). Cyanogenic Glycosides: synthesis, physiology, and phenotypic plasticity. Annu. Rev. Plant Biol. 65, 155–185. Groves, R.A. et al. (2015). Transcriptome Profiling of Khat (Catha edulis) and Ephedra sinica reveals gene candidates potentially involved in amphetamine-type alkaloid biosynthesis. PLoS ONE. 10, e0119701 Hagel, J.M. and Facchini, P.J. (2013). Benzylisoquinoline alkaloid metabolism: a century of discovery and a brave new world. Plant Cell Physiol. 54, 647–672. Hagel, J.M., Krizevski, R., Marsolais, F., Lewinsohn, E. and Facchini, P.J. (2012). Biosynthesis of amphetamine analogs in plants. Trends Plant Sci. 17, 404–412. Havemann, J., Vogel, D., Loll, B. and Keller, U. (2014). Cyclization of Dlysergic acid alkaloid peptides. Chem. Biol. 21, 146–155. Ishida, M., Hara, M., Fukino, N., Kakizaki, T. and Morimitsu, Y. (2014). Glucosinolate metabolism, functionality and breeding for the improvement of Brassicaceae vegetables. Breed. Sci. 64, 48–59. Jakubczyk, D., Cheng, J.Z. and O'Connor, S.E. (2014). Biosynthesis of the ergot alkaloids. Nat. Prod. Rep. 31, 1328–1338. Khan, M.I. and Giridhar, P. (2015) Plant betalains: chemistry and biochemistry. Phytochemistry. 117, 267–295. Liscombe, D.K. and Facchini, P.J. (2008). Evolutionary and cellular webs in benzylisoquinoline alkaloid biosynthesis. Curr. Opin. Biotechnol. 19, 173–180. Liu, D.H. et al. (2007). Terpenoid indole alkaloids biosynthesis and metabolic engineering in Catharanthus roseus. J. Integr. Plant Biol. 49, 961–974.

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Liu, J., Ng, T., Rui, Z., Ad, O. and Zhang, W. (2014). Unusual acetylationdependent reaction cascade in the biosynthesis of the pyrroloindole drug physostigmine. Angew. Chem. Int. Ed. 53, 136–139. McKeague, M., Wang, Y.H., Cravens, A., Win, M.N. and Smolke, C.D. (2016). Engineering amicrobial platform for de novo biosynthesis of diverse methylxanthines. Met. Eng. 38, 191–203. O'Connor, S.E. and Maresh, J.J. (2006). Chemistry and biology of monoterpene indole alkaloid biosynthesis. Nat. Prod. Rep. 23, 532– 547. Pan, Q., Mustafa, N.R., Tang, T. Choi, Y.H. and Verpoorte, R. (2016). Monoterpenoid indole alkaloids biosynthesis and its regulation in Catharanthus roseus: a literature review from genes to metabolites. Phytochem. Rev. 15, 221–250. Polturak, G. et al. (2016) Elucidation of the first committed step in betalain biosynthesis enables the heterologous engineering of betalain pigments in plants. New Phytol. 210, 269–283. Schmidt, G.W. et al. (2015). The last step in cocaine biosynthesis is catalyzed by a BAHD acyltransferase. Plant Physiol. 167, 89–101. Sonderby, I.E., Geu-Flores, F. and Halkier, B.A. (2010). Biosynthesis of glucosinolates - gene discovery and beyond. Trends Plant Sci. 15, 283– 290. Ullrich, S.F., Hagels, H. and Kayser, O. (2017). Scopolamine: a journey from the field to clinics. Phytochem. Rev. 16, 333–353. Young, C.A. et al. (2015). Genetics, genomics and evolution of ergot alkaloid diversity. Toxins. 7, 1273–1302. Zagrobelny, M., Bak, S. and Moller, B.L. (2008). Cyanogenesis in plants and arthropods. Phytochemistry. 69, 1457–1468. Zhang, G.h. et al. (2016). De novo sequencing and transcriptome analysis of Pinellia ternata identify the candidate genes involved in the biosynthesis of benzoic acid and ephedrine. Front. Plant Sci. 7, e1209. Zhang, Z.-X. et al. (2016). Discovery of putative capsaicin biosynthetic genes by RNA-Seq and digital gene expression analysis of pepper. Sci. Rep. 6, e34121. Ziegler, J. et al. (2009). Evolution of morphine biosynthesis in opium poppy. Phytochemistry. 70, 1696–1707.

UNIT III BIOTECHNOLOGY OF BIOACTIVE PLANT MOLECULES

CHAPTER TEN IN VITRO PRODUCTION OF BIOACTIVE PLANT MOLECULES

In the two previous units we have discussed why bioactive metabolites are important for plants’ chemical defence against phytophagous and pathogenic attacks and for survival from abiotic stress. Furthermore, the second unit analysed the biochemical mechanisms that underlie the production of bioactive molecules produced by the secondary metabolism. The aim of this third and final unit is to tackle the problem of production of secondary metabolites from a biotechnological point of view. In this chapter, we will explore the relationships that exist between the vital metabolic processes that allow all organisms to live (the primary metabolism) and the dependence of the production of bioactive metabolites on the availability of basic molecules provided by the primary metabolism. After discussing the basic procedures for the preparation of cell and tissue cultures, we will analyse the biotechnology of production of secondary metabolites. We will focus on the mechanisms of accumulation of bioactive molecules in cell systems cultivated in vitro and on the possibility of regulating their biosynthetic production. We will conclude the chapter with an overview of some cellular systems developed for the production of metabolites of particular economic interest. One of the main characteristics of cell cultures is their lack of autotrophy; once dedifferentiated, cells lose their photosynthetic capacities and become totally dependent on external organic food supply sources. We will begin by analysing the relationships that exist between the primary metabolism and the production of bioactive molecules.

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10.1. Interaction between the Primary and Secondary Metabolisms Plants have the ability to fix carbon dioxide into complex molecules such as sucrose and glucose. However, this capacity is lost when tissues are cultivated under conditions that induce dedifferentiation. Under such conditions, plant cells need a source of sucrose to survive, just as those non-photosynthetic cells present in non-green parenchymatic tissues do. As we have discussed, secondary metabolites are molecules formed by carbon skeletons deriving from intermediates of the primary metabolism, which are variously substituted with hydrogen and oxygen and, in some classes of compounds, by nitrogen and sulphur atoms. If we consider the metabolic origin of secondary metabolites, we see that there is a clear and unequivocal interaction between the primary and secondary metabolisms. Starch, a polysaccharide produced in the chloroplasts and in the amyloplasts, is one of the most important plant energy molecules. This substance is the food base for the seed embryo, but it is also the most important food resource for humans and animals, being present in cereals. In plants, starch accumulates in storage tissues and is hydrolysed and variously degraded to glucose. Sucrose is also an important food source. Sucrose is made up of glucose and fructose and is easily hydrolysable into its two components. Fructose is enzymatically converted into glucose, which is phosphorylated to glucose 6-phosphate. This molecule enters a sequence of catabolic reactions known as anaerobic glycolysis, the first step in the demolition of sugar in the catabolic process of respiration. Glucose 6-phosphate is also degraded by the oxidative pentose phosphates pathway, which produces metabolic intermediates used by the secondary metabolism for the production of phenolic compounds via the shikimic acid pathway (see Chapter 6). From glycolysis, some triose phosphates, such as glyceraldehyde 3phosphate and pyruvic acid, represent the basic molecules to feed the MEP metabolic pathway that produces IPP (the precursor of all terpenes, Chapter 7). Phosphoenolpyruvate is used, along with erythrose 4phosphate, for the synthesis of phenolic compounds. Furthermore, glycolysis produces building blocks for the production of amino acids. Acetyl-CoA is the final product of anaerobic glycolysis. This compound is used by several metabolic pathways (such as fatty acids synthesis)

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including the MVA pathway, which produces terpenoids. The Krebs cycle catalyses the complete oxidation of acetyl-CoA and is characterized by decarboxylation and oxidation reactions that produce important metabolic intermediates known as Į-ketoacids. Transamination of Į-ketoacids generates amino acids which are used to synthesize nitrogen-containing secondary metabolites. Aromatic amino acids are formed during the biosynthesis of phenolic compounds and provide molecules for the synthesis of some alkaloids (see Chapter 9) (Figure 10.1).

Figure 10.1 Sucrose and starch are primary metabolism products used by the plant cell as an energy source by the catabolic process known as respiration. During

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glycolysis, some intermediate compounds are used as a substrate for the formation of bioactive metabolites, such as terpenoids, alkaloids and phenolic compounds. Glucose 6-phosphate enters the oxidative pentose phosphates pathway which provides erythrose 4-phosphate for the shikimic acid pathway, which synthesizes the aromatic amino acid phenylalanine, tyrosine and tryptophan. Acetyl-CoA, produced by the decarboxylation of pyruvic acid, enters the Krebs cycle, but also serves as a substrate for the MVA pathway for terpenoid production. The Įketoacids Į-ketoglutarate and oxaloacetate produced in the Krebs cycle can be transaminated to amino acids, which can be used for the synthesis of alkaloids and other nitrogen-containing secondary metabolites.

The production of secondary metabolites depends directly or indirectly on the availability of primary metabolites (e.g., starch or sucrose) and on the metabolic capacity to use the products of primary catabolism for the building up of biotic and abiotic stress-related molecules. In the following paragraphs we will discuss how plant cells can be grown indefinitely in artificial systems able to reproduce the ideal environment for plant life. We will note that the fundamental premise is to provide the cells with a specific “diet” consisting of a mixture of sugars, mineral elements, vitamins and, in some cases, synthetic plant hormones or phytostimulants. We will start by analysing the nutrients essential to nourish cell and tissue cultures.

10.1.1. Carbon as a Nutritional Source The carbon source that is most easily used by cultured cells is sucrose, which is hydrolysed by a series of isoenzymes (invertases) to glucose and fructose. Sucrose is therefore the most effective molecule to deliver glucose to the cell respiratory process even though other glucosecontaining molecules, such as maltose and lactose, can be used as a reduced carbon nutritional source. Fructose catabolism requires its phosphorylation to fructose 6-phosphate and then the enzymatic transformation into glucose 6-phosphate.

10.1.2. Nitrogen as a Nutritional Source Providing nitrogen as nitrate imposes a substantial energy expenditure by the cells, while the administration of nitrogen as ammonium is more efficient in energy terms, although its utilization from the culture medium by the cells causes a pH lowering. Other sources of nitrogen that can be used in culture media are urea, glutamine and glutamate, alanine and

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casein hydrolysate. Organic nitrogen sources have the advantage of supplying the cell both nitrogen and carbon (as happens, for instance, with amino acids). In addition, some organic compounds containing nitrogen may serve as precursors for the production of nitrogen-containing secondary metabolites (see Chapter 9).

10.1.3. Other Nutritive Elements The complete dependence of in vitro cultures on the chemical composition of the culture medium requires the addition of other nutrients, such as K, Mg, S, I, B, Mn, Zn, Na, Cl, Mo, Cu, Co, Fe, Ca and P, in the form of either hydrated or non-hydrated salts. In addition to inorganic compounds, other organic supplements such as myo-inositol, nicotinic acid, pyridoxine, thiamine and glycine are also required. In the sixties, Murashige and Skoog developed an ideal formulation, which can be modified in various ways, to allow the in vitro culture of plant cells.

10.1.4. The Culture Cycle Considering the energy demand, the culture cycle is characterized by three phases defined by completely arbitrary time limits. In the first phase, there is the accumulation and the “loading” of nitrogen and carbon sources with a rapid absorption from the culture medium. In this phase, the oxidation of compounds is low and monosaccharides are accumulated as starch. In the second phase, the cell divisions begin, accompanied by oxidative processes that catabolise stored nutrients accumulated in the previous phase and make carbon sources available for cell growth. The oxygen demand increases along with the increase in cellular respiration. In the third phase, the energy stores are mobilized to form carbon skeletons for both the oxidation and synthesis of structural components and bioactive metabolites. At this stage, there is an inverse relationship between the synthesis of secondary metabolites and the rate of growth. Usually, the production of secondary metabolites occurs at the end of the cell division phase, the so-called stationary phase, in which cells cease to divide and start their expansion. There is a possible link between the stationary phase and the use of stored substances for the production of secondary metabolites (Figure 10.2).

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An alternative way to produce secondary metabolites from heterotrophic cells in vitro is the use of photoautotrophic cell cultures. Photoautotrophism is a peculiar characteristic of chloroplasts and, as we have mentioned, this characteristic is not usually expressed in cell cultures. It is therefore necessary to use cells that possess the ability to remain in the “green” state when cultivated, which means keeping the chloroplasts active. The main technique for obtaining these cultures is to enrich the culture medium with carbon dioxide (1–2%). In the presence of chloroplasts, it is possible to obtain a higher quantity of secondary metabolites, because most of the enzymes involved in their production are located in these organelles. By comparing the data obtained from heterotrophic, photomixotrophic and photoautotrophic cultures, we can clearly show the increase in the production of certain secondary metabolites in the presence of chloroplasts. However, the concept cannot be extended to all secondary metabolites. For example, some indole alkaloids are commonly produced in heterotrophic cultures.

Figure 10.2 Cell growth in in vitro culture can be represented by three phases. In the first phase, also defined as the loading phase, growth is reduced and the storage of carbon sources is much higher than their oxidation. In the second

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phase, cells divide quickly and the biomass increases. The oxidation of carbon sources increases and their storage decreases; at this stage there is still no production of secondary metabolites. In the third phase, the stationary phase, cells stop dividing and the culture reaches a stable (and slowly decreasing) level in the production of biomass. At this stage there is a marked decline in the storage and oxidation of carbon sources in favour of the production of secondary metabolites. From Cresswell et al., 1989, modified.

10.2. Cell and Tissue Cultures The terms cell cultures and tissue cultures refer to the ability to grow isolated plant portions or tissue fragments in aseptic and predominantly heterotrophic conditions. The idea of using aseptic cultures to grow plants was taken from the cultivation techniques for the isolation and cultivation of fungi and bacteria. Plant cells can be grown in vitro and every cell is able to reproduce the whole organism from which it derives. Two terms are often used when talking about tissue or cell cultures: in vivo and in vitro. From a metabolic point of view, we define as in vivo those processes that require cellular organization, whereas in vitro are those processes in which cellular organization is not necessary. Yet a root explant (e.g., root apex) grown in a culture medium under aseptic conditions in a plastic capsule is still an organized system, therefore in vivo, but many authors and most of the public opinion considers these conditions to be in vitro. An internationally recognized concept is to define as in vitro those techniques that refer to the culture of explants, tissues or plant cells in artificial conditions (e.g., in a test tube), whereas the term in vivo mostly refers to the growing of plants in natural and not sterile conditions (as in pots or in the field). To give a general picture on the potentiality of the use of cell and tissue cultures it is necessary to analyse some basic concepts related to in vitro culturing. Tissues grown on culture media which are rich in nutrients such as sugars, vitamins and peptides are the ideal substrate for the growth of microorganisms such as fungi and bacteria. It is therefore necessary that plant explants be thoroughly sterilized, in order to avoid the proliferation of undesirable organisms. The ideal environment to operate is the sterile (vertical flow) hood, while solutions containing sodium hypochlorite or antibiotics or oxidizing agents are the most appropriate means by which to eliminate any microorganism. Once the explants have been sterilized, they are placed on a nutritive medium that can be either a liquid or a gel. In the case of liquid cultures, cell or tissue growth occurs on orbital agitators,

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which allow the continuous contact of the culture medium with the explant/cell. The presence in the culture medium of plant hormones, such as auxins or cytokinins, can induce tissue dedifferentiation, with the loss of tissue organization. Dedifferentiated cells cluster to form a cellular mass defined as a callus (plural calli). The callus, obtained either in liquid or gels, can be grown indefinitely through repeated series of sub-culturing, thus becoming a genetic reserve of plant material. The success of an in vitro technique is not limited only to the formation of calli starting from differentiated tissues, but also to the capability of performing the inverse process, that is, regenerating a whole plant starting from an undifferentiated callus cell. This process is called “regeneration”; some plant species are particularly suited to this process, while in others (such as many monocotyledons) the regenerative process is extremely difficult to achieve. The regeneration of plants through in vitro cultures can be obtained through three methodologies: embryo culture, somatic embryogenesis and somatic organogenesis. Embryo culture is the in vitro cultivation of zygotic embryos obtained in aseptic conditions by explant of the embryo from seeds or ovules and cultivation in culture conditions designed to replace the endosperm (the nutritive medium) environment. The development and germination of the embryo takes place in the culture medium just as it would in the seed. Unlike embryo culture, somatic (or asexual) embryogenesis is the production of embryo-like structures from somatic cells. Somatic embryos are independent structures, which have a bipolarity and are not physically attached to the tissue of origin. The capacity of these embryos is to germinate and develop into seedlings just like during a zygotic development. The development of plants through organogenesis includes the formation of a shoot from an undifferentiated mass of cells and the subsequent formation of a root system. The sprout that is formed is, unlike what is described for somatic embryogenesis, unipolar and is physically connected to the tissue of origin (Figure 10.3). Protoplasts are what remain of a plant cell when it is deprived enzymatically or mechanically of the cell wall. Their shape is spherical and, depending on the tissue from which they are isolated, they can show different colours. Protoplasts obtained from plant organs, calli, cell suspensions, shoot and root apexes and flower petals can regenerate the whole plant from which they were extracted. However, the regeneration of plants from protoplasts is still limited to some families; above all, the Solanaceae are particularly suitable for plant regeneration.

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Figure 10.3 Callus induction and plant regeneration from leaf tissues of Vetiveria zizanioides showing the proliferation of a white transparent callus on a basal, non-differentiated chlorophyll-free leaf fragment. Brownish and friable calli are then subcultured on a maintenance medium and embryo-like structures (arrows) on the callus appear when grown on the regenerating medium. Shoot regeneration from the embryo-like structures allows the transfer of plantlets to culture tubes containing a rooting medium. After several weeks, rooted plants are finally transferred to pots for in vivo culturing.

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The possibility of protoplast fusion, even between different species, and the possibility to regenerate the products of cell fusion have allowed the creation of new hybrids and new plant species, by the process known as cellular hybridization. When protoplasts isolated from two sources are allowed to fuse, their cytoplasms mix. The nuclei of the fused protoplasts may fuse and these nuclei are called heterokaryons and the resulting cells are defined as heterokaryocytes. Through this technique, it is possible to combine the best plant traits, even in the same species or in the same genus, thus obviating reproductive incompatibilities associated with the normal process of fertilization. For instance, it is possible to merge the protoplasts of two different species using a technique called “electroporation”. This technique uses an electric field which, when applied to protoplast suspensions, causes micro-holes in the plasma membrane. Through these holes a cytosolic material exchange occurs between two contiguous protoplasts, resulting in the fusion of two cells and the formation of a somatic hybrid. This fusion product is called a cybrid or cytoplasmic hybrid. Some genetic factors are carried in the cytoplasm, and hence the formation of cybrids has practical applications in plant breeding. The fused protoplasts begin to build the cell wall and organize into clusters of undifferentiated cells, the calli. Calli can therefore be regenerated to plants with intermediate characters in between those of the parental plant species.

10.3. Bioactive Molecules from Cell Cultures Many bioactive molecules are produced via cell culture techniques and the versatility of plant cell cultures to produce such molecules is remarkable. If we consider the economic importance of some metabolites and that sometimes their concentration in plant tissues can be very low, the importance of biotechnological methods for the production of these bioactive molecules is evident. The development of plant cell cultures as an alternative source of secondary metabolites has been encouraged by a number of factors, including: x x x x

independence from growing seasons; the lack of diseases; the avoidance of pesticide practice; the ability to produce compounds at any time and in the desired quantities;

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x the limiting of deforestation; x the conservation of biodiversity (see also Chapter 1). However, cell cultures rarely produce quantities of secondary metabolites comparable to those produced by plants in vivo, although there are some exceptions to this rule. An important point in the in vitro production of secondary metabolites is the connection between secondary metabolism and the level of tissue differentiation. Some metabolites (e.g., phytosterols and some flavonoids) are not associated with any particular level of structural organization, whereas some alkaloids and terpenoids are synthesized and accumulated in specialized secreting structures that require a high level of tissue organization. Even in this case, the rule is not so strict and there are numerous examples of the accumulation of alkaloids (e.g., nicotine) and terpenoids (e.g., sesquiterpenes) in undifferentiated cultures. Two factors are extremely important when considering the in vitro production of secondary metabolites: secretion and storage of bioactive molecules. In general, the main site of storage of secondary metabolites is the cell vacuole, in which a high water content allows the solubilization of polar and water-soluble metabolites. Cell suspensions offer an ideal system as they are composed of a uniform cell population formed by rapidly growing cells. On the other hand, callus cultures are made up of cells of different developmental stages, where fastgrowing cells and necrotic cells are clustered together. This involves the formation of gradients comprising nutrients, growth regulators and secondary metabolites. Nevertheless, it is more convenient to use calli for the in vitro production of secondary metabolites. In general, there are four nutrient categories that promote the in vitro production of secondary metabolites: minerals, organic compounds, hormones and carbon sources. Once the correct formulation for the preparation of the culture medium has been established, it is possible to further increase the production of secondary metabolites by acting on environmental growth conditions. For example, both the intensity and duration of light, as well as the appropriate wavelength, are important factors. Certain flavonoids are produced in vitro if the cell cultures are subjected to intense light radiation. Light can also be indispensable for the

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formation of phototrophic cultures, which is indispensable for the production of secondary metabolites synthesized by chloroplasts. Also, the stirring speed of the cell suspension cultures and the quality of the gas composition (O2, CO2) have a significant influence on product formation. Finally, growth rate and enzyme activities are strictly dependent on the temperature, which can usually vary according to species from 15 to 35 °C. With the same culture media, different environmental conditions can induce or suppress the in vitro production of secondary metabolites, and the correct combination of nutritional and environmental factors is the secret to optimal yield. Despite a large number of scientific publications related to the production of secondary metabolites from cell cultures, only a few examples can be cited with regard to commercial applications. Berberine (1) and shikonin (2) are two molecules produced industrially by cell cultures. Plants that contain berberine are important because berberine and some similar alkaloids have anti-inflammatory, antileukemic and antineoplastic properties. Unfortunately, plants that produce berberine (Menispermaceae, Ranunculaceae, Rutaceae and Berberidaceae) have rather slow growth and take five to seven years before they can be used for the extraction of this compound. A fundamental assumption for the in vitro synthesis of a secondary metabolite is the understanding of the biochemical mechanisms that underlie its production. Only a detailed investigation of the enzymatic processes involved in the synthesis of secondary metabolites allows the proposing of strategies to increase their production. One of the advantages of in vitro systems is their high enzyme activity with respect to the corresponding in vivo systems. In particular, enzymes that act as a link between primary and secondary metabolism are considered key points for optimal production. But one of the major difficulties in the industrial application of cell cultures is the instability and variation of productivity. Although the so-called “somaclonal variability” is one of the focal points in the selection of high-throughput cell lines (see below) it also determines a lack of production constancy. Berberine production from some clones of the genus Thalictrum showed considerable content variations, depending on the cellular subculturation. Only a few cell lines were stable, while for most of the remaining ones berberine concentrations were subject to consistent qualitative and quantitative variations. Naphthoquinones, which include shikonin (2), alkannin (3) and their derivatives, present a larger group of quinone pigments. Shikonin and its derivatives are commercially the most important of the naphthoquinone

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pigments. The first successful production of shikonin derivatives in vitro was made from callus cultures of Lithospermum erythrorhizon in the early seventies. Later on, cell suspension cultures were shown to produce several shikonin derivatives as found in the roots of the intact plant and a Japanese company became successful in producing shikonin from cell cultures by introducing the first example of commercial production of a secondary metabolite from a cell culture.

A final consideration on the production of secondary metabolites by cell cultures is related to the level of toxicity that such molecules may exert when produced in artificial systems. It is indeed a common fact that the release of secondary metabolites in the culture medium causes a decrease in the cell viability with a consequent reduction of product formation. Two fundamental points are therefore necessary to grant production capacity: x It is necessary to avoid accumulation of secondary metabolites in the culture medium, and at the same time it is important to provide cells the time necessary for the metabolite production. This goal can be achieved through the development of systems able to operate a turnover of nutritive elements and metabolites produced in the culture medium; x It is necessary to be able to isolate the secondary products quickly and easily, separating them from the culture medium without wasting the nutrients that are still present. At the same time, it is important to ensure cell survival and efficiency.

10.4. Bioactive Molecules from Tissue and Organ Cultures The main causes of the lack of productivity in in vitro systems depend on:

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x lack of expression in non-specialized cells of those genes controlling the main biosynthetic steps; x subtraction of substrates from the biosynthetic pathways for the formation of secondary metabolites; x lack of transport mechanisms for the removal of any toxic compound from the production sites; x impossibility of forming structures for the storage of secondary metabolites; x lack of catabolic regulation of the synthesized compounds. These factors are most likely under the control of developmental processes and can operate independently for each metabolite or class of secondary metabolites both in the plant and the single cell. On the contrary, the organization of undifferentiated tissues has the capacity to regenerate the synthetic and storage capacity and, when referring to the organ-specific differentiation, allows the production of biomass (carbon flow) towards the metabolic pathways of secondary compounds. For this reason, it is sometimes more convenient to grow in vitro either tissues or organs instead of cells. Although the industrial use of tissue and organ cultures has proved to be feasible in only a limited number of cases, it is appropriate to describe the potential that this technique has for the large scale formation of secondary products. Roots and shoots are the most used organs for this purpose.

10.4.1. Root Cultures The ability to grow roots in the absence of soil is a technique that was acquired at the beginning of the last century, when plant physiologists were using hydroponic cultures to study the mechanisms of nutrient absorption and assimilation, thus establishing the criteria of nutrient essentiality. The ability of roots to grow and develop in liquid nutritional systems and the capacity of these organs to produce a large quantity of secondary metabolites have been used mainly for the production of some classes of alkaloids, even if interesting results have been obtained for other classes of compounds such as the terpenoids, steroids and phenolic compounds. Root hydroponic cultures of Atropa belladonna produce a higher amount of alkaloids than the roots of soil cultivated plants, although the amount of apoatropine (4) was lower. In Hyoscyamus niger, the differentiation of roots from cell cultures with a high content of hyoscyamine (5) and scopolamine (6) allowed the obtaining of comparable quantities and in some cases higher contents than roots of soil grown

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plants. The same results have been obtained for the production of many other alkaloids produced by medicinal plants such as Catharanthus roseus, Cinchona sp., Papaver somniferum and Senecio spp. Root cultures of Valeriana and Centranthus species produced amounts of secondary compounds similar to the content of soil cultivated plants. In particular, root cultures of V. officinalis produced valerenic acid (7), whose content was enhanced by elicitation with Fusarium gramineum extract, whereas root cultures of C. ruber produced the valepotriates valtrate (8) and isovaltrate (9) at concentrations very similar to that found in the roots of field-grown parent plants. Ginger (Zingiber officinale) root cultures accumulate a large quantity of monoterpenes such as neral (10) and geranial (11), which are responsible for the pungent taste of this plant. However, in this case the concentration obtained in the root culture was lower than that produced by the plant cultivated in vivo. In some cases, the concentration and the quality of secondary metabolites produced by root cultures is not constant. For example, in Digitalis purpurea root cultures, the initial production of cardenolides was similar in quality and quantity to that of plants grown in the field. However, during subculturing the root culture lost its biosynthetic capacity and after only two months the cardiac glycosides were only present in traces. The coumarin umbelliferone (12) is produced from the roots of some Apiaceae, such as Pimpinella anisum and Anethum graveolens; however, in this case also the in vitro production is lower than in in vivo plants.

An alternative technique to obtain root cultures in vitro is the transformation of explants following inoculation with the soil bacterium Agrobacterium rhizogenes. This technique allows the formation of a large root system with numerous ramifications originating from the point of

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infection. The bacterium transfers DNA segments to infected tissue and induces root formation. The nucleus of transformed cells begins to express the bacteriumtransferred genes and the newly formed roots can be severed and subcultured (after treatment with antibiotics to kill the bacteria that are still present) to form so-called hairy-roots (hr). These hr cultures grow faster than conventional root cultures, show a highly heterogeneous structure with the presence of many meristematic tissues and also display a steady and stable growth even in the absence of phytohormones (auxins and cytokinins). This happens because hormone production occurs endogenously due to transformation by A. rhizogenes. One of the advantages of this technique is that there is no antagonism between fast tissue growth and the production and accumulation of secondary metabolites. Hr cultures are very resistant, they can be grown in suspension cultures and can be easily cryopreserved; however, they are still scantly used for the production in fermenters (see Chapter 11). The application of hr culturing for the production of secondary metabolites has been successfully tested using cultures of Atropa belladonna, with production of hyoscyamine (5) and scopolamine (6) superior to that obtainable with root cultures or plants grown in open field. In this case, the production remained stable even after 120 subculture cycles over more than 10 years. The use of A. rhizogenes is not limited to the production of hr; in fact, this bacterium can be used as a vector to introduce genes for the production of secondary metabolites. For example, the introduction of ornithine decarboxylase genes in Nicotiana rustica roots using A. rhizogenes as a vector allowed an overproduction of ornithine (13) and a consequent increase in the concentration of putrescine (14) and nicotine (15). These and many similar results have highlighted the enormous potential of hr for the production of valuable bioactive molecules.

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10.4.2. Shoot and Bud Cultures Callus differentiation in the presence of a high content of cytokinins leads to the formation of shoots, which can be grown in vitro even without root formation. In vitro shoot cultures can be produced also through the explant of apical or axillary buds from plants grown in the field, after tissue sterilization. Alternatively, young shoots produced in vitro from seedlings can be used as starting material. Regardless of the origin, multiple shoots can be proliferated in high-capacity containers, or in particular bioreactors, and can produce biomass three to four times higher than those obtained from cell cultures. As for root cultures, there are numerous examples for the production of the main classes of secondary metabolites by shoot cultures. Shoot cultures have been obtained from buds, seeds and leaf discs of the medicinal plants Atropa belladonna, Rauwolfia serpentina, Catharanthus roseus, Cinchona spp. and Papaver somniferum. However, the production of alkaloids from these plants has proved to be considerably lower compared to that obtained using hr or root cultures. As previously mentioned, the choice of an organ for the production of secondary metabolites is closely linked to its biosynthetic capacities; in the case of many classes of alkaloids, shoot cultures proved to be not very productive, whereas completely opposite results were obtained for the production of volatile substances such as some terpenoids. As described in Chapter 2, mono- and sesquiterpenes are produced in particular secretory tissues which are present both on the epidermis (glandular trichomes) and within the leaf and shoot parenchyma (lysigenous and secretory cavities and resin ducts). It is therefore clear that the cultivation of organs capable of producing such structures can be extremely effective in increasing productivity. There are many examples of aromatic plants grown in vitro and in the cases of many bioactive molecules the results obtained allowed a high productivity to be ascertained. The interest is so high that we will discuss this aspect in a paragraph of this chapter. As regards the production of phenolic compounds, studies carried out on grapefruit plants (Citrus paradisi) and on two Apiaceae, Pimpinella anisum and Foeniculum vulgare, have shown that phenylpropane derivatives can also be produced in shoot cultures. In the case of grapefruit, the bitter compound naringin (16) is not produced from cell cultures, but its content increases considerably in shoot cultures until it reaches the concentrations found in plants grown in the field. The

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concentrations of pseudoisoeugenol (17) and anethole (18), produced by P. anisum and F. vulgare respectively, were lower than in in vivo plants, despite the tissues being able to differentiate the resin ducts. In vitro shoot cultures of Mentha piperita showed a progressive change in their monoterpene percentage and content with an increasing concentration of limonene (19) and a progressive reduction of menthone (20) and menthol (21) with subculturing. These results clearly indicate the potential, but also the limits of secondary metabolite production from shoot cultures. These considerations, together with the high costs of the in vitro production of volatile terpenoids, have limited the development of these technologies to laboratory smolecular studies on gene expression or biochemical evaluations of biosynthetic capacity.

10.5. In Vitro Turnover, Regulation and Storage of Plant Bioactive Metabolites Every cell of a plant contains the information necessary for the formation of any organ (i.e., totipotency) and for the production of all the metabolites that characterize the adult plant. Yet, as we have noted in the previous paragraphs, this is not always true, or always possible. In order to activate genes, it is necessary to access those switches capable of triggering the metabolic processes that prompt the activation, transcription, translation and implementation of their products. In some cases, it is essential to stress the cells and this derives from the fundamental fact that most of the secondary metabolites are produced as a result of stresses imposed by the plant’s surrounding environment, whether they are abiotic or biotic in nature. In this regard, abiotic stresses are mainly related to the nutrition factors and environmental conditions in which cells grow. Although tissue and organ cell cultures are able to produce secondary metabolites at certain stages of their development, there are numerous experimental data indicating that some molecules remain at the stationary concentration. This fact depends on the ability of the cell to measure the flow of primary

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metabolites to secondary metabolites while maintaining a certain metabolic control. In plants cultivated in soil, the flow of molecules between primary and secondary metabolism and the storage, turnover and degradation of secondary products are regulated by precise patterns of plant development, depending on nutritional and environmental factors (alternating seasons, abiotic and biotic stresses). In in vitro systems, stress conditions are strictly under the control of the operator who can use them for precise and pre-established ends. Under these conditions, the influence of nutrients and phytohormones present in the culture medium on cell metabolism becomes of paramount importance. In plant cell cultures, metabolic turnover and the storage of secondary metabolites are strictly dependent on spatial and functional compartmentalization. There are three general compartments: (i) the internal environment (consisting of the cytosol and cytoplasmatic organelles); (ii) the external environment (formed by the cell wall and the medium in which cells are grown); and (iii) the storage environment (mainly represented by the vacuoles). The separation between these compartments is guaranteed by the presence of biological membranes that control the inter-compartmental exchange of secondary metabolites. Precursors, and intermediate and final secondary metabolites are produced in the internal environment and, along with the products of the metabolic turnover, are stored permanently or momentarily in the storage environment or excreted into the external environment. A large part of these molecular displacements is not unidirectional, but reversible, thus contributing to the general dynamics of secondary metabolism.

10.5.1. Metabolic Turnover To better define metabolic turnover we can use the following product definitions: x Interconversion reaction products, which transform molecules that accumulate momentarily into products that are metabolically distant from the initial ones; x Products obtained as a result of conjugation with molecules not necessarily belonging to the same class of compounds. In the case of conjugation with sugars, a change in the compound polarity may occur with relative higher or lower ease in crossing the membrane;

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x Compounds formed as a result of oxidative polymerizations that lead to insoluble structures with a higher molecular weight, often bound to membranes or encrusted in the cell wall; x Degradation and catabolism products. Understanding the mechanisms of regulation and implementation of metabolic turnover is a fundamental condition for the study of secondary metabolism in vitro. In many cases, the metabolic turnover is regulated by a progressive change in the relationship between the activities of anabolic and catabolic enzymes. Some authors hypothesize that the enzymes involved in the turnover of secondary metabolites are expressed at a late stage of the growth curve. In the case of the production of betalains from cultures of Phytolacca and Chenopodium, the turnover of these pigments depends on the growth phase, while the presence of peroxidase enzymes has been repeatedly linked to the discolouration of these compounds. In Chenopodium spp., the ability of cell suspensions to bind to the cell wall products of the phenylpropane metabolism, such as benzoic and cinnamic acids, indicates that the cell wall can be an ideal sink for the storage of phenolic compounds. The glucosylation of molecules belonging to different classes, such as the isoflavones of the genus Cicer, the terpenoids in numerous species belonging to the Lamiaceae family or the alkaloids of the genera Coffea and Nicotiana, identified the vacuole as one of the main storage sites for metabolic turnover. In addition to the presence of numerous molecules with low and medium molecular weight, there is also the presence of macromolecular constituents such as polysaccharides, peptides and enzymes. In particular, the last are directly involved in the extracellular transformation processes that contribute to the metabolic turnover of excreted molecules. From a metabolic point of view, this extracellular environment is closely linked to the cell wall, so changes in the chemical composition of the culture medium or the presence of secondary metabolites excreted by the cell can significantly influence not only the cell growth processes, but also the enzymatic activities related to primary and secondary metabolism. The metabolic turnover in the extracellular space is guaranteed by the presence of numerous enzymes including: acid phosphatase, phosphodiesterase, esterase, glucosidase, mannosidase, lipase, amylase, proteinase, peroxidase and nuclease. A direct comparison between these extracellular enzymes and those present inside the cell indicates the

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existence of a metabolic relationship between the internal and the external environments of the cell. In many cases, a disappearance of secondary metabolites from the culture medium occurs and this phenomenon is attributable to the activity of extracellular enzymes, capable of annulling the potential in vitro production of bioactive molecules. This phenomenon has been observed in cellular systems producing isoflavonoids, pterocarpanes, thiophenes, polyacetylenes and coumarins.

10.5.2. Transport and Storage of Bioactive Molecules In general, secondary metabolites are accumulated in compartments different from those in which they are produced, and the vacuole represents the overall accumulation site of excellence for hydrophilic substances. It is therefore important to understand the mechanisms by which metabolites produced by other cell compartments can pass through the tonoplast (the membrane that delimits the vacuole), accumulate in the vacuole and be specifically retained or released from it. One of the proposed mechanisms for the transport of secondary metabolites within the vacuole is simple diffusion, caused by a concentration gradient between the outside and the inside of the organelle. The tonoplast is more or less permeable to a certain number of secondary metabolites including cardenolides, anthocyanins and alkaloids. In some cases, the presence of proteins was found both for the facilitated diffusion and for the active transport (ABC transporters) of some secondary metabolites, as in the case of morphine (22). Lipophilic compounds possess a superior diffusion potential when compared to polar metabolites and charged molecules; nevertheless, the reversibility of simple diffusion does not allow any increase in concentration. The presence of passive transporters is more easily demonstrated through saturation kinetics, specificity of transport and competition between analogous molecules. The active transport of secondary metabolites has been demonstrated for several metabolite classes including alkaloids, acylated anthocyanins, cardiac glycosides and glucosylated flavonoids. A clear indication of the active transport of substances comes from studies with labelled substances, inhibition of active proton pumping, the use of uncouplers and protonophores or with the use of reagents capable of blocking the SH-groups of proteins. However, the ability of the vacuole to retain secondary metabolites is largely due to the presence of active transport mechanisms. This is mainly due to the cell’s need to keep potentially toxic secondary metabolites away for the metabolic functions of the cytosol and in an isolated environment. There is also the possibility that secondary metabolites stored in the

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vacuole lose the ability to cross the tonoplast. This may occur if a substance is dissociated in the neutral environment of the cytosol and then protonates once accumulated in the acidic environment of the vacuole. Other factors may also be involved, for example the alkaloids contained in the vacuoles of Cinchona cells are poorly released even after the administration of dimethyl sulfoxide, a decoupling agent, indicating that the efflux from the tonoplast does not depend on the presence of a translocator. Changes in conformation, isomerization, complexation with ions, binding to other molecules or to the tonoplast itself may be limiting factors for the release of secondary metabolites from the vacuole. A special case of molecule entrapment in the vacuole is covalent modification: covalently linked forms of the alkaloids morphine (22) and codeine (23) have been identified in the vacuoles of Papaver somniferum cultured in vitro, while intravacuolar modifications, such as glycosylation, are known for many cellular systems.

10.5.3. Regulation of Secondary Metabolism in Cell Cultures The availability of nutrients in an in vitro culture system is a critical factor for the growth or production of secondary metabolites. In general, a lower content of sugars or a reduction of carbon sources may have a positive influence on the production of certain secondary metabolites. The regulation in the production of secondary metabolites in vitro starts from a correct management of the nutrients that are supplied to the culture during the growth and production phases. Phenolic metabolism shows that the positive effect of glucose for in vitro production is strictly linked to the availability of nitrates and phosphates. This relationship can shift the balance in the flow of carbon from glycolysis to the secondary pathways, such as the oxidative pentose phosphate pathways, that form precursors for the synthesis of phenols and that generate NADPH useful for their biosynthetic processes. As for nitrogen, it was noted that Lithospermum cultures produced shikonin (2)

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exclusively in the presence of nitrates, while its synthesis was inhibited by the presence of ammonium. In general, the nitrogen deprivation has proved to be useful for the production of naphthoquinones, polyphenols, depsides, anthocyanins and anthraquinones. An inhibiting action on the synthesis of phenols is caused by the presence of organic sources of nitrogen such as casein, yeast extract and peptone. High concentrations of phosphorus inhibit the synthesis of coumarins, polyphenols, rosmarinic acid (24) and anthocyanins, probably due to the inhibition of phosphatases responsible for the release of phenolic compounds in the culture medium. An important role in regulating the production of phenols is given to the synthetic and natural plant hormones present. While the presence of growth stimulants is crucial in the second stage of development of the culture, their absence or limitation becomes an important requirement for the synthesis of flavonoids in the stationary phase (see Figure 10.2). This requirement is particularly valid for auxins, while it seems to be less restrictive for cytokinins. The quality of light plays an equally important role in the regulation of phenolic compounds. With the exception of carrot cells, that produce anthocyanins in culture only when kept in the dark, in many plant cell systems white light stimulates the production of flavonoids. Blue light is particularly suitable for the formation of polyphenols, but it inhibits the synthesis of podophyllotoxin (25) and naphthoquinone (26), whereas UVB light stimulates the production of some flavonoids, including quercetin (27). Stilbenes are stimulated by UV-C light. The stimulation of the phenolic metabolism by light is related to its effects on the regulation of genes involved in the transcription of shikimic acid pathway enzymes, as well as those regulated by phytochrome. Another important regulation point for the secondary metabolism is the availability of precursors. Attempts to increase the production of secondary metabolites through the introduction into the culture media of precursors has given encouraging results. The addition of phenylalanine (28) and tyrosine (29) to Coleus blumei cell cultures allowed the formation of considerable quantities of rosmarinic acid (24), whereas the administration of luteolin (30) and coniferyl alcohol (31) to Silybum marianum cultures increased the production of flavolignans. The failure to induce or increase the yield of a secondary product often depends on a lack of knowledge regarding the time and method for the administration of precursors.

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10.6. The Search For and Selection of Cells with a High Production of Plant Bioactive Molecules The results obtained so far by the industrial production of secondary metabolites from cell cultures have been achieved thanks to the careful study of the regulation of secondary metabolism. However, the simple evaluation of the metabolic potential of a culture would have no use if it were not combined with the selection of genetically suitable sources to generate the cell lines able to respond constantly and efficiently to the cultivation techniques used. For this reason, one of the basic rules for the formation of secondary products in vitro is to follow a strategy based on some fundamental steps: x selecting high yield plants; x obtaining cell cultures from selected plants starting from: o different plants, o different cultivars, o different plant species, o different parts of the plant; x developing an optimal culture media without considering the production of secondary metabolites starting from: o cell aggregates, o cloning of single cells; x developing methods to induce the formation of secondary metabolites; x selection cloning of highly inducible cellular strains;

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x optimizing the nutrient media for the production of secondary metabolites. Selection can be made on any type of coloured or colourless, fragrant or odourless molecule provided the possibility exists to develop a suitable test to quantify the molecules of interest. Several methods have been developed to identify high producing cells. Coloured cells (due to the accumulation of alkaloids or phenolic compounds) can be selected visually and subcultured, or can be identified and selected automatically by the use of special fluorochromes combined with cell separators. Immunological assays have prevailed over all other techniques, not only because of their extreme sensitivity, but also because of the large number of individual cell clones that can be analysed. An alternative technique for selection is to use toxic agents to localize cell types capable of detoxifying them due to the presence of high concentrations of enzymes involved in the secondary metabolism. p-Fluorophenylalanine (32) is a toxic compound which is rendered harmless by deamination; in this way cells producing cinnamylputrescine (33) or rosmarinic acid (24) can be selected. Likewise, fluorinated tryptophan was used to select indole alkaloids by isolating cells capable of decarboxylating the amino acid to tryptamine (33). The most frequent problem with plant cell lines is inversion, which usually exceeds the rate of spontaneous mutation. Therefore, many of the selected cell lines are epigenetic variants rather than true genetically different subspecies. However, with a certain number of selection cycles, clonal selection allows the isolating of both mutants and variants in which the epigenetic state is stabilized, as shown by some examples of the industrial exploitation of cell cultures. As we have mentioned, the industrial selection of high yield strains has been succesful for berberine (1) and shikonin (2), while for more than eighty molecular species the results obtained did not allow industrial production. Work in progress for the industrial production of bioactive molecules includes that for capsaicin (34), the active ingredient of red pepper (the second species in the world after black pepper), rosmarinic acid (24) from Coleus cell suspensions, anthraquinones (35) obtained from Morinda’s callus cultures, diosgenin (36) from cell suspensions of Dioscorea deltoidea, sanguinarine (37) from cell cultures of Papaver somniferum and ginsenosides (38) from ginseng roots.

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Selection criteria also concern the metabolic characteristics of a given culture and, in addition to what has been defined at the beginning of this paragraph on selection criteria, we can include the following requirements: x selection of a reaction catalysed by one or a certain number of enzymes; x availability in relatively large quantities of the substrate for the identified reaction(s); x difficulty in obtaining chemically the final product of the reaction; x considerable commercial value of the final product, due to the scarcity of the material in nature or due to geographical or political difficulties in obtaining the plant material for extraction. Analytical and biochemical selection are indispensable tools in obtaining high yield cell lines for secondary metabolite production, but the real variants obtained from selection can be recognized only after a long period of repeated cloning. For this reason, the industrial production of high yielding strains is a long and costly process. A classic selective system uses a group of cells from which increasingly more and more productive strains are selected in continuous phases, until reaching the maximum of the physiological and biochemical potential that the crop can produce (Figure 10.4).

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Figure 10.4 The selection process is the starting point for obtaining cell lines with high and constant yields of secondary metabolites. The figure shows a classic selection model in which an initial cell is separated from other cells because of a higher content of a given bioactive molecule. The process (curved arrows) can be repeated tens of times until a high-yield cell line is obtained. The cell that demonstrates production stability is finally selected and cloned to obtain mass cultures for industrial production.

10.7. Elicitation of In Vitro Production of Plant Bioactive Molecules In natural systems, the attack of a pathogen on a plant triggers a sequence of responses that lead to the production of defence molecules defined as phytoalexins (see Chapter 3). Phytoalexins are produced as a result of elicitation from substances released by the pathogen or following the degradation of the plant cell wall; these substances are defined as elicitors. Responses to biotic stimulators and abiotic stresses can also be elicited in cell and tissue cultures and occur with an increase in the accumulation of secondary metabolites in the vacuole and subsequent excretion into the culture medium.

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In the case of biotic elicitation, short exposures to minimum amounts of elicitors (about 20 ȝg per gram of fresh weight) are sufficient to increase the enzymatic activity of a certain number of enzymes involved in the production of specific chemical defence molecules. The newly formed molecules released into the culture medium are then metabolized and disappear within a few tens of hours from the time of production. The most used biotic elicitors are fragments of fungal mycelium, autoclaved fungal spores or in some cases fragments (pellets) of viable fungi. Biotic stimulation can greatly increase the in vitro production of certain secondary metabolites; for example, in Papaver somniferum, sanguinarine (37) shows a 26-fold increase in the presence of biotic elicitors, while the cell culture undergoes a dry weight increase of about 3%. In Gossypium arboreum biotically-elicited cell cultures, there is a 100-fold increase in the production of gossypol (39), while the dry weight yield of the crop increases by 2%. Studies on plant-microbe interaction mechanisms have led to the identification of four types of interaction: x direct release of an elicitor from the microorganism and recognition by the plant. There is a receptor for this type of elicitor on the plasma membrane of many plant species (e.g., soybeans); x the enzymes produced by the pathogen degrade the cell wall of the host plant with the formation of molecular debris that act as elicitors. Some examples are endopolygalacturic acid lyase and pectolytic enzymes produced by various fungi; x the enzymes produced by plants in response to pathogen attacks can degrade the microbial cell wall and produce debris that act as elicitors that induce the production of phytoalexins. In many plants it was possible to demonstrate the presence of pathogenesis related proteins such as cutinases and endopolygalatturonases; x biotic elicitors may be present in the plant as constitutive defences, released by the plant cell in response to biotic or abiotic stimuli. For example, the mechanical or chemical fragmentation of the cell wall gives rise to oligosaccharides that have a powerful stimulating action on the production of phytoalexins. The addition of enzymes that degrade the cell wall of carrot and parsley to cellular suspension stimulate the production of 6-methoxymellein (40) and furanocoumarins (41) respectively, with an increase comparable to that obtained by fungal biotic elicitors.

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In many cases, there is a close link between the mechanical action that a biotic agent imparts on the cell wall of the host cell and the production of molecules capable of stimulating a physiological response. But the interaction between a biotic agent and the host response can be much more complex. In cell cultures of Phaseolus vulgaris, the release of an endogenous stimulator has been demonstrated following treatment with a biotic stimulator. In many cases, the presence of a second messenger directly involved in the stimulation mechanism has been hypothesized. This compound would have the task of transmitting the perceived signal from the plasma membrane to the nucleus, by triggering the transcription and translation mechanisms. In soy cell suspensions, the synthesis of callose is stimulated by the presence of chitosan and depends on the presence of Ca2+. The polycation chitosan damages the membrane and allows the influx of Ca2+ into the cells; this ion acts as a second messenger, probably interacting with activation of calmodulin-dependent and other calcium-dependent systems. Methodologically, the appropriate choice of a biotic agent able to produce elicitors is undoubtedly fundamental. There are specific microorganism– plant interactions, but there are also microorganisms able to stimulate responses in a wide range of plant hosts. For the elicitation of secondary metabolites production, viruses, bacteria, blue algae and more frequently fungi can be used. The problem of inoculum quantity can be solved by using conidiospores, zoospores or sporangia although their eliciting properties may be different from those of the relative mycelia. The final selection will be of the microorganism which leads to the best response with the smallest quantities. Once the ideal conditions for co-culture are established, it is essential to ascertain the degree of specific elicitation of a given secondary metabolite. For example, co-culture with the yeast Rhodotorula rubra stimulates the production of acridone (42) and its derivatives in Ruta graveolens cell cultures, the formation of indole alkaloids in Chataranthus roseus and the formation of sanguinarine (37) in Papaver somniferum, whereas glucans produced by Phytophthora megasperma stimulate the production of isoflavans in many species belonging to the Fabaceae family. These results indicate that the same biotic elicitor is capable of inducing the synthesis of an array of secondary metabolites in different cell cultures and that the products formed in response to the stimulus are specific to the cell culture and are not altered by the presence of the elicitor. Conversely, it is possible to obtain the production and storage of the same compound by eliciting a plant cell culture with different biotic agents.

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An important factor in inducing the formation of secondary products is an exact knowledge of the period in which the elicitor is to be administered. In general, the accumulation of secondary metabolites begins within a few hours after administration of the elicitor and is preceded by an increase in the enzymes’ biosynthetic capacity. The maximum activity is reached in a period of time ranging from 12 hours to 5 days, usually followed by a rapid decline. The time required for activation of the biosynthetic pathways is plant-specific, but there is experimental evidence that the amount and type of the elicitor can affect or modulate the activation period. Although it has been established that the ideal period for producing secondary metabolites is during the stationary growth phase, the response to biotic stimulation is most pronounced during the exponential growth phase (second phase of Figure 10.2). The stimulation of a culture in the phase where production of secondary metabolites is already activated does not lead to significant yield increases and in some cases it may even suppress the biosynthetic capacity of the culture. Some authors consider that during the stationary phase of many cell systems, the production of secondary metabolites is the result of a growth phase elicitation, during which the lysis of compartments containing endogenous elicitors would trigger the biosynthetic mechanism. Moreover, biotic and abiotic elicitation during the growth phase leads to an instantaneous and permanent arrest of the cell growth; in many cultures the rapid change of physiological cell status triggers the secondary metabolism expression. The use of cell cultures for the study of biotic agents-induced phytoalexins production is of extreme importance for the understanding of the mechanisms of disease resistance. For example, the production of pterocarpan (43) and isoflavones in Cicer arietinum cultures in the presence of the pathogenic deuteromycete Ascochyta rabiei served to elucidate the molecular basis of both plant resistance and fungal virulence. Among primary metabolism enzymes, only glucose 6-phosphate dehydrogenase (from the oxidative pentose phosphates pathway) shows an increased activity after elicitation by the pathogen, whereas a higher number of secondary metabolism enzymes are activated, including phenylalanine ammonia lyase, cinnamic acid 4-hydroxylase, chalcone synthase and isoflavone glucosyltransferase. In Mentha piperita, stimulation by an imperfect fungus probably belonging to the genus Acremonium causes an increase in the biomass of in vitro plants and the transition of the plant oil chemical composition

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from oxidizing conditions to more reducing conditions, with an increase of menthol (21) and other reduced monoterpenes. Furthermore, the field application of this growth-promoting fungus reduces the attack by plant pathogenic fungi.

10.8. In Vitro Production of Plant Bioactive Molecules of Economic Importance The purpose of this last paragraph is to give some examples on the production of some bioactive molecules from plant cell and tissue cultures. Essential oils are an interesting group of secondary metabolites with important commercial applications in the perfume industry, for aromas for food and beverages, and for cosmetics and pharmaceuticals. In some cases, the cost of distillation of essential oils from plant materials grown in the soil is extremely high, and for this reason numerous studies and pieces of research have been dedicated to in vitro production. Thuja occidentalis cell suspensions produce numerous mono- and diterpenes, the latter being present both in cell extracts and in the culture medium. However, as found for many other species, grown in vitro, the chemical composition of the terpenoids produced differs consistently from that extracted from the leaves of field plants. The typical monoterpenes present in this species (Įpinene, 44; ȕ-pinene, 45; myrcene, 46; and limonene, 19) can be identified in Thuja cell suspensions only after the addition to the culture medium of Miglyol®, a non-toxic triglyceride that forms a layer that covers the cells without affecting either the availability of nutrients or gaseous exchange. The presence of this additional phase in the culture medium proved to be indispensable for the production of terpenoids, and in some cases, such as for Pelargonium cell cultures, has led to up to 500-times yield increases. Other substances that can be used as monoterpene traps are mixtures of nhexadecane (47), polyethylene glycol (48), activated carbon, ion-exchange resins and adsorbent resins such as Amberlite XAD. Amberlite XAD is a copolymer of styrene-divinyl benzene with a characteristic microreticular structure, which is responsible for its high adsorbent capacity. These molecules are widely used to adsorb organic substances present in the

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aqueous phases. The possibility of desorbing organic molecules from the Amberlite XAD makes it possible to recover the secondary products synthesized by plant cell cultures. Lavender (Lavandula spp., Lamiaceae) are aromatic ornamental plants that are widely used in the food, perfume and pharmaceutical industries. Largescale lavender production requires efficient in vitro propagation techniques to avoid over-exploitation of natural populations and to enable biotechnology-based approaches for plant improvement and for the production of valuable secondary metabolites. Micropropagation methods have been developed in several species of lavender based mainly on meristematic proliferation and organogenesis. Other aromatic substances are produced by the degradation of precursors; such is the case, for example, of the S-alkyl-L-cysteine (49) produced from garlic that, as a result of tissue lesions, is degraded by the enzyme alliinase giving rise to the characteristic aroma of garlic. Studies carried out on tissue cultures have shown that root cultures of Allium spp. are able to produce both the aroma and the compound that causes tearing, the latter being absent in shoot and callus cultures. Catharanthus roseus is one of the most important sources of secondary metabolites, with particular reference to indole alkaloids. The Vinca alkaloids have been studied intensively and more than 60 molecular structures are known. Ajmalicine (50) and serpentine (51) are used to treat hypertension, while vindoline (52) and catharanthine (53) are the precursors of vinblastine (54), a potent antileukemic agent (see also Chapters 4 and 9). Numerous alkaloids have been produced from C. roseus cell cultures and about 75% of the 400 isolated cell lines so far have produced yields of more than 0.1% on a dry weight basis. The root alkaloids ajmalicine (50) and serpentine (51) dominate the chemical composition of the cell cultures, whereas the leaf alkaloids vindoline (52) and catharanthine (53) are obtained only from intensive cultivation of shoots maintained on a hormone-free culture medium under intense light. The synthesis of cataranthine (53) and other indole alkaloids can be stimulated through the induction of tryptophan decarboxylase (see discussion above). Paclitaxel (55) is another molecule of extreme interest and subjected to intense clinical tests for its healing effects on breast and uterine cancer (see Chapter 4). Derivatives of this compound have been isolated from cell cultures and from calluses of species belonging to the genus Taxus. Plant

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cell cultures are used for the biotechnological industrial-scale production of important secondary bioactive metabolites including paclitaxel. In the last two decades there have been numerous empirical attempts to improve the biotechnological production of taxanes, which have led to the conclusion that treatment of Taxus spp. cells with methyl jasmonate or other elicitors is the most effective strategy. Once the biochemical and molecular mechanisms have been identified to regulate the biosynthetic pathway of taxanes, it will be possible to design cell lines of Taxus spp. bearing overexpressed genes that control key metabolic steps, thus increasing the in vitro productivity of taxanes. Other anticancer compounds such as camptothecin (56) have been produced by Captotheca acuminata cell cultures, while podophyllotoxin (57), a strategic compound for the synthesis of numerous anticancer pharmaceutical products, is produced up to 1.5% on a dry weight basis from calli of species belonging to the genus Podophyllum. The main purpose of producing plant cell and tissue cultures is their industrial exploitation for the production of bioactive molecules of applied interest. The enormous potential of these bioactive molecules is clear in worldwide market estimates, where aroma and perfume compounds amount to a turnover of over 2 billion dollars a year, while revenue exceeds 50 million dollars for opium derivatives. A list of top natural substances proposed for the development of in vitro production includes: diosgenin (36), codeine (23), atropine (58), reserpine (59), hyoscyamine (5), digoxin (60), scopolamine (6), digitoxin (61), pilocarpine (62), quinidine (63), colchicine (64), emetine (65), morphine (22), quinine (66), vinblastine (54), paclitaxel (55), artemisinin (67) and hyperforin (68). The drive towards the in vitro production of secondary metabolites has originated from the excellent results obtained from bacterial and fungal cultures. However, comparison between in vitro microorganism culturing and plant production of secondary products shows that plant cells are about 200 times larger than microorganism cells and have production times from two to three weeks, compared to the two to three hours of some bacterial strains. In addition, plant cultures pose a higher number of problems, mainly linked to the need for continuous removal of produced products and the maintenance of aseptic conditions.

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Suggested Reading Benedito, V.A. and Modolo, L.V. (2014). Introduction to metabolic genetic engineering for the production of valuable secondary metabolites in in vivo and in vitro plant systems. Rec. Pat. Biotechnol. 8, 61–75. Georgiev, V., Ilieva, M., Bley, T. and Pavlov, A. (2008) Betalain production in plant in vitro systems. Acta Physiol. Plant. 30, 581–593. Georgiev, M.I., Weber, J. and Maciuck, A. (2009) Bioprocessing of plant cell cultures for mass production of targeted compounds. Appl. Microbiol. Biotechnol. 83, 809–823. Goncalves, S. and Romano, A. (2013). In vitro culture of lavenders (Lavandula spp.) and the production of secondary metabolites. Biotechnol. Adv. 31, 166–174. Granicher, F., Christen, P. and Kapetanis, I. (1995) Production of valepotriates by hairy root cultures of Centranthus ruber DC. Plant Cell Rep. 14, 294–298. Gupta, S.K. et al. (2012). In vitro propagation and approaches for metabolites production in medicinal plants. London: Academic Press Ltd-Elsevier Science Ltd. Kirakosyan, A., Sirvent, T.M., Gibson, D.M. and Kaufman, P.B. (2004). The production of hypericins and hyperforin by in vitro cultures of St John’s wort (Hypericum perforatum). Biotechnol. Appl. Biochem. 39, 71–81. Malik, S., Bhushan, S., Sharma, M. and Singh Ahuja, P. (2016) Biotechnological approaches to the production of shikonins: a critical review with recent updates. Crit. Rev. Biotechnol. 36, 327–340. Marchev, A. et al. (2014). Sage in vitro cultures: a promising tool for the production of bioactive terpenes and phenolic substances. Biotechnol. Lett. 36, 211–221. Mucciarelli M., Gallino M., Scannerini S. and Maffei M. (1993). Callus Induction and Plant Regeneration in Vetiveria zizanioides, Plant Cell Tiss. Org. Cult. 35, 267–271. Murashige T. and Skoog F. (1962). A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15, 473– 497. Murthy, H.N., Lee, E.J. and Paek, K.Y. (2014). Production of secondary metabolites from cell and organ cultures: strategies and approaches for biomass improvement and metabolite accumulation. Plant Cell Tiss. Organ Cult. 118, 1–16.

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Murthy, H.N., Dandin, V.S. and Paek, K.Y. (2017). Tools for biotechnological production of useful phytochemicals from adventitious root cultures. Phytochem. Rev. 15, 129–145. Onrubia, M. et al. (2013). Bioprocessing of plant in vitro systems for the mass production of pharmaceutically important metabolites: paclitaxel and its derivatives. Curr. Med. Chem. 20, 880–891. Sivakumar, G. (2006). Bioreactor technology: a novel industrial tool for high-tech production of bioactive molecules and biopharmaceuticals from plant roots. Biotechnol. J. 1, 1419–1427. Smetanska, I. (2008). Production of secondary metabolites using plant cell cultures. Berlin: Springer-Verlag Berlin. Torkamani, M.R.D., Jafari, M. Abbaspour, N., Heidary, R. and Safaie, N. (2014) Enhanced production of valerenic acid in hairy root culture of Valeriana officinalis by elicitation. Cent. Eur. J. Biol. 9, 853–863. Vanisree, M. et al. (2004). Studies on the production of some important secondary metabolites from medicinal plants by plant tissue cultures. Bot. Bull. Acad. Sin. 45, 1–22.

CHAPTER ELEVEN BIOTECHNOLOGY OF BIOACTIVE PLANT MOLECULES

Biotechnology is defined as the exploitation of biological processes for industrial and other purposes, with particular reference to the genetic manipulation of microorganisms for the production of valuable products. It is therefore an applied concept that uses biochemical, physiological and in many cases microbiological knowledge in close relationship with chemical and engineering techniques. The term biotechnology implies the presence of living organisms used in ideal conditions to grow, develop and produce those metabolites for which they have been selected or genetically transformed. The term biotechnology implies the use of any living organism: from bacteria to animal cells.

11.1. Plant Biotechnology If we consider all the changes that humans have made to plants in order to increase their yield or resistance to disease then we realize that the term plant biotechnology refers to an old concept, at least ten thousand years old, from the moment when humans invented agriculture. By restricting the meaning to technological applications in which living organisms are entirely responsible for the production of certain products, this technique goes back to about three thousand years B.C., to the use of microorganisms for the fermentation of wine and beer. However, if we exclude microorganisms from biotechnological processes and we consider only plant cells, plant biotechnology is a very recent science, born in the middle of the twentieth century. In general terms, a biotechnological process can be reduced to a simple equation in which a substrate is put in contact with plant cells to give rise, through a technological and biochemical process, to products. The technological process makes use of equipment designed and built to create

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the ideal conditions for cell culture, but it also includes procedures for the extraction and isolation of all the metabolites produced by the culture. The biotechnological process for the production of secondary metabolites is anything but simple and requires the control of a large quantity of cultural conditions, sometimes impossible without the help of computerized systems. Starting from the concepts of selection developed in Chapter 10, the next step is the preparation of a technological model for the control of plant cultures. A variety of methods are used to develop a biotechnological process, but when it comes to industrial production the goal is to reduce the number of methods to a minimum. The final production must start from low-cost materials, with economic costs that should not exceed 50% of the total cost of production. In general, optimistic estimates for a biotechnological production plant start from manufacturing costs of about US$ 500 per kg of natural substance if one gram of substance per litre of culture medium is formed every 15 days in a fermenter (see below) of 100 m3. High fermentation costs are the typical hurdle that a biotechnological process has to face and explain why, after half a century of technological developments, there are still very few industrially produced in vitro bioactive molecules (for example, shikonin and berberine, discussed in Chapter 10). It is therefore clear that only plant products produced in low quantities from valuable species can be considered for biotechnological production. When discussing the feasibility of the in vitro production of bioactive molecules, it is common to compare the costs of production in open field with those incurred biotechnologically. In fact, the impossibility of inducing the production of all bioactive molecules in in vitro systems shows the limits of the current productive potential of plant cell cultures. The focal point is that the limitations imposed by the cost/benefit ratio restrict research and industrial applications to a limited number of natural products and therefore of plant organisms. This often deprives in the search for the funds necessary to expand knowledge on less profitable but no less interesting systems. Plant cells differ greatly from microorganisms, due first of all to their larger size and also to their lower ability to divide. This latter point leads to extremely long production times compared to those obtained with microorganisms (see Chapter 10) and therefore this implies a higher financial commitment to keep the culture sterile and vital for these longer times. To move from ten to one thousand litres of cells in culture, between six and eight weeks are needed, depending on the culture. Despite this,

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technical problems have often been solved, as in the case of 200 litre fermenters with Digitalis cultures, 750 litre fermenters with Catharanthus and Lithospermum cultures, and up to 20,000 litre fermenters with tobacco cell cultures. Before proceeding with the technological analysis it is useful to describe the basic process of biotransformation on which the main biotechnological applications are based.

11.2. Biotransformation of Plant Bioactive Molecules Biotransformation is a technique that uses enzymes contained in cultured cells to alter the chemistry of metabolite functional groups which are supplied in the culture medium. It can also be defined as the series of chemical changes occurring in a compound, especially a bioactive molecule, as a result of enzymatic or other activity by a living organism. The main chemical reactions that characterize biotransformation are: reduction, oxidation, hydroxylation, epoxidation, glycosylation, hydrolysis, esterification, methylation, demethylation and isomerization. Usually, one of these reactions is used to increase the biological activity of a given chemical structure and often it involves the action of one or more enzymes in a sequence of specific chemical reactions. The enormous biochemical potential of cell cultures in the biotransformation of natural or synthetic products is the production of metabolites of higher commercial value. In the case of synthetic compounds, we can use molecular analogues or intermediate metabolites present in other species and that normally are not present in the plant to produce new compounds with unique biological activity. As most of the reactions are intracellular, it is essential that the substrate absorption occurs in the enzymatic compartment; furthermore, it is desirable that the biotransformed product can be quickly released into the culture medium. Studies performed on plants that biotransform alkaloids or cardenolides suggest that transport can be either active or passive (by diffusion). In some cases, extracellular biotransformation activity has also been noted, due to the presence in the culture medium of enzymes extruded by the cells. Usually biotransformation occurs in two ways: x through the use of whole cells, where cells are suspended in a culture liquid or immobilized by fixing them to a solid support;

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x through the use of enzymes isolated and purified from plant cells and immobilized on an external support. In general, the conversion rates are so rapid and efficient that if the precursor is a low-cost molecule (compared to the biotransformed product) and if the process can run continuously there is a good chance for the industrial exploitation of the process. In any case, it is necessary that the biotransformation product be a molecule with a high commercial value and that cannot be further biotransformed. In this regard, most of the biotransformation systems developed so far have been based on a one step procedure, where the compound (usually a drug) is subjected to a single enzymatic reaction. In order to obtain a system capable of efficient biotransformation, there are some basic rules to follow: x cultures must possess the enzymes necessary for the biotransformation of the precursor into the desired product; x the speed of formation of the new product must be considerably higher than its further transformation into another compound of lower value; x cultures must be able to tolerate the presence of both the precursor and the biotransformed product. Furthermore, to obtain a single product from the biotransformation of a generic precursor, it is necessary to: x select a reaction catalysed by one or more enzymes; x provide a relatively large amount of substrate; x use a substrate with almost no physiological or pharmacological activity, but capable of being transformed into a product with a high physiological and pharmacological activity; x ensure that biotransformation is a difficult reaction to achieve by normal chemical reactions. A look at the scientific literature offers an interesting view on the ability of plant cells to biotransform synthetic substances or metabolic intermediates to form molecules of high commercial value, as shown in Table 11.1.

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Table 11.1. Some examples of the biotransformation of valuable products Species

Anisodus tanguticus

Type of culture CS

Artemisia annua

CS

Berberis stolonifera

CS

Cannabis sativa

CS

Catharanthus roseus

CS

Cheiranthus cheiri

CS

Cinchona spp.

CS

Citrus paradisi Coffea arabica

CS CS

Coronilla varia

CS

Curcuma zedora

CS

Cymbidium spp.

CS

Datura innoxia

CS

Daucus carota

CS

Dendrobium phalaenopsis Digitalis purpurea

CS

Duboisia myoporoides Epidendrum ochraceum Eucalyptus perriniana

CS CS

Gardenia jasminoides

CS

CS

CS

Substrate or biotransformation products Hyoscyamine Æ Hydroxyhyoscyamine Artemisinic acid Æ glucosylic ester of hydroxyartemisinic acid Reticuline Æ protoberberine Cannabinol Æ cannabielsoine Anhydrovinblastine Æ vinblastine Cholesterol Æ cholestenone Tryptophan Æ ȕcarboline Naringenin Æ prunin Phenylpropionic acid Æ glucoside Nitropropanoic acid Æ glucopyranoside Germacrone Æ curcumenone Menthyl acetate Æ menthol Umbelliferone Æ glucoside Gitoxigenin Æ hydroxygitoxigenin Testosterone Æ androstenedione Digitoxigenin Æ hydroxygitoxigenin Rhamnose Æ glucose Menthyl acetate Æ menthol ȕ-thujaplicin Æ isopropyltropolone glucoside Catechol Æ glucoside

Type of chemical reaction Hydroxylation Hydroxylation, glucosylation Multiple reactions Multiple reactions Hydrogenation Oxidation Multiple reactions Glucosylation Glucosylation Esterification Cycling, hydroxylation Hydrolysis of the ester glucosylation hydroxylation Oxidation hydroxylation Isomerization Hydrolysis of the ester glucosylation glucosylation

Biotechnology of Bioactive Plant Molecules Species

Glycine max Glycyrrhiza glabra

Type of culture CS CS

Jasminum officinale

CS

Lactuca sativa

CS

Lavandula angustifolia Lonicera tatarica

CS CS

Lycopersicum esculentum Mahonia nervosa Mallotus japonicus

CS

Medicago sativa

CS

Mentha spp.

CS

Mentha Canadensis

CS

Mentha piperita

CS

Nicotiana tabacum Ochrosia elliptica

CS CS

Panax ginseng Petroselinum crispum Phaseolus vulgaris

CS CS CS

Rauwolfia serpentina

CS

Rosa centifolia Rosmarinus officinalis

CS CS

Salvia officinalis

CS

Silene alba

CS

Solanum tuberosum

CS

CS CS

Substrate or biotransformation products Geraniol Æ geranial Papaverine Æ various compounds Isopiperitenol Æ piperitenone oxide Chlortolurone Æ various compounds Citronellal Æ citronellol Loganin Æ secologanin Teasterone Æ dehydroteasterone Thebaine Æ oripavine Hydroxybenzoic acid Æ glucoside Carvone Æ neoisodihydrocarveol Pulegone Æ isomenthone Menthyl acetate Æ menthol Isopiperitenone Æ hydroxysopiperitenone Borneol Æ camphor Papaverine Æ papaverinol Panaxatriol Æ glucoside Geraniol Æ nerol Tryptophan Æ norarmane Ajmalicine Æ new indole alkaloids Geraniol Æ geranial Linalool, menthol, geraniol, farnesol Æ glucosides sucrose Æ rosmarinic acid Papaverine Æ demethylpapaverine Solavetivone Æ rishitin

389 Type of chemical reaction Oxidation Multiple reactions Epoxidation Multiple reactions Reduction Oxidative degradation Oxidation demethylation glucosylation Reduction Reduction Hydrolysis of the ester hydroxylation Oxidation hydroxylation glucosylation Isomerization Multiple reactions Methylation, acetylation, isomerization Oxidation glucosylation Multiple reactions demethylation hydroxylation

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Spirodela oligorrhiza

Type of culture CS

Stevia rebaudiana Strophanthus amboensis Strophanthus intermedius Thuja occidentalis

CS CC, CS

Weigelia japonica

CS

CS CS

Substrate or biotransformation products Benzyl acetate Æ benzyl alcohol Steviol Æ stevioside Digitoxin Æ purpureaglicoside Digitoxigenin Æ hydroxygitoxigenin Hexadecane Æ terpinolene Loganin Æ secologanin

Type of chemical reaction Hydrolysis of ester glucosylation glucosylation hydroxylation Multiple reactions Oxidative degradation

CS= cell suspension; CC = callus culture.

In all biotransformation, it is important that the committed enzyme be expressed in the selected cell lines. Usually, the substrate is incubated in a period ranging from eight hours to eight days and the conversion into the desired product may show an efficiency that in some cases reaches 90%, although the average percentage is around 10–15%. The main objective is to convert a biologically inactive compound into an active one. For example, digitoxin and digoxin are two cardenolides of considerable importance (see also Chapter 7), but plants such as Digitalis lanata or Strophanthus spp. mainly produce the first compound, which has a lower biological action. The biotransformation of digitoxin into digoxin is possible using cell lines of both species that express a high activity of the enzyme responsible for the hydroxylation of the beta carbon. The development of ideal biotransformation conditions led to conversion yields higher than 50% with a 90% release of digoxin in the culture medium. Biotransformation of digitoxin can also be carried out by cell lines of species that normally do not accumulate this compound. This is the case with Daucus carota cell suspensions in which the enzymes present are not part of the biosynthetic pathway leading to the synthesis of digoxin, but simply react with digitoxin due to a lack of substrate specificity. On the other hand, many species belonging to the genus Papaver are able to transform tyrosine into thebaine, but only cultures of Papaver somniferum and P. setigerum are able to perform the additional biosynthetic steps to produce codeinone, codeine and morphine (see Chapter 9). The choice of species and cell lines to perform a given biotransformation remains the focal point for each of the steps that follows. In order to increase the

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production of a biotransformation system it is also possible to use the strategies described in Chapter 10, such as biotic and abiotic stimulation. The future of industrial biotransformation depends fundamentally on strategies that will allow an increase in yield and a reduction in production times and costs. To fulfil these principles, it is necessary to give importance to some basic research areas such as: x production of cells subjected to abiotic stress; x gene activation and induction using biotic stress and stimulators; x identification, isolation and characterization of key biosynthetic enzymes; x optimization of production through high yield crops or immobilization of cells or enzymes. Many authors agree with the idea that in the future it is the manipulation of cells, genes and plant enzymes, rather than the intervention on plants themselves, that will provide the basis for the biotechnological production of secondary metabolites.

11.3. Bioreactors and Fermenters As we described in Chapter 4, despite major advances in synthetic chemistry, plants are still the source of bioactive molecules for at least 25% of all prescribed medicines. Because it is possible to grow plant cells in aseptic conditions, plant cell cultures represent an ideal system for the production of phytochemicals of high commercial value. An efficient industrial process requires the large scale production of plant cells, with volumes suitable for the obtaining of profitable quantities of secondary metabolites. A bioreactor is a manufactured or engineered device or system that supports a biologically active environment. Cell growth in bioreactors allows the precise control of parameters such as oxygen and carbon dioxide concentration, pH and nutrients. Bioreactors allow the mass production of plant cells in containers of from 2 to several thousand litres. Large-scale cultivation began at the end of the twentieth century and the first attempts sought systems capable of producing food for space applications. The first 300-litre fermenters were developed in the mid1970s, and in the early 1980s they reached volumes of over a thousand litres. Nowadays, cells are grown in 80–100,000-litre bioreactors which have been designed for the production of valuable phytochemicals.

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Numerous bioreactor configurations can be used for aerobic cell suspension cultures and the choice of the ideal system depends on a number of factors. The most important is certainly the availability of oxygen, its mixing in the culture medium and the ability of cells to use it. In the specific case of plant cell suspensions, the bioreactor must ensure adequate mixing and generate the least amount of stress factors. In general, there are three types of bioreactor, characterized by different air supply systems. The first type of system is equipped with a device that ensures the agitation of the culture medium: these are blades or cylinders that rotate at a certain speed (usually from 60 to 100 revolutions per minute) ensuring the mixing of the culture. In the second and third systems there is no agitation and the mixing is guaranteed by the movement of the air bubbles entering the culture solution. The difference between the last two is in the way of managing the air mixing. Bioreactors can be made using a closed container in which the culture starts at a certain time and is stopped at a later time – the so-called “batch systems”. Batch systems can be unlimited, limited by the substrate or limited by oxygen. Another type of system is designated the “extended culture system”, where the concentration of the limiting substrate is kept constant by supplying the system continuously. The last system type is the “continuous operation system” in which, unlike the batch system, a continuous flow of nutrients is supplied to the bioreactor. This system is characterized by the continuous discharge of produced molecules. In this case processes may be either unlimited, limited by the substrate or limited by oxygen processes. In this type of system the steady state of production is reached with a density and concentration of theoretically constant cells. In general, the plant cells are less tolerant of elastic forces created in a bioreactor with mechanical agitation than microbial systems because of the damage inflicted by the blades on the plant cell walls. Therefore, the best system for plant cells is the airlift system, in which a flow of air generates the culture mixing. However, when using high biomass systems, airlift systems might not be adequate because, beyond certain dimensions, the air flow is not able to guarantee an adequate mixing without creating areas in which there is a stagnation of reaction products. In these cases, increasing the air flow is discouraged as it can generate foam, which reduces the bioreactor capacity. Thus, it is necessary to choose the ideal agitation system according to the quantity of biomass used. Furthermore, several varieties of plant cell culture have shown a particular preference for the culture agitation system. For example, a large-scale production of

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Coleus blumei cells reached a maximum yield with a helical stirring system, whereas in the case of Maclura tricupidata cultures cells grew better in the presence of a rotating blade. An important factor in the production of plant cells in bioreactors is the preparation of the inoculum, that is, the known amount of plant cells to be inserted to start the bioreactor culture. Bioreactors have an inoculum density below which there is no growth, and usually this corresponds to 10% of the overall culture volume. This means that in order to start a culture in a 20-litre bioreactor it is sufficient to introduce an inoculum prepared with a 2-litre flask. Likewise, to start a culture in a 10,000-litre bioreactor an inoculum of about 1,000 litres is required, and the latter will be obtainable only with another bioreactor. It is clear that industries working with 100,000-litre bioreactors require a battery of scalar-sized bioreactors that are used in series to produce the inoculum for the next bioreactor. As discussed in Chapter 10, the production of secondary metabolites occurs mainly in the stationary phase, implying that the bioreactor continues its activity even when cells stop dividing. Most often, cells retain the secondary metabolites they produce in the stationary phase, which requires the arrest of the bioreactor to allow the extraction of metabolites. In general, bioreactors are connected to a filtering and extraction system. The first step is usually the separation of the liquid phase from the cells. The filtrate may contain secondary metabolites, possibly released into the culture medium; therefore, the bioactive molecules are extracted using a suitable solvent. Next, bioactive molecules are extracted from the cell culture. The extracted metabolites are either separated or merged and then purified, while the remaining biomass finds different applications, being still rich in proteins and carbohydrates (e.g., cellulose and polysaccharides).

11.3.1. Photobioreactors Photobioreactors are a class of bioreactors in which microorganisms or plant cells use sunlight or artificial light to convert different substrates into desired compounds, including numerous bioactive metabolites such as astaxanthin (see Chapter 7). The shape of these devices is either planar or tubular. The engineering of photobioreactors for the economical and effective use of algae and their bioactive products has made impressive and promising progress. Nowadays, the photobioreactors in use are open systems because of the considerable minor costs; however, other types of

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photobioreactors have recently attracted considerable attention for the production of valuable biochemicals. For high density cultures in photobioreactors the optimization of environmental factors is required, factors including the availability of light, the transfer of gases such as CO2 and O2, and the nutrient culture media, mixing and temperature. Within the next decade, it is expected that advanced photobioreactor engineering will open new and profitable markets for the production of high value bioactive products from microalgae. Despite their cost, closed photobioreactors have several important advantages over open systems; they: x minimize contamination and allow the cultivation of axenic algal monocultures; x offer better control of conditions such as pH, temperature, light, and CO2 and O2 concentrations; x reduce the loss of CO2 to the environment; x prevent the evaporation of water; x allow higher cell concentrations; and x allow the production of complex biopharmaceuticals. When designing a photobioreactor, the following points must be considered: x the reactor should allow the cultivation of various species of microalgae; x the design of the reactor must provide uniform illumination of the culture area and fast mass transfer of CO2 and O2; x microalgae cells are highly adhesive, which results in rapid misting of the surfaces and a reduction in light penetration. This leads to frequent shutdown and the mechanical cleaning and sterilization of photobioreactors; x high rates of mass transfer must be achieved without damaging the cultured cells or suppressing their growth; x the photobioreactor must be able to work in conditions of intense foam formation, as often happens in bioreactors with high mass transfer rates; x the photobioreactor should have at least a small non-illuminated part.

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Different types of photobioreactors have been designed and developed for the production of microalgae: Vertical column photobioreactors. These consist of transparent vertical tubes that allow light penetration. A diffuser is positioned at the bottom of the reactor and generates small CO2 bubbles. The system also removes the O2 produced during photosynthesis. Vertical tubular photobioreactors, depending on the way they flush the liquid, can be divided into column, bubble and airlift reactors. In the first case the mixing of algae and the mass transfer of CO2 take place through a gas exchanger. The photosynthetic efficiency can increase substantially by increasing the flow rate of the gas which leads to a shorter cycle of light and darkness. Airlift reactors are reservoirs with two communicating zones. One of the tubes is called the “rising column”, where the gaseous mixture is stripped, while the other is called the “descending pipe” and does not receive the gas. Flat panel photobioreactors. These reactors are cubic in shape with the smallest possible optical path. They can be made from transparent materials such as glass, plexiglass, polycarbonate, etc. They are characterized by a high surface/volume ration and are provided with an open gas disengagement system. Stirring is provided either by blowing air or by both peristaltic and circulating pumps that ensure the flow of liquids inside the photobioreactor. Horizontal tubular photobioreactors. The horizontal tubular reactors are positioned horizontally with a parallel series of transparent tubes, arranged in a ring, with an inclined or horizontal tubular shape to the tubular reactor. Its shape gives advantages regarding its orientation towards sunlight, with a consequently higher efficiency of light conversion. The CO2 mixture is introduced into the connecting pipe by a gas exchange system. However, the oxygen accumulated during photosynthesis can cause photooxidation and reduce photosynthetic efficiency. When exposed to sunlight, in order to cool the system, water is sprayed onto the tube’s surface. Alternatively, tubes can be overlapped or the temperature can be adjusted by a cooling system. Helical-type photobioreactors. These consist of a transparent and flexible small-diameter tube which is rolled up with separate or attached degassing units. A centrifugal pump connected to the degassing unit is used to drive the culture through the tube. However, the energy required by the centrifugal pump for culture recirculation limits its commercial use.

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The occurrence of deposits inside the reactor is another disadvantage of this type of system. Mixed tank photobioreactors. These are the most conventional models, where agitation is provided mechanically with the help of helices of different sizes and shapes. Deflectors are used to reduce cavitation and CO2-enriched air is bubbled from below to provide the carbon source for photosynthesis. This type of bioreactor has been transformed into a photobioreactor by illuminating it externally with fluorescent lamps or optical fibres, but the main disadvantage of this system is the low surface/volume ratio which in turn reduces the efficiency of light harvesting. Hybrid photobioreactors. These are widely used and exploit the advantages of two different types of reactor; an airlift transport system is used along with a tubular transport system. On the one hand, the outer rings act as a light collecting unit; on the other, the airlift system acts as a degassing system. Its advantages include better control of cultural variables, enabling higher productivity and reducing energy consumption.

11.4. Immobilized Plant Cell Cultures One of the major problems in the cultivation of high plant cell biomass is the stress condition to which cells are subjected as a consequence of cell wall contact with the agitation system. This problem can be solved by immobilizing the cells on a solid substrate and sliding the culture liquid between them. The methods and procedures used for the immobilization of plant cells are similar to those that have been successfully adopted for microorganisms. The most frequently used technique is the entrapment of cells or chloroplasts in various sorts of polymerized gels, such as agar, polyacrylamide, agarose and gelatine. The main reasons for the immobilization of plant cells as an alternative to using suspended cultures for the production of bioactive molecules can be summarised as the: x re-utilisation of biomass through the retention of cells in a bioreactor and the recovery of the product from the culture medium; x physical separation of cells from the culture medium and then from the released products;

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x use of low-cost and efficient bioreactors through the use of highbiomass and low-volume containers; x use of continuous systems. The requirements to develop an efficient immobilized cell system are: x to obtain a cell culture with definite properties, for example high yield and low growth rate; x to define an adequate immobilization method with a well-defined geometry in order to guarantee the cell viability and the production of the desired metabolites; x to obtain the metabolite release in the culture medium while keeping the cells alive, especially in instances where products are usually accumulated in the vacuoles. Furthermore, to be used in industrial applications, the system must be: x x x x x x

safe for operators and for product consumers; usable for long periods while maintaining sterility; adequately gentle in order to ensure cell viability; stable and durable; extremely active and specific; inexpensive.

The physiological requirements include, inter alia, cell-to-cell contact, which allows the intercellular transfer of substances. Furthermore, such contact induces cytodifferentiation, which is directly linked to the production of secondary metabolites. In an immobilized system, the growth and production phases can be accurately controlled through physical and chemical stresses, which allow cells to be kept in bioreactors for extended periods by alternating growth and production cycles. The main limitation of immobilized cultures is the ability to excrete in the culture medium bioactive metabolites. Methods that use chemical agents that permeabilize membranes often significantly reduce the cells vitality, thus lowering the ability to biosynthesize or biotransform secondary metabolites. The remarkable sensitivity of plant cells to environmental changes requires the use of moderate immobilization methods.

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11.4.1. Plant Cell Immobilization Techniques Calcium alginate is one of the compounds most widely used to trap plant cells. The polymer lends itself well to autoclaving (remember that cells must be grown in a sterile environment) and the polymerization process can be easily reversed by adding a calcium chelating agent such as ethylenediamine tetraacetic acid (EDTA) or citrate, which destroy the gel by disrupting the bond with Ca2+. Alginate is a polysaccharide that forms a stable gel in the presence of cations. In practice, the technique consists in forming pellets of alginate that contain plant cells by dripping a solution of alginate and cells into a calcium chloride solution. During the alginate pellet formation, there is a progressive shrinkage due to the exclusion of water as the calcium ions spread inside by binding to the polymer. The shrinkage varies depending on the polymer concentration. Some red algae, as in the genus Chondrus and particularly C. crispus, are a foodstuff known as carrageen, used in the food and medicinal industries. K-carrageen is a polysaccharide containing sulphate esters (more than 20% of the sugar component is sulfonated) and is insoluble in cold water. It can instead be solubilized in hot water, and when it cools it forms a gel. Also in this case, the quality of the gel depends on the concentration of the polymer as well as on the quantity and type of cations present. Kcarrageen is often used to trap plant cells with a polymer that requires a temperature of 25–30 °C to be solubilized in water. Another way to trap and immobilize cells is through the use of agar and agarose (a purified form of agar). One of the advantages of agarose is that it does not require the presence of ions for gel stability, although the mechanical stability of the agarose is lower than that of alginate or kcarrageen used at the same concentration. Furthermore, the commercial value of agarose is far higher than that of the two above mentioned polysaccharides. The gel entrapment by polymerization is also obtained using polyacrylamide. Although the chemical compounds used to polymerize acrylamide are toxic, new methods have been developed recently that reduce toxicity and increase the vitality of immobilized cells. The technique consists in suspending plant cells in an aqueous solution of polyacrylamide which has been pre-polymerized and partially substituted with groups of acrylhydrazine. The polymer chains are then linked together through the use of a dialdehyde. Another method is to mix the

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cells with a viscous alginate solution to ensure cell protection before trapping them in polyacrylamide. Another support used to trap cells is a solidified polyurethane foam. Cells remain trapped in the polyurethane matrix due both to physical retention and bonding with the foam. The efficiency of this system depends on the correlation between the internal spaces and the size of the cells. Urethane polymers with terminal isocyanate functional groups are ideal for immobilizing biocatalysts. When these polymers are mixed with an aqueous solution containing a cell suspension, a gel is formed and the polymer reacts to form urea bonds. Another way to trap cells is through the use of hollow fibres. Bioreactors with hollow fibres were the first tools for the entrapment of plant cells. In practice, the system works by distributing oxygen through the fibre lumen. Cells aggregate to the fibres and remain trapped between the meshes.

11.4.2. Viability of Cells A successful cell immobilization depends on the biosynthetic capacity of culture cells and the possibility of absorbing the precursors and releasing the products of the biochemical reactions. Excluding simple biotransformation, the formation of a compound may depend on the prolonged vitality of the immobilized cells. Various methods are used to evaluate the viability of an immobilized cellular system through the use of dyes, by measuring the growth rates, cell division, respiration, substrate absorption and the release of metabolites. The most commonly used dye is fluorescein diacetate (FDA). The dye, used at 0.5%, is absorbed by the cells and the cytosolic esterases break down the ester bond by deacetylating the molecule, that then becomes fluorescent when irradiated by an ultraviolet irradiation. This analysis is performed with an epifluorescence optical microscope through which it is possible to distinguish and count viable cells. While the method is suitable for cell protoplasts and suspensions, it becomes less usable when cells tend to form aggregates. In this case, only the outer layer of cells is observed, while it is difficult to control the viability within the clusters. Another colorimetric method uses triphenyl tetrazolium chloride (TTC), which is particularly useful for immobilized cells, based on the production of red formazan. Another indicator of cell viability is the mitotic index, the percentage of the cell population that at a certain time is in a given stage of

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mitosis. Carbolfuchsin, a mixture of phenol and basic fuchsin, is used as a dye as an indicator of the mitotic index because it is able to colour the chromosomes following fixation. The increase in cell mass and cell count is an excellent indicator of viability, but while it is difficult to establish the number of cells in an immobilized system it is much easier to evaluate the total weight (or biomass) variation of cultured cells. Another method for assessing viability is the isotopic method. A certain amount of a non-metabolizable 14C-labeled sugar is introduced into the immobilized culture together with a diffusible molecule such as 3H-labeled water. The method discriminates the living cell volume from the dead one based on the presence of intact or disrupted plasmalemma. The “vital” volume is the percentage of space that excludes a non-metabolizable sugar (e.g., mannitol) and that allows marked water to pass through. A commonly used method is the determination of the dry and fresh weight of the culture, by assuming that the dry weight of the immobilizing matrix does not vary.

11.4.3. Biosynthetic Capacity The biosynthetic capacity of immobilized plant cells can be divided into three categories: biotransformation, de novo synthesis and synthesis from precursors. Bioconversion or biotransformation of a compound is carried out by a multitude of enzymes belonging to several enzymatic classes. The best example of the biotransformation of secondary metabolites in immobilized systems is the conversion of a derivative of digitoxin into digoxin (see Chapter 7) using Digitalis lanata cells trapped in alginate. But comparable results were obtained with strains of Daucus carota, Mentha piperita, Papaver somniferum and many other species (Table 11.2). In many cases, the synthesis of a given metabolite does not occur due to a low concentration of the relative precursor. A considerable increase in the biosynthesis of secondary metabolites in immobilized cells can be achieved by adding to the culture medium adequate quantities of those precursors or compounds limiting the speed of synthesis. A classic example is the production of indole alkaloids in immobilized cultures of Catharanthus roseus using agar, agarose or k-carrageen as inert phases. In these studies, the addition of a precursor caused a yield increase of up to

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12 times when compared to that obtained solely with the nutrients available in the culture medium. Datura innoxia cells immobilized in alginate are able to produce high amounts of scopolamine when the culture medium has ornithine added to it. Likewise, with Capsicum frutescens cells immobilized in polyurethane, the addition of phenylalanine or synaptic acid to the culture medium significantly increases the formation of capsaicin. De novo synthesis of complex secondary metabolites involves numerous components of the cellular metabolism, which can function efficiently only in viable cells. Table 11.2 Some examples of biotransformation in immobilized plant cells. Species Catharanthus roseus Digitalis lanata Daucus carota Mentha spicata Mucuna pruriensis Ochrosia elliptica Panax ginseng Papaver somniferum

Substrate Æ biotransformed product Cathenamine Æ ajmalicine Digitoxin Æ hydroxydigitoxin Gitoxigenin Æ hydroxygitoxigenin Menthone Æ neomenthol Tyrosine Æ diidrossifenilalanina Papaverine Æ papaverinol Panaxatriol Æ glucoside Codeinone Æ codeine

About one hundred new compounds have been produced by about thirty plant species grown in immobilized systems. De novo synthesis of indole alkaloids has been carried out in cell cultures of Catharanthus roseus trapped in alginate and polyacrylamide, with yields higher than those obtained with cell suspensions. Similar results have been obtained for many other species (Table 11.3).

11.4.4. Release of Bioactive Molecules Most cells accumulate bioactive molecules in the vacuole or in specialized structures such as glandular trichomes or other secretory tissues (see Chapter 2). This requirement significantly limits the exploitation of immobilized cell systems for the production of such substances. Since one of the major advantages of immobilization of a biocatalyst is the possibility of a continuous production process, it is important that the compound produced by the immobilized cells be released quickly into the culture medium. Most of the studies on immobilized cultures are dedicated to the possibility of inducing the release of metabolites from the

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immobilized cells and in many cases the result is achieved by membrane permeabilization. In some cases, immobilization induces the spontaneous release of the metabolites, as in the case of immobilized C. roseus cells, which quickly release indole alkaloids. In other cases, immobilization slightly damages the cell culture without reducing its biosynthetic capacities, but induces a lower degree of permeability. However, the spontaneous release of products is not a common feature and it is often necessary to operate either with a selection of strains with less permeability or using permeabilising agents. Promising results have been obtained with an intermittent permeabilisation technique, which allows the programmed release of products without bringing the membrane permeability alteration process to cell lethal limits. Table 11.3 Some examples of the de novo synthesis of secondary metabolites in immobilized plant cells. Species Apium graveolens Capsicum frutescens Catharanthus roseus Coffea arabica Dioscorea deltoidea Ginkgo biloba Lavandula vera Morinda citrifolia Nicotiana tabacum Salvia miltiorrhiza Solanum aviculare Thalictrum minus Vicia faba

Biosynthesized product Phtalides Capsaicin Serpentine Methylxanthine Diosgenin Gingkolides Pigments Anthraquinones Epiandrosterone Cryptotanshinone Steroid glycosides Berberine Ethane

11.5. Cryopreservation The maintenance of cell cultures in a continuous state of cell division through repeated subcultures often leads to the onset of anomalies such as: x x x x

increased ploidy; accumulation of spontaneous mutations; decrease or loss of morphogenetic potential; decline or loss of the biosynthetic capacity to form metabolites;

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x inversion of selected varieties or mutants in the original (wild) phenotype; x unwanted selection of unwanted phenotypes; x somaclonal variation. Each of these anomalies can greatly reduce the ability to exploit a cellular system for the production of secondary metabolites and there are many examples of loss of biosynthetic capacity due to prolonged culturing. To ensure the industrial production of secondary metabolites over time it is necessary to use safe plant genetic stores, but maintenance through subculturing is not a safe method. In simplistic terms, it is necessary to find methods able to considerably reduce the culture growth kinetics by lengthening the subculture time. In some cases, the dilatation of time may be of several months, and in others it may be years or even centuries. While it is not yet possible to obtain for cultured cells the dormancy observed in some seeds, physical methods must be adopted to slow down cell growth. Temperature control is one of the most effective methods. Growth arrest can be achieved by freezing plant cells at ultra-low temperatures such as those obtained with liquid nitrogen (-196 °C). The technique that uses deep cold to conserve biological material is called cryopreservation. Since no cell would resist immersion in liquid nitrogen without being killed, it is necessary to prepare the cells for freezing. Before subjecting cells to cryopreservation, special handling must be carried out to obtain the cells with the highest degree of tolerance to freezing. Calli generally tolerate freezing. Approximately one hour before freezing, selected calli or cells are absorbed in solutions able to alter membrane permeability, cell freezing point and cold stress responses, which are essential for maintaining viable cells. Polyethylene glycol solutions are usually used, although there are protocols that make use of other molecules such as dimethyl sulfoxide (DMSO) and glycerol. These substances are called cryoprotectants and are usually added to the culture medium at twice the concentration, to be diluted at the time of application, which usually occurs slowly and at low temperature. In the fast freezing preparation procedure, to temperatures between zero and down to -100°C, ice crystals form both inside and outside the cell and the cooling rate is an extremely critical parameter. Slow cooling will result in cell dehydration, while rapid cooling could create ice crystals in the cytosol. An ideal procedure for a given sample is to cause the least possible damage to the cell. To prevent ice re-crystallization, it is

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necessary to keep cells at temperatures below -100 °C. Frozen samples are usually stored in liquid nitrogen. The return to natural conditions (for example at room temperature) must be as quick as possible, again to avoid the formation of intracellular crystals. Once brought back into natural conditions, the frozen cell requires particular care through the use of specific culture media, preferably of semi-solid nature. The cryopreservation of cells capable of producing secondary metabolites has been achieved for many medicinal plants. In Catharanthus roseus, sorbitol was used along with DMSO as a cryoprotectant and the optimal freezing speed was 0.5 °C min-1 down to -40 °C, and the final preservation was obtained in liquid nitrogen. For Digitalis lanata a solution of sucrose, glycerol and DMSO was used as a cryoprotectant and a cooling rate of 2 °C min-1 down to -60 °C was used; cells were then stored in liquid nitrogen. For the cryopreservation of Panax ginseng cells it was necessary to harden the cells by gradually increasing the sucrose concentration from 3 to 25%, while simultaneously reducing the culture temperature to 2 °C. The use of the cryoprotectants sucrose, glycerine and DMSO allowed the lowering of the temperature by 0.5 °C min-1 down to -70 °C, followed by the final preservation in liquid nitrogen. It is important that after thawing, cells can quickly regain normal growth rate, mitotic index and, above all, the ability to biosynthesise or biotransform as before freezing. In the case of C. roseus, cryopreservation does not alter the growth capacity of thawed cultures, nor the biosynthetic capacity to produce indole alkaloids. The same result was obtained for hundreds of strains of Daucus carota selected to produce anthocyanins or Lavandula vera calli for the production of biotin. Numerous experiments carried out on cell lines selected for the production of secondary metabolites have shown that cryopreservation is one of the most reliable methods for the long-term preservation of the genetic and biosynthetic properties of plant cells. A critical point remains the ideal moment during the cellular development at which to operate the freezing process so as not to cause cell damage. The demonstration that thawing of cells cryopreserved for several years in liquid nitrogen allows the complete recovery of cell biochemical and molecular capacities makes cryopreservation an indispensable tool for the maintenance over time of the biosynthetic potential of in vitro systems.

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Suggested Reading Archambault, J., Volescky, B. and Kurz, W. (1990). Development of bioreactors for the culture of surface-immobilized plant cells. Biotechnol. Bioeng. 35, 702–711. Benson, E.E. (2008). Cryopreservation of phytodiversity: a critical appraisal of theory practice. Crit. Rev. Plant Sci. 27, 141–219. Cascaval, D., Galaction, A.I. and Blaga, A.C. (2007). Photobioreactors. Rom. Biotechnol. Lett. 12, 3377–3388. Fickel, J., Wagener, A. and Ludwig, A. (2007). Semen cryopreservation and the conservation of endangered species. Eur. J. Wildlife Res. 53, 81–89. Ginai, M. et al. (2013) The use of bioreactors as in vitro models in pharmaceutical research. Drug Disc. Today. 18, 19–20. Gonzalez-Arnao, M.T., Panta, A., Roca, W.M., Escobar, R.H. and Engelmann, F. (2008). Development and large scale application of cryopreservation techniques for shoot and somatic embryo cultures of tropical crops. Plant Cell Tiss. Organ Cult. 92, 1–13. Kawamoto H., Asada Y., Sekine H. and Furuya T. (1998). Biotransformation of Artemisinic acid by cultured cells of Artemisia annua, Phytochemistry. 48, 1329–1333. Miele, L. (1997). Plants as bioreactors for biopharmaceuticals – regulatory considerations,.Trends Biotechnol. 15, 45–50. Nguyen, T.K.O., Dauwe, R. Bourgaud, F. and Gontier, E. (2013) From bioreactor to entire plants: development of production systems for secondary metabolites. Adv. Bot. Res. 68, 205–232. Olivieri, G., Salatino, P. and Marzocchella, A. (2014). Advances in photobioreactors for intensive microalgal production: configurations, operating strategies and applications. J. Chem. Technol. Biotechnol. 89, 178–195. Pandolfi, V., Pereira, U., Dufresne, M. and Legallais, C. (2017) Alginatebased cell microencapsulation for tissue engineering and regenerative medicine. Curr. Pharm. Design. 23, 3833–3844. Park, S.-H., Chae, Y.-A., Lee, H.J., Lim, Y.-H. and Kim, S.-U. (1994). Production of (-)-7-hydroxyisopiperitenone from (-)-isopiperitenone by a suspension cell culture of Mentha piperita, Planta Med. 60, 374–375. Pulz, O. (2001). Photobioreactors: production systems for phototrophic microorganisms. Appl. Microbiol. Biotechnol. 57, 287–293. Singh, R.N. and Sharma, S. (2012). Development of suitable photobioreactor for algae production – a review. Ren. Sust. En. Rev. 16, 2347–2353.

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Suh, I.S. and Lee, C.G. (2003). Photobioreactor engineering: design and performance. Biotechnol. Bioproc. Eng. 8, 313–321. Vasumathi, K.K., Premalatha, M. and Subramanian, P. (2012). Parameters influencing the design of photobioreactor for the growth of microalgae. Ren. Sust. En. Rev. 16, 5443–5450. Wang, B., Lan, C.Q. and Horsman, M. (2012). Closed photobioreactors for production of microalgal biomasses. Biotechnol. Adv. 30, 904–912. Wang, S.K., Stiles, A.R., Guo, C. and Liu, C.Z. (2014). Microalgae cultivation in photobioreactors: an overview of light characteristics. Engin. Life Sci. 14, 550–559. Warrmann, U. and Knorr, D. (1993). Conversion of menthyl acetate or neomenthyl acetate into menthol od neomenthol by cell suspension cultures of Mentha canadensis and Mentha piperita, J. Agric. Food. Chem. 41, 517–520. Wilhelm, R. and Zenk M.H. (1997). Biotransformation of thebaine by cell cultures of Papaver somniferum and Mahonia nervosa, Phytochemistry. 46, 701–708.

CHAPTER TWELVE GENETIC ENGINEERING OF BIOACTIVE PLANT MOLECULES

The achievement of cellular systems capable of producing secondary metabolites in vitro is undoubtedly an unquestionable result. However, although revolutionary and sometimes highly productive, these techniques do not allow absolute control of the final production of a given metabolite or the obtainment of a hybrid with an a priori definable character. Under physical-chemical control, the production of bioactive molecules depends on the natural laws of genetics. Sometimes it is necessary to act on the basic process that allows enzymes to be expressed in a given culture, at a certain moment of development and with certain kinetic characteristics. Therefore, it is necessary to act on genes, by modifying, transferring, reconstructing or activating them, or making them remain silent. The set of techniques that allows the manipulation of genetic information is defined as genetic engineering and we will discuss this topic with particular reference to the transformation of cells for the production of plant bioactive molecules. We need first to introduce some basic concepts of genetic manipulation. We will begin with some definitions and examples of transgenic plants. Then we will consider gene manipulation and the related methods. Because aromatic and medicinal plants are an integral part of the human and animal food system, they are not excluded from considerations of safety issues when genetically transformed.

12.1. Transgenic Plants Transgenic plants are organisms in which a gene or more genes from unrelated species or organisms have been introduced through genetic engineering techniques. The genes determine specific characters such as colour, height, tolerance to biotic or abiotic agents, transformation of a

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substrate or production of a particular molecule. Adding a new gene to a plant means adding new characters and new abilities. The amazing thing is that through genetic engineering we can move genes from any biological source (animal, bacterial or plant) into plant cells. In practice, a genetic engineer considers any organism, even a butterfly or a firefly, as a source for the introduction of a given character; once identified and isolated, the gene can be introduced into any organism. In many cases, transgenic plants contain combinations of genes that neither nature nor conventional genetics can produce. This is the case, for example, with the cold resistance introduced in Arabidopsis thaliana plants, by transferring this character from a fish from the North Sea. The enormous potential of this technique has caused quite a few ethical and social perplexities, as we will discuss later on in this chapter. Advances in genetic engineering can be traced back to recombinant DNA technology, which involves the cloning of hybrid DNA molecules. One organism which is present in nature and capable of acting as a vector for the introduction of foreign DNA into plant cells is the soil bacterium Agrobacterium tumefaciens, known as the agent of the crown gall tumour. This soil bacterium penetrates wounds at the boundary between the root and the stem of the plant, and inserts a fragment of the plasmid DNA (called T-DNA) into the DNA of the infected plant cells. Infected and transformed cells express the transferred genes by producing a higher amount of plant hormones (auxins and cytokinins) and of other substances not normally synthesized by plants (opines). The result is the uncontrolled proliferation of cells to form an undifferentiated mass very similar to the callus described in Chapter 11. When compared to a callus obtained by dedifferentiation of an explant, the tumour has the ability to continue to grow and multiply even in the absence of plant hormones or growth promoters, as it possesses the endogenous capacities (transferred by the bacterium) of overproducing the growth factors. In Chapter 11 we mentioned another Agrobacterium, A. rhizogenes, when we discussed hairy roots. This Agrobacterium, unlike A. tumefaciens, transfers a T-DNA that instead of causing tumours induces the formation of hairy roots. In addition to numerous applications in agronomy for the creation of transgenic plants resistant to herbicides, diseases or phytophages, or tolerant to drought, cold or heat, or used as bioindicators of atmospheric pollution, there are numerous applications for this technology for the production of plant bioactive molecules.

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In general, to increase productivity, the metabolic pathway can be triggered by reducing the catabolism of the compound of interest or by increasing the number of cells involved in the biosynthetic process. To achieve these goals, it is necessary to eliminate all possible metabolic hurdles, such as retroactive inhibition or the presence of competitive reactions. In biomolecular terms, this can be solved by introducing a gene under the influence of a powerful promoter or through the use of a foreign gene that encodes a more efficient enzyme. Antisense gene technology is used to reduce the presence of competitive or parasitic reactions. Normally the messenger RNA (mRNA) has a sequence of nucleotides defined as “sense” since the translation of the sequence gives rise to a gene product (a protein). The reading of mRNA occurs with transfer RNA (tRNA) as the ribosomes read and translate the nucleotide sequence. But just as when DNA is duplicated to form RNA, duplication can also occur for RNA, leading to the formation of a complementary copy. This complementary copy is defined as antisense and when produced it blocks translation, because it cannot be read by the ribosomes or is rapidly degraded by ribonuclease. With recombinant DNA techniques it is possible to introduce sequences that encode antisense RNA. With these techniques it is possible to reduce by up to 90% the expression of some competing genes in certain biochemical reactions. Increasing cell proliferation is more complex because it requires the involvement of many genes that work in the growth and development processes. There are numerous examples of transgenic plants engineered for the production of bioactive molecules. The crucial point is always the identification of a key enzyme and the cloning of the gene responsible for the formation of that enzyme. In addition to the discussion of pros and cons (see below) it is important to consider the economic and legislative aspects related to the creation of transgenic plants. The nations that are the main growers of genetically modified plants are, in order of cultivation levels, the United States, Brazil, Argentina, India, Canada and China with sizes of cultivation ranging from 4 to tens of millions of hectares. With an increase from 36.7 to 40.3 million hectares, Brazil has had the most consistent increase. In India, the cultivation of genetically modified (GM) cotton has increased to 11 million hectares, corresponding to about 95% of Indian cotton production, while in Canada the cultivation of GM plants has decreased.

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Despite the obvious advantages that recombinant DNA technology offers there are still many doubts about human consumption. First of all, is it safe to eat transgenic plants? Do the advantages they offer outweigh potential dangers? Is a GM plant the property of the company that created this new organism? With regards this last question, the European Patent Convention (EPC) allows the patenting of new inventions, which include the invention of methods and procedures and which can be applied industrially (including in the agricultural sector). There are specific restrictions on patenting, but none is generally related to biological material. Discoveries, as such, are not patentable. Article 53.B of the EPC specifies the prohibition of the patenting of varieties of plants and animals, as well as biological processes for the production of plants and animals. According to Article 2 of the EU Directive, a biological material is defined as any material containing genetic information and able to reproduce itself or be reproduced in a biological system. Article 3 specifies that this may include material isolated from its natural environment or produced through a technological process. This last sentence provides an important confirmation of the validity of existing practices in the patent offices of many nations which for many years had considered purified and isolated natural products as patentable materials, not to be automatically rejected as the creations of “Mother Nature”.

12.2. Genetic Manipulation and the Regulation of Gene Expression In an attempt to alter cell metabolism, in addition to the techniques described in Chapter 11 such as somaclonal variation or somatic fusion of protoplasts, there is the possibility of transforming DNA and thus extending genetic variability. Since one of the basic purposes of in vitro cell culturing is the production of bioactive molecules or otherwise of functional products, the application of molecular biology techniques takes on a particularly important significance, especially for the commercial production of these metabolites. Considerable progress has been made in the technology for stable integration and for the expression of foreign genes in aromatic and medicinal plants allowing, among other things, the clarification of

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numerous mechanisms involved in the regulation of gene expression and improvements in the production of numerous cultivated plants. A multigenic system is difficult to control, even with the most modern molecular biology techniques. A fundamental requirement for genetically manipulating plants that produce secondary metabolites is to understand the molecular mechanism of regulation of the biosynthetic pathways, with particular reference to gene expression and the steps that limit the speed of reaction. Advances in the production of secondary metabolites in genetically modified plants can be divided into two main categories: x cultures of transgenic organs, such as the hairy roots described in Chapter 11, for the production of specific compounds; x transfer and manipulation of artificially manipulated foreign genes, including genes capable of changing metabolite properties. In nature, bacteria belonging to the genus Agrobacterium transform host cells by inducing the production of particular metabolites defined as opines. These molecules can be grouped into five families of compounds: octopine, nopaline, agropine, agrocinopine and cucumopine. Octopines and nopalines are imino acids formed by the reduction of Į-keto acids and amino acids, whereas agropines are formed from amino acids and mannose. Agrocinopines are phosphodiesters of sugars commonly found in cells, while cucumopines are formed by the condensation/cyclisation of Į-ketoglutarate and histidine. Opines do not have a metabolic significance for the host cells, but are the main source of nitrogen for the infecting bacteria. There are still few studies on the pharmacological properties of opines and their interaction with the secondary metabolism of cultured cells, but the data obtained suggest a possible involvement in cell proliferation processes. In Chapter 11 we described the potentialities of hairy roots (hr); now we can investigate the mechanism by which the genetic transformation of roots occurs after infection by A. rhizogenes. The plasmid of A. rhizogenes that induces roots (Ri) stably transfers a specific DNA (T-DNA) fragment into the genome of the host cell. The interaction between the bacterium and the plant that occurs in nature can be exploited in the laboratory. The natural process of gene transfer carried out by A. rhizogenes begins with the release of phenolic compounds from the wound cell of a plant tissue. This initiates a series of events such as chemotaxis and adhesion of the bacterium to the host cell wall; furthermore, phenolic compounds

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induce the expression of virulence (vir) genes of the Ri plasmid. The expression of some genes present in the chromosome of the bacterium allows the binding of the bacterium to the cell wall of the host cell and the formation of cellulose fibrils that help the bacterium to aggregate to the plant cell. The products of some vir genes are then involved in the subsequent steps involving the transfer of a fragment of the T DNA, with a process analogous to the transfer of plasmids during bacterial conjugation. Little is known about the process of integration with the plant genome; it is known, however, that T-DNA integrates preferably with regions of the plant DNA that have the potential to be transcribed. Following integration, the T-DNA genes are expressed and the promoters that direct the expression of the genes encoded by T-DNA work perfectly in the plant cells, although the level of expression they direct appears to be modulated by small changes in those substances that promote cell growth. The rolA, B and C genes of the Ri plasmid T-DNA modify the cellular growth responses to molecules such as auxin and induce root differentiation, while the expression of other genes induces the production of the abovementioned opines. An important factor in the procedure adopted to insert foreign genes is the control of gene integration. Many model genes have been used to investigate the different elements that regulate gene expression in transgenic plants. Genes such as those that encode chloramphenicol acetyltransferase (cat), neomycin phosphotransferase II (kan) or ȕglucuronidase (gus) are widely used as model reporter genes to evaluate the transformation of a plant, because the translation products of these prokaryote genes are stable in most plant cells. Furthermore, their enzymatic activities can be easily detected in the transformed tissues both in vitro and in vivo. These model genes have been used in several medicinal plants for the production of bioactive molecules. For the integration of foreign genes, the binary vector system based on the Ri plasmid has often been used, because this technique allows selection of the transformed cells without using antibiotics. In addition, the technique produces hr that grow rapidly and produce secondary metabolites in abundance. Some essential priorities for the exploitation of genetic manipulation techniques for the production of secondary metabolites concern: x the isolation and characterization of enzymes and genes involved in the various steps of the secondary metabolism biosynthetic pathways;

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x the understanding of the regulation of the expression of secondary metabolism genes at the tissue and development level; x the identification of the cis and trans factors able to regulate the temporal and spatial expression of genes involved in the different secondary metabolism biosynthetic pathways; x the development of tissue-specific promoters; x the development of reproducible methods for plant regeneration from cells of any plant capable of producing bioactive or commercial molecules.

12.3. Molecular Engineering and the Production of Plant Bioactive Molecules The commercial significance of numerous secondary metabolites, combined with the possibility of their use in agronomy (as defence molecules against pathogens and pests) makes these molecules an ideal target for genetic engineering protocols. The introduction of foreign genes allows the expression of secondary metabolites in plant species that normally do not produce them. To give an example of how it is possible to “engineer” the secondary metabolism we will consider the results obtained on some important classes of secondary metabolites.

12.3.1. Terpene Engineering Studies carried out on the characterization and purification of several enzymes involved in the monoterpene metabolism make possible the biomolecular approach for the manipulation of the metabolism both in plants that produce monoterpenes and in those that are not capable of their synthesis. To tackle the problem of introducing foreign genes for: (i) the production of monoterpenes to increase the content of essential oil; (ii) the synthesis of chemical defences based on terpenoids; (iii) the alteration of the aroma of fruits or vegetables; and (iv) the creation of new fragrances in ornamental plants, it is necessary to operate on: composition, yield and production methods in space and time. In Chapter 2 we discussed the secondary metabolite secretion sites and we noticed how monoterpenes are accumulated in glandular trichomes, in lysigenous cavities and pockets and in resin ducts. The genetic manipulation of plants for the production of secretory structures is one of the main objectives if the aim is to introduce the ability to produce monoterpenes into plants unable to do so. To bioengineer plants and make

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them capable of producing secretory structures, it will be necessary to deepen knowledge on the multigenic systems that regulate the development of such highly differentiated tissues, which implies the identification of transcription factors involved in the metabolism of secretory tissues, as well as knowledge of the mechanisms of secretion and allocation for the formation of secondary metabolites. At the moment there is not yet an ideal model on the production of secreting structures. Before applying recombinant DNA technology efficiently for the production of essential oils in plants that produce terpenoids, it is important that the integrated genes be expressed in the right way, without any potential complication (which could cause cytotoxicity) and by using promoter elements that allow the trichome-specific expression of the genes of interest. Although several specific promoters for epidermal cells have been identified, including promoters that direct the expression in nonglandular trichomes in Arabidopsis thaliana and promoters for glandular and non-glandular trichomes in Nicotiana tabacum, a specific epidermal promoter for glandular tissues remains to be discovered. Promoter regions of a specific number of genes that encode enzymes involved in the biosynthesis of monoterpenes located in the trichomes are being investigated in order to identify regulatory elements that allow the obtaining of the desired tissue level specificity. Another goal to be achieved is the control and increase of the photosynthetic metabolic flow towards the production of secondary metabolites. Knowledge is still missing on both the various development factors that control the formation of essential oil and the potential importance of metabolic branching points where metabolic flow is controlled. Likewise, control over the important mechanisms of inhibition or retroactive activation of metabolic intermediates and over secretion mechanisms is still lacking. In many cases, attempts to increase the metabolic flow towards the production of secondary metabolites have caused metabolic and sometimes structural imbalances in the modified organism. For example, in attempting to overexpress the gene for phytoene synthase in tomato transgenic plants, a dwarf phenotype was obtained. This phenotype appeared to be correlated to the diversion of the metabolic flux at the expense of the production of gibberellins, as well as the shortage of lipids for the biosynthesis of phytol contained in the chlorophyll molecules. Another area of interesting research is the manipulation of the aroma of some aromatic plants. In the case of M. piperita, once control of the gene that converts menthone to menthol is obtained, it will be possible to increase the content of the latter compound

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with a consequent increase in the commercial value of the essential oil. In the same way, it will be possible to suppress the synthesis of other less valuable molecules that reduce the production of menthol such as menthofuran or the isomer neomenthol (see Chapter 7). Another interesting point is the dependence of the expression of some genes on the plant responses to abiotic and biotic factors. We have repeatedly stressed in this book that secondary metabolites are produced by plants as a response to external stimuli. An increased pressure from environmental factors or the presence of pathogens triggers signal transduction pathways that activate genes or gene families that produce specific responses. In addition to gene control for the expression of one or more genes linked to the metabolism of a given molecule, it will be important to either control the multigenic response factors or trigger a series of reactions that, from the primary metabolism, convey photosynthates to the secondary metabolism. This will allow both the desensitizing of plants to environmental factors – by ensuring a more homogeneous production of secondary metabolites – and the introduction of chemical defence factors in plants that do not possess them. Numerous microorganisms are used to produce a substantial number of natural products of high commercial value, including vitamins and antibiotics. Numerous terpenoids have commercial applications important enough to justify the development of methods for their biotechnological production using prokaryote genetic engineering techniques, provided that the introduction of foreign genes involves a limited number of biomolecular steps. Furthermore, the use of transgenic microorganisms offers the control of products’ stereochemistry, which can be an advantage in the case of synthetic chemistry. In addition, the biotransformation of a compound by an organism is considered a natural process that leads to the synthesis of “natural” compounds, an important factor both for the labelling of a product and for consumers’ acceptance. As regards monoterpenes, prokaryotes do not possess the enzyme geranyl diphosphate synthase, a key enzyme for the condensation of IPP with DMAPP (see also Chapter 7). It is clear that any intervention aimed at producing monoterpenes from prokaryotes must start from the insertion of this enzyme. Moreover, three other steps are necessary to complete the biosynthetic process, and these are the introduction of genes for: (i) cyclization (cyclase) of geranyl diphosphate; (ii) the transformation of the cyclized product into hydroxylated compounds (cytochrome P450dependent hydroxylase); and (iii) any further oxidation on the hydroxyl

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group or desaturing/saturing system working on carbon-carbon bonds (oxidoreductase). Any toxicity of the products obtained by transgenesis in the prokaryotic culture will have to be evaluated, together with the possibility of removing the synthetic products as described in Chapter 11.

12.3.2. Phenolic Compounds Engineering As we discussed in Chapter 4, resveratrol, a plant phenolic compound of considerable interest, is found in red wine, but it is not widely distributed in other common food sources. The benefits of resveratrol for health include the prevention of cardiovascular disease and cancer and, as recently discovered, the promotion of longevity in different animal systems. The enzymes of resveratrol biosynthesis are well characterized (see Chapter 6) and the metabolic engineering of this compound has been demonstrated in plants, animals and microbes. In grapes, UV irradiation and fungal attacks induce resveratrol biosynthesis in leaves and fruit teguments. The expression of vine stilbene synthase (STS) in tobacco leaves deviates the typical substrates of chalcone synthase (CHS) towards the production of resveratrol, with yields of up to 300 ȝg g-1 on a fresh weight basis. These transgenic plants have shown an increased resistance to the tobacco fungal pathogen Botrytis cinerea, suggesting that the heterologous expression of a new phytoalexin is toxic to the native pathogens that did not develop countermeasures against it. Although the mechanism of the toxicity of resveratrol regarding fungal pathogens is unclear, the strategy of expressing STS genes has been used to generate transgenic resistance to pathogens in plant species such as lettuce, tomato and several other plants. Interestingly, most of the resveratrol produced in transgenic plants is conjugated with a glucose residue, forming piceid (see Chapter 6), which is then sequestered into vacuoles. Resveratrol as a food supplement is relatively inexpensive. It can be isolated from cheap grape seeds or from Polygonum cuspidatum roots; various methods for the chemical synthesis of this compound have also been described. Considering the huge cost of introducing GM genes into GM crops and the regulatory and public resistance to the acceptance of these food products, the commercial future of resveratrol engineering in plants remains to be demonstrated. However, a possible commercial application has been documented in recent research, in which the introduction of resveratrol into hops (Humulus lupulus) through the expression of peanut STS has proved useful in improving the nutritional value of beer and, at the same time, the hop resistance to diseases.

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To produce phenolic acids that are beneficial for medical and commercial purposes, researchers have carried out studies to improve the levels of naturally reduced salvianolic acid B (Sal B) produced by Salvia miltiorrhiza. A combinatorial genetic manipulation strategy was used to enrich the precursors available for the biosynthesis of Sal B. This approach, involving the biosynthetic pathway of lignin, requires the simultaneous ectopic expression of an Arabidopsis transcription factor (AtPAP1) for anthocyanin combined with the co-suppression of two genes of the key endogenous enzymes: cinnamyl-CoA reductase (SmCCR) and calleic acid O-methyltransferase (SmCOMT). Compared to the unprocessed control, a higher accumulation of Sal B was obtained (up to 3 times higher) accompanied by a reduction in the the lignin content. Lignin is also the main cause of the presence of recalcitrant lignocellulosic biomass during the industrial transformation of wood. It is desirable that bioenergy cultures possess a lignin easily degradable through chemical pre-treatments, without altering its plant biological role. Because plants can tolerate large variations in lignin composition (often without apparent side effects), the substitution of some traditional monolignol fractions with alternative monomers through genetic engineering is a promising strategy for lignin use in bioenergy cultures. However, the success of lignin engineering in incorporating alternative monomers requires knowledge of the plant phenolic metabolism and the coupling properties of these alternative monomers. An increasing number of studies have indicated that the genetic engineering of lignin can improve the efficiency of plant biomass transformation into pulp, the digestibility of fodder and the production of biofuels. Systemic approaches, in which the plant’s response to the engineering of a single gene in the biosynthetic pathway is studied at the organism level, are beginning to highlight the interaction between lignin biosynthesis and other pathways and metabolic processes. Flavonoids also show a protective effect against colon and breast cancer, diabetes, hypercholesterolemic atherosclerosis, lupus nephritis and immune and inflammatory reactions. Thus overproduction of these compounds in plants through genetic engineering can enhance the biotechnological applications of these plant products. For instance, the third-generation of field grown flax (Linum usitatissimum) that overexpresses some key genes of the flavonoid pathway has been used to demonstrate that the introduced genes have been stably inherited and expressed through successive generations. The overproduction of flavonoids has led to the increase of fatty acid accumulation in the oil of transgenic seeds due to the protection against oxidation offered during the synthesis and maturation of the seed. Biochemical analysis of extracts of

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transgenic flax seeds revealed a significant increase in flavonoids (kaempferol), phenolic acids (coumaric, ferulic and synaptic acids) and lignan content. All products analysed in the generated transgenic plants are enriched with antioxidant compounds derived from the shikimate pathway. These transgenic plants provide a valuable source of flavonoids, phenolic acids and lignans for biomedical applications. Phenolic compounds are the most common antioxidant molecules in the diet, and among these chlorogenic acid (CGA) accumulates at high levels in some cultivated plants (see also Chapter 4). CGA acts as an antioxidant in plants and protects animals from age-related degenerative diseases. cDNA clones encoding the enzyme that synthesizes CGA, hydroxycinnamyl-CoA chinate:hydroxycinnamyl transferase (HQT), have been characterized in tomato and tobacco. Gene silencing has shown that the activity of HQT is the main biosynthetic route for the accumulation of CGA in the Solanaceae. Overexpression of HQT in tomato causes the accumulation of high levels of CGA, without side effects on the levels of other soluble phenolic compounds, and shows a greater antioxidant capacity and resistance to infections by bacterial pathogens. Tomatoes with high levels of CGA could be used in foods with specific benefits for human health.

12.3.3. Alkaloid Engineering Nornicotine is an undesirable secondary alkaloid present in cultivated tobacco, because it serves as a precursor of N'-nitrosonornicotine (NNN), a specific tobacco nitrosamine with suspected carcinogenic properties. During senescence and the processing of tobacco leaves, nornicotine is produced by the oxidative N-demethylation of nicotine through the enzyme nicotine N-demethylase. Silencing the gene encoding nicotine Ndemethylase, CYP82E4, with an RNAi-induced technology is an effective way of suppressing the conversion of nicotine to nornicotine. Transgenic plants transformed with the RNAi construct were morphologically indistinguishable from empty vector or wild-type controls and have shown that the genetic transformation of tobacco with the 82E4Ri298 construct is an effective strategy for reducing nornicotine and, finally, NNN levels. Opium poppy, Papaver somniferum, and its narcotic and analgesic alkaloids have an ancient history of use (and abuse) by humankind. The metabolic engineering of morphine biosynthesis is able to block the formation of morphine and to accumulate a potentially valuable intermediate in the metabolic pathway, (S)-reticuline. These findings

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highlight the potential of changing the plant’s drug production, but also raise questions about the complex regulation of the biosynthetic pathways. Benzylisoquinoline alkaloids such as morphine, sanguinarine and berberine are synthesized from tyrosine through reticuline in the Magnoliaceae, Ranunculaceae, Berberidaceae, Papaveraceae and many other species (see Chapter 9). There are several strategies for the improvement of the yield and quality of these alkaloids, such as: x overexpression of a limiting enzyme in the early pathway to increase the overall yield of alkaloids; x introduction of a new metabolic variant in the biosynthesis that has been shown to produce new metabolites; x accumulation of an intermediate compound from the knock-down of a fundamental biosynthetic step. These metabolic changes can be further modified in cell cultures to increase chemical diversity through somatic variation. In addition to these events, bioactive molecules are produced by direct metabolic engineering with single biosynthetic genes, by regulation of the biosynthetic activity with transcription factors and/or by reconstruction of the entire biosynthetic pathway. Tea is a rich source of antioxidants that contribute substantially to the promotion of health and the prevention of various chronic diseases. Despite the fact that tea has various important compounds, it also contains a purine alkaloid, caffeine (see Chapter 9). Therefore, a high consumption of tea leads to an increase in the caffeine level that can cause various health problems. Reduction or elimination of caffeine from the tea plant can be accomplished through two biomolecular techniques: the overexpression of genes involved in the degradation of caffeine or the silencing of genes involved in caffeine biosynthesis. The identification and cloning of genes involved in the biosynthesis of caffeine in tea and genes present in degrading microorganisms opens up the possibility of using genetic engineering to produce naturally decaffeinated tea. The alkaloids of Catharanthus roseus comprise a group of about 130 indole terpenoid alkaloids (see Chapter 9). The efficient genetic engineering of the medicinal plant is essential to the improvement of the production of pharmaceutically important anticancer compounds such as vinblastine and vincristine. Given the pharmaceutical importance and the low content of vinblastine and vincristine alkaloids in plants, C. roseus has

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become one of the most studied medicinal plants. These alkaloids can be genetically manipulated (through metabolic engineering) with strategies that aim to achieve higher levels of production. Another approach is to produce alkaloids (or their precursors) in other organisms such as the yeast Saccharomyces cerevisiae. Despite the availability of a limited number of biosynthetic genes, research on C. roseus has already led to very interesting results, and many more will be achieved when more genes become available. Through Agrobacterium-mediated transformation of C. roseus, and after induction with 5 mM 17 beta-estradiol, the transgene expression was strongly and rapidly induced in the tips of hairy roots and was sustained for 5 days in the presence of estradiol. Coptis japonica rhizomes are used as extracts to treat gastroenteritis, as they accumulate the antimicrobial alkaloid berberine. Unfortunately, C. japonica is a slow-growing plant and takes more than 5 years to provide a crude extract suitable for pharmacological purposes. To improve alkaloid productivity, the N-methylcoclaurine 3'-hydroxy 4'-O-methyltransferase (4'OMT) gene was overexpressed in the plant by Agrobacterium-mediated transformation. The overexpression allowed berberine content to increase 2.7- and 2-fold in leaves and roots respectively, compared to wild-type plants. These results indicate that 4'OMT is one of the target enzymes for berberine biosynthesis and is useful for the metabolic engineering of C. japonica.

12.4. Plant Molecular Pharming Molecular pharming is a new and promising branch of plant biotechnology that refers to the use of plants for the production of biofuels, pharmaceutical and industrial recombinant proteins and other secondary metabolites. It is based on the production of high value proteins and other valuable substances from transgenic plants. A large group of scientists is working on the development of transgenic foods that can both provide a better nutritional contribution and prevent illness. One of the main goals is to prevent the premature death of the millions of children who still die from infectious diseases. Historically, the production of the first recombinant pharmaceutical protein of plant origin took place in 1986; it was the human growth hormone, and in 1989 the first recombinant antibody was produced. However, it was necessary to wait until 1997 for the first commercial production of avidin (an egg protein), expressed in transgenic maize. Over

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the years, the ability of plants to produce complex mammal proteins with therapeutic activity has often been demonstrated, examples being the human serum proteins and the growth regulators, antibodies, vaccines, hormones, cytokines and numerous enzymes. This has been made possible thanks to the post-translational modifications present in the plant cells, which allow the correct folding of the recombinant polypeptides and the maintenance of their structure and functional integrity. For this and other advantages, including low production costs, practicality and high safety, plants could replace the most conventional systems existing today, which include the use of yeasts, bacteria, animal cells and transgenic animals. Table 12.1 contains a comparison between different systems for the recombinant production of human pharmaceutical proteins. There are different methods for the production of recombinant proteins, each with both advantages and disadvantages. For example, stable nuclear transformation allows the insertion of one or more foreign genes of interest within the plant nuclear genome, generally by using A. tumefaciens or by particle bombardment. In this way the genome is altered, allowing the expression of the transgene after its integration into the host genome. Another method is the stable transformation of the plastid, which causes the gene of interest to integrate into the genome of the organelle. This method can be considered an alternative route to nuclear transformation. In most species, in fact, plastid genes are inherited through maternal tissues and the pollen does not contain chloroplasts. The problem of potential crossover with other plants is thus solved, since the transgene cannot be transferred. It is also possible to obtain an enormous level of expression, up to 70% of the total soluble proteins. The transient expression system is perhaps the fastest and most convenient method for the production of biopharmaceutical molecules. In this way the gene does not integrate itself in a stable way with the host genome and does not form inheritable traits, which is why it is ideal for proteins that are required in small quantities. A disadvantage of this technique is that the product must be processed immediately due to the degradation of plant tissue. For this method there are several ways to express a gene, including:

Very low

Long

Very high

High

Little difference Low

Economic

Production time

Scale-up capability

Product quality

Glycosylation

Contamination

Storage cost

Transgenic plants

Cost

Specification

Moderate

Low

Little difference

High

Medium

Medium

Medium

Cell cultures

Correct

Virus, prions and oncogenic DNA Expensive

Virus, prions, oncogenic DNA Expensive

Very high

Very low

Long

High

Mammalian cells

Correct

Very high

Low

Very long

High

Transgenic animals

System

Low

Short

High

Low

NO

Endotoxins

Moderate

Medium

High

Medium

Incorrect

Low

Moderate

Bacteria

Medium

Yeast

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Table 12.1 Comparison of systems for the production of human recombinant proteins for pharmaceutical purposes

x agroinfiltration, which involves the infiltration of a suspension of A. tumefaciens inside the tissue; x virus infection, which involves the use of certain plant viruses, such as the tobacco mosaic virus (TMV) and the potato X virus (PVX),

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which are used as vectors to transport foreign particles into plants, without integration; x magnifection technology, which, unlike the two above mentioned methods, allows the co-expression of two or more polypeptides necessary for the assembly of hetero-oligomeric proteins. Among the most used plants for molecular pharming studies are tobacco, canola, corn and potatoes, even though agronomists are investigating the possibility of exploiting fodder plants or non-food plants to avoid the possibility of unwanted crossings with the consequent transfer of foreign genes (see discussion in Chapter 1). The expression of vaccines within plants involves a number of advantages. For example, intrinsic risks due to production are reduced by the traditional use of live attenuated or inactivated viruses. Other advantages are large-scale production potential, cost reduction and the option of oral intake of these proteins through the ingestion of fruit or vegetables. One of the plants is potato in which genes are being introduced to produce vaccines that help fight one of the main causes of infant mortality, diarrhoea. Another plant with similar applications is banana. Although the road ahead is still long to the point where we can talk about food vaccines, many studies are already under way looking to obtain food containing vaccines. Hepatitis B antigen has been expressed in transgenic potato, tomato and banana and in tobacco cell culture, and the Escherichia coli enterotoxin B subunit has been produced in potato tubers, corn seed, tobacco and soy. The variety of substances produced ranges from the above vaccines to chemical compounds with pharmacological action to biodegradable plastics. Other molecules that can be obtained through this technique are antibodies, which are used for the prevention, diagnosis and treatment of diseases. There are two experimental approaches that are used for their production. The first consists of the cross-pollination of transformed plants that individually express light or heavy chains, and the second involves the co-transformation of the genes of both chains onto one or more expression cassettes. An example of application is the secretory antibody against a surface antigen of Streptococcus mutans, which proved to be as effective as the original mouse IgG in protecting against colonization of S. mutans on teeth. Molecular pharming of cell wall lythic enzymes, such as cellulase, hemicellulase, xylanase and ligninase, has potential for applications in the

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biofuels industry. With these enzymes, starting from wood residues, wheat ears and other agricultural waste, it is possible to produce cellulosic ethanol, a biofuel that could have the potential to reduce greenhouse gas emissions by 100%, compared to petrol. Two important applications in molecular pharming are rhizosecretion and phyllosecretion. In the first case, the ability of roots to secrete (or rhizodeposit) is exploited in the environment in which substances of applicative interest are produced. Phyllosecretion, on the other hand, exploits leaf guttation, the elimination of fluids from leaves through secretory tissues defined as hydatodes. In some applications, the guttation flow is combined with the secretion of proteins in transgenic plants and has been used by some industries for the production and extrusion of recombinant proteins. Many companies have changed their production strategy using recombinant DNA technology to produce natural products that combine the value of substances produced by organisms (such as cotton) to the qualities and manageability of synthetic substances (such as plastics). Cotton has been transformed in such a way as to produce cellulose fibres similar to polyester, extending the concept of recombinant DNA in agriculture to plants not directly involved in human or animal nutrition. Transgenic plants capable of producing biodegradable plastics, industrial lubricants, basic substances for soaps and detergents and a large quantity of pharmaceutical products have been obtained. Among transgenic plants that will probably undergo a significant increase in cultivation, there are those that produce oils for industrial applications, polymers and biodegradable plastics. There are already transgenic rapeseed plants capable of accumulating lauric acid, a 12 carbon atom fatty acid used for the manufacture of soaps and detergents. To obtain these transgenic plants, it was sufficient to introduce a gene taken from Umbellularia californica (Lauraceae). This gene prevents the lengthening of the lipid chain beyond 12 carbon atoms and besides being present in this plant is also expressed in other species such as oil palm. In addition to lauric acid, there are many other lipids produced by canola following the introduction of new genes. There are variants of transgenic rape capable of producing Nylon 13-13 and erucic acid, while in Arabidopsis thaliana bacterial genes have been introduced which allow the transgenic plant to produce polyhydroxybutyrate (PHB). The best results were obtained by introducing a sequence that directed the biosynthesis within the plastids, thus reducing the degree of toxicity of the molecule and ensuring a higher quantity of substrate for the synthesis.

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The main purpose of research in transgenic plants for the production of industrial chemical compounds, such as plastics, is aimed at reducing the current dependence on oil derivatives, but also to re-directing world agriculture in the case of overproduction of food plants, with the obligation to allocate part of the arable land to alternative crops. This will significantly lower government costs for subsidies and help to obtain tax relief, using bioengineering as a technological source for the obtaining of new crops.

12.5. Food Safety, Recombinant DNA and Bioethics Plants that accumulate bioactive molecules are subject to the same rules and controls as those that produce primary foods (starch, fats, oils and plant proteins), as part of the general food system. The issue of safety is one aspect that should not be left out; for this reason, numerous regulations are being developed, in order to increase the biological safety of plant products of transgenic origin. The main concern is the impact that molecular agriculture could have on the existing ecosystem, in particular through transgene diffusion and unintentional exposure (see also Chapter 1). There are precise regulations and legislations regarding molecular pharming, so improper and dangerous uses are avoided. In the European Union there are directives and regulations that govern the development, production and trade of transgenic plants. The Directive 2001/18/EC, specific for each state, controls their placing on the market, while the European Agency for the Evaluation of Medicinal Products (EMEA) regulates products derived from GMOs which can be used for human or veterinary use. The European Food Safety Authority (EFSA), on the other hand, is an independent agency that takes care of the release of GMOs and their products into the environment. In the United States, the production of proteins from transgenic plants is regulated by two agencies which focus on the containment of GMOs – the U.S. Department of Agriculture (USDA) and the Animal and Plant Health Inspection Service (APHIS) – and the Food and Drug Administration (FDA) which focuses more on the production system. Finally, it is expected that all these developments may attract new subjects willing to use this innovative technique, particularly in the clinical development of large amounts of proteins, which at present, with production by conventional methods, proves to be very expensive.

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What is the relationship between nutrition and biotechnology? As we have defined in Chapter 11, biotechnology is nothing more than the use of living organisms to provide goods and services and the term can be applied to the past ten thousand years of agricultural development even if we understand it better as the development of new technologies. The novelty lies in the fact that with recombinant DNA we can create new varieties more quickly and with a higher control on the genetic variation. Foods obtained from transgenic plants are sold all over the world and the quality of food nowadays has an extra control term, bioethics. An important part of bioethics is risk assessment, which includes risk analysis and risk prediction. Risk management, on the other hand, is a different concept. In practice it is the process that evaluates the alternatives in the selection of the most appropriate strategies or actions and integrates the results of the risk assessments of different alternatives. When evaluating the possibility of marketing a transgenic plant from which a transgenic food will be obtained, a risk assessment must be carried out. First of all, the identification of a potential risk and the risk estimate must be carried out. The benefits of introducing a transgenic plant are part of the risk management, but have nothing to do with risk assessment. In practice, bioethics combines the assessment of risk (the concept of avoiding harm) with the evaluation of benefits (the concept of doing good). What are the risks in introducing foreign genes? One of the most frequent risks is to change genes in an organism unintentionally, as we discussed in Chapter 1. In biotechnology, the concept of risk involves both potential change and potential damage. A very important problem is, for example, the contamination of the food chain. This can be solved with the use of non-food products and crops, such as tobacco, Arabidopsis, microalgae or mosses. However, when the use of edible plants is appropriate, for instance for the production of oral vaccines, it is possible to adopt strategies such as physical confinement in isolated areas or greenhouses, the growing of plants at different times from other crops intended for food use, or the use of non-tradable varieties, so that contamination does not occur. Since it is very difficult to avoid the mixing of transgenic and nontransgenic crops, accidental contamination threshold limits are being introduced. For example, in 2003 the European Parliament allowed the presence of 0.5% GMOs in non-GMO plants or feed, while for nonhazardous and non-pharmaceutical products, the threshold limit was set to 0.9%. Gene transfer technology has taken faster steps than our understanding of the true meaning of plant biotechnology itself and of other important plant

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traits of agronomic interest. Therefore, attention has been focused on those characters that can result from single genes. It is also important to consider the potential risks of disease on organisms such as insects, birds and soil microorganisms, which can come into contact with pharmaceutical proteins through accidental ingestion. In Chapter 4 we explored the vastness of natural compounds present in plants. Many molecules used as dietary supplements are now produced by recombinant DNA technology. For instance, a group of non-primary metabolism substances are non-sugar sweeteners. Taumatins are a class of highly sweetening proteins isolated from the fruits of the tropical tree Thaumatococcus danielli. The use of taumatin is approved in many countries and the protein is used both as a sweetener and as a flavour enhancer. The gene that encodes taumatin has been introduced into plants (potatoes) and microorganisms under the transcriptional control of heterologous gene promoters. At the moment, yields are still low when compared to plant extracts, but the expectations are great, even considering the fact that the taumatin gene can be inserted into other fruits to enhance flavour. There are dozens of examples like this and all are based on the transfer of quality genes from a plant or organism to other plants of wider commercial interest. The purpose of genetic engineering is not limited to the introduction or in some cases deletion of a single gene, but to the manipulation of the metabolism. In general terms, increasing the production of plants that produce secondary metabolites makes the use of the land more efficient, and this is particularly important considering that about a quarter of the world’s arable land area is rendered unproductive by losses due to diseases, herbivores and weeds and adverse weather conditions, including salinity and desertification. Through the introduction of foreign genes, it is possible to make transgenic plants more resistant to these adversities, thus improving and increasing productivity and land use. In addition to introducing genes to add characters that are not present in a given plant, it is also possible to remove any toxic secondary metabolites, making some plant species more usable for human nutrition both in terms of energy and nutrients. For example, caffeine content can be significantly reduced, if not cancelled, by the deletion or silencing of one or more genes involved in the metabolic pathway. This solves the problem upstream, avoiding additional expenses needed to remove caffeine with chemicalindustrial processes. Furthermore, the genetic removal of caffeine does not

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alter the taste and aroma of the product, as might happen during chemical removal. We can discuss and present a myriad of applications in which the recombinant DNA technique proves to be a valid and ideal tool for improving productivity and production efficiency, but, as we have mentioned, scientific developments go hand in hand with the acceptance by people of the moral principles on which they are based, and this is particularly true in the case of food substances. Despite the approval by many governments, the use of transgenic food always depends on the consumers’ desire to buy and on the positions that food distributors must consequently take. In the United States, despite the millions of hectares of land cultivated with transgenic plants, a group of about two thousand restaurateurs declared that they will never serve foods from genetically modified organisms, while in the UK a supermarket chain has released a document entitled “Your right to know” stating that they will not sell plant and animal foods in which foreign genes have been introduced. There are various strategies for getting to know the public’s views and in Europe the EU uses a widespread service among member states called the Eurobarometer. The basic problem in evaluating people’s reactions is that the concept of transgenic plants and animals cannot be easily assimilated. There are also differences between countries, and further within countries. In general, concern is focused on: interference with nature, environmental risk and health risk. We must also consider that being a relatively new technology it has not yet fully entered the basic educational systems (elementary and lower secondary). This creates family debates set exclusively on opinions transmitted by the media and not by comparison on direct experiences. Usually, the ability of a population to judge fairly the good and the bad of a technology is an indicator of the bioethical maturity of a society. According to some authors it is not the act itself of creating new organisms that is wrong, but the way in which an organism can be created. It is necessary, however, to reduce the problem to the reality of the facts, because the technologies we possess allow us to integrate one or two genes into an individual that contains hundreds of thousands of genes that still remain unchanged. Making an absurd reasoning, the action of a molecular biologist on an organism is comparable to the change in colour of a stroke of Michelangelo in the Sistine Chapel. Nobody could say that a new Sistine Chapel has been created, but by dint of touch-ups the initial information could be lost and then we could not talk any more about the Sistine Chapel as it was originally.

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With regard to human health, there are two types of effects that transgenic plants potentially possess, the first concerns the individual, while the second affects groups of people or the community. The first type of effect is focused on toxic substances or pleiotropic or allergic effects in a particular person, while the second involves the spread of a gene transfer vector among the consumers, which is highly unlikely in the case of viruses that infect plants. Some plant foods, such as fruits and vegetables, contain intact DNA that is ingested, especially if these foods are consumed raw, whereas in cooked foods the DNA is completely degraded. We must consider that every day we ingest vegetables containing hundreds of thousands of plant viruses and it is extremely unlikely that a plant genetic vector can be transmitted among people, even if it enters the bloodstream of an individual. In plants resistant to viruses in which part of the viral protein (the analogue of human vaccines) is expressed, the plant remains the same, except for the extra anti-virus protein. Since there are no plant viruses that infect humans, many scientists claim that there is no reason to worry about the presence of anti-virus proteins in the plants we eat. However, optimism is never good science and in many cases it has been proven that the a priori exclusion of the possibility of a particular gene transfer in the digestive tract is not always provable. The most common transfer is among the microorganisms that inhabit the digestive tract. In a mouse study, the transfer of DNA by conjugation between a donor strain of Lactobacillus lactis and a strain of Enterococcus faecalis isolated from human faeces occurred. In vivo and in vitro, plasmid transfer from one bacterium to another has been demonstrated. There are numerous initiatives regarding the regulation of food safety. The EU Ethics Committee on Biotechnology recommends that foods be labelled to indicate whether there has been a modification using genetic engineering techniques. The recommendations of the EU committee indicate that information to the consumer must contain “transparent” information which must be: x x x x

useful, adequate and instructive; clear, understandable and non-technical; honest, unequivocal and suitable for the prevention of any fraud; evaluable, i.e., verifiable.

The answer to the question of whether the global effects of biotechnology are positive or negative on the environment and food safety, based on current technologies, would undoubtedly be positive. This is because

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biotechnological methods allow better monitoring of both environmental and food health and safety effects, apart from the advantages of increasing productivity. The ultimate goal of technology must be to improve and protect human health through the ongoing assessment of risks and benefits. Worldwide, UNESCO and other UN organizations will have to engage on various fronts to: x promote research on the socio-economic implications of plant biotechnology in different cultures and countries, encouraging studies on bioethics; x increase the degree of independence and credibility of information, so that people can trust the information they receive. World organizations, independent and not directly involved, must be the depositors of the information, information that must not remain only in the hands and under the management of those industries that produce transgenic organisms; x educate the population at every educational level on the benefits and risks of using transgenic technologies. This includes every educational level including the bioethical and normative aspects; x increase control over the eventuality of producing transgenic foods with potential toxicity, including various allergenic forms; x promote research on transgenic plants applicable to developing countries, in addition to already well-funded projects for industry; x assess the potential risk of “gene leaks” into the environment, especially regarding pesticide and herbicide resistance traits; x finance publicly available databases on food, environmental and technological security. Some of these databases are already in operation at the USDA (US Department of Agriculture), the EU and UNIDO; x protect intellectual property for both traditional and biotechnological genetic studies, in order to distinguish discoveries from inventions.

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Fray, R.G., et al. (1995). Constitutive expression of a fruit phytoene synthase gene in transgenic tomatoes causes dwarfism by redirecting metabolites from gibberellin pathway. Plant J. 8, 693–701. Gavilano, L.B. et al. (2006). Genetic engineering of Nicotiana tabacum for reduced nornicotine content. J. Agric. Food Chem. 54, 9071–9078. Gomez-Galera, S., Pelacho, A.M., Gene, A., Capell, T. and Christou, P. (2007). The genetic manipulation of medicinal and aromatic plants. Plant Cell Rep. 26, 1689–1715. Gruzza, M., Langella, P., Duval-Iflah, Y. and Ducluzeau, R. (1993). Gene transfer from engineered lactococcus lactis strains to Enterococcus Faecalis in the digestive tract of gnotobiotic mice. Microb. Rel. 2, 121–125. Halls, C. and Yu, O. (2008). Potential for metabolic engineering of resveratrol biosynthesis. Trends Biotechnol. 26, 77–81. Horn, M. E., Woodard S. L. and Howard, J. A. (2004) Plant molecular farming: systems and products, Plant Cell Rep. 22, 711–72. Hu, Z.B. and Du, M. (2006). Hairy root and its application in plant genetic engineering. J. Integr. Plant Biol. 48, 121–127. Inui, T. et al. (2012). Improvement of benzylisoquino-line alkaloid productivity by overexpression of 3'-hydroxy-N-methylcoclaurine 4'O-methyltransferase in transgenic Coptis japonica plants. Biol. Pharmac. Bull. 35, 650–659. Karami, O., Esna-Ashari, M., Kurdistani, G.K. and Aghavaisi, B. (2009). Agrobacterium-mediated genetic transformation of plants: the role of host. Biol Plant. 53, 201–212. Lau, O.S. and Sun, S.S.M. (2009). Plant seeds as bioreactors for recombinant protein production. Biotechnol. Adv. 27, 1015–1022. Leonard, E., Runguphan, W., O’Connor, S. and Prather, K.J. (2009). Opportunities in metabolic engineering to facilitate scalable alkaloid pro-duction. Nat. Chem. Biol. 5, 292–300. Niggeweg, R., Michael, A.J. and Martin, C. (2004). Engineering plants with increased levels of the antioxidant chlorogenic acid. Nat. Biotechnol. 22, 746–754. Page, J.E. (2005). Silencing nature’s narcotics: metabolic engineering of the opium poppy. Trends Biotechnol. 23, 331–333. Richter, L., Mason, H.S. and Arntzen, C.J. (1996). Transgenic plants created for oral immunization against diarrheal diseases. J. Travel Med. 3, 52–56. Rizvi, N.F. et al. (2015). An efficient transformation method for estrogeninducible transgene expression in Catharanthus roseus hairy roots. Plant Cell Tiss. Organ Cult. 120, 475–487.

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