Wheat: chemistry and technology [4th ed] 9781891127557, 1891127551

Table of contents :
Front Cover......Page 1
Wheat: Chemistry and Technology......Page 2
Copyright Page......Page 3
Table of Contents......Page 10
Contributors......Page 4
Preface to the Fourth Edition......Page 6
Preface to the Third Edition......Page 7
Preface to the Second Edition......Page 8
Preface to the First Edition......Page 9
WHEAT AND PEOPLE......Page 14
WHEAT TAXONOMY......Page 17
WHEAT IN A HUNGRY WORLD......Page 19
WHEAT IN THE GRAIN CHAIN......Page 21
WHEAT AND HUMAN HEALTH......Page 27
REFERENCES......Page 28
CHAPTER 2. The Wheat Crop......Page 32
ORIGINS......Page 34
GROWTH AND DEVELOPMENT......Page 38
WHEAT IMPROVEMENT......Page 45
AGRONOMY OF WHEAT......Page 51
REFERENCES......Page 58
TERMINOLOGY AND WHOLE-GRAIN CONSIDERATIONS......Page 64
MICROSTRUCTURE OF THE MATURE AND DEVELOPING WHEAT GRAIN......Page 69
MOLECULAR AND BIOCHEMICAL STUDIES OF GRAIN DEVELOPMENT......Page 90
STRUCTURE AND GRAIN MECHANICAL PROPERTIES......Page 93
REFERENCES......Page 100
WHEAT CLASSIFICATION......Page 110
GRADING FACTORS THAT AFFECT MILLING YIELD AND END-USE QUALITY......Page 112
CHEMICAL CHARACTERISTICS......Page 117
INTRINSIC CHARACTERISTICS......Page 119
PROCESSING QUALITY......Page 120
WHEAT CLASSES—PRODUCING NATIONS......Page 122
REFERENCES......Page 129
RELEVANT WEBSITES......Page 131
CHAPTER 5. Wheat Flour Milling......Page 132
WHEAT RECEIVING AT THE MILL......Page 133
WHEAT STORAGE......Page 137
THE MILL'S WHEAT-CLEANING SYSTEM......Page 139
WHEAT CONDITIONING FOR MILLING......Page 142
MILLING MACHINERY......Page 143
THE MILLING PROCESS......Page 149
END-PRODUCT QUALITY AND HANDLING......Page 153
MILL TECHNICAL PERFORMANCE ANALYSIS......Page 154
RECENT DEVELOPMENTS IN COMMERCIAL MILLING......Page 160
REFERENCES......Page 161
SUGGESTED WEBSITES FOR FURTHER INFORMATION......Page 165
STRUCTURE-FUNCTION MODELS OF GLUTEN......Page 166
MIXING FLOUR INTO DOUGH......Page 168
WHEAT PROTEINS DURING PROOFING......Page 174
WHEAT PROTEINS DURING BAKING......Page 179
REFERENCES......Page 185
B VITAMINS......Page 192
TOCOPHEROLS AND TOCOTRIENOLS......Page 198
CAROTENOIDS......Page 202
MINERALS AND TRACE ELEMENTS......Page 206
PHYTOSTEROLS......Page 209
PHENOLIC COMPOUNDS......Page 214
CHOLINE AND BETAINE......Page 222
REFERENCES......Page 223
OVERVIEW......Page 236
PROTEIN AND AMINO ACID COMPOSITIONS OF WHOLE-GRAIN FRACTIONS......Page 240
GLUTEN PROTEINS......Page 249
LOW MOLECULAR WEIGHT PROTEINS RELATED TO PROLAMINS......Page 278
OTHER GROUPS OF PROTEINS WITH FUNCTIONAL PROPERTIES......Page 285
OTHER BIOLOGICALLY ACTIVE PROTEINS......Page 288
CONCLUSIONS AND A FORWARD LOOK......Page 290
REFERENCES......Page 291
MONO-, DI-, AND OLIGOSACCHARIDES......Page 312
FRUCTANS......Page 313
WHEAT STARCH.STRUCTURE, SYNTHESIS, AND FUNCTIONALITY......Page 315
CELL WALL POLYSACCHARIDES......Page 332
CARBOHYDRATES IN ALEURONE PROTEIN BODY INCLUSIONS.PHYTIN GLOBOIDS AND NIACYTIN GRANULES......Page 349
IMPACT AND APPLICATIONS OF CELL WALL POLYSACCHARIDES IN GRAIN UTILIZATION, END-USE QUALITY, AND NUTRITION......Page 350
REFERENCES......Page 356
WHEAT LIPID CLASSIFICATION......Page 376
ANALYTICAL METHODS......Page 380
LIPIDS IN WHEAT FRACTIONS AND WHEAT CLASSES......Page 384
FUNCTIONAL ROLES OF WHEAT LIPIDS......Page 396
GENETIC RESEARCH ON LIPID-RELATED COMPONENTS......Page 402
FUTURE RESEARCH......Page 403
REFERENCES......Page 404
HYDROLYTIC ENZYMES......Page 414
INHIBITORS OF HYDROLYTIC ENZYMES......Page 423
OXIDOREDUCTASES AND THEIR INHIBITORS......Page 427
REFERENCES......Page 436
DEVELOPMENT OF WHEAT TRANSFORMATION METHODS......Page 450
APPLICATION OF TRANSGENESIS TO IMPROVE GRAIN QUALITY......Page 454
REFERENCES......Page 459
Index......Page 466

Citation preview

Fourth Edition

WHEAT

CHEMISTRY AND TECHNOLOGY

Edited by

Khalil Khan

North Dakota State University Fargo, North Dakota, U.S.A. and

Peter R. Shewry

Rothamsted Research Harpenden, Hertfordshire, U.K.

Front cover photographs: bread loaf and wheat heads, copyright © Rothamsted Research Ltd.; molecular model, from Parchment et al, Cereal Chemistry, 2001; grain section, courtesy VTT Technical Research Centre of Finland; wheat field, PhotoDisc; wheat kernels, adapted from Fig. 4.2 on page 101 Reference in this publication to a trademark, proprietary product, or company name by personnel of the U.S. Department of Agriculture or anyone else is intended for explicit description only and does not imply approval or recommendation to the exclusion of others that may be suitable. Library of Congress Control Number: 2009920375 International Standard Book Number: 978-1-891127-55-7 ©1964, 1971, 1988, 2009 by AACC International, Inc.

Published 1964. Fourth Edition 2009 All rights reserved. No part of this book may be reproduced in any form, including photocopy, microfilm, information storage and retrieval system, computer database or software, or by any other means, including electronic or mechanical, without written permission from the publisher. Copyright is not claimed in any portion of this work written by United States government employees as part of their official duties. Printed in the United States of America on acid-free paper AACC International, Inc. 3340 Pilot Knob Road St. Paul, Minnesota 55121, U.S.A.

• Contributors

J. Abecassis, L’Institut National de la Recherche Agronomique— Unité Mixte de Recherche, 1208 Montpellier, France D. B. Bechtel (Retired), Bechtel Consulting, Manhattan, Kansas 66503, U.S.A. Ferenc Békés, CSIRO Plant Industry, Canberra, ACT, Australia Kristof Brijs, Laboratory of Food Chemistry and Biochemistry, Katholieke Universiteit Leuven, 3001 Leuven, Belgium Gordon R. Carson, Canadian International Grains Institute, Winnipeg MB R3C 3G7, Canada Okkyung Kim Chung (Retired), Grain Marketing and Production Research Center, USDA, Agricultural Research Service, Manhattan, Kansas 66502, U.S.A. E. N. Clare Mills, Institute for Food Research, Colney Business Park, Norwich NR4 7UA, U.K. Christophe M. Courtin, Laboratory of Food Chemistry and Biochemistry, Katholieke Universiteit Leuven, 3001 Leuven, Belgium Sylvie Davidou, ENSIA, F-75241 Paris 03, France Jan A. Delcour, Laboratory of Food Chemistry and Biochemistry, Katholieke Universiteit Leuven, 3001 Leuven, Belgium Renato D’Ovidio, Universita degli Studi della Tuscia, Vai San Camillo de Lollis, Viterbo 01100, Italy Nancy M. Edwards, Canadian Grain Commission, Winnipeg MB R3C 3G8, Canada Päivi Ekholm, Department of Applied Chemistry and Micro­ biology, FIN-00014 University of Helsinki, Finland A. D. Evers (Retired), Ascus Ltd., Markyate, Herfordshire AL3 8HY, U.K. Rebeca Garcia, ENSIA, F-75241 Paris 03, France Kurt Gebruers, Laboratory of Food Chemistry and Biochemistry, Katholieke Universiteit Leuven, 3001 Leuven, Belgium Hans Goesaert, Laboratory of Food Chemistry and Biochemistry, Katholieke Universiteit Leuven, 3001 Leuven, Belgium Michael J. Gooding, Department of Agriculture, The University of Reading, Berkshire, U.K. Rob J. Hamer, Wageningen Centre Food Sciences, Wageningen, Netherlands Robert J. Henry, Southern Cross University, Centre for Plant Conservation Genetics, Lismore NSW 2480, Australia Crispin A. Howitt, CSIRO Plant Industry, Canberra ACT 2601, Australia

John A. Jenkins, Institute for Food Research, Norwich NR4 7UA, U.K. Huw D. Jones, Rothamsted Research, Harpenden, Hertfordshire AL5 2JQ, U.K. Domenico Lafiandra, Universita degli Studi della Tuscia, Viterbo, 01100, Italy Anna-Maija Lampi, Department of Applied Chemistry and Microbiology, FIN-00014 University of Helsinki, Finland Kirsi-Helena Liukkonen, VTT Technical Research Centre of Finland, FIN-02044 VTT, Finland Finlay MacRitchie, Department of Grain Science and Industry, Kansas State University, Manhattan, Kansas 66506, U.S.A. Matthew K. Morell, CSIRO Food Futures Flagship, Canberra, ACT, Australia Jacques Nicolas, ENSIA, F-75241 Paris 03, France Jae-Bom Ohm, Northern Crop Science Laboratory, USDA, Agricultural Research Service. Fargo, North Dakota 58105, U.S.A. Seok-Ho Park, Grain Marketing and Production Research Center, USDA, Agricultural Research Service, Manhattan, Kansas 66502, U.S.A. Vieno Piironen, Department of Applied Chemistry and Microbiology, FIN-00014 University of Helsinki, Finland Elieser S. Posner, ESP International, Savyon, Israel Jacques Potus, ENSIA, F-75241 Paris 03, France M. S. Ram (Retired), Grain Marketing and Production Research Center, USDA, Agricultural Research Service, Manhattan, Kansas 66502, U.S.A. Marjatta Salmenkallio-Marttila, VTT Technical Research Centre of Finland, FIN-02044 VTT, Finland Peter R. Shewry, Rothamsted Research, Harpenden, Hertfordshire AL5 2JQ, U.K. Caroline A. Sparks, Rothamsted Research, Harpenden, Hertfordshire AL5 2JQ, U.K. Bruce Stone (Deceased), Department of Biochemistry, La Trobe University, Bundoora 3086, Victoria, Australia Peter L. Weegels, Sonneveld Group BV, Papendrecht, Netherlands C. W. Wrigley, Food Science Australia and Wheat CRC, North Ryde (Sydney), NSW 1670, Australia

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• Preface to the Fourth Edition

It has been an honor and pleasure, as well as an immense responsibility, to preside over the revision of this much loved and widely used volume. Y. Pomeranz noted in his preface to the third edition that it had become a “bible” for wheat chemists, and this position has been consolidated during the 20 years in which his meticulously presented volume has been in print. However, the past 20 years have seen many changes, in publication technology as well as in wheat science, which means that this new edition has a new look as well as new science. First, the range of topics is more focused, covering the wheat plant and grain (essentially Volume 1 of the third edition) but not some of the more detailed aspects of processing, which are now covered in other volumes published by AACC International. Second, most of the chapters have been wholly rewritten and focus on more-recent developments that were not included in previous editions. It is therefore intended to complement rather than wholly replace the third edition, which will remain in regular use by many.

Third, recent advances in publication technology mean that we are able to produce the volume in an attractive large, twocolumn format with liberal use of color. We hope you will agree that it is a pleasure to handle and read as well as a mine of information. Finally, we wish to thank the many people who have made this possible: the authors, who have treated the project as a labor of love rather than a duty, and the AACC International staff, who have guided our efforts. We would also like to thank Helen Jenkins and Lynda Castle at Rothamsted Research for their help with formatting and checking chapters and preparing figures. We especially would like to thank all our colleagues who willingly devoted their valuable time to review relevant chapters to improve the quality of this book. We hope you enjoy using the book and will continue to do so for the next 20 years. Khalil Khan Peter R Shewry

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Preface to the Third Edition

But the images of men’s wit and knowledge remain in books, exempted from the wrong of time and capable of perpetual renovation. Neither are they fitly to be called images because they generate still, and cast their seeds in the minds of others, provoking and causing infinite actions and opinions in succeeding ages. Francis Bacon Almost a quarter of a century has passed since the publication of the first edition of this monograph and almost two decades since the second edition. During those years, the book became accepted worldwide as the “wheat bible.” I have seen it in practically every place that I have traveled around the world, and it invariably made me feel “at home” even in the most distant and isolated places. The task of revising a “bible,” even in the field of wheat chemistry and technology, is not a simple one. In conversations with the 27 coauthors (22 of whom are new ones) from six countries, I have been asked repeatedly whether the book should be a scientific primer, a textbook, a reference book, an overview of applications, a review of the state of the art, or a review of potential developments and projections of the future. My answer was “Yes” to all of these. The coauthors were asked to review critically the important material in the previous edition(s); to update and emphasize the new in information and interpretation, while making sure that the “classical” was retained, not merely out of respect but as landmarks in development; and to include new frontiers in biotechnology and genetic engineering while covering well-established and mature technologies. For decades, much of what was accomplished in our field was based on rules of thumb. This has provided the basis for a tradition sanctified by time and strengthened by much reluctance and opposition to change. The motto has been “If it works, don’t fix it.” We have seldom asked how well it works, whether it works consistently or merely sometimes, whether it can be made to work better. To answer those questions, it is not enough to have know-how; we must also have know-why. Just as there is a need to make sure that students gain in college a wide range of technical skills and a broad spectrum of knowledge, there is a need to provide cereal chemists and technologists with a good data base

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with which to understand, interpret, and perform well in their field of expertise. To provide that data base, the third edition of Wheat: Chemistry and Technology is a set of two books; the first one covers mainly the science (chemistry) and the second one mainly the technology. Every chapter has been revised extensively and most are completely new. The new edition has separate chapters on the microscopic structures and the composition of the wheat kernel. Two new chapters were added: one on nutritional aspects of wheat and wheat products and one on flat breads (including those produced in the Far and Middle East). Much space is devoted to, and information provided on, the theory and practice of evaluating wheat and wheat products around the world. All this required a considerable expansion (almost doubling) of the text and publication of the book in two volumes. I would like to end this preface on a somewhat personal note. In discussions with the authors, many have expressed the view that a chapter should be either applied-pragmatic or basic-theoretical. I have always believed that the two are intertwined, that they reinforce and drive each other. In some of the positions I have held, there has been much pressure to complete the routine work first and engage in research only after that. Somehow the time that all routine work is done never comes and, consequently, some people never get to engage in research. This is unfortunate, because you really do not do satisfactory routine work if you do not, in a limited way at least, do some of your own exploratory work—and that is, after all, research. And finally, we often rely too much on machines, store reams of data, and do not take enough time to find out about their meaning and consequences. Let us let the modern automated instruments and computers do what they do best, generate and compute data, and let us see what they cannot see and, based on information in good books, interpret the uniqueness and research dimension of those data. If we do, we will be happier, more qualified, more equal and respected partners, and we can provide better services and cooperation in evaluating wheat in plant breeding, genetics, production, storage, marketing, processing, and utilization. It is my hope that the new, revised edition of Wheat: Chemistry and Technology will help you attain that objective. Y. Pomeranz

• Preface to the Second Edition

The present edition of the Wheat Monograph contains material that has been updated to include developments and advances in the last decade. This has resulted in a substantial increase in the size of the book. The main objective of the revision has been to describe, in detail, the present status of our knowledge in wheat chemistry and technology and to discuss critically significance and relevance of recent biochemical findings or new technological processes. Extensive changes were made in Part Three of the Monograph, on the composition and role of principal chemical components and elucidation of their structure by modern biochemical techniques. Part Four has been enlarged by addition of a chapter on relation between composition and functionality, and the chapter on dough properties has been revised extensively. The three chapters in Part Five have undergone major revisions to reflect the great changes that have taken place in recent years in processing of end products of wheat. The net result is a new book. It is based on the general outline and objectives of the previous edition, but some of the older material has been deleted and almost half has been revised.

Editors of multi-authored monographs are faced with two major problems: delays in submission of manuscripts and differences in scope and depth of presentation. It is, therefore, a pleasure to acknowledge the excellent cooperation of all contributors to the monograph. The contributors have prepared their manuscripts in a way that did not detract from their creative originality and individuality; yet, they conformed to a general basic approach; they sent in the material promptly so that all contributions could be processed for publication at approximately the same time. It is a particular pleasure to acknowledge the generous help and useful suggestions of Dr. B. S. Miller, Past President of the American Association of Cereal Chemists. Mrs. Eunice R. Brown who had given nearly twenty years of devoted editorial service to the Association and was in charge of the technical editing for this edition, passed away before it was completed. Her work was carried on by Mrs. Joy McComb. They, with the aid of the Association’s staff, have made every effort to produce an attractive and useful book. Mrs. Eleanore V. Neu is thanked for excellent secretarial help. Y. Pomeranz

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Preface to the First Edition

The plant breeder who develops new varieties of wheat, the producer who grows the wheat, and grain inspector who grades it, the miller who mills it into flour, the baker who bakes the flour into bread, and the cereal chemist who studies the chemical composition and properties of the substances that make up the wheat kernel—all are preoccupied with some aspect of the same question: what constitutes quality in wheat. There is, of course, no simple answer to this question. Nevertheless, considerable progress has been made by cereal chemists over the years and recorded in widely scattered literature. The present volume, the third in the Monograph Series sponsored by the American Association of Cereal Chemists, is an attempt to provide a coherent set of reviews of our present knowledge on the cereal chemistry of wheat. It is a multiauthor work, with the advantage that each subject is dealt with by an expert in that field, but also with some disadvantages inherent in this type of book. The quality of wheat is a function of the composition and properties of the basic material of wheat. But it is also a function of the technological methods by which wheat is transformed into intermediate, and ultimately into consumer, products. Accordingly, in the organization of this monograph both these

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aspects were included. The chapters on wheat, flour, dough, and end-use products emphasize the technological aspects, while the chapters on proteins, carbohydrates, lipids, etc., stress the chemical aspects. It is hoped that this book will serve as a useful reference for the cereal chemist and, in a secondary way, as a guide to original literature. For the appearance of this book the credit must go to the individual authors. They gave generously of their time and knowledge both within and beyond their normally busy schedule of duties. Co-operation of the Monograph Committee helped to guide the Monograph especially through its earlier stages. The Committee comprised Dr. W. B. Bradley, the late Dr. W. F. Geddes, Dr. Lawrence Atkin, Mr. W. G. Bechtel, Drs. B. M. Dirks, J. W. Pence, J. A. Shellenberger, and Majel M. MacMasters. Thanks are also due to R. J. Tarleton, Executive Secretary of the Association, who was in charge of production, and to his assistant, Mrs. Eunice R. Brown, who was responsible for the technical editing. Finally, acknowledgment goes to F. D. Kuzina and Mrs. Mary Kilborn for technical assistance, and to others not mentioned but who assisted in many ways to make this Monograph a reality. I. Hlynka

• Contents

Chapter

  1. Wheat: A Unique Grain for the World.  C. W. Wrigley  x  1 Wheat and People  ♦  1

Wheat and Human Origins  |  Wheat and Human Food

Wheat Taxonomy  ♦  4

Wheat and Its Cereal Relatives  |  Wheat as a Grain Genus  |  Wheat and Its Ancestral Relatives

Wheat in a Hungry World  ♦  6

Wheat Around the World  |  Wheat Harvesting Around the Globe  |  Wheat in World Trade  |  Wheat—How We Measure It

Wheat in the Grain Chain  ♦  8

Wheat Breeding—Then and Now  |  Wheat Production  |  Wheat Harvesting and Segregation  |  Wheat Storage and Transport  |  Wheat Marketing  |  Wheat Processing

Wheat and Human Health  ♦  14

Advantages and Limitations of Wheat in the Diet  |  Allergy and Intolerance to Wheat

Conclusion  ♦  15

  2. The Wheat Crop.  Michael J. Gooding  x  19 Origins  ♦  21

Progenitors  |  Early Use, Then Domestication  |  Free-Threshing and Hexaploid Wheats  |  Wheats of Today

Growth and Development  ♦  25

Seed Structure and Germination  |  Vegetative Growth  |  Reproductive Growth

Wheat Improvement  ♦  32

The Decline of Stature  |  The Increase of Biomass  |  Resistance to Disease  |  Tolerance of Abiotic Stresses  |  Modern Breeding Methods

Agronomy of Wheat  ♦  38

Sowing  |  Nutrition  |  Disease Control

  3. Development, Structure, and Mechanical Properties of the Wheat Grain.  D. B. Bechtel, J. Abecassis, P. R. Shewry, and A. D. Evers  x  51 Terminology and Whole-Grain Considerations  ♦  51

Terminology  |  Structure of the Inflorescence  |  External Appearance of the Wheat Grain  |  Whole-Grain Development

Microstructure of the Mature and Developing Wheat Grain  ♦  56 Caryopsis Coats  |  Endosperm  |  Embryo

Molecular and Biochemical Studies of Grain Development  ♦  77 Gene Expression Analysis  |  Proteomic Studies

Structure and Grain Mechanical Properties  ♦  80

Properties of the Outer Layers  |  Endosperm Mechanical Properties  |  Integration of Data

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  4. Criteria of Wheat and Flour Quality.  Gordon R. Carson and Nancy M. Edwards  x  97 Wheat Classification  ♦  97 Technological Classification of Wheat  |  End-Use Classification of Wheat

Grading Factors That Affect Milling Yield and End-Use Quality  ♦  99

Grading Factors That Affect Milling Yield  |  Grading Factors That Affect End-Use Quality  |  Grading Factors That Affect Food Safety

Chemical Characteristics  ♦  104

Moisture Content  |  Ash  |  Flour Color  |  Fiber Content  |  Protein Content  |  Enzymatic Activity

Intrinsic Characteristics  ♦  106

Protein Quality  |  Starch Quality

Processing Quality  ♦  107

Milling Quality  |  Bread-Baking Quality  |  Noodle and Asian Product Quality

Wheat Classes—Producing Nations  ♦  109

Major Exporters  |  Other Producing and Exporting Countries

  5. Wheat Flour Milling.  Elieser S. Posner  x  119 Wheat Receiving at the Mill  ♦  120 Evaluation and Testing

Wheat Storage  ♦  124

Precleaning of Wheat Before Storage  |  Storage Bins  |  Preserving Wheat in Storage  |  Other Means to Preserve Wheat  |  Issue of Wheat and Flour Blending in the Mill

The Mill’s Wheat-Cleaning System  ♦  126

Handling of Wheat from Storage Bins  |  Wheat-Cleaning Machines and Work Principles  |  Wheat Screenings

Wheat Conditioning for Milling  ♦  129 Milling Machinery  ♦  130

Grinding and Reduction Equipment  |  Sieving and Separation Equipment  |  Purification of Endosperm  |  Air as a Tool and a Transport Medium in the Milling Process

The Milling Process  ♦  136

The Mill Flow Sheet (Diagram)  |  Break System  |  Grading System  |  Purification System  |  Sizing System  |  Reduction System  |  Milling of Hard Wheat  |  Durum Wheat Milling  |  Soft Wheat Milling

End-Product Quality and Handling  ♦  140

Flour and Semolina Color  |  Bran Content in Mill End Products  |  Flour Particle Size  |  Ash as a Flour Evaluation Parameter  |  Starch Damage

Mill Technical Performance Analysis  ♦  141

Flour Streams in the Mill and Their Blending  |  Flour Extraction  |  Fluctuation in Mill Performance  |  Flour Mill Sanitation  |  Air Classification of Flour: Methods and Objectives  |  Heat Treatment of Flour  |  Whole-Wheat Products  |  Mill By-Products  |  Flour Storage, Blending, Additives, and Flour Enrichment  |  Loading, Packing, and Shipping

Recent Developments in Commercial Milling  ♦  147

Wheat Debranning Before Milling  |  Light-Out Mills  |  Controlling Ambient Conditions in the Mill Space  |  ISO, HACCP, and GMP and Their Effect on the Industry

Future Developments in Wheat Milling  ♦  148

  6. Structure and Functional Properties of Gluten.  Rob J. Hamer, Finlay MacRitchie, and Peter L. Weegels  x  153 Scope  ♦  153 Structure-Function Models of Gluten  ♦  153 Models Based on the Structure of Glutenin  |  Models Based on Polymer Science  |  Models Based on Rheology  |  Summary of Models

Mixing Flour into Dough  ♦  155

Mixing-Induced Changes  |  How Glutenin Proteins Affect Mixing  |  The Glutenin Protein Network as a Guide to Understanding Mixing  |  Chemical Reactions During Mixing  |  Nondisulfide Cross-Links in Dough  |  Synthesis

Wheat Proteins During Proofing  ♦  161

Protein Functionality in a Proofing Dough  |  Bulk Rheological Properties of Wheat Proteins  |  Surface Rheological Behavior of Wheat Proteins

Contents  Wheat Proteins During Baking  ♦  166

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Transformation of Dough to Bread  |  Chemical Changes  |  Changes in Extractability  |  Conformations of Gluten Proteins During Heating  |  Physicochemical Changes of Gluten Proteins During Heating  |  Conclusions: Mechanisms of Denaturation of Gluten Proteins

  7. Micronutrients and Phytochemicals in Wheat Grain.  Vieno Piironen, Anna-Maija Lampi, Päivi Ekholm, Marjatta Salmenkallio-Marttila, and Kirsi-Helena Liukkonen  x  179 B Vitamins  ♦  179

Significance of Wheat in B Vitamin Nutrition  |  Bioavailability of B Vitamins from Wheat Products  |  B Vitamin Levels in Wheat Grains  |  B Vitamin Contents in Grain Fractions  |  Effect of Other Processing Methods on B Vitamin Contents

Tocopherols and Tocotrienols  ♦  185

General Aspects  |  Significance of Wheat as a Source of Tocopherols and Tocotrienols  |  Tocopherols and Tocotrienols in Wheat Grains  |  Tocopherols and Tocotrienols in Milling Fractions of Wheat  |  Effects of Other Processing Methods and Storage on Tocopherols and Tocotrienols

Carotenoids1  ♦  189

General Aspects of Carotenoids in Wheat  |  Significance of Wheat as a Source of Carotenoids  |  Carotenoids in Wheat Grains | Carotenoids in Wheat Milling Fractions  |  Effects of Other Processing Methods and Storage on Carotenoids

Minerals and Trace Elements  ♦  193

Wheat as a Source of Minerals and Trace Elements  |  Mineral and Trace Element Contents of Wheat  |  Bioavailability of Mineral and Trace Elements from Wheat

Phytosterols  ♦  196

Phytosterols as Functional Food Components  |  Phytosterols in Wheat Grain  |  Phytosterols in Milling Fractions  |  Free Phytosterols and Steryl Conjugates

Phenolic Compounds  ♦  201

Phenolic Compounds in Wheat  |  Phenolic Acids  |  Alkylresorcinols  |  Lignans  |  Other Phenolic Compounds

Choline and Betaine  ♦  209

General Aspects  |  Significance of Wheat and Wheat Products as a Source of Choline and Betaine

Future Research Needs  ♦  210

  8. Wheat Grain Proteins.  Peter R. Shewry, Renato D’Ovidio, Domenico Lafiandra, John A. Jenkins, E. N. Clare Mills, and Ferenc Békés  x  223 Overview  ♦  223

Wheat Seed Proteins: A Historical Perspective  |  Impact of “-omics” Technologies  |  Classification of Wheat Grain Proteins  |  Seed Protein Superfamilies  |  Proteomic Analyses

Protein and Amino Acid Compositions of Whole-Grain Fractions  ♦  227

Grain Protein Content  |  Amino Acid Composition of Wheat Grain  |  Nutritional Quality  |  Analysis of Grain Fractions  |  Extraction and Analysis of Protein Fractions

Gluten Proteins  ♦  236

Polymorphism and Genetics  |  Sequences and Relationships of Gluten Proteins  |  Structures  |  Gluten Proteins and Grain Processing Properties

Low Molecular Weight Proteins Related to Prolamins  ♦  265

Puroindolines and Grain Softness Protein  |  α-Amylase Inhibitors (CM Proteins)  |  Lipid Transfer Proteins  |  α-Globulins  |  7S Storage Globulins  |  11S Globulins/Triticin

Other Groups of Proteins with Functional Properties  ♦  272

Proteins Involved in Allergy and Intolerance  |  Surface-Active Proteins

Other Biologically Active Proteins  ♦  275

Wheat Germ Agglutinin  |  Wheatwin1 (PR4)  |  Ribosome-Inactivating Proteins  |  Thionins

Conclusions and a Forward Look  ♦  277

  9. Carbohydrates.  Bruce Stone and Matthew K. Morell  x  299 Carbohydrates in Grain: Classification  ♦  299 Mono-, Di-, and Oligosaccharides  ♦  399 Fructans  ♦  300 Occurrence and Structure  |  Biosynthesis  |  Analysis

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Wheat Starch—Structure, Synthesis, and Functionality  ♦  302

Starch Structure  |  Physicochemical Functionality of Wheat Starch  |  Synthesis of Wheat Starch  |  Relationships Between Starch Synthesis, Structure, and Functionality  |  Starch and the Staling of Bread  |  Starch and Nutrition  |  Future Directions

Cell Wall Polysaccharides  ♦  319

Occurrence, Composition, and Organization in Walls of Cell Types in Grain  |  Cellulose  |  Glucomannans  |  Arabinoxylans 

|  (1→3,1→4)-β -D-Glucans  |  Arabinogalactan-Peptides

Carbohydrates in Aleurone Protein Body Inclusions—Phytin Globoids and Niacytin Granules  ♦  336 Phytin Globoids  |  Niacytin Granules

Impact and Applications of Cell Wall Polysaccharides in Grain Utilization, End-Use Quality, and Nutrition  ♦  337 Milling and Conditioning  |  Bread and Other Baked Products  |  Applications of Arabinoxylans  |  Nonstarch Polysaccharides in Human and Animal Nutrition

10. Wheat Lipids.  Okkyung Kim Chung, Jae-Bom Ohm, M. S. Ram, Seok-Ho Park, and Crispin A. Howitt  x  363 Wheat Lipid Classification  ♦  363 Saponifiable Lipids  |  Nonsaponifiable Lipids  |  Lipid Class by Location of Lipids in Wheat Structural Parts  |  Lipid Class by Extraction Method and Lipid Solubility

Analytical Methods  ♦  367

Methods for Extraction of Lipid  |  Fractionation and Characterization

Lipids in Wheat Fractions and Wheat Classes  ♦  371

Wheat Flour Lipids  |  Carotenoids  |  Sterols  |  Tocol Derivatives  |  Wheat Straw Lipids and Waxes

Functional Roles of Wheat Lipids  ♦  383

Nutritional Roles  |  Roles as a Wheat Quality Index  |  Functional Roles in Wheat Products  |  Effects on Cookie Quality  |  Effects on Cake Quality  |  Effects on Spaghetti and Noodles  |  Effects on Other Wheat Products

Genetic Research on Lipid-Related Components  ♦  389

Manipulating the Carotenoid Content of Wheat  |  Manipulating the Tocochromanol Content  |  Manipulating the Fatty Acid Content of Seeds

Future Research  ♦  390

11. Enzymes and Enzyme Inhibitors Endogenous to Wheat.  Kristof Brijs, Christophe M. Courtin, Hans Goesaert, Kurt Gebruers, Jan A. Delcour, Peter R. Shewry, Robert J. Henry, Jacques Nicolas, Jacques Potus, Rebeca Garcia, and Sylvie Davidou  x  401 Hydrolytic Enzymes  ♦  401

Starch-Degrading Enzymes  |  Nonstarch Polysaccharide-Degrading Enzymes  |  Protein-Degrading Enzymes  |  Esterases

Inhibitors of Hydrolytic Enzymes  ♦  410

Inhibitors of Starch-Degrading Enzymes  |  Inhibitors of Nonstarch Polysaccharide-Degrading Enzymes  |  Inhibitors of Protein-Degrading Enzymes  |  Inhibitors of Esterases

Oxidoreductases and Their Inhibitors  ♦  414

Lipoxygenase  |  Polyphenol Oxidase  |  Peroxidase  |  Catalase  |  Ascorbic Acid Oxidase  |  Glutathione-Dehydroascorbate Oxidoreductase

Conclusion  ♦  423

12. Transgenic Manipulation of Wheat Quality.  Huw D. Jones, Caroline A. Sparks, and Peter R. Shewry  x  437 Development of Wheat Transformation Methods  ♦  437 Transgenics for Up- or Down-Regulation of Gene Expression  |  Targeting and Tagging Transgenes in the Endosperm  |  Transgenic Approaches to Crop Improvement

Application of Transgenesis to Improve Grain Quality  ♦  441

Production of Lines Expressing HMW Subunit Transgenes  |  Effects of HMW Subunits on Functional Properties of Gluten  |  Substantial Equivalence of Lines Expressing HMW Subunit Transgenes  |  Production and Properties of Lines Expressing LMW Subunit Transgenes  |  Silencing Gliadins by RNAi Technology  |  Improvement of Nutritional Quality  |  Reduction of Preharvest Sprouting  |  Prospects

Index  x  453

•

CHAPTER  1

Wheat: A Unique Grain for the World C. W. Wrigley Food Science Australia and Wheat CRC North Ryde (Sydney), NSW, Australia

Wheat is unique. Of all the seeds in the plant kingdom, the wheat grain alone has the gluten proteins capable of forming the fully elastic dough required to bake leavened bread. These gluten proteins are also needed to make the great variety of foods that are associated with wheat around the world. This unique property is the reason why about 1014 wheat plants are grown annually on all continents (except Antarctica), producing well over 600 million tonnes (metric tons, t) of grain (Table 1.1) from about 220 million hectares (ha), with an average yield of nearly 3 t/ha. Worldwide,

this level of wheat production is equivalent to nearly 300 g of grain per person per day. However, in practice, this theoretical estimate is meaningless, since the regions of wheat production differ from the populations in need of the grain. Furthermore, although human food is the main use of wheat, a significant proportion also goes to animal feed and to industrial uses. Wheat and bread are integral to human life as well as to human food. Wheat and bread have entered our vocabulary as symbols of food and of social interaction. It is an Eastern European custom to offer a loaf of bread as a symbol of welcome to a guest. Biblical mentions of wheat and bread, such as “Man shall not live by bread alone,” “Cast thy bread upon the waters,” and “Give us this day our daily bread” have entered common usage. The motto of the United Nations’ Food and Agriculture Organization is “Fiat panis,” meaning “Let there be bread.” On the other hand, hunger has traditionally been depicted as the absence of wheat and bread. The dilemma of the world’s “have-nots” is depicted graphically on an old German platter by wheat heads and slices of bread and the words “Altes Brot ist nicht hart – kein Brot, das ist hart!” (“Old bread is not hard—no bread, that is hard”) (Fig. 1.1).

WHEAT And People

Fig. 1.1. Old German platter, depicting the plight of the world’s hungry. (Reproduced, with permission, from Bushuk 1992)

Wheat and Human Origins Wheat is among the oldest and most extensively grown of all grain crops. The period over which people have influenced the cultivation of wheat is, however, short in terms of human existence on earth. It is widely accepted that wheat was first grown as a food crop about 10,000–8,000 B.C.E. Presumably, wheat’s unique dough-forming property was seized upon by early people, so that wheat grain was treasured above other grain species for baking. Along with other cereal grains, wheat became a major

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reason for the transition from the hunter-gatherer nomad to the settled agriculturalist. The cultivation of storable grains meant that the family or tribe did not need to keep moving in search of whatever plant and animal food could be found. Instead, it was able to settle in one place, growing crops that could be stored safely for the long period after harvest. This major change in attitude led to a changed life style, leaving time for the development of cultural exploits beyond the day-to-day necessity of seeking food (Diamond 1997). Deities OF wheat

The ancient civilizations of Babylonia, Egypt, Crete, Greece, and Rome were based on wheat as a principal food plant. The

TABLE 1.1 Wheat Production and Yield in 2005 for Countries Producing More Than One Million Tonnesa Country

Algeria Argentina Australia Austria Bangladesh Belgium Brazil Bulgaria Canada Chile China Czechoslavakia, former area of Denmark Egypt Ethiopia, former area of France Germany Greece Hungary India Iran Italy Mexico Morocco Nepal Netherlands Pakistan Poland Romania Russian Federation Saudi Arabia South Africa Spain Sweden Syrian Arab Republic Tunisia Turkey UK USA USSR, former area of a Source: FAOSTAT

Production (millions of tonnes)

Yield (t/ha)

3 16 24 1 1 2 5 4 26 2 96 6 5 8 2 37 24 2 5 72 15 8 3 3 1 1 22 9 7 45 2 2 38 2 5 1 21 15 57 92

1.4 2.6 2.1 5.0 2.0 8.3 2.2 3.2 2.6 4.4 4.2 5.5 7.2 6.5 1.4 7.0 7.4 2.1 4.5 2.7 2.3 3.5 5.0 1.0 2.1 8.7 2.6 3.8 2.9 … 5.2 2.5 1.7 6.3 2.6 1.6 2.6 8.0 2.8 2.0

data (2006), accessed via the web site at www.fao.org.

Egyptians believed wheat to have been a gift of the god Osiris (Buller 1919). The Greeks attributed the provision of wheat to Demeter (Fig. 1.2), the “goddess of the earth and its fruits, a deity presiding over or representing the generative powers of nature” (Fowler 1908). Demeter may have been adopted by the Romans under the name Ceres, but there is evidence that in her earliest form, Ceres was strictly of Roman origin. The first temple to Ceres was built at the foot of the Aventine, one of the seven hills of Rome, and dedicated to Ceres on (our equivalent of) April 19, 493 B.C.E. (Fowler 1908). According to tradition, the temple to Ceres was a consequence of a famine in the year 496 B.C.E. that limited the supply of grain. A divine source of help was sought, and Ceres was the obvious source. The name “Ceres” came from the verb creare, to create, but the meaning became narrowed to relate to agriculture and subsequently to grains, especially wheat. The blessing of Ceres was invoked to bring to maturity the seed sown in the autumn, by preserving it from all pests and hurtful events. The time of autumn sowing extended from the equinox to the winter solstice, but there was also a spring sowing, which began on (our equivalent of) February 7 (Fowler 1908). The festival of Cerealia at the end of the Roman summer involved the offering of ripe ears of wheat to Ceres (Fowler 1908). Today, the term cereals refers to the family of grains derived from the monocotyledonous grasses. The main cereal species are listed in the upper half of Table 1.2, where they are contrasted with the dicotyledonous grains—oilseeds and legumes.

Fig. 1.2. The Greek goddess, Demeter, the goddess of wheat. Awned wheat heads and stalks form her headdress, the sheaf in her arm, and the contents of the basket at her feet. Drawn from a painting found in Pompeii, Naples. (Reproduced from Buller 1919)

Wheat: A Unique Grain  Wheat and other cereals

In the Mediterranean region, centuries before recorded history, various species of grain played a major role in feeding the population. Weaver (1950) and Takahashi (1955) have reported that, for a time, barley was grown more extensively than wheat; apparently, barley was more highly regarded as human food. Wheat was, however, always prized by the Romans. Indeed, it played such a dominant role in the Roman Empire that this area was described as a “wheat empire.” Subsequently, migrations from the north caused wheat to be partly replaced by rye. Throughout the Middle Ages, rye was a more important food in Europe than were the different species of wheat then available. However, the proportion of wheat to total grain used for food, as mentioned by Jasny (1944), was considerably larger than indicated by production, because barley and rye were used as animal feed, whereas almost all wheat was used for human food. Likewise, wheat dominated domestic and international trade almost to the exclusion of all other cereals. After a period of slow progress, wheat became commonly regarded as the best of the cereal foods, and the availability of wheat for food was considered a sign of a high stage of civilization. The quality of wheat for bread production improved, and bread in one form or another became an important food for the Western world. In fact, Boals (1948) stated that wheat had become a world symbol, reflecting the food situation for many millions of people and, equally important, indicating economic prosperity and political stability. Furthermore, wheaten bread has today assumed sociological status in developing countries, often preferred to rice-, sorghum-, and millet-based foods. Reflecting these trends, the production of

Monocotyledonous plants Triticeae Triticeae Triticeae Triticeae Triticeae Aveneae Andropogoneae Andropogoneae Oryzeae Oryzeae Eragrostideae and Paniceae Dicotyledonous plants Asteraceae Asteraceae Brassicaceae Fabaceae Fabaceae Fabaceae Fabaceae Linaceae Malvaceae a Source

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wheat has increased significantly in developing countries in the past few decades. Furthermore, wheat production and consumption are projected to rise in developing countries, whereas the consumption of rice is expected to remain constant (Marathée and Gomez-MacPherson 2001). On the other hand, the production of oilseeds has increased in both developed and developing regions during recent decades.

Wheat and Human Food Wheat, through the millennia, has been intimately associated with human food uses. Gradually, Homo sapiens learned by ingenuity to develop tools, to make use of fire, and to react to climatic changes by adjustment of habits rather than by passive acceptance of what nature supplied. A need arose for establishing more-or-less permanent settlements to supplant the temporary abodes used in the roaming life in the search for food. Humans became food gatherers in addition to hunters, and all classes of plant and animal life, including the seeds of wild grasses, became sources of food. Among the difficult problems to be solved in using grass seeds for food was securing enough grain in one place to justify the effort needed to isolate the edible inner portions of the kernels from the outer glumes and the branny outer coat. Solving the problems of cultivating cereal grains, developing crude methods for grinding, and processing the ground meal into useful foods led to the beginnings of civilization. The history of the development of grinding implements and the part these advances have had in the progress of civilization are recorded by Jacob (1944), Starck and Teaque (1952), and

TABLE 1.2 The Main Grain Species, Showing Their Significance in Terms of Annual Production in 2005 Family or Tribe

x 

Genus and Species

Common Names

Triticum aestivum Triticum durum xTriticosecale sp. Secale cereale Hordeum vulgare Avena sativa Sorghum bicolor Zea mays Oryza sativa Zizania aquatica Several genera and species

Common (bread) wheat Durum (macaroni) wheat Triticale Cereal rye Barley Oats Sorghum Corn, maize Rice Wild rice Millet

Helianthus annuus Carthamus tinctorius Brassica napus Cicer arietinum Lupinus angustifolius and Lupinus albus Glycine max Several genera and species Linum usitatissimum Gossypium spp.

Sunflower Safflower Canola, oilseed rape Chickpea Blue and white lupin Soybean Bean Linseed, flax Cottonseed

of production statistics: FAOSTAT data (2006), accessed via the website at www.fao.org.

World Productiona (millions of tonnes)

  626 (all wheat) 13 15 138 25 57 692 615 (as paddy) 27 31 1 45 9 1 210 19 3 41

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Lockwood (1960). In the Stone Age, grains were crushed between flat stones; the crushed material was moistened with water and made into a flat cake, which was dried in the sun. At some later period, the practice of placing the cakes on hot stones or covering them with hot ashes was developed. Early efforts in the art of baking involved only meal, water, and heat, producing bread without fermentation, which we designate as “unleavened.” Such breads are common today in the oatcakes of Scotland, the Passover cakes of the Jewish people, and the chapatti of India and Pakistan. Written references to bread date back to about 2600 B.C.E. The history of the development of bread from its early form to the modern varieties has been described by Starck and Teaque (1952) and by Pyler (1958). The ancient Egyptians perfected the art of baking. Monuments from antiquity reveal that white bread was produced in many different shapes, from small, round loaves to the elongated loaves, often decorated with seeds, characteristic of modern Vienna bread. Baking became a most important occupation during the classical period of Greek and Roman domination of Western civilization. There were many large public bakeries during this period, but much bread was also baked in the home.

The Industrial Revolution also brought mechanization to the baking industry. Machinery superseded manual labor in many bakery operations (Matz 1960). New and different baked products were developed. The shape of baked products has not changed much since ancient times, but great diversity in baked goods has been provided by the use of baking pans and a variety of ingredients (shortening, sugar, milk, eggs, spices), as well as mixtures of wheat with other flours (barley, oats, rye, rice, potato, soy, and sorghum) and the incorporation of vital dry gluten. Certain types of baked products became associated with particular countries or places, such as the dark breads of old Russia, French bread, and Vienna bread. These products probably had some degree of standardization at one time, but at present none of them is well defined. For example, Vienna bread originally was a light crusty loaf, usually in the form of rolls, made from matured Hungarian flour, with a liberal amount of yeast, baked quickly in a hot oven containing steam. French bread was crisp, contained little crumb, and was usually made without the addition of sugar or fat. The dough was formed into sticks about 50 cm (18 in.) long. These and other types are now included under the general classification of hearth breads.

Urbanization and mechanization

Wheat, unique as a food source

With the advent of the Industrial Revolution, many people moved from agricultural regions to the cities. This meant that increased agricultural production was required to feed the city people. The farm’s production no longer had to produce for the resident family; extra wheat was also needed for city flour mills and bakeries. The trends of the Industrial Revolution have continued progressively worldwide, so that, in the next few years, more of the world’s population will live in urban environments than live in rural situations (Fig. 1.3). These ongoing demographic changes put more pressure on rural areas to produce grain for city dwellers, and the future will see a need for more processed foods with greater emphasis on food quality.

Wheat is a major component of most diets of the world because of its agronomic adaptability, ease of storage, nutritional goodness, and the ability of its flour to produce a variety of palatable, interesting, and satisfying foods. Doughs produced from wheat flour differ from those made from other cereals in their unique viscoelastic properties. This property is responsible for the universal use of wheat for a wide range of products. Among these are pan bread, noodles, cakes, biscuits/cookies, steamed bread, doughnuts, croissants, bagels, pizza, flat breads, and chapatti. Each of these products is ideally produced from a wheat selected to provide flour with the required characteristics. Moss (1973) summarized the requirements for the balance of grain hardness and protein content for several common products (Fig. 1.4).

WHEAT TAXONOMY

Fig. 1.3. Rates of urbanization. (Courtesy CIMMYT, Mexico)

Wheat and Its Cereal Relatives Wheat is one of the three most important grain species in the world, based on annual volume of production (Table 1.1). It shares this distinction with corn (maize) and rice. The production figure for rice (Table 1.2) is for paddy rice, the form of rice as it is initially harvested; this figure should be reduced by about 20% to indicate the production of rice grain, after removal of the outer hulls. Volumes of production are much less for the wider range of agricultural seed crops (Table 1.2); the species of next significance are barley and soybeans. Of all these, wheat has the distinction of being the most important in world trade

Wheat: A Unique Grain 

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and also the grain for which quality specifications are the most critical. The seed crops of economic value are divided into two major taxonomic groups, namely, the “monocots” and the “dicots” (Table 1.2), referring to the presence of one or two embryonic leaves (cotyledons) in the seed and young seedling. Although

both groups are sometimes referred to as “grains,” this botanical term should strictly be applied only to cereals in which the grain is a single-seeded fruit called a “caryopsis” (see Chapter 3). Further taxonomic groupings lead down through order, family, and tribe to genus and species (Heywood 1993; Morrison and Wrigley 2004).

Fig. 1.4. Types of wheat required for the diverse uses of wheat. (Re­ produced, with permission, from O’Brien and Blakeney 1985)

Wheat as a Grain Genus The genus name for wheat, Triticum, comes from the Latin tero (I thresh). Triticum vulgare is the old (no longer accepted) species name for bread wheat, in which vulgare means “common.” The current binomial name, Triticum aestivum, refers to hexaploid bread wheat (genomes A, B, and D), distinguishing it from tetraploid macaroni wheat (Triticum durum) (genomes A and B), which is used primarily for pasta production. Most of the wheat grown worldwide (>90%) is the aestivum species; despite its being referred to as “bread” wheat, it is used for the full range of applications, even including pasta production in some regions. In addition, T. monococcum (including “small spelt” wheat as a subspecies) and T. timopheevii (including “Georgian” wheat) are cultivated to a limited extent, the former in Yugoslavia and Turkey, and the latter in the former Soviet Union (Feldman and Sears 1981). The main cultivated form of spelt is the hexaploid T. aestivum var. spelta, also classified as T. spelta (Fig. 1.5) (Morrison and Wrigley 2004). The ancestral diploid wheat species are T. monococcum, Aegilops speltoides, and T. tauschii and a wild Aegilops species that is probably most closely related to the modern A. speltoides. Each of these species has seven pairs of chromosomes (2n = 14).

Fig. 1.5. Variations in the appearance of heads of wheat species, one of many morphological characteristics used for their taxonomic classification. The wheat species are (including their genome assignments and common names) a, Triticum boeoticum (2x: wild einkorn); b, T. monococcum (2x: einkorn); c, T. dicoccoides (4x: wild emmer); d, T. dicoccum (4x: emmer); e, T. durum (4x: macaroni wheat); f, T. carthlicum (4x: Persian wheat); g, T. turgidum (4x: rivet wheat); h, T. polonicum (4x: Polish wheat); i, T. timopheevii (4x: Timopheev’s wheat); j, T. aestivum (6x: bread wheat); k, T. sphaerococcum (6x: shot wheat, Indian dwarf wheat); l, T. compactum (6x: club wheat); m, T. spelta (6x: spelt wheat); and n, T. macha (6x: macha wheat). The diploid A-genome species, T. urartu, is not shown here. 2x = diploid; 4x = tetraploid; 6x = hexaploid. (Adapted from Mangelsdorf 1953)

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T. durum (also named T. turgidum ssp. durum) is tetraploid (2n = 14), having been derived from the natural hybridization of T. monococcum (A genome) and the ancestral A. speltoides (B genome). Common bread wheat (AABBDD) is a hexaploid (2n = 42) resulting from the natural hybridization of Triticum dicoccoides (AABB) and T. tauschii (DD) (Mangelsdorf 1953, Feldman 2001, Shewry et al 2003). The diversity of head morphology for these and other ancestral wheats is shown in Figure 1.5. Although it is not clear from the head illustrations, these various primitive wheats differ greatly in the ease of threshing out grains from the heads, an important characteristic for successful cultivation and harvesting of any grain species.

Wheat and Its Ancestral Relatives The origins of wheat and these hybridization events are believed to have occurred in the Middle Eastern region of Ancient Egypt, the Levant, and Mesopotamia, watered by the Nile, Jordan, Euphrates, and Tigris rivers. Wild emmer, the progenitor of cultivated wheats, was first known to the Western world from the work of the Austrian botanist T. Körnicke. In 1873, emmer was shown in the National Museum of Vienna among samples of wild barley collected by Körnicke in 1855 on the slopes of Mount Hermon, in southeastern Lebanon. It was not until 1889 that Körnicke reported this discovery to the Botanical Society of the Lower Rhine and Westphalia (Feldman 2001). Wild emmer carries the full scientific name of T. turgidum L. ssp. dicoccoides (Körn. ex Asch. and Graebn.) Thell., or just T. dicoccoides (Fig. 1.5). It was later found in many sites across Israel, Jordan, Lebanon, and Syria, thus confirming Körnicke’s hypothesis that this wild progenitor still grows in the Near East. In fact, it grows in a wide range of conditions, from the Jordan Valley, 200 m below sea level, to the slopes of Mount Hermon, up to 1,600 m, and in a wide range of soil types. Stories about the discovery of other wheat progenitors in the Fertile Crescent and beyond are told by Feldman (2001). They have proved to be valuable sources of novel genes for use in the improvement of cultivated wheats. Wheat remnants, unearthed in the Levant (southeastern Turkey south to the Jordan), have been dated to about 10,000 B.C.E. Neolithic humans are presumed to have cultivated emmer wheat, einkorn (T. monococcum ssp. aegilopoides), T. urartu, and T. timopheevii. Of these four ancestral wheats, all except T. urartu

evolved into domesticated forms with nonbrittle spikes. This important characteristic meant that the head did not readily shatter and fall to the ground, making it very difficult to harvest. These nonbrittle species gradually spread throughout Southwest Asia (Feldman 2001). Wild einkorn suffered from the disadvantage that, when threshed, the glumes (hulls) still remained attached. A freethreshing form of einkorn has been found in Central Asia; this would have been easier to harvest and thus it would have been propagated in preference to other forms. Similarly, the early wild heads of emmer were brittle, and it may have been 9,000 B.C.E. before nonbrittle forms appeared. In about 6,000 B.C.E., this grain spread to Egypt, India, and Central Asia. It is still cultivated to a small extent in parts of India, Iran, eastern Turkey, and the Balkans. Durum wheat (T. turgidum ssp. durum), related to emmer (T. turgidum ssp. dicoccoides), has been found in pottery dated to about 7,000 B.C.E., but it does not appear to have been established as a major crop until about 2,000 B.C.E. This apparent delay in its adoption would not be expected, as durum has free-threshing large grains, but may have been because of the limited agronomic adaptability of durum wheat (which has a narrower genetic basis than bread wheats) and because of the greater difficulty in milling the very hard durum grain. Today, however, durum wheat is cultivated worldwide to the extent of about 10% of wheat production. Common hexaploid wheat first appeared in about 9,000 B.C.E., presumably in northwestern Iran or northeastern Turkey, resulting from natural hybridizations between tetraploid wheat (probably not wild emmer) and the diploid species T. tauschii (A. tauschii) (Feldman 2001). This event probably occurred more than once (Morris and Sears 1967).

WHEAT IN A HUNGRY WORLD Wheat Around the World Wheat-production regions of the world (Fig. 1.6 and Table 1.1) range through many diverse climates and through countries of varying degrees of “development.” The figures in Table 1.1 provide a “snapshot” of one year’s production. Because the volume of production fluctuates from year to year, 10-year averages

Fig. 1.6. Wheat-production regions of the world. (Adapted from Trethowan et al 2005, courtesy CIMMYT, Mexico)

Wheat: A Unique Grain  (1993–2002) may be more indicative. On this basis, the production volumes in millions of tonnes for the top seven countries are China (93.9), the European Union (91.7), India (68.8), the United States (53.3), the Russian Federation (46.9), Canada (20.6), and Australia (19.4) (Worden 2004). The larger areas of production are in the northern hemisphere. Wheat is grown as a winter or a spring crop. In severely cold regions, spring-wheat types are sown in spring, to develop and mature quickly for harvest before the onset of autumn snows. In more moderately cold regions, wheats of winter habit are sown before the arrival of winter snows, which overlay the seedlings, causing them to vernalize and permitting prompt development as soon as the snow melts in the spring. In these areas, a winter wheat can flourish more rapidly than a spring wheat, due to the delay caused by the difficulty of sowing a spring wheat in thawing fields. In warmer climates, the distinction between spring and winter wheats is almost meaningless; the more important distinction is the maturity—late or early. Thus, in many Mediterranean climates, spring wheats (or those with only a small vernalization requirement) may be sown in autumn and are thus grown as if they were winter wheats.

Wheat Harvesting Around the Globe Sowing and harvest times continue throughout the year. These times depend most obviously on whether a particular region is in the northern or southern hemisphere but also on distance from the equator and height above sea level. For cold regions, sowing times also depend on whether winter or spring types are being sown. Harvest times around the world are listed in Table 1.3 for a large number of countries, showing that wheat is being harvested somewhere around the world at any time of the year. Nearly half of the global wheat area is in developing countries, and about 20% is in the emerging economies of Eastern Europe and in the Russian Federation. Most wheat (more than 80% of what is produced) is consumed within the source country, mainly as human food. The remainder, about 110 million tonnes annually, enters into international trade. This volume makes wheat the most-traded grain in the world. Less than 20% of the traded wheat is grown in developing countries, since most

Wheat in World Trade The major factors used to distinguish wheats in trade are the hardness or softness of the grain, winter or spring habit, red or white bran color, and protein content (Cracknell and Williams 2004). Within the resulting classes, wheat is further described according to bushel weight or test weight (a measure of bulk density), cleanliness (i.e., absence of contamination with foreign materials, including other cereal grains and weed seeds) and the level of screenings (i.e., small foreign seeds and broken or shriveled wheat kernels), the degree of soundness (i.e., absence of sprouted grain), moisture content, and dough-quality attributes that determine suitability for end-product processing. These properties are influenced by a combination of genetic and environmental factors (genotype [G] and environment [E] and the interaction G × E). Attributes, such as grain hardness, are largely determined by genotype (variety), whereas the more significant contributors to aspects of quality, such as protein content, are the result of growth conditions—soil fertility, rainfall, and temperature during the growing season, at harvest, and during storage and transport. Some varieties are more influenced by growth conditions than others; thus interactions between genotype and environment (G × E) are also significant determinants of processing quality. Wheats can be closely matched to many different end uses according to their grain hardness and protein content (Fig. 1.4) (Faridi and Faubion 1995, Posner and Hibbs 2005). Durum wheats are preferred for pasta; soft (hexaploid) wheats for ­biscuits/­cookies, cakes, and pastries; and hard-grained bread wheats for Asian noodles, flat breads, pan bread, and other products. By preference,

Month

Region

January February March April May June July August September October November December

Argentina, Southern Australia, Chile, Uruguay Upper Egypt, Southern India Egypt, India, Libya Southern Egypt, India, Iran, Iraq, Mexico, Syria Algeria, Morocco, Central and Southern Asia, Tunisia, Southern USA Central China, Southern France, Greece, Italy, Portugal, Spain, Turkey, Central USA Austria, Bulgaria, Northern China, France, Southern Germany, Hungary, Japan, Romania, Southern Russia, Central USA Belgium, Britain, Southern Canada, Denmark, Germany, Holland, Poland, Central Russia, Northern USA Canada, Sweden, Northern Russia, Northern USA Northern Canada, Northern Russia, Northern Scandinavia Northern Argentina, Brazil South Africa, Argentina, Central Australia

from Posner and Hibbs (2005).

7

of that production is consumed domestically. The 80% of traded wheat that is produced in developed countries comes from the United States (28% market share, 1993–2002), Canada (16%), the European Union (EU) (15%), Australia (14%), and Argentina (8%) (Worden 2004). Nevertheless, wheat production has increased in India in recent years, to the extent that India exported 3 million tonnes of wheat in 2003/04 (Pena 2007). In recent years, the buying countries importing more than 3 million tonnes were Algeria, Brazil, China, Egypt, the EU, Indonesia, Iran, Japan, South Korea, and the Russian Federation (Worden 2004).

TABLE 1.3 Times of Wheat Harvest Around the Worlda

a Adapted

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harder-grained wheats are deliberately cultivated in areas known to produce high protein contents, because the combination of high protein and hard kernels results in the flour most suited to products such as pan breads and Cantonese noodles. High protein content is also required for durum-wheat semolina. At the other end of the scale, lower levels of protein and soft kernels go hand in hand, and the flour from these wheats is well suited to biscuits/cookies, cakes, and other pastry goods. It is for this reason that certain wheat types are dominant in each wheat-growing region of the world. The combination of quality characteristics genetically built into varieties and those influenced by environment is carefully balanced to produce wheats with particular end uses in mind. The classification of wheat into specific grades is essential for pricing and trading purposes because it allows customers to specify their requirements according to the particular products they manufacture (Cracknell and Williams 2004). The large number of combinations of wheat variety and environment ensure a broad range of wheat quality types in the world. Much research effort has been directed at determining which types from which regions are best suited to individual products. This research on the many types of noodles, flat breads, chapatti, and other non-Western foods has been initiated by countries involved in international trade (especially the United States, Canada, and Australia), even though domestic use in these countries has traditionally been dominated by pan-bread products. More recently, these non-Western foods are starting to achieve popularity in these countries, so that their research efforts have also proved valuable domestically.

Wheat—How We Measure It Despite the long-standing arrangements of international trade in wheat and other grains, there is still no general agreement on the “language” to describe units of measurement. Much of the world has adopted the International System (SI) of units (see http://www.iso.ch), but the British and U.S. versions of the Imperial System are still in use (Wrigley 2004a). For example, the U.S. bushel (35.24 L) is equivalent to 32 dry quarts or 8 U.S. gallons, but it differs from the British Winchester bushel (36.37 L), which is equivalent to 8 British gallons. The bushel is a measure of volume, yet it is used as a measure of mass to indicate yield (e.g., bushels per acre). Accordingly, the U.S. government has defined Imperial mass units for a U.S. bushel of each of the major grains, namely, 60 lb for a bushel of wheat, 48 lb for a bushel of barley, 32 lb for a bushel of oats, and 56 lb for a bushel of maize. Conversion of yield figures from these Imperial units to metric is complex; for wheat, the equivalence is that 100 bu/acre = 6.73 t/ha. The equiva­lence is different for each of the cereal crops. On the farm, the common unit of yield may be bags per acre; 10 (U.S.) bags to the acre = 1.235 t/ha. This last measure is used in Table 1.1 for average wheat yields by countries in 2005. The “tonne” (metric ton [t], 1,000 kg, 2,204 lb) has become the basic unit of international trade, but the “ton” can have more than one meaning. The U.S. short ton (2,000 lb) equals 0.9072 t, and the U.S. long ton is 1.016 t, equivalent to the traditional British ton (2,240 lb). This confusion is partly due to historical

differences in the hundredweight (20 to the ton), which is 100 lb in the United States and 112 lb in Britain. One tonne is equivalent to 36.73 U.S. bushels of wheat.

WHEAT IN THE GRAIN CHAIN An appreciation of the wheat industry requires understanding of the “value chain”—the various transactions from “farm to fork.” There are many steps in this process, and at each stage, value is added. Simplistically, this increase in value can be seen by comparing the price of a few seeds of wheat, ready to sow, with the price of a glossy Danish pastry ready for consumption in an expensive restaurant. The chain starts with the breeder, who is responsible for the development of new varieties that suit the agronomic and quality needs of the steps throughout the chain. Next come the seed producers and the farmers, who use appropriate management to maximize dollar returns, represented by the combination of grain yield and grain quality (market value) (see Chapters 2 and 4). Beyond the farm gate are the stages of segregation, buying, storage, and transport. Further downstream are the many forms of processing (which generally involve milling) and food manufacture (see Chapters 5 and 6). The consumer, at the end of the chain, is the key factor responsible for driving the demand for specific foods and thus for specific raw-material qualities. Consumer issues range from pricing, through perceived quality (taste, mouthfeel, and texture), to nutritional factors (see Chapter 7). Consumer demands and reactions are communicated back to the breeder and to others in the chain, in terms of the range of quality attributes needed by the markets. The “chain” is thus better seen as a series of circles, with feedback from consumer to those responsible for grain and flour quality throughout the process. The lines of communication may be extremely long, both in distance and in time. Given the extent of world trade, the distance factor may involve the flow of information from a bakery in the Middle East or in China to the cereal chemist in a breeding program in Canada or Australia. The time factor may involve many years, due to the lag necessary for a breeder to change the direction of quality objectives, alter the selection of parent lines, revise crossing strategies, and introduce new methods to select for grain quality at early and late generations.

Wheat Breeding—Then and Now Early attempts at wheat improvement involved the selection of heads or plants that appeared to be better than the rest of the crop; the seed from these was propagated separately in hopes of better performance. This approach was limited by the lack of genetic diversity. Admittedly, crops of some centuries ago were probably quite heterogeneous, thus allowing the breeder scope to select the better plants, but this approach certainly had limitations. The need for genetic diversity

Wheat does not naturally outcross. Thus, its genotype remains stable from one crop to the next (a distinct advantage for

Wheat: A Unique Grain  retaining varietal purity), but as a result, there is little opportunity for improvement. The cross-breeding approach, first used for wheat in the latter part of the nineteenth century, offered the advantages of greatly increasing genetic diversity and of doing this with specific objectives in mind. Appropriate choice of parents permits the generation of a range of progeny, some of which are expected to display a combination of the desirable attributes of both parents. Early cross-breeding activities were undertaken in Europe, North America, and Australia in the 1880s and later. One of the notable Canadian successes was the production of the variety Marquis (Hard Red Calcutta × Red Fife) by the combined efforts of William Saunders and his sons Charles and Percy (Buller 1919). A key to this achievement was the recognition of the variety Red Fife as a source of yield and grain quality. Red Fife came from a sample of wheat introduced into Ontario in about 1842 by a farmer, David Fife, who received it from a friend in Glasgow, Scotland. Its actual origin was the port of Danzig, and it is likely to have been the European variety Galician (Buller 1919). Good teeth—a breeder requirement

The other parent of Marquis was an introduction from India, Hard Red Calcutta, which was used with the aim of providing earlier maturation in the progeny. One of the lines resulting from this cross was named Markham. From a crop of Markham, in turn, the variety Marquis was selected by Charles Saunders in 1903 (Morrison 1960). The basis for his selection was the excellent quality of the selection. This judgment was based on his “chewing test” to test grain hardness and gluten quality: In testing the value of these crosses, I began by chewing the grain to determine the elasticity of the gummy substance produced. … It requires some patience and a fairly good set of teeth, but these two attributes may be considered essential to all breeders of wheat. (Morrison 1960)

This approach to quality assessment was known to wheat buyers, so it was a reasonable extension to use it to select breeding lines. Describing Saunders’s chewing test, Buller (1919) wrote: To obtain sufficient gluten for a test, he usually chewed about ten or a dozen kernels from the crop of each individual plant. … He found it to be a general rule that the strongest flour is obtained from those wheats which produce gluten having the greatest ability to recover its shape. … The chewing test is certainly of great value although it should always be confirmed by actual baking trials as soon as sufficient wheat is obtainable for that purpose.

In about 1905, the Saunders laboratory was set up with “a small experimental mill, a fermenting cupboard, and an oven.” The breeder-chemist collaboration

At about this same period, William Farrer was also experimenting with cross-breeding in Australia, based on wheats introduced from Africa, India, and the Middle East, as well as high-quality germplasm such as Red Fife. He enlisted the assistance of the chemist Frederick Guthrie to provide quality-testing

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facilities, thereby initiating the breeder-chemist association that has continued as an integral part of wheat breeding programs (Blakeney and Wrigley 1993). Guthrie devised small-scale rollermilling equipment with which to evaluate milling quality, as well as facilities for testing gluten quality and baking performance (Guthrie 1898). Wheat breeding—now

A century or so later, wheat breeding has made great advances, but the aims are similar, namely, to choose parent lines that carry the desirable genetic attributes and to combine these in wheats adapted to the target region, thereby providing increased yield, improved resistance to pathogens, tolerance to abiotic stresses, and grain quality suited to market requirements (Bonjean and Angus 2001). (See Chapter 12.) Genes have been identified that control many specific attributes—aspects that contribute to yield potential, resistance genes for various pathogens, and grain-quality attributes. As they are discovered and characterized, genes are listed in a gene catalog, which previously was published annually as supplements, with five-yearly revisions, but which is now available online (McIntosh et al 1998, or go to http://wheat.pw.usda.gov/ggpages/wgc/2003/ Catalogue.pdf). There is also much more information on the Graingenes site, operated by the U.S. Department of Agriculture (http://wheat.pw.usda.gov), and in a list of genetic stocks, run by the Wheat Genetics and Genomic Resources Center, Kansas State University (www.k-state.edu/wgrc). After the initial cross is made, the most valuable progeny must be selected from the large number of lines produced. There are great advantages to the breeder if it is possible to identify specific traits in these lines on the basis of gene presence or absence. Appropriate selection at an early stage of the process avoids the need for ongoing propagation of undesirable lines. Simply inherited traits are selected early. Attributes such as yield and grain quality that involve many genes (quantitative traits) are more difficult to select for, so they have traditionally been part of the mid- to late-generation selection schedule, determined largely on the basis of actual phenotype (measurable aspects of yield and quality performance), rather than according to the presence or absence of relevant genes. Selection at the gene level

Marker-assisted selection (MAS) offers a new approach to selection based on genotype, moving the basis for selection from the phenotype to the gene level and thus removing the confusing effect of environment (at least at this stage of selection). However, MAS has the obvious limitation that it requires adequate polymorphism (genetic diversity) with respect to the mapped regions relevant to the traits of interest. This approach is particularly useful for some traits that are difficult to determine, such as resistance to some pathogens, and for tolerance to defects that appear only under specific environmental conditions, i.e., those with a strong genotype × environment (G × E) influence. An example is late-maturity α-amylase (LMA), also termed “pre-maturity α-amylase” (Mrva and Mares 2001). This defect is difficult to detect because the phenotype (elevated levels

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of α-amylase in the sound mature grain) is evident only after certain growth conditions (cool temperatures [namely, 12–18°C] at 25–35 days after flowering) have been present. Yet, breeders have been trying to eliminate lines that carry this defect, as it has proved to be the “downfall” of many otherwise excellent varieties. In an attempt to simplify the detection of LMA, efforts have been made to map the location of the gene(s) involved. A physical map is being developed to be linked to a genetic map, initially focusing on getting a physical map of chromosome 3B, which carries quantitative trait loci (QTLs) for a diseaseresistance locus (Sr2, on 3BS) and for LMA. Quantitative trait loci

The search for LMA genes has made use of the recent method of finding QTLs, defined as genetic loci that, in combination with several additional loci, contribute to the control of specific phenotypes. This approach is most appropriate for quantitative traits, such as grain yield or baking quality, and less relevant for LMA, since the approach has indicated that only two genes are involved for LMA. Nevertheless, QTL analysis indicated that the two independent genes involved are on chromosome arms 3BL and 7BL for the progeny of a specific cross between the varieties Halberd and Cranbrook (Mrva and Mares 2001). The next step is to show that these conclusions are also relevant for a wider range of genotypes. Thus, an even wider spread of genotypes must still be screened. So far it appears that the presence of either one of the genes is sufficient to produce the LMA defect; the presence of both is more serious. These findings provide a basis for MAS— screening for these genes at the DNA level. This has proved to be possible using microsatellite markers that flank the 7B locus or are linked to the 3B locus. QTLs are being identified for many traits, making the task of gene-based selection feasible for many aspects of phenotype. These QTLs are mapped to regions on the relevant chromosome, so that nearby known markers can be used to select for the respective trait. Many techniques have been developed to detect DNAsequence variation between individuals; they include Southern hybridization-based methods, polymerase chain reaction (PCR)based methods, and techniques based around single-nucleotide polymorphisms (Langridge et al 2001). Traditional gel-based methods, which are labor-intensive, are being replaced by chipbased methodologies capable of simultaneous analysis of large numbers of markers, rendering DNA analysis cost-effective and feasible for use with the large numbers of samples generated in breeding programs. An exciting prospect is to integrate specific QTLs with the actual parts of the genome and thus identify the actual gene sequences for the active genes involved. This prospect should become a reality as a result of the work of the International Wheat Genome Sequencing Consortium. This lengthy project aims to produce a freely available sequence of the wheat genome, as a basis for twenty-first-century research—an “encyclopedia of genes.” Work is focusing on the variety Chinese Spring, a genotype that has long been the subject of many aneuploid and genetic studies. A physical map is being developed to be linked to a genetic map, initially focusing on getting a physical map of chromosome 3B, which carries one of the QTLs for LMA. With

all results in the public domain, there will be no restraints on intellectual property. Details are available at the website www. wheatgenome.org. Protein markers

Individual proteins may also act as markers to select for specific traits, provided that reliable associations can be identified between the presence of the protein and the trait. The high molecular weight subunits (polypeptides) of glutenin are an example of successful protein markers. These proteins of the endosperm contribute to dough strength in the formation of the gluten complex (Shewry et al 2006). The pair of subunits designated 5 and 10 is consistently associated with the wheat that forms stronger dough compared to wheats with the pair of subunits numbered 2 and 12 (Payne 1987), depending partly on what other glutenin subunits are present (see also Chapter 8). For many years, breeders have taken advantage of these protein-based markers to select for dough properties. Now, sophisticated computer programs are available to select parents and progeny for dough-quality attributes, based on the presence and amounts of the glutenin subunits (Cornish et al 2006). Gelbased methods of analysis have traditionally been the methods for determining protein composition, but (as for DNA analysis) chip-based methodologies are being developed to identify and quantify glutenin subunits, using capillary electrophoresis (Uthayakumaran et al 2006). The suitability of wheat varieties for white-salted noodles (udon) is related to the granule-bound starch synthase (GBSS) enzymes, coded by Wx genes. These involve three complementary loci on chromosome arms 7AS (Wx-A1), 4AL (Wx-B1), and 7DS (Wx-D1). Some varieties are “null” for the second of these, giving their starch properties advantages for noodle production. This characteristic is termed the “Wx-B1b” allele; the presence allele being “Wx-B1a” (Zhao et al 1998). In place of gel electrophoresis, antibody-based methods can detect the presence of such marker proteins more efficiently (Gale et al 2001). Protein synthesis is a few steps away from the gene level, so growth environment can have an effect on the presence and amount of a potential marker protein. This G × E interaction may cause complications of interpretation that are not an issue for gene-based selection. On the other hand, knowledge of the effect of environmental factors may also provide the breeder with some advantages, because ultimate selections must be based on phenotype, and phenotype must be understood for the target regions where the new variety is planned for commercial production.

Wheat Production Wheat production worldwide has doubled in recent decades, partly (20%) due to increased area of production but mainly (80%) due to improved varieties and agronomic practices (Paulsen and Shroyer 2004). Grain yield obviously depends on the number and mass of grains per unit of growth area. The components of this measure include plant density (plants per square meter), tillers (thus heads per plant), spikelets and grains per head, and the mass distribution of the grains. These factors are partly determined by genotype (variety, designed by the breeder) but largely

Wheat: A Unique Grain  by agronomic practice, which aims to optimize these components. Maximizing all these components is not an option, as they are interactive; for example, the number of tillers per plant is reduced if plant density is too high, and excessive numbers of tillers may cause fewer grains per head. Managing the growth environment

Some environmental factors cannot be manipulated by management practice; climatic influences are in this category, although climate history should limit the expected range of such factors, temperature and rainfall being the main ones. Wheat is best suited to modest growth temperatures of about 15°C, but it is grown in many regions where the temperature range goes higher than 30°C, and sometimes higher than 40°C. These heat stresses are likely to reduce grain yield and modify quality, especially dough strength. In other growth regions, cold hazards can reduce yield due to winterkill, freeze injury, and frost at flowering. The adaptation of genotypes to these extremes is the main management technique for these abiotic stresses. Other climatic extremes are drought (reducing yield drastically), hail and/or wind (causing damaged plants to “lodge”—fall over, making harvest difficult), and rain at harvest (causing preharvest sprouting). Plant nutrition is more open to management, mainly by the application of fertilizer at various stages of growth. Obviously, soil type is a fixed factor that has limited possibilities for manipulation. Biotic factors include a wide range of pathogens, especially fungal organisms and root diseases, as well as competition from weeds. Breeding for pathogen resistance is a valuable means of combating some of these factors, but the application of appropriate fungicide and herbicide sprays is common in regions where intensive agriculture is warranted. This type of approach is common in many parts of Europe where modest temperatures and adequate rainfall warrant these intensive measures. For example, wheat grown in England enjoys a daily temperature of about 17°C during grain filling and about 50 mm of rain per month. Grain yields are increased commonly to 8 t/ha by the application of nitrogen-based fertilizer (routinely at 200 kg of N per hectare) and two or three fungicide sprays, especially at the times of emergence of the flag leaf and the ear (Ruske et al 2004). Insecticide spraying may also be used. Genetic resistance to pathogens may also be relied upon, but varieties may be selected for their overall yield potential and ability to provide high test weight. The fungicide spray may also extend the life of the flag leaves, thereby also increasing the period of grain filling and grain yield. However, these high yields may come at a price with respect to grain quality. For example, high applications of nitrogen fertilizer lead to the risk of sulfur deficiency. As a result, some European millers require analyses of sulfur as well as nitrogen on the grain that is offered for sale (the latter to determine protein content). Low-sulfur grain is associated with reduced protein quality due to the low content of sulfur-rich gluten proteins, with consequent poor dough extensibility (Ruske et al 2004). Other quality problems include black-point damage to kernels (likely to reduce flour yield on milling) and the presence of LMA, which is inherent in some high-yielding varieties (Wrigley 2006b). Both of these last defects involve G × E interactions, that

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is, the variety involved has genes that become active under specific environmental conditions. In another environment, the wheat industry of Argentina relies heavily on double cropping of wheat in combination with soybeans. The Pampas region is classed as prairie, with good depth of soil. Soy is the main crop, at about 70% of grain production, followed by wheat (20%) and maize (10%). Much of the maize is genetically modified (GM) with the “Bt” gene. Wheat is planted in May or June, with heading in September and harvest in November and December (Table 1.3). Soy is then planted for harvest in late autumn, and the crop rotation continues with the planting of wheat or maize. The wheat crops are particularly free of weeds, due to the use of herbicide for the alternate crop of GM soy in the double-cropping system. Wheat production in the Argentinean Pampas is reasonably intensive; conditions are generally warm, with daily maxima of 30–33°C during grain filling. Fertilizer use involves nitrogen (40–60 kg/ha) and phosphorus (10–20 kg/ha). The alternate soy crops may also contribute some fixed nitrogen, but apparently not sulfur deficiency. Low-input systems

Other regions of the world have the combination of low rainfall and high temperatures. As a result, grain yields are low (Table 1.1), and farmers must rely on broad-acre production with inputs that are much less than for the examples described above. In some parts of these regions, where irrigation water is available, more intensive inputs can be provided to increase yields. In these situations, wheat may be “rotated” in the same year with other crops such as cotton, sorghum, or rice (Paulsen and Shroyer 2004). In Western Australia, wheat may be grown in “rotation” (from one year to the next) with other grain crops, especially legumes such as lupins, which provide fixation of nitrogen from the atmosphere. It is also common in regions of low rainfall and poor soil fertility for fields to be left fallow (unused) for a year or for rotations to involve oilseeds, such as canola, which may provide valuable control of soilborne diseases of wheat. Much of the wheat produced in the drier regions of the North American Great Plains is grown in wheat-fallow rotations. One of the significant changes in wheat production in recent decades is the change from deep plowing of the soil before sowing to direct drilling of the seed into narrow furrows that are cut simultaneously with sowing. This approach of “zero tillage” is claimed to increase grain production, while also preventing soil degradation and even repairing degraded soils (Pieretti 2007). Organic farming

An alternative to intensive farming practices is organic farming, which involves the avoidance of artificial fertilizers and synthetic fungicides and herbicides. To increase soil fertility, organic wheat producers use crop rotations, composted green manure crops, legume-based pastures, and mineral-based fertilizers, such as rock phosphate. Weed control may involve controlled grazing, cultivation, mulching, and crop rotations. Organic farming is likely to achieve lower yields than intensive practices, but organic grain commands higher prices (Gélinas and David 2004).

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Precision agriculture

A recent approach to increasing the efficiency of crop production is known as “precision agriculture.” This term has become associated with a scientific approach to grain production that involves monitoring inputs (seed, fertilizer, etc.) and outputs (grain yields) on a small scale (e.g., per square meter, rather than per hectare) (Long et al 2000). It is usual for grain yield to be mapped across the field using a global-positioning system (GPS) mounted on the combine during harvesting, to record the yield of grain as it is discharged into the hopper (Pringle et al 2003, Ehlert et al 2004). The resulting map of yield indicates those parts of the field that are producing well or poorly, so that diagnostic and remedial actions may be taken on a localized level. These actions may involve providing better drainage to waterlogged areas, soil sampling to determine fertility deficiencies or soil acidity, and weed control. In the next season, the producer has the opportunity to vary seed rate and fertilizer treatment, again using GPS to identify specific places of need. Precision agriculture therefore offers the potential advantages of reducing unnecessary inputs, permitting the application of fertilizers, herbicides, and pesticides at the points where they are needed, rather than indiscriminately. Thus, the advantages of precision agriculture go beyond economic considerations to include pollution reduction and sustainability.

Wheat Harvesting and Segregation Reaping, threshing, and winnowing (cutting off the stalks, removing the grain from the husks, and separating out the clean grain, respectively) are the three stages of grain harvesting. Early farmers used a scythe or sickle for the first step. At this stage, it may have been necessary to stand the stalks in bundles, called “shocks,” “stooks,” or “ricks.” Drying in this way was necessary to ensure ease of threshing and winnowing in the wind. Mechanization of the harvesting process was an essential part of the advance of wheat farming. The McCormick reaper, patented in 1834, revolutionized American wheat production. Later developments led to the modern combine harvester, which combines the three steps of harvesting. In regions of subsistence agriculture, the harvested grain is kept on-farm. In such cases, grain quality is targeted to suit the needs of the extended family, which uses the grain throughout the year until the next harvest. On-farm storage may also occur for the produce of large farms in developed countries, where it offers the advantage of relieving the rush of harvesttime, also giving the farmer extended time to negotiate the sale of the grain. In contrast, the produce of this latter example may cross the seas to feed people in distant lands. For this reason, the targeted quality attributes must reflect the needs of the export market. Typically, in developed countries, grain from many farms is delivered, immediately after harvest, to a flour mill or to a regional storage and transport center—a grain “elevator” or “silo.” This delivery center offers the initial opportunity for segregation of grain according to the foreseen quality demands of the marketplace. In many regions, this market relates primarily to the domestic milling industry. Knowledge of the variety provides information about several genotypically determined attributes,

especially grain hardness, starch properties, milling quality, dough-strength potential, and possible tolerance to some defects. For this reason, premium grades of wheat are often restricted to selected varieties, although this is not generally so for U.S. grades. Such classes and grades also require that standards be met for test weight (bulk density), moisture content, the absence of contaminants, and protein content. As many as possible of these grain characteristics should be analyzed when the grain is delivered, so that the class or grade is determined before the grain load is dumped into the appropriate storage cell. In general, the time frame for making this determination is a matter of minutes, so rapid test methods are needed. Grain samples are taken from several parts of the truckload; they are combined, sieved, and tested for bulk density, protein content, and several other aspects of quality, as described in Chapter 4. The principles of precision agriculture may be extended to include harvesting. This approach involves the need for detailed knowledge about the quality of the grain before harvest so that selective harvesting can be performed to maximize the value of the crop. This principle is particularly appropriate following rain at harvest, which may have caused sprout damage to some parts of the crop. Selective harvesting of the sound grain without the damaged grain improves returns, compared with the alternative of harvesting the crop as a whole and thus having a small proportion of damaged grain cause the whole crop to be downgraded. Another example of “precision harvesting” is to use prior knowledge of the protein-content distribution of grain across a field, thus allowing selective harvesting to achieve a specific protein-content premium. This approach obviously involves the testing of grain samples taken from various points across the field or the use of a protein monitor on the combine harvester to permit real-time monitoring of protein content.

Wheat Storage and Transport In many countries, the wheat-production regions are distant from the wheat-processing and wheat-export centers. Longdistance transport is thus an essential part of the grain chain in these countries. Rail transport is the common means of moving wheat across these distances. Figure 1.7 shows an example of a country “silo” in Australia. The rail line is seen at the top of the photo, running close to three types of grain-storage structures— horizontal, vertical, and circular. Barge transport is also used on the waterways of North America. Wheat-export centers are obviously located at harbors, as sea transport is the most costeffective means of transporting grain around the world. Even for barge transport on waterways, such as the Mississippi and Columbia rivers and the Great Lakes in North America, grain must be transferred to ocean-going ships, often via storage in “terminal” storage elevators. Arrival at the seaport of destination may not be the end of the journey for grain shipments, but in many cases, flour mills are located adjacent to port facilities to avoid the need for further transport before milling. Storage is thus an essential part of transport, both during grain movement and at intermediate stages. These activities take advantage of an important characteristic of grains, namely, that they remain virtually unchanged for long periods provided they

Wheat: A Unique Grain  are kept dry (e.g., under 14% moisture) and free of destructive pests, especially rodents and insects (Heaps 2006). A critical requirement for bulk-storage facilities is that they should be sealable, to permit fumigation for insect disinfestations as well as to prevent the entry of vertebrate and invertebrate pests. Although storage structures are usually made of masonry and steel, it has also proved possible to use “bunker” storage, formed by placing stored grain straight onto the ground, with layers of plastic sheeting underneath and over it. The provision of many separate storage cells is an advantage at a storage site to permit the segregation of wheats of different quality classes, as well as to avoid any admixture with grains of other species. Silotype storage facilities may be equipped to aerate the stored grain. By various means, air is drawn upward through the grain to help dry or to cool it and thus reduce the risk of insect infestation (Heaps 2006) or microbial infection (e.g., Reed 2006). Maintenance of grain quality during transport and storage is a high priority, because spoilage in its many forms can reduce the value of the grain (Reed 2006). Yet, long-term storage is a necessary part of the wheat industry because grain must be stored from one harvest to the next for use throughout the year. Furthermore, carryover from one year well into the next is common. Accordingly, the use of pesticides with residual effects has been an attractive approach to grain protection. However, the use of treatments that leave no residue is greatly preferred. Controlled atmospheres such as 40% carbon dioxide or 99% nitrogen have proved effective for insect control, but this approach usually requires that these elevated concentrations of carbon dioxide or nitrogen be maintained for a few weeks. Fumigants such as phosphine or methyl bromide are much faster acting, but methyl bromide use is now minimal because it is an ozone-depleting substance. Some new fumigants (carbonyl sulfide, ethyl formate, and sulfuryl fluoride) offer promise, but further testing is needed. Other methods of protection include the use of inert and abrasive dusts and mechanical impact. Merely the act of pneumatic conveyance of grain can lead to the elimination of a significant proportion of adult insects, this stage being particularly

Fig. 1.7. Aerial view of GrainCorp’s country grain terminal at Bellata in New South Wales, Australia. (Courtesy GrainCorp, Australia)

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sensitive to mechanical impact. Cooling of grain to below 15°C is also effective in reducing the growth of insects, but there is the risk that the cooler temperature, especially if there is excessive moisture, may encourage other pests such as mites. Alternative methods of disinfestation include irradiation with gamma-rays or X-rays, and heating with microwaves, radio frequency, or light radiation.

Wheat Marketing In today’s competitive world, wheat growers must be aware of market movements, so as to be continually conversant with the most profitable strategies at all stages of their parts of the production process. Various critical decisions relate to the marketing of the crop, and many of these must be made well before sowing. The choice of variety to sow relates to the local environment and anticipated climate. In addition, choice of variety probably affects possible premium payments. The selection of pre-sowing tillage and strategies for fertilizer use will determine the protein content of the harvested grain and thus its value. Harvest timing is likely to determine grain soundness and its moisture content. The timing of the actual sale of grain is also likely to be critical, and the possibility of on-farm storage offers flexibility in respect to this aspect of marketing for the local grower. Wheat growers have long faced a cost-price squeeze, forcing them to increase productivity and efficiency to be able to remain financially viable. World wheat prices have shown a progressive decrease over many decades. In about 1916, wheat sold in North America for about $2 per bushel (Buller 1919). This is equivalent to $73/t (1916 dollar value) or $1,344/t (2006 dollar value), making allowance for inflation. Compared to the present value of about $200/t (2006 dollar value), the price of wheat has dropped by a factor of between 6 and 7. A 10-fold decrease in wheat prices can be calculated, if increases in wages since that time are also taken into account. More recent trends in world prices for wheat, rice, and maize (Fig. 1.8) show a degree of volatility as well as downward movements. A projected world deficiency in wheat supply is expected to contribute to a gradual reversal of the historic trend of decreasing wheat prices. A part of this expected deficiency is the improved economies of developing nations and the consequent demand for more wheat-based foods in their diets. International wheat pricing and payments are affected by various geopolitical influences. Governments in some countries provide subsidies to farmers, even to those producing wheat where it may not otherwise be profitable. The wheat-marketing and buying systems of the trading nations include the involvement of government agencies, reliance on private milling companies (some vertically integrated), and calls for tenders to supply specific quality classes, to the use of the “spot market” (Worden 2004), Wheat Processing The pricing of wheat reflects market demand for quality aspects that determine suitability for processing. The first stage of most wheat processing is milling. This is generally a dry process in which the bran and germ sections of the grain are separated

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from the white floury endosperm, which is then reduced to a fine powder (flour), as described in Chapter 5. The production of semolina, generally milled from durum wheat for pasta manufacture, does not require this second stage of reduction of particle size. The range of foods made from wheat is enormous, ranging from bread in its many forms (the most obvious wheat-based food), through pastries, cakes, and biscuits/cookies to noodles, Chinese steamed breads, and the many types of flat breads (Quail 1996). Beyond this range are products such as the many confectionaries and snack bars that contain a high proportion of wheat flour, although its presence may not be obvious. Whole wheat is an important contributor to breakfast cereals in their many forms (Fast and Caldwell 2000). Couscous and burghul (bulgur) are further forms of wheat-based foods; they do not involve complete milling, but pearled or kibbled wheat is used. In the case of burghul, cracked wheat is parboiled or steamed and is used in dishes such as tabbouleh, kofta, and kibbeh (Bayram 2000).

WHEAT AND HUMAN HEALTH In some developing countries, cereal grains provide more than 70% of the energy in the diet and a large part of the protein intake. As a country becomes more affluent, the consumption of cereals tends to decline, although they still make important contributions to nutrition. The nutritional value of cereal grains can be illustrated by using a least-cost rations computer program, such as is normally used to formulate animal feed. For this novel purpose, inputs to the program were human recommended dietary intakes for all nutrients plus the nutrient contents and retail prices of supermarket foods. The least-cost combination of foods amounted to just under 1 kg in weight and only a few dollars in cost per day. It indicated that the cereal grains provide cost-effective delivery of good nutrition. A major part of the hypothetical diet was 390 g of breakfast cereals, based on whole wheat and oats. For about half of the daily cost, the cereals provided 77% of the protein requirement, half of the required energy, 35% of the calcium, and most of the

iron, riboflavin, and niacin. On the other hand, this exercise showed that cereals are lacking as a source of vitamins A and C; these were most cheaply provided by a small amount of liver and 190 g of potatoes, respectively.

Advantages and Limitations of Wheat in the Diet As a prominent member of the cereal family, wheat is recognized as an important source of essential nutrients, providing energy, fiber, carbohydrate, protein, B vitamins, iron, calcium, phosphorus, zinc, potassium, and magnesium. Some of these nutrients are concentrated in specific structural parts of the grain, as described in Chapter 3. The fractionation of the crushed grain during milling thus has critical implications for the distribution of many nutrients. The wholemeal product thus provides better nutrition than does white flour (Marquart et al 2002). Cereal grains are rich in carbohydrate, the main component of the endosperm being starch, which is embedded in a protein matrix. In addition, nonstarch polysaccharides contribute as an important source of dietary fiber; see Chapter 9. The fat content of wheat flour is low compared to that of most foods, and of course it does not contain cholesterol (Chapter 10). Lysine is the first limiting amino acid of wheat protein (Chap­ ter 8). Most civilizations, however, have developed traditional food patterns based on a staple cereal supplemented with small quantities of legumes, nuts, fish, or milk. These diets are nutritionally adequate for protein when energy requirements are met. Publicity for the nutritional benefits of low-fat, high-fiber diets favors the grains, including wheat-based foods, so that the cereal grains are now regarded as essential components of a healthy diet. Nutritional guidelines have been developed by the governments of most countries, as a means of recommending food choices that promote good health. Many of these guidelines are diagrammatic, reflecting the culture of the country (Painter et al 2002). The pyramid diagram is familiar to many Westerners. Displays have also taken the form of circle, wheel, plate, and pie diagrams. Canada’s diagram is a half rainbow, whereas an Asian diagram is a pagoda. A common factor in these guidelines is the recommendation that grain-based foods should form the greatest part of the diet, preferably in the form of whole-grain foods. Some of these recommendations are quantitative, recommending the numbers of servings of grain-based foods per day and the actual composition of those servings (Painter et al 2002). Options obviously vary according to national and cultural preferences, but in most cases, foods made from wheat flour are prominent.

Fig. 1.8. Trends in world prices for wheat, rice, and maize. (Courtesy CIMMYT, Mexico)

Allergy and Intolerance to Wheat Despite their generally valuable contribution to our diet, wheat-based foods present health problems for a minority of people, due in particular to celiac disease or, alternatively, to various other forms of

Wheat: A Unique Grain  intolerance (Anderson and Wieser 2006). Celiac disease (CD, also known as “celiac sprue” and “gluten-sensitive enteropathy”) is an inflammatory disease of the duodenum and jejunum with a range of symptoms, including malabsorption of nutrients generally, diarrhea, and loss of appetite. In children, there may be abdominal distension, vomiting, and muscle wasting, accompanied by impaired ability to thrive. Because of the diversity in symptoms, diagnosis has been difficult, leading in the past to an underestimation of prevalence. However, the current availability of serological screening tests for CD permits better diagnosis, indicating that CD may affect about 1% of Caucasians and South Asians (Anderson and Wieser 2006). Strict diagnosis of CD involves gastroscopy with biopsy of the small intestine while the individual is consuming gluten, with flattened mucosa of the gut lining being a positive sign of CD. Currently, the essential and only therapy is maintaining a lifelong gluten-free diet. In this case, “gluten” means not only the gluten of wheat (Triticum species) but also the storage proteins of rye, triticale, barley, and possibly oats. Adherence to a glutenfree diet is difficult, due to the common occurrence of wheat and related cereals in processed foods. A list of food ingredients according to the presence of gluten has been published to assist people with CD in devising a suitable diet (Wrigley 2004b). Occupational exposure to wheat flour by inhalation may lead to “bakers’ asthma,” a type-1 immediate hypersensitivity, mediated by the E class of immunoglobulins. Bakers’ asthma differs from CD, which is T-cell mediated, a type-4 delayed hypersensitivity. Dietary allergy to wheat gluten is less well defined and may be exacerbated by various forms of inhalant allergy to cereal pollens during the flowering season (Armentia et al 2002, Rasanen et al 1994). Allergy and intolerance to wheat grain proteins are discussed in more detail in Chapter 8.

CONCLUSION In the second half of the last century, global population grew by 90%, whereas our food supply grew by 115%, representing a food-supply increase of 25% per person (Dixon 2007). Wheat is a part of the latter triumph, with wheat now accounting for more than a quarter of the calorie intake in developing countries. Wheat production has risen steadily during this period, due to improvements in varieties and agronomic practice, as well as to changes in marketing and government involvement. International wheat breeding for increased productivity has been claimed to be the major reason for an additional 14–41 million tonnes of wheat per year, representing a cost-benefit ratio of 1:12 (Dixon 2007). Ongoing improvements are expected from further research, but significant problems loom. New pathogens are causing new concern, especially the Ug 99 race of stem rust. There are further concerns about the availability of and price rises for nitrogen fertilizers, especially urea. Rising oil prices will also have repercussions for the cost-price squeeze. Other inputs are also getting scarce, including the essential commodity of water. Subsidies to encourage wheat growing are paid in some countries, but these are expected to decrease, making wheat less attractive for the farmers involved. Efforts to increase returns to growers may involve further emphasis on grain-quality attributes, targeting niche markets

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for wheats with qualities that suit specific products (e.g., certain noodle types, snack foods, and certain types of biscuits/cookies and cakes, possibly even “ingredient wheats” with extreme qualities that may be blended with “normal” wheats to suit processing needs). Price premiums are expected for wheats with specific nutritional advantages, such as genotypes that can provide higher fiber levels and low-glycemic-index foods. Global warming (Wrigley 2006a) will, in turn, bring its own set of problems and opportunities for wheat production. Global demand for wheat is projected to increase at 1.2% per year for food and 0.8% per year for feed. By 2030, this will require the global average wheat yield to increase to 3.5 t/ha (Dixon 2007). Much of the increase is expected to come from developing countries, especially as a result of increased area and yields for irrigated wheat (Marathée and Gomez-MacPherson 2001). Thus, wheat may be expected to make its contribution to solving the world’s continuing crisis in hunger and poverty, as well as possibly contributing to the contrasting problems of obesity. Dyson (1999) concluded a review of world cereal-grain production and the prospects for the year 2025 with the optimistic view: The world food situation has been improving.…This trend probably will continue during the next few decades. World food output will continue to rise, although there will be a growing degree of mismatch between the expansion of food demand and the capacity to supply that demand. Accordingly the balance will be met by a considerable expansion of the world food trade. As a result, most people probably will be better fed in 2025 than is the case today.

Norman Borlaug (1970 Nobel Peace Laureate) has given the Human Challenge for the Twenty First Century: “To be able to keep increasing the agricultural output, but at the same time, conserving the natural resources.” Hopefully, the current century will see fewer and fewer people experiencing “kein Brot” (Fig. 1.1). REFERENCES

Anderson, R. P., and Wieser, H. 2006. Medical applications of glutencomposition knowledge. Pages 387-409 in: Gliadin and Glutenin: The Unique Balance of Wheat Quality. C. W. Wrigley, F. Bekes, and W. Bushuk, Eds. AACC International, St Paul, MN. Armentia, A., Rodriguez, R., and Callejo, A. 2002. Allergy after ingestion or inhalation of cereals involves similar allergens in different ages. Clin. Exp. Allergy 32:1216-1222. Bayram, M. 2000. Bulgur around the world. Cereal Foods World 45:80-82. Blakeney, A. B., and Wrigley, C. W. 1993. One hundred years of cereal chemistry: The centenary of a chemical discipline. Chem. Aust. 60:459-460. Boals, G. P. 1948. Wheat—A world symbol. Foreign Agric. 12:27-31. Bonjean, A. P., and Angus, W. J., Eds. 2001. The World Wheat Book. A History of Wheat Breeding. Lavoisier Publishing, Paris. Buller, A. H. R. 1919. Essays on Wheat. The Macmillan Company, New York. Bushuk, W. 1992. Significant research achievements in cereal chemistry and technology in the past decade. Pages 1-4 in: Cereal Chemistry and Technology. A Long Past and a Bright Future. P. Feillet, Ed. Institut de Recherches Technologiques Agroalimentaires des Cereales (IRTAC), Montpellier, France.

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Cornish, G. B., Bekes, F., Eagles, H. A., and Payne, P. I. 2006. Prediction of dough properties for bread wheats. Pages 243-280 in: Gliadin and Glutenin: The Unique Balance of Wheat Quality. C.  W. Wrigley, F. Bekes, and W. Bushuk, Eds. AACC International, St. Paul, MN. Cracknell, R. L., and Williams, R. M. 2004. Wheat: Grading and segregation. Pages 355-363 in: Encyclopedia of Grain Science, Vol. 3. C. Wrigley, C. Walker, and H. Corke, Eds. Elsevier Ltd., Oxford, UK. Diamond, J. 1997. Guns, Germs, and Steel. The Fates of Human Societies. W. W. Norton and Co., New York. Dixon, J. 2007. The economics of wheat: Value chains from research to field to fork. Pages 9-22 in: Proc. 7th Int. Wheat Conf. H. T. Buck, J. E. Nisi, and N. Salomón, Eds. Springer, The Netherlands. Dyson, T. 1999. World food trends and prospects to 2025. Proc. Natl. Acad. Sci. USA 96:5929-5936. Ehlert, D., Schmerler, J., and Voelker, U. 2004. Variable rate nitrogen fertilisation of winter wheat based on a crop density sensor. Precision Agric. 5:263-273. Faridi, H., and Faubion, J. M., Eds. 1995. Wheat End Uses Around the World. Am. Assoc. Cereal Chem., St. Paul, MN. Fast, R. B., and Caldwell, E. F., Eds. 2000. Breakfast Cereals and How They Are Made, 2nd ed. Am. Assoc. Cereal Chem., St. Paul, MN. Feldman, M. 2001. Origin of cultivated wheat. Pages 3-53 in: The World Wheat Book. A History of Wheat Breeding. A. P. Bonjean and W. J. Angus, Eds. Lavoisier Publishing, Paris. Feldman, M., and Sears, E. R. 1981. The wild gene resources of wheat. Sci. Am. 244:98-109. Fowler, W. W. 1908. The Roman Festivals of the Period of the Republic. MacMillan and Co. Ltd., London. Gale, K. R., Panozzo, J. F., Eagles, H. A., Blundell, M., Olsen, H., and Appels, R. 2001. Application of a high-throughput antibody-based assay for identification of the granule-bound starch synthase WxB1b allele in Australian wheat lines. Aust. J. Agric. Res. 52:14171423. Gélinas, P., and David, C. 2004. Organic growing of grains. Pages 386-396 in: Encyclopedia of Grain Science, Vol. 2. C. Wrigley, C. Walker, and H. Corke, Eds. Elsevier Ltd., Oxford, UK. Guthrie, F. B. 1898. Wheat testing. Description of mill. Agric. Gaz. N.S.W. 9:713-716. Heaps, J. W., Ed. 2006. Insect Management for Food Storage and Processing, 2nd ed. AACC International, St. Paul, MN. Heywood, V. H. 1993. Flowering Plants of the World. Oxford University Press, Oxford, UK. Jacob, A. E. 1944. Six Thousand Years of Bread. Doubleday, Doran, Garden City, NY. Jasny, N. 1944. The Wheats of Classical Antiquity. Univ. Stud. Hist. Pol. Sci., Ser. 62, No. 3. Johns Hopkins Press, Baltimore, MD. Langridge, P., Lagudah, E. S., Holton, T. A., Appels, R., Sharp, P. J., and Chalmers, K. J. 2001. Trends in genetic and genome analyses in wheat: A review. Aust. J. Agric. Res. 52:1043-1077. Lockwood, J. 1960. Flour Milling. Henry Simon Ltd., Stockport, Cheshire, England. Long, D. S., Engel, R. E., and Carlson, G. R. 2000. Method for precision nitrogen management in spring wheat: II. Implementation. Precision Agric. 2:25-38. Mangelsdorf, P. C. 1953. Wheat. Sci. Am. 189:50-59. Marathée, J. P., and Gomez-MacPherson, H. 2001. Future world supply and demand. Pages 1107-1116 in: The World Wheat Book. A History of Wheat Breeding. A. P. Bonjean and W. J. Angus, Eds. Lavoisier Publishing, Paris. Marquart, L., Slavin, J. L., and Fulcher, R. G. 2002. Whole-Grain Foods in Health and Disease. Am. Assoc. Cereal Chem., St. Paul, MN. Matz, S. A. 1960. Bakery Technology and Engineering. AVI Publ. Co., Inc., Westport, CT.

McIntosh, R. A., Hart, G. E., Devos, K. M., Gale, M. D., and Rogers, W. J. 1998. Catalogue of gene symbols for wheat. In: Proc. Int. Wheat Genetics Symp., 9th. Univ. Saskatchewan Extension Press, Saskatoon, SK, Canada. Morris, R., and Sears, E. R. 1967. The cytogenetics of wheat and its relatives. Pages 19-87 in: Wheat and Wheat Improvement. K. S. Quisenberry and L. P. Reitz, Eds. Am. Soc. Agron., Madison, WI. Morrison, J. W. 1960. Marquis wheat—A triumph of scientific endeavour. Agric. Hist. 34:182-188. Morrison, L. A., and Wrigley, C. W. 2004. Taxonomic classification of grain species. Pages 271-280 in: Encyclopedia of Grain Science, Vol. 3. C. Wrigley, C. Walker, and H. Corke, Eds. Elsevier Ltd., Oxford, UK. Moss, H. J. 1973. Quality standards for wheat varieties. J. Aust. Inst. Agric. Sci. 39:109-115. Mrva, K., and Mares, D. J. 2001. Quantitative trait loci analysis of late maturity α-amylase in wheat using the doubled haploid population Cranbrook x Halberd. Aust. J. Agric. Res. 52:1267-1273. O’Brien, L., and Blakeney, A. B. 1985. A census of methodology used in wheat variety development in Australia. Cereal Chem. Div., R. Aust. Chem. Instit., Melbourne. Painter, J., Rah, J. H., and Lee, Y. K. 2002. Comparison of international food guide pictorial representations. J. Am. Diet. Assoc. 102:483-489. Paulsen, G. M., and Shroyer, J. P. 2004. Wheat: Agronomy. Pages 337-347 in: Encyclopedia of Grain Science, Vol. 3. C. Wrigley, C. Walker, and H. Corke, Eds. Elsevier Ltd., Oxford, UK. Payne, P. I. 1987. Genetics of wheat storage proteins and the effect of allelic variation on bread-making quality. Annu. Rev. Plant Physiol. 38:141-153. Pena, R. J. 2007. Current and future trends of wheat quality needs. Pages 411-424 in: Proc. 7th Int. Wheat Conf. H. T. Buck, J. E. Nisi, and N. Salomón, Eds. Springer, The Netherlands. Pieretti, R. A. 2007. The global need for sustainable agriculture. Pages 1-7 in: Proc. 7th Int. Wheat Conf. H. T. Buck, J. E. Nisi, and N. Salomón, Eds. Springer, The Netherlands. Posner, E. S., and Hibbs, A. N. 2005. Wheat Flour Milling, 2nd ed. Am. Assoc. Cereal Chem., St. Paul, MN. Pringle, M. J., McBratney, A. B., Whelan, B. M., and Taylor, J. A. 2003. A preliminary approach to assessing the opportunity for site­specific crop management in a field, using a yield monitor. Agric. Syst. 76:273-292. Pyler, E. J. 1958. Our Daily Bread. Siebel Publ. Co., Chicago. Quail, K. J. 1996. Arabic Bread Production. Am. Assoc. Cereal Chem., St. Paul, MN. Rasanen, L., Lehto, M., Turjanmaa, K., Savolainen, J., and Reunala, T. 1994. Allergy to ingested cereals in atopic children. Allergy 49:871876. Reed, C. R. 2006. Managing Stored Grain to Preserve Quality and Value. AACC International, St. Paul, MN. Ruske, R. E., Gooding, M. J., and Dobraszczyk, B. J. 2004. Effects of triazole and strobilurin fungicide programmes, with and without late-season nitrogen fertiliser, on the baking quality of Malacca winter wheat. J. Cereal Sci. 40:1-8. Shewry, P. R., Halford, N. G., and Lafiandra, D. 2003. Genetics of wheat gluten proteins. Adv. Genet. 49:111-184. Shewry, P. R., Halford, N. G., and Lafiandra, L. 2006. The high­molecular-weight subunits of glutenin. Pages 143-170 in: Gliadin and Glutenin: The Unique Balance of Wheat Quality. C. W. Wrigley, F. Bekes, and W. Bushuk, Eds. AACC International, St. Paul, MN. Starck, J., and Teaque, W. D. 1952. A History of Milling: Flour for Man’s Bread. Univ. Minn. Press, Minneapolis, MN. Takahashi, R. 1955. The origin and evolution of cultivated barley. Adv. Genet. 7:227-266.

Wheat: A Unique Grain  Trethowan, R., Hodson, D. P., Braun, H-J., Pfeiffer, W., and van Ginkel, M. 2005. Wheat breeding environments. Pages 4-11 in: Impacts of International Wheat Breeding Research in the Developing World, 1988–2002. M. Lantican, J. Dubin, and M. Morris, Eds. Centro Internacional de Mejoramiento de Maiz y Trigo (CIMMYT), Mexico City. Uthayakumaran, S., Listiohadi, Y., Baratta, M., Batey, I. L., and Wrigley, C. W. 2006. Rapid identification and quantitation of highmolecular-weight glutenin subunits. J. Cereal Sci. 44:34-39. Weaver, J. C. 1950. American Barley Production. Burgess Publ. Co., Minneapolis, MN. Worden, G. C. 2004. Wheat: Marketing. Pages 375-383 in: Encyclopedia of Grain Science, Vol. 3. C. Wrigley, C. Walker, and H. Corke, Eds. Elsevier Ltd., Oxford, UK.

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Wrigley, C. W. 2004a. Units of grain science. Pages 475-482 in: Encyclopedia of Grain Science, Vol. 3. C. Wrigley, C. Walker, and H.Corke, Eds. Elsevier Ltd., Oxford, UK. Wrigley, C. W. 2004b. Appendix: Foods for celiac diets. Pages 449-457 in: Encyclopedia of Grain Science, Vol. 3. C. Wrigley, C. Walker, and H. Corke, Eds. Elsevier Ltd., Oxford, UK. Wrigley, C. W. 2006a. Global warming and wheat quality. Cereal Foods World 51:34-36. Wrigley, C. W. 2006b. Late-maturity α-amylase: Apparent sprout damage without sprouting. Cereal Foods World 51:124-125. Zhao, X. C., Batey, I. L., Sharp, P. J., Crosbie, G., Barclay, I., Wilson, R., Morell, M. K., and Appels, R. 1998. A single genetic locus associated with starch granule properties and noodle quality in wheat. J. Cereal Sci. 27:7-13.

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CHAPTER  2

The Wheat Crop Michael J. Gooding Department of Agriculture The University of Reading Berkshire, United Kingdom

Wheat, maize, and rice dominate world grain production (Fig. 2.1). These grasses (family Poaceae, syn. Gramineae) are grown primarily for their grain (or caryopsis), and are thus “cereals.” The term wheat describes a number of species and subspecies in the genus Triticum, but today the most important are common, or bread, wheat (T. aestivum subsp. aestivum), which accounts for more than 90% of world wheat production, and durum wheat (T. turgidum subsp. durum), which is responsible for a further 5%. Wheats are members of the tribe Triticeae Dumort, alongside other important cereal genera, such as barley (Hordeum) and rye (Secale). For 10,000 years, cereal cultivation has been a major factor supporting community settlement, cultural development, and population growth. The word cereal derives from Ceres, the Roman goddess associated with agriculture. The importance of cereals appears easy to explain: relative to other grain crops, yields are both high and stable; compared to root and tuber crops, cereal grain is easier to grow, transport, and store (Evans 1993). While the grain has been used for diverse foodstuffs, the exploitation of straw for roofing, livestock fodder, and bedding has also contributed to the popularity of cereal farming over the millennia (Sinclair 1998). Grain’s initial status and economic importance have justified further investment in research, plant breeding, fertilizer and irrigation technology, agrochemical discovery, machinery improvement, and advisory services—all contributing to increased productivity per unit area (Fig. 2.2B), further competitive advantage, improved processing methodology, and trading efficiency (Wibberley 1989). Wheat is the primary cereal of temperate areas, but it is also the most widely adapted, being grown from the Arctic Circle to the Equator, from sea level to 3,000 m, and in areas with between 250 and 1,800 mm of rainfall. Wheat is cultivated on more land than any other food crop (Fig. 2.2C), half of the wheat area being

in developing countries. The top 10 producers include countries from Asia (China, India, Pakistan, and Turkey), North America (the United States and Canada), Europe (France and Germany), the Russian Federation, and Australia. The majority of South American wheat is grown in Argentina. Most African wheat is produced in the north (e.g., Egypt and Morocco) or in highland areas, such as in Kenya and Ethiopia, although breeding advances have extended production into the warmer tropics. Average wheat yields are lower than those of maize and rice (Fig. 2.2B), reflecting the extensive cultivation of wheat over large

Fig. 2.1. Production of the world’s most important grain crops (average values 2000–2004). (Data from FAOSTAT 2008)

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areas where production is limited by water shortage. Even in much more productive areas, wheat yield can be compromised by short growing duration when it is a component of rotations that have more than one crop in a year. These systems include the rice-wheat rotations of South and East Asia and the wheatsoybean-maize systems in South America. Wheat cultivation can exploit an opportunity to maximize production and increase efficiency of land and labor use over the cropping cycle despite wheat’s lower yields in such circumstances. In temperate, moist, and long-season areas where intensive production derives from responsive use of fertilizers and agrochemicals, grain production in commercial rain-fed fields can commonly exceed 10 tonnes per hectare (t/ha) and may be more limited by light interception than by water availability. Such conditions are typical in the countries where average national wheat yields exceeded 7 t/ha between 2003 and 2005 (FAOSTAT 2008), namely Ireland, the Netherlands, Belgium, the United Kingdom, Germany, New Zealand, and Denmark. The comparatively easy storage and transportation of cereals renders them suitable for trade, and wheat is the most important grain crop in this respect (Fig. 2.2D). Total world wheat exports were valued at US$20,000 million in 2004, with more than 75% being accounted for by just five countries: the United States (>25%), Australia, Canada, France, and Argentina, where wheat exports accounted for between 0.04% of gross domestic product in the United States and 0.75% in Argentina. The greatest importers of wheat included China, Japan, Italy, Algeria, and Indonesia. Wheat therefore forms a significant component of the balance of trade of national economies, and the movement of wheat (and security of supply) is a major issue in political and economic relationships between governments. Historically, wheat supply has underpinned democracy and has supported or broken govern-

ments, the bread riots at the beginning of the French Revolution in 1789 being an oft-cited example. Wheat, like other cereals, is multifunctional, providing a concentrated source of carbohydrate (mostly starch), with useful amounts of protein, fat, minerals, vitamins, and fiber. Wheat provides between a fifth and a quarter of the energy and protein in the worldwide human diet. Two thirds of the crop is used directly in products for human consumption, and wheat is the main source of calories for 1.5 thousand million people (Reynolds et al 1999). Varieties of wheat are preeminent among cereals in the ability of doughs made from their flours to trap carbon dioxide liberated from fermentation. This allows leavened foods to be baked, bread in diverse forms being by far the most important such food. Yeast spores can be found naturally on the surface of cereal grains, so fermentation occurs readily in wheat dough left to rest. It is likely, therefore, that leavening is of prehistoric origin. Certainly wheat doughs have been baked to produce primitive flat or dense breads from at least the Neolithic era (10,500–7,500 B.P.). Bread continues to be the “staff of life” and is arguably the world’s oldest convenience food. Many types of bread have been developed and have become associated with their region of origin. Low-density pan and hearth breads are familiar worldwide but particularly in the West. In China and surrounding countries, rolls of fermented dough are steamed (rather than baked) to produce a bread with a dense crumb and thin white skin. Flat breads include chapatis (India and Pakistan), naan (N. India), roti or rotta (India), injera (Ethiopia), and in Arabian countries, khoubs or pitta. In Central and South America, products that are more commonly made from maize, such as tortillas and tacos, can also be partly or wholly made from wheat. Biscuits (cookies) are a common outlet for wheat varieties not suited to leavened bread production. In many Asian countries, different types of noodle are made by cutting sheeted dough into thin strips that are then boiled and dried, ready for subsequent cooking. Pastes produced from the semolina of durum wheat are extruded to form different kinds of pasta. Alternatively, semolina grains can be steamed to produce couscous, or durum wheat can be harvested prematurely and the grains parched to produce frekah (Middle East, North Africa) or Gruenkern (Germany). Other processes lead to a plethora of breakfast cereals. Some wheat is simply prepared by soaking for use in porridge, broth, or pudding. Bulgur and “wurld” wheat can be prepared by removing part or all of the bran, respectively, from whole grain. Of the remaining third of the wheat harvest, i.e., that which is not used directly for human consumption, much is included in livestock rations. While less important than maize as an animal feed, wheat has a different geographical range than maize, can equal maize in energy value, often betters maize with respect to protein concentration, and is particularly important for non-ruminants. Wheat starch, like starch from other cereals, can be fermented for use in alcoholic beverages. Wheat that has a low nitrogen content, and thus is less suited to breadmaking, can give a high spirit yield for grain whisky Fig. 2.2. World production (A), yield (B), area (C), and exports (D) of wheat production or be used in the barley-brewing industry (1), paddy rice (t), and maize (!) since 1961. (Data from FAOSTAT 2008)

The Wheat Crop  to aid head retention in pasteurized beers. In areas of the world where local supply exceeds demand for wheat-based foods and livestock feed, fermentation of low-nitrogen-content grain is increasingly used to produce bioethanol and hence reduce reliance on nonrenewable sources of energy. Experimentally, wheat grain has produced up to 46.5 L of ethanol per 100 kg of grain (Rosenberger 2005), while commercial plants have approached 40 L, representing significant positive energy balances when full life-cycle analyses have been performed (Malça and Freire 2006). Industrial exploitation also includes using the starch as a stiffening or surface coating agent in the manufacture of paper and board; as an adhesive in the manufacture of corrugated boxes (Jones 1987); as a loading agent in the production of resins and plastics; as a fermentation substrate in the production of antibiotics, vitamins, and hormones; as a gelling agent or emulsifier in paint; as well as in a host of other more-minor uses.

ORIGINS Progenitors Pollen grain analyses show that grasses were present 55 million years ago (MYA), in the Paleocene, although their first appearance may well have been earlier than this (Kellogg 2001). Genetic sequences (Huang et al 2002, Gill et al 2004) indicate that the three major cereals (wheat, rice, and maize) diverged from a common ancestor about 40 MYA. Divergence of wheat from barley and wheat from rye is estimated to have occurred at 10–14 MYA and 7 MYA, respectively. The most recent divergence is that of the goat grasses (Aegilops spp.) from Triticum, which appear to have radiated 2.5–4.5 MYA. All members of the Triticeae tribe have similar, comparatively large chromosomes in multiples of seven (x = 7). Chromosomes that are from different species but are similar to each other are termed “homeologous.” Despite this homeology, the chromosomes of different species within the Triticeae have diverged significantly from each other such that they do not pair regularly in hybrids, so diploids remain biologically isolated. The genome groups relevant to wheat evolution are labeled A, B, D, G, and S (Kihara 1929, 1954; Dewey 1984; Fig. 2.3), although recently it has been considered that genomes B, G, and S could be sufficiently similar to be classed as the same group (Dvořák et al 2006). The different homeologous groups are denoted with the numerals 1–7, i.e., 1A is homeologous with 1B. The wild diploid wheats (wild ein­korn) found today are T. urartu Tum. (genome

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formula AuAu) and T. monococcum subsp. boeoticum (Boiss.) Mk. (AbAb). Despite the genetic similarities, and their overlapping distributions, T. urartu and T. monococcum produce sterile hybrids and so are isolated genetically, the lineages diverging from each other possibly 0.5–1 MYA (Huang et al 2002), justifying the u and b superscripts in their respective genome formulas. Despite the biological isolation of diploids within Triticeae, hybridization within the tribe does occur in the form of amphiploidy, for example, by the addition of the diploid chromosome complement of two species. This is nowhere more apparent than in the evolution of wheats, where it occurred long before domestication, with the appearance of tetraploid wheats (2n = 4x = 28), i.e., wild emmer (Fig. 2.3), possibly between 0.2 and 0.5 MYA (Huang et al 2002, Gill et al 2004). Hybridization through amphiploidy seems to have contributed greatly to the morphological and ecological adaptation of the allopolyploid species. Environmental and geographic distributions are often much larger for the hybrid species than for the diploid parents

Fig. 2.3. Proposed lineages among wheats adapted from Dvořák et al (2006). Scientific names adapted from MacKey (1988) except for the promotion of several tetraploid wheats from variety groups to subspecies as described by van Slageren (1994) and the use of Triticum sinskajae from Dorofeev et al (1979). Common names and genome labels from Miller (1987).

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(Miller 1987). The multiplicity of genomes would be expected to confer “fixed” hybrid vigor, genetic buffering, and evolutionary adaptability (Simmonds and Smartt 1999). The emmer associated with our most common cultivated forms, T. turgidum subsp. dicoccoides Korn. Thell. (AuAuBB), is a hybrid of T. urartu and an ancestral goat grass (Huang et al 2002, Brandolini et al 2006). The putative goat grass, donor of the B genome, has been the subject of much research, a process complicated by the time and divergence since the hybridization took place. Morphological and some cytological studies have supported Aegilops speltoides Tausch as the closest modern day relative. There is also evidence from organellar DNA that Ae. speltoides contributed the cytoplasmic DNA of polyploid wheats (Wang et al 1997). However, that Ae. speltoides is the sole source of the B genome was not confirmed by some studies of chromosome banding, in situ hybridization, and isoenzyme- and chromosome-pairing under­taken in the 1970s (Miller 1987). More recent gene sequence analyses have both rejected (Huang et al 2002) and supported (Peterson et al 2006) Ae. speltoides as the donor of the B genome. Nonetheless, it is widely accepted that the Sitopsis (S genome) section of Aegilops has contributed at least part of the B genome in polyploid wheats. The current distributions of T. urartu and Ae. speltoides overlap considerably, while other members of the section such as Ae. bicornis (Forsk.) Jaub. and Sprach., Ae. longissima Schweinf. and Muschl., and Ae. searsii are found in more-restricted areas. The fertility of the amphiploid is highly dependent on the Ph1 gene, which suppresses pairing of chromosomes from within the same homeologous groups; i.e., Ph1 confers regular segregation of material in a diploid fashion, and thus fertility and genetic stability (Feldman et al 1995), and has been fundamental to the evolution and utilization of wheats. Ph1 occurs on chromosome 5B, but it is not present in any of the wild diploids of the Triticeae. The gene, therefore, appears to have arisen subsequent to, or possibly as part of, the polyploidization event (Griffiths et al 2006).

Early Use, Then Domestication Wheat is among several species that were first utilized, domesticated, and farmed in the Fertile Crescent, an area that extends northward along the eastern coast of the Mediterranean, bordered by the River Jordan, then curves around the north of the Syrian Desert, into southeastern Turkey, and then southeast toward the Persian Gulf, along the valleys of the Tigris and the Euphrates Rivers and the hilly flanks surrounding the deserts and steppe of Iraq and Iran. As well as wheat and barley, other plants used and domesticated in the area include lentil (Lens culinaris), pea (Pisum sativum), chick pea (Cicer arietinum), broad bean (Vicia faba), and flax (Linum usitatissimum). Wild emmer and einkorn can still be found throughout large areas of the Fertile Crescent (Nesbitt 2001). Dating of samples in the Fertile Crescent has been problematic, as radiocarbon dating has probably underestimated the age of objects (Nesbitt 2001). Dates calibrated to account for these discrepancies are labeled cal. There are also difficulties with positive identification of wheats and their relatives from charred and fragmentary remains (e.g., Nesbitt 2001). However, one of the earliest dates, with reasonable consensus for positive identification of wheat utilization, from the archaeological site Ohalo

II, a hunter-gathering camp on a lake shore, is 23,500–22,500 cal. B.P. (Nadel et al 2006). Ohalo II is just off the present-day southwestern shore of the Sea of Galilee in Israel. The site is routinely submerged, but lower water levels in periods of drought or heavy pumping have allowed intermittent excavations. Ancient flooding of the site appears to have contributed to preservation, and earlier utilization is likely. In early collections excavated from Ohalo II (Kislev et al 1992), charred remains of wild cereals were dominated by barley (H. spontaneum) with just a few grains and ear fragments typical of wild emmer. The gathering of the seed of wild cereals may have been favored over gathering of seeds of other grasses because of their larger grains and more compact inflorescences (i.e., in the case of wheat and barley, the ear or “spike”). However, a wide range of smaller-seeded grasses also appear to have been gathered at Ohalo II, possibly to expand the food base and particularly to extend the harvesting period (Weiss et al 2004). The time from the apparent gathering and use of wheat at Ohalo II to the time when wheat was consciously sown and cultivated probably spanned about 10,000 years. In the Fertile Crescent, there appears to have been a shift from the foraging of the “hunter-gatherers” to farming in the early Neolithic, a period known as the Pre-Pottery Neolithic A. Radiocarbon dating places this period at 10,500–9,500 B.P. (12,600–10,700 cal. B.P.). Wild emmer and einkorn have many characteristics that favor natural dispersal but that would greatly hinder efficient harvesting by humans. Most importantly, the rachis, the main axis or stem of the ear, is brittle in the wild types such that it breaks easily into segments at maturity, with the ear falling apart from the top. Each rachis segment has an attached spikelet, i.e., a cluster of one or more grains with associated chaff of glumes, lemmas, and paleas (Fig. 2.4). As the rachis disarticulates, the spikelets are shed, each one having a rachis segment with a smooth abscission scar at its base. The abscission scar provides a smooth semicircular surface, i.e., the point of an arrow-shaped spikelet designed (together with springy awns, elongated glumes, and backwardpointing barbs and hairs) to penetrate into surface litter and soil cracks. These wild-type characters would improve natural establishment and survival against seed predation from birds, rodents, and ants. Human gatherers, however, would only have harvested partial or immature ears and/or gathered seed from the soil. Of great benefit to humans would have been einkorn and emmer plants with a less fragile, or “semi-tough,” rachis, produced then and now as recessive mutants within wild populations. In such plants, the ear remains intact until after maturity; seeds are not dispersed, or buried, and are highly likely to be eaten. In domesticates, however, the nonbrittle rachis would extend the period over which the ear could be harvested and, in so doing, would significantly reduce grain loss. When manual threshing breaks up the ear, the spike still disarticulates, with entire spikelets again being attached to rachis segments, but instead of having a smooth abscission scar, the base of the rachis segment is frayed and angular, a feature normally diagnostic of domesticated forms. In domesticated emmer and einkorn, the grain is still enclosed within the lemma, palea, and glumes, tightly held to the rachis segment, so the grain is still “hulled” rather than free-threshing or naked. Domestication also leads to an increase in mean grain size. This would confer a number

The Wheat Crop  of potential advantages, including more efficient harvesting and threshing, improved crop establishment, and an increase in the starch-to-bran ratio. Cultivation of wild wheats may have preceded that of the nonbrittle rachis forms, but, given that the semi-tough rachis can be the result of mutation at only one locus and given the relative fitness of the character in cultivation, it may have taken only 20–100 generations for domesticated forms to reach 99% prevalence within a crop (Hillman and Davies 1990). Comparative DNA analysis of domesticated emmer and einkorn and of modern wild populations suggests that, based upon several assumptions, the original site of wheat domestication was in a rather confined region of southeastern Turkey (Heun et al 1997, Dvořák et al 2006). Early archaeological evidence of cultivation is found at sites in Syria, Jordan, Turkey, Palestine, and Israel. Genetic flows from wild to domesticated forms appear to have occurred in more than one location after initial domestication, but it is notable that wheat is one of the six out of the seven Fertile Crescent crops listed above that is predominantly self-fertilized. It has been suggested that “selfing” was advantageous to the development of cultivated types in areas where the wild types were still common because it would have limited the genetic swamping of domesticated forms by the wild population. It is also probable, however, that occasional out-crossing (probably less than 1%) would have produced new types recurrently, thereby aiding further selection.

Free-Threshing and Hexaploid Wheats Many archaeological sites dating to the Pre-Pottery Neolithic B (PPNB, 10,700–8,300 cal. B.P.) period show evidence of cultiva-

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tion of domesticated forms of both einkorn and emmer. How­ ever, hulled emmer appears to have been the dominant cultivated wheat in the Fertile Crescent during the Neolithic period and was the principal wheat of subsequent dispersal (Nesbitt and Samuel 1996). Despite the prevalence of emmer, however, two further developments in the PPNB, i.e., the appearances of hexaploid wheat and free-threshing wheat, have had major significance for the wheat crop as currently grown. Free-threshing describes the way in which “naked” grain can be readily released from glumes, lemma, and palea. This character is conferred by the major gene Q, the dominant allele over q, on the long arm of chromosome 5A. Sequencing and cloning studies (Simons et al 2006) suggest that Q is a transcription factor and that it differs only by a single amino acid from q. As well as the free-threshing character, Q has a number of pleiotropic effects relevant to domestication. Rachis fragility is further reduced, such that it is classed as “tough” rather than as “semi-tough,” and the rachis remains largely intact during threshing. Q also imparts a shorter, squarer spike, reduced plant height, and delayed ear emergence (Simons et al 2006). Hexaploid wheat arose with the addition of the D genome from Ae. tauschii to T. turgidum. Combining DD with the AABB tetraploid to form the AABBDD hexaploid wheat (T. aestivum) had a major impact on the utilization and geographic distribution of wheat. Genes contained on the D genome, for example, have profound influences on the breadmaking characteristics and dough rheology of wheat flour. Ae. tauschii has a much wider range of environmental adaptation than the proposed donors of the A and B genomes, as it is naturally distributed from northern China westward to the west and southwest of the Caspian Sea (Nesbitt 2001). The role of the D genome in hexaploid wheats has

Fig. 2.4. Wheat growth and development. Drawn by Rebecca Kiff with reference to Tottman (1987) and Wibberley (1989). Only main stem shown from stem-erect growth stage. Horizontal bars indicate when yield components are determined (adapted from Slafer 2003), numerals in brackets denote decimal growth stage (Zadoks et al 1974; see text for more details).

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therefore been considered a main contributory factor, together with the efforts of plant breeders, to the present wide geographic distribution of cultivated hexaploid wheats. Hexaploid wheat does not occur in the wild, and it is assumed that the addition of the D genome occurred within cultivation, with domesticated, rather than wild, emmer being the donor of AABB. This is supported by more recent DNA analysis (with the possible exception of T. aestivum subsp. macha) and by the fact that the distribution of wild emmer and the distribution of the specific gene pool of Ae. tauschii that donated the D genome do not overlap (Dvořák et al 2006). Analysis of DNA suggests that either Armenia or southwestern Caspian Iran was the site of origin of T. aestivum (Dvořák et al 1998). It is not certain whether the AABB donor to hexaploid wheat was free-threshing or whether Q occurred in T. aestivum shortly after hybridization. Hulled hexaploids do occur (Fig. 2.3), but socalled “spelt” wheats appear in the archaeological records after the appearance of free-threshing forms, and modern spelt forms (T. aestivum subsp. spelta) may have arisen after introgression of free-threshing hexaploids with emmer. Where ploidy level can be judged from rachis morphology in archaeological remains, free-threshing tetraploids and hexaploids appear at about the same time and often at the same sites in Turkey and Syria in the PPNB. If Q did appear first in T. aestivum (and we concur with Simons et al [2006] that the mutation from q occurred only once), then free-threshing forms of T. turgidum such as durum must have arisen following introgression with the hexaploid. It appears likely that introgression has occurred between hexaploid and tetraploid wheats (both wild and domesticated) since the initial appearance of T. aestivum (Dvořák et al 2006). Gene flow between tetraploids and hexaploid wheats is possible because pentaploid hybrids from 4x × 6x hybridization backcross to either ploidy level. Dispersal of wheat, principally as emmer, proceeded to Greece and Pakistan by 8,000 B.P., Spain by 7,000 B.P., the Netherlands by 6,000 B.P., and to England and Scandinavia by 5,000 B.P. As described above, naked wheats, both tetraploid and hexaploid, were present from early in domestication, but there was a significant lag before they became dominant. In eastern Turkey, einkorn and emmer appear to have been replaced by free-threshing forms from 5,000 B.P., with their dominance within certain communities moving gradually westward to the Aegean Sea by 2,500 B.P. It appears that hulled and free-threshing forms were being cultivated alongside each other for millennia. The greater effort required to process hulled wheat must have been countered by benefits. It is difficult to be certain about what these benefits were, although hulledness may have protected the grain in the field and in storage, and the absence of Q and the associated shortening may have increased tolerance and/or competitiveness with weeds. In addition, socioethnic food preferences may have helped maintain hulled wheat cultivation. Emmer dominated in Egypt until Roman times. Harlan (1981) describes the Romans as initially consumers of emmer but suggests that a taste for baked products developed as the city increased in size and standards of living rose. This demand, together with the development of a controlled food supply and processing, encouraged more hexaploid wheat to be grown, first in Egypt and then throughout the Mediterranean Basin. Replacement of hulled

wheat with free-threshing forms was virtually complete over most of Europe by about 1,000 B.P., but in isolated areas, such as southern Germany and northern Switzerland, this did not occur until the twentieth century C.E. (Nesbitt and Samuel 1996).

Wheats of Today HULLED WHEATS

Einkorn, emmer, and spelt are still cultivated in mountainous regions, often on poor soils, where hardiness, particularly in the seedling stages, is recognized. Emmer remains an important crop in the central and northern highlands of Ethiopia, after durum and bread wheat, and is a minor crop in regions of India, Italy, Turkey, and Iran. It is considered to be generally higher yielding than einkorn and more tolerant of weed infestations. Einkorn has, therefore, always been less important than emmer; yet, it has traditionally been grown in cooler environments and more marginal land in the Middle East and in southwestern Europe. Today einkorn is still grown in small, isolated areas on poor soils and in harsh environments in France, India, Italy, Turkey, and the former Yugoslavia. Spelt remains a locally important cereal crop in isolated, mountainous regions of southeastern Europe, principally in Germany and Switzerland, and in Iran. Interest in spelt, and to a lesser extent emmer, has recently increased in many areas of the European Union, as it apparently grows relatively well in production systems using lower levels of nitrogen fertilizer and crop-protection chemicals. This interest is also being driven by a demand for traditional products such as specialty breads and wheat beers that can be made from spelt. Other cultivated, hulled hexaploid wheats include T. aestivum subsp. vavilovii (Armenia) and T. aestivum subsp. macha (western Georgia). FREE-THRESHING TETRAPLOID WHEATS

While the hulled wheats persist as remnants in isolated areas, or to supply niche markets, naked wheats have become dominant throughout the world. Durum covers about 8% of the wheat area and often does relatively well when maturation coincides with hot and dry conditions. Such environments lead to high concentrations of protein in the grain, which is a requirement for pasta production. Nearly 45% of durum wheat is cultivated in West Asia and North Africa. It is also widely grown in Mediterranean countries, parts of eastern Europe, the north central states (particularly North Dakota) and southwestern part of the United States, Mexico, and areas in and surrounding Saskatchewan in Canada. In South America, durum wheat is cultivated in Chile, Argentina, and Andean regions. Other tetraploids such as Polish wheat (T. turgidum subsp. polonicum) and rivet wheat (T. turgidum subsp. turgidum) are closely related to durum wheat and are found in similar Mediterranean and European locations but in much smaller amounts. Minor cultivated forms of the AABB genome in the Middle East and Asia include the Persian wheat (T. turgidum subsp. carthlicum; Iraq, Iran, Turkey, and Southern Transcaucasia) and the Khorasan wheat (T. turgidum subsp. turanicum; central Asia and northern parts of the Middle East).

The Wheat Crop  FREE-THRESHING HEXAPLOID WHEATS

T. aestivum includes some minor subspecies such as club wheat (T. aestivum subsp. compactum), which is grown principally in Washington, Oregon, Idaho, and California in the United States, with a very small amount grown in Canada, and Indian dwarf or shot wheat (T. aestivum subsp. sphaerococcum), found in northwestern India and Iran. However, as already stated, by far the most important wheat is the naked hexaploid bread or common wheat (T. aestivum subsp. aestivum). Hereafter this subspecies is referred to as “common” wheat, recognizing that not all varieties are particularly suited to making bread. Not only does end use vary considerably among varieties, but there is also great diversity in agro-ecological adaptation. Much of this variation is relatively simply inherited and has contributed to common wheat varieties being classified on the basis of characteristics relevant to the growing, breeding, and marketing of the crop and the utilization of the grain. That is, varieties are often distinguished on the basis of seed coat color, endosperm texture, dough strength, and sowing season. Red and White Wheats. The intensity of red pigmentation (phlobaphene) in the seed coat is principally determined by three functionally equivalent genes (R/r) at homeologous loci on the long arms of chromosomes 3A, 3B, and 3D. Red varieties carry one or more of the red (dominant) alleles, and intensity of pigmentation increases as “gene dosage” increases to three (Flintham 2000). Red varieties exhibit more dormancy than white varieties and so are favored in climates conducive to preharvest sprouting. White wheats are more suited to areas that are dry during ripening and harvest and are favored for the manufacture of certain types of flat bread and noodles. Hard and Soft Wheats. The hardness of a variety or seed relates to the resistance encountered when it is milled, i.e., the harder the wheat, the greater the force required. When they are milled, hard wheats break down along the outlines of endo­ sperm cell walls, or through the cells across the starch grains and proteins alike, to yield coarse, smooth-sided granules that flow easily over surfaces and through sieves. Conversely, milling soft wheat produces a mass of fine, angular-shaped particles of cell debris. Hardness is inherited comparatively simply and is mostly under the control of a major gene encoded on the short arm of chromosome 5D. Friabilin is a surface protein complex occurring on the surface of starch granules; it is abundant in soft wheats, scarce in hard wheats, and absent in durum wheat. Two of the main components of friabilin are the isoform polypeptides puroindoline-a and puroindoline-b (Oda and Schofield 1997). The presence of both isoforms appears to be necessary for the expression of softness, and mutation in either can be associated with hardness. Hard wheats are favored for breadmaking, not only because they are easier to mill, but because the higher grinding pressures that can be tolerated cause more damage to starch granules, which, in turn, leads to a greater potential for water adsorption. Soft wheats are desired for products where dryness is a favored characteristic, i.e., biscuits (cookies). Strong and Weak Wheats. Leavened bread production is largely limited to the genomes coding for the proteins necessary to generate an elastic, strong dough suitable for the capture of gas in bubbles during fermentation, thus allowing the dough to rise. Precise dough rheological properties result from the inter-

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actions among all major constituents of flour (i.e., starch, lipid, and protein) and water. However, the unique elastic properties of doughs made from wheat flours are largely the result of the type and amount of gluten present. The gluten contains the proteins insoluble in water and alcohol, i.e., the prolamin storage proteins. These prolamins are divided into the monomeric gliadins and the polymeric glutenins (Shewry et al 1986). Varieties with a high gliadin-glutenin ratio tend to have viscous, extensible doughs that are often suitable for cookie (biscuit) making. Those having a low gliadin-glutenin ratio have more elasticity and strength, which are desired for breadmaking. Allelic variation in the high molecular weight (HMW) glutenins is closely linked with breadmaking quality and dough resistance. These subunits are composed of 580–730 amino acid residues and are controlled by genes at loci located on the long arms of chromosomes 1A, 1B, and 1D, notated as Glu-A1, Glu-B1, and Glu-D1. Varieties of bread wheat contain between three and five major HMW glutenin subunits. Two of these are coded by genes at Glu-D1, one or two by Glu-B1, and one or none by Glu-A1 (Payne et al 1987). More than 50% of the variation in baking potential and/or dough rheology within wheat collections of several countries has been explained on the basis of HMW glutenin subunit composition. Winter and Spring Wheats. Varieties differ in their requirement for a cold period to hasten, or permit, normal development toward reproductive development. This need for vernalization (literally, “making ready for spring”) is strongly affected by variation at the Vrn-1 loci located on each of the long arms of the group 5 chromosomes (i.e., Vrn-A1, Vrn-B1, and Vrn-D1) and their apparent regulation by minor vernalization genes (Loukoianov et al 2005). A dominant Vrn-1a allele on any of the three wheat genomes results in a spring habit, and the presence of recessive Vrn-1b alleles on all three genomes results in a winter habit. For example, Acevedo et al (2002) found that spring wheats require 7–18°C for five to 15 days for floral initiation, while winter wheats require 0–7°C for 30–60 days. Wheats with a significant vernalization requirement are thus maintained in a vegetative state until the requirement has been met. This in itself may contribute to the avoidance of cold damage if reproductive development is not initiated until after the harshest weather has passed. However, Vrn-1 genes are closely linked to, and also interact with, other genes conferring cold tolerance (Reddy et al 2006) and, therefore, survival over winter.

GROWTH AND DEVELOPMENT Wheat plants develop through recognizable phases, typical of many annual grasses (Fig. 2.4). The vegetative phase commences with germination, followed by the appearance of leaves from the apical meristem stem or growing point and the appearance of more stems, or tillers, on each plant. The reproductive stage commences when the stem apex or growing point, while still close to ground level, starts producing the ear, or spike. The structures of the ear develop as it is simultaneously elevated through the leaf sheaths of the canopy by the extending stem. Booting describes the swelling of the sheath of the ultimate leaf, the flag leaf, as the developing ear expands within it. This is soon followed by emergence of the ear above the flag leaf, after which flowering (anthesis) precedes fertilization. Soon after ­fertilization, the

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grain fills with water, which is replaced with dry matter until a maximum quantity of dry matter per grain is reached (i.e., mass maturity), after which the grain dries to harvest maturity. The development of the wheat plant is said to be “determinate” because of the distinction between the vegetative and reproductive phases; i.e., once the stem apex has moved to the reproductive phase (producing the structures of the ear), it does not revert to producing leaves and tillers. The distinction between the vegetative and reproductive stages is less precise for a crop than for an individual stem because the maturity of tillers can be delayed compared with that of the main stem. In extreme situations (for example, when only a few plants are established but conditions are nutritious and moist until late in the season), tillers can still be appearing while the main stem is at anthesis (Fig. 2.5F). Nonetheless, variations in growth stage between different tiller generations become less as the crop proceeds toward maturity; i.e., later-appearing tillers have faster maturation rates, such that the optimal timing of harvest is better defined for crops of wheat than for less determinate crops. The rate of wheat development depends largely on variety, temperature, the need for a cold period (vernalization), and day length (photoperiod). As already described, maturation of winter wheat varieties is hastened following vernalization, i.e., exposure to low temperatures, typically 3–10°C, for six to eight weeks. Development can also be accelerated by exposure to long days; i.e., photoperiod-sensitive varieties are quantitative longday plants, although short days can sometimes substitute for vernalization. Major genes controlling photoperiod sensitivity in wheat (i.e., Ppd-D1, Ppd-B1, and Ppd-A1) are found on the short arms of group 2 chromosomes, with dominant (notated a) alleles conferring insensitivity to photoperiod. The presence of Ppd-D1a has, for instance, been associated with plants flowering up to 14 days earlier than photoperiod-sensitive genotypes in typical U.K. field conditions (Snape et al 2001). Even when vernalization and photoperiod requirements are fully met (e.g., by growing plants from vernalized seedlings with continually long days in growth cabinets), developmental rates still vary among varieties. These differences can be ascribed to variations in earliness per se. Genes affecting this character have been reported to be on the group 2 chromosomes (loci Eps-2) as well as on chromosomes 3A and 5A. Many more relevant genes are postulated, partly because of expected homeology with identified loci on closely related species (Snape et al 2001). Because varieties vary in their response to temperature, vernalization, and photoperiod; in the extent to which these factors interact; and in relative sensitivity to them at different growth stages, varieties vary, apparently continuously, in their rates of maturation, thus contributing to the wide adaptation and distribution of wheat in world agriculture. Variation in development rate among varieties, not just because of vernalization requirements, also impacts the ideal sowing date of a genotype.

Seed Structure and Germination The wheat grain, or caryopsis, consists of not only the true seed but also the pericarp, or “fruit.” The grain has two distinct sides, one that is dissected by a crease, known as the “ventral” side, and the rounded back or “dorsal” side. One end of the

grain is covered in a fine layer of hairs, known as the “brush” end, opposite the germ end, which encases the embryo. Grain dimensions vary greatly depending on growing conditions, variety, and position within the ear, but typically, lengths range between 3 and 8 mm and maximum widths between 2 and 5

Fig. 2.5. Effects of sowing 50 (!), 100 (O), 200 (U), 350 (S), or 600 (P) seeds of Hereward winter wheat per square meter on plants per square meter surviving the winter and thus on other factors. a, grain yield fitted with eq. 7 (see text); b, harvest index, eq. 7/eq. 5; c, aboveground biomass, eq. 5; d, radiation use efficiency (RUE), eq. 5/eq. 6; e, interception of photosynthetically active radiation (PAR), eq. 6; f, tiller numbers per plant during the cropping season. Seeds were sown on October 14 and received N at 200 kg/ha at the start of stem extension. Arrows and numerals in parentheses denote growth stage of main stem (Zadoks et al 1974). Curves in a–e fitted simultaneously. (Data from Gooding et al 2002)

The Wheat Crop  mm. Individual grain weights are often between 20 and 60 mg of dry matter (DM). Surface color ranges from golden brown with a reddish hue to creamy, depending largely upon variety. The endosperm may either be glassy and vitreous or white and floury, depending on variety and growing conditions. On imbibition of water, the grain can swell by more than 40% at the start of germination. Germination can proceed once dormancy has been broken and environmental requirements have been met. Some hormonally controlled dormancy is desirable to prevent grain germinating in the ear before harvest. Such dormancy is readily expressed, even by some modern varieties, at temperatures above 20°C (Kindred et al 2005) and may delay germination in warm seedbeds (Cook and Veseth 1991). This dormancy can be broken by exposure to low temperatures, declines during storage, and is often lower in modern varieties compared with their predecessors. The minimum moisture content and temperature for germination are 35–45% and 4°C, respectively, although the speed of germination is faster at higher moisture contents and temperatures, with 20–25°C being optimal for nondormant seed (Evans et al 1975, Kindred et al 2005). The coleorhiza is the first part of the embryo to emerge from the seed coat layers, which are ruptured by the extension. This is shortly followed by the appearance of the seminal, or seedling, roots. A primary root and then two pairs of lateral roots break through the coleorhizae, followed by the plumule. The scutellum is the modified seedling leaf, or cotyledon, of the wheat plant. This remains in the germinating seed, protecting the developing seedling from the hydrolytic reactions occurring in the endosperm. The plumule elongates to the soil surface, and the coleoptile, a sheath protecting the first true leaf, emerges as a single pale tubelike structure (Fig. 2.4). Coleoptile elongation generally stops when it is exposed to light, but its maximum length, and hence ultimate ability to reach the surface and permit successful seedling emergence from deep sowing or through surface litter, depends on variety (Rebetzke et al 2007) and on carbohydrate and/or protein reserves in the seed (Evans et al 1975). The apical meristem, or growing point, is raised to the soil surface by expansion of an underground stem (rhizome) as the subcrown internode lengthens. The first true leaf then emerges from a slit in the top of the coleoptile and is quickly followed by others.

Vegetative Growth LEAVES

The apical meristem produces leaf primordia, which are attached at nodes or joints on the stem. During the vegetative stage, the stem is less than 5 mm long, and, therefore, the nodes are compressed, with very short internodes. The leaves are pushed upward and consist of two main sections, the leaf sheath and the leaf blade, or lamina. The leaf sheaths are usually hairy (nonglabrous). The leaf blades are narrow, with about 12 veins. The junction between the sheath and blade is known as the “collar,” where the ligule and auricles can be found. The auricles are commonly 1–3 mm long and are usually hairy (Fig. 2.4). The ligule is blunt and between 2 and 4 mm long. New leaves emerge from within the leaf sheath of the next-youngest leaf. The rate at

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which leaves are formed on the meristem, emerge, expand, and unfold (as well as the size and shape of the leaf lamina) depends on temperature, light intensity, day-length, nutrient availability, and variety (Evans et al 1975). The day degrees (sum of mean daily degrees Celsius above a base temperature) between successive emerging leaves can be calculated as the “phyllochron” (base temperature = 0°C). The total number of leaves formed on the main stem depends greatly on the environment but commonly ranges between 7 and 12. The number of green leaves on the stem at any one time, however, is often two to five, depending on nutrition, moisture, and the incidence of pests and diseases. TILLERS

Nodes can also give rise to side shoots, or tillers. These develop from buds in the axils, where the leaf sheath joins the stem, and rate of appearance is therefore also related to the phyllochron. During initial growth, the tiller is entirely enclosed in the leaf sheaths of the parent shoot and is dependent on the parent shoot for nutrition. Independency does not normally occur until the tiller has three mature leaves of its own. Tillering allows the plant to expand and to produce more ears and therefore more grains per plant when interplant competition is low. Tillers thereby permit the wheat crop to partly compensate for adverse conditions such as poor germination and establishment, frost or hail injury, pest attack, and grazing damage. Tillering increases with increasing light and nitrogen availability during the vegetative phase and also depends greatly upon variety and plant density (Bunting and Drennan 1966, Evans et al 1975, Gooding et al 2002; Fig. 2.5F). It is delayed and reduced by deep planting and is generally less in spring wheats than in winter wheats. Tillering from a single plant in a fertile, low-competition environment can be substantial. The main stem gives rise to primary tillers, but primary tillers can also produce secondary tillers and, in some cases, even tertiary tillers. Excessive tillering, however, is undesirable. Maturity of the late-produced tillers tends to lag behind that of the main-stem tillers, resulting in an uneven crop, which is difficult to manage and harvest at optimum times. Not all tillers produced are fertile and reach maturity. A tiller is likely to die as a consequence of intraplant competition for assimilates, particularly competition from developing ears within more robust tillers. The start of tiller death can be associated with the ratio of red to far-red light at the base of canopies, which in turn can be related to leaf nitrogen content (Sparkes et al 2006) and therefore nitrogen availability. Tiller mortality causes differences between varieties to become smaller toward the flowering stage, when the number of tillers per unit area is more a function of environmental conditions, although the genetic influence is still significant. Tiller death may represent a waste of resources, particularly as 90% of the dry matter is not translocated to surviving tillers (Berry et al 2003). Therefore, much research has been done on the genetic basis of tillering, which has revealed numerous quantitative trait loci (QTL) associated with tiller production and death in wheat (Li and Gill 2004). A few major genes conferring tiller inhibition (tin) have been identified (e.g., Richards 1988), which can also be associated with other potentially useful traits such as increased mean grain weight and improved harvest index, as discussed later. In other

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situations, however, early tillering, even when followed by tiller death, can be useful in, for instance, competition against weeds and improved nutrient capture. Much of the nitrogen is retranslocated from senescing tillers, so infertile tillers may sometimes have a role in ensuring continuous nutrition to grain-bearing stems (Evans et al 1975). ROOTS

As well as the seminal roots, adventitious or nodal roots also derive from the coleoptile node or lower nodes of the main stem and tillers. These roots develop about one to two months after germination and generally overtake the seminal roots, occupying more than 90–95% of the total root volume of a fully grown crop. The adventitious roots are generally thicker (0.3–0.7 mm) than seminal roots (0.2–0.4 mm). Roots can grow faster than shoots at low temperatures, but this situation reverses at higher temperatures such that the ratio of root dry matter to shoot dry matter declines greatly as the crop develops. In winter wheat-growing areas, the plant typically spends more time at lower temperatures, and therefore, more root biomass and length are produced than for spring-sown crops. In commercial, rain-fed, autumn-sown winter wheat, root biomass can increase exponentially until the end of tillering in spring (Gregory et al 1978), reaching root-shoot ratios of 0.3–0.5. After this, net root biomass accumulates approximately linearly but at a slower rate than that of shoot growth, such that root-shoot ratios often decline to 0.1 or less by anthesis. In deep soils, roots may reach a depth of 2 m. Nitrogen availability can increase root mass and length (Fig. 2.6), but the effect of nitrogen is typically much less on roots than on the canopy, so nitrogen application can reduce root-shoot ratios. During grain filling, root biomass and/or length can

remain rela­t ively constant, degenerate, or, in well-fertilized, high-yielding situations, increase (Fig. 2.6; Ford et al 2006). In terms of vertical distribution, rooting is affected by soil structure, moisture availability, nutrients, and variety (Clarke 2002, Ford et al 2006). In relatively homogeneous soil, however, the decline in both root dry matter (Clarke 2002) and length (Gale and Grigal 1987) with depth can be approximated by an exponential model:

Y = 1 – βd

(1)

where Y describes the cumulative proportion of the total root system to any depth (d). Modeling approaches have suggested that root lengths as low as 0.1–1 cm/cm3 are sufficient to exploit available water and mobile ions, such as nitrate (van Noordwijk 1983). These models do not account for the heterogeneity of the soil environment or the varying rate of ion uptake depending on root age or location. It is also likely that higher root length densities are appropriate for exploiting less-mobile nutrients such as phosphorus and also for capturing resources when the plant is in competition with other plants. Nonetheless, modern wheat crops often have much larger root systems than 1 cm/cm3 (Fig. 2.6), particularly in the upper soil profile. It has been predicted, therefore, that there should be advantage for wheat crops that invest more in roots at depth, rather than in the surface layers, i.e., have a value of β in the above model that is closer to unity (King et al 2003). Although elite lines of modern wheats do vary in the relative distributions of roots down soil profiles (Ford et al 2006), this characteristic is not always easy to correlate with improved yields or with late-season uptake of nitrogen (Gooding et al 2005a, 2007). Indeed, in some environments, wheat roots are the last vegetative organs to senesce and may ultimately compete with grains for photosynthate and nitrogen (Andersson et al 2005).

Reproductive Growth SPIKELETS

Fig. 2.6. Root distribution of rain-fed Hereward winter wheat. Open circles indicate that no nitrogen was applied. Otherwise, 200 kg of N per hectare was applied at the start of stem extension. (Adapted from Clarke 2002)

The reproductive development of the apex begins as it elongates from 0.1 to 0.3 mm with the appearance of primordia as single ridges (Fig. 2.4). At this stage, the stem apex is still close to the ground and surrounded by subtending leaves. The buds in the axils of the apex ridges are spikelet primordia and, with their leaf initials, form double ridges as the developing spike elongates to between 0.8 and 1 mm. The spike continues to elongate, and as it does so, the central spikelets swell while additional double ridges are formed acropetally until the terminal spikelet is formed at the apex in a plane at a right angle to the other spikelets. The number of spikelets formed on each ear depends on both duration and rate of initiation. Rapid initiation is often associated with shorter durations of spikelet production. Negative correlation is only

The Wheat Crop  partial, however, and varieties can vary in both duration and rate, causing genetically determined variations in spikelet number and ear lengths. In long-day environments, photoperiod insensitivity can reduce the duration of spikelet production and hence reduce spikelets per ear (Snape et al 2001). As temperature increases, the duration of spikelet formation declines, but, up to about 25°C, the rate of production increases such that there may be little variation in spikelets per ear produced between 10 and 20°C (Kirby and Appleyard 1987). Reduced light intensity can reduce the number of spikelets per ear through lowering the rate of production (Fischer 1985), while salinity appears to reduce spikelet numbers through reducing duration of production (Grieve et al 1993). Spikelet number per ear usually increases with increased nitrogen availability, apparently resulting from an increase in rate of spikelet initiation (Whingwiri and Kemp 1980). FLORETS

When the terminal spikelet has been formed, the embryonic spike may be 1.5–4 mm long, and, depending on variety and conditions, may be held 10–30 mm above the ground. This stage can often coincide with the growth of one to two distinct nodes that can be detected manually on the extending stem (Fig. 2.4), although this also depends on variety (Wibberley 1989). Differentiation of the spikelets continues, having started before the terminal spikelet stage and being most advanced in the lower mid-part of the spike. Glume initials (basal bracts) are seen first, and, as these elongate, often seven to 11 florets are initiated acropetally on alternate sides of a lengthening rachilla. Floret initiation continues until about flag leaf emergence. The floret differentiates from a primordium in the axil of a floret bract, the lemma, to form an opposing floret bract, the palea, which encloses the stamens (filament and anthers), carpel, and lodicules. The floret apex, surrounded by the carpel, develops into the ovule. A single-egg (megaspore) mother cell is formed from one archesporial cell in each ovule and undergoes meiosis. Each anther contains many archesporial cells, each forming four pollen mother cells, which each undergo meiosis while the anthers are green and about 1 mm long, i.e., usually at booting while the spike is expanding within the flag leaf sheath (Fig. 2.4). The duration from the terminal spikelet stage to ear emergence and then anthesis is dependent on both temperature and photoperiod. Growth of the spike and its component organs is exponential until it stops at anthesis, when the spike is typically 80–150 mm long. Despite the large numbers of florets initiated, only some develop green anthers, still fewer reach anthesis, and still fewer set grains (Evans et al 1975). Competent florets with a carpel, three stamens, and two lodicules, all enclosed in a lemma and palea, may number only two to four per spikelet by anthesis. Floret survival may be limited by assimilate availability to the ear and thus also by the duration from the start of the reproductive phase to anthesis (Reynolds et al 1999, Miralles et al 2000). This has been supported by comparing different genotypes with different photoperiod sensitivities or by modifying photoperiods within controlled environments. The plant hormones abscisic acid (negative) and cytokinin (positive) have strong influences on floret development and fer-

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tility, possibly by influencing sucrose availability to the developing ear (Waters et al 1984). Increasing assimilate supply during the rapid phase of ear growth can increase mean ear dry weight at anthesis, which is often correlated with the number of fertile florets per ear (Gonzalez et al 2003). It has also been argued that floret numbers and subsequent grain set are also related to nitrogen concentration in the spike (Sinclair and Jamieson 2006). FERTILIZATION

Anthesis commences typically between three and eight days after ear emergence, depending on temperature and variety. Flowering starts in the basal florets of central spikelets and proceeds basipetally and acropetally within the ear and acropetally within the spikelet. Flowering within a spike is usually complete within two to three days, while over a whole plant or crop, it may extend over five to 10 days due to variations in tiller maturity. Wheat is generally self-pollinated, but some cross-pollination is possible. Successful seed set is dependent on the production of viable pollen grains, transfer of pollen to the stigma, germination of the pollen grains and growth of pollen tubes down the style, union of the male gamete with a viable oocyte, and normal development of a zygote following fertilization. All these steps are temperature sensitive. The optimum temperature for fertilization is 18–24°C, and, when nutrition is also adequate, grain set in competent florets is usually more than 80% (Subedi et al 2000). Sterility, particularly in distal spikelets, can be caused by both heat and cold stress during ear emergence and anthesis. Similarly, sterility can be increased by nutrient deficiency, particularly of boron; droughts and waterlogging; extremes of humidity; and alkalinity. Most of these factors show strong interactions with variety (e.g., Subedi et al 2001). GRAIN DEVELOPMENT

Grain filling with dry matter (and nitrogen) from anthesis to harvest maturity can often be adequately described with an ordinary logistic function with the constant omitted (Gooding et al 2005b; Fig. 2.7C). Physiologically, however, grain development proceeds in three phases (Jenner et al 1991). The first phase is one of grain enlargement as cells multiply and expand, with rapid accumulation of water into the grain (Pepler et al 2006; Fig. 2.7D). Division of the endosperm nucleus occurs within a few hours of fertilization. The first cell walls appear about three days later. The rate of cell division slows until a maximum cell number (typically around 105) is attained 15–20 days after anthesis, at about the time when rapid water accumulation stops. A-type granules, the largest class of starch granules at maturity, are initiated during the first week after anthesis and typically attain a lenticular shape of 20–50 µm diameter by maturity. B-type (~5–10 µm) granules are spherical and are initiated during the second and third weeks after anthesis (Stoddard 2003). Storage protein appears about 10 days after anthesis in membrane-bound spherical bodies (0.5–1.5 µm) derived from the Golgi apparatus, closely associated with rough endoplasmic reticulum. The second phase of endosperm development is a near-linear increase in grain dry matter, starting between 10 and 15 days after anthesis and continuing, depending on temperature, for

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15–30 days. The mass of water per grain during this phase is relatively constant (Pepler et al 2006; Fig. 2.7D); i.e., water entry to the developing grain and water loss are approximately equivalent. Many protein bodies fuse, forming a continuous protein matrix that embeds the starch granules. The third phase describes processes subsequent to the attainment of maximum dry matter per grain. The time of maximum dry matter is often taken as physiological maturity, rather than harvest maturity, and it coincides with the start of rapid

Fig. 2.7. Effect of fungicide on disease progress (A) and senescence (B) of the flag leaf and on the dry matter (C) and water content (D) of medial grain of Malacca winter wheat. ! = nil fungicide, 1 = epoxiconazole (63 g/ha) at start of stem extension, p = epoxiconazole (63 g/ha) at start of stem extension followed by epoxiconazole (63 g/ha) plus azoxystrobin (250 g/ha) at flag leaf emergence. Fitted curves are exponential (A); modified Gompertz (B) (Gooding et al 2000); logistic, constant omitted (C) (Zahedi and Jenner, 2003); and smoothed with splines (D). Dashed lines connect time to 37% green leaf area (Gompertz time scalar, m) to end of grain filling, estimated from a simultaneous fit of time of maximum grain dry matter and onset of net rapid water loss (Pepler et al 2006).

net water loss from the seed (Pepler et al 2006; Fig. 2.7D) and the acquisition of dormancy. The end of grain filling is also sometimes (Hanft and Wych 1982), but not always (Kindred and Gooding 2005, Pepler et al 2005), associated with flag leaf senescence. Final grain weight under good growing conditions, i.e., without significant drought stress or pathogen infection, has often been correlated with the water mass per grain and/or the number of endosperm cells attained during the two to three weeks after anthesis (Brocklehurst 1977, Borrás et al 2004). Further evidence that this is a particularly important stage for determining potential final grain mass includes the observations that environmental treatments applied during the two to three weeks after anthesis have a much larger impact on final grain size than treatments applied later (Gooding et al 2003). However, late-season nutrient deficiency, moisture stress, and disease can significantly reduce mean grain weight and therefore grain yield. Often 40% or more of the photosynthate ultimately found in the grain derives from photosynthesis in the flag leaf. The flag leaf is the leaf in closest vascular proximity to the ear and is usually the most important leaf for light interception during grain filling; held above the other leaves, it is often the longest-lived leaf and the last to senesce. During the linear phase of grain growth, 80% of the photosynthate produced in the flag leaf is translocated to the ears (Thorne 1982). Grain filling with carbohydrate is thus mostly a function of photosynthesis occurring concurrently; other major contributions derive from photosynthesis in the ear and penultimate leaf. Despite the importance of concurrent photosynthesis to grain filling, stems can also hold reserves of nonstructural carbohydrates, which can potentially be remobilized to support grain growth. These reserves may constitute up to 40% of the stem dry weight but may account only for 5–15% of final grain yield under favorable conditions (e.g., Austin et al 1977). When postanthesis photosynthesis is curtailed by stress, however, the proportionate contribution of remobilized stem reserves to final grain yield may increase to more than 50% (Blum et al 1994). The nitrogen accumulated as protein in the growing grain derives from a combination of nitrogen remobilized from senescing tissues and nitrogen taken up after anthesis. In good growing conditions, 70–75% of the nitrogen accumulated by anthesis is subsequently remobilized to the grain. This remobilized nitrogen typically accounts for 50–70% of grain nitrogen at harvest. The remaining 50–30% of grain N derives from postanthesis uptake by the plant, for which partition rates to the grain can reach 90% (e.g., Gooding et al 2005b, 2007). Enhanced grain N recovery is important for maintaining protein concentrations (N × 5.7) in high-yielding crops and thus the marketability of wheat for a number of end uses (Dimmock and Gooding 2002). However, high grain-nitrogen concentrations may indicate that the crop is less efficient at producing grain dry matter per unit of N accumulated (Foulkes et al 1998). Improved nitrogen utilization efficiency to produce grain (NUtEg), i.e., the ratio of grain dry matter to nitrogen in the aboveground crop (OrtizMonasterio et al 1997, Kindred and Gooding 2004), could assist in maintaining yields with reduced nitrogen fertilizer applications and/or increasing the energy balance of the crop (Rosenberger et al 2001).

The Wheat Crop 

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TABLE 2.1 The Zadoks Growth Stage Score a Score Growth Stage

Explanation

0n 1n 2n 3n

Germination Leaf production on the main stem Tiller production Stem elongation

4n 5n 6n 7n 8n

Booting Ear emergence Anthesis Grain expansion Dough development

9n

Ripening

n indicates preleaf emergence development from dry seed (n = 0) to first leaf at coleoptile tip (n = 9) n = number of unfolded leaves, to maximum of 9 n = number of tillers per plant, to maximum of 9 n = number of nodes (Fig. 2.4) detectable, to maximum of 6, then n = 7 for flag leaf just visible, and n = 9 for flag leaf collar visible n indicates degree of swelling n indicates proportion of ear emerged n indicates degree of completion i.e., the grain fluid exuded when the caryopsis is squeezed changes from watery (n = 1) to milky (n = 7) i.e., no droplet exuded from squeezed caryopsis, thumbnail imprint not retained (n = 3) to thumbnail imprint retained (n = 5+) describes harvest ripeness (n = 2), to seed with no primary dormancy (n = 7)

a The

substage (0–9) is represented by n.

EMPIRICAL ANALYSIS OF GRAIN YIELD

TABLE 2.2 The Feekes Growth Stage Score Stage

Description

1

Seedling emergence to three leaf stage Start of tillering End of tillering Leaf sheath lengthens Leaf sheaths strongly erect First node detectable Second node detectable Flag leaf just visible Flag leaf emerged Booting Full ear emergence Start of anthesis Kernel watery ripe Kernel milky ripe Soft dough Harvest ripe

2 3 4 5 6 7 8 9 10 10.5 10.51 10.54 11.1 11.2 11.4

Approximately Equivalent Zadoks Stage

10–13 21–25 26–29 Early 30s Late 30s 31 32 37 39 45 59 60 71 75 85 92

GROWTH STAGE SCORES

Much agronomic research and advice is based on identifying specific growth stages at which to assess the crop and/or to apply inputs. Growth stage scores have thus been devised, based on the external appearance of the crop, to help define developmental periods and standardize reporting. Commonly used systems are those of Zadoks et al (1974) and Feekes (1941). Some of the key growth stages are illustrated (Fig. 2.4), and more detailed comparison is available elsewhere (e.g., Cook and Veseth 1991). The Zadoks scale (Table 2.1) is said to be decimal because it describes 10 main phases of growth (0–9), each divided into 10 further stages. The Feekes scale (Table 2.2) describes 11 main phases of growth but has useful subdivisions of stages to describe ear emergence and grain filling.

The grain yield of wheat, in terms of mass per unit area, can be described empirically in many ways, but the literature reveals two main approaches, one based on the sink and the other based on the source. For reasons that are explained below, the two approaches do not necessarily contradict each other, but they do alter the way agronomic inputs are justified and also how advances from breeding efforts are explained and targeted. The first approach, based on the sink, is to understand yield in terms of yield components. Here, grain yield is most simply a function of grain numbers produced and mean grain weight: Grain yield/m 2 = plant no./m 2 × ears/plant × spikelets/ear × grains/spikelet × mass/grain

(2)

Further expansion is, however, possible. For example, plant number per square meter is a function of the number of seeds sown, the proportion that establish (which is itself affected by both seed viability and seedling vigor), and the proportion that survive to harvest. Ear number per plant is a function of tillers produced per plant, the proportion that survive, and the proportion of surviving stems that produce fertile ears. Grains per spikelet depends on florets per spikelet, the proportion of florets that are competent, the proportion of competent florets that are fertilized, and the proportion of fertilized florets that are not aborted. It is even possible to think of the mass per grain as being related to the numbers of endosperm cells produced and the mean weight per endosperm cell. Using this approach, yield can be thought of as being formed throughout the growth of the crop, with the environment and agronomic inputs at different growth stages influencing yield through their impact upon specific yield components, susceptible to modification at particular times (Fig. 2.4). Research and agronomy aimed at influencing grain components are often said to concentrate on the sink because they deal with the capacity of the grain population to contain yield. The size of the sink can be thought of as important in determining yield for several reasons. To start with, the potential size of individual grains is probably constrained before the end of grain

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filling (e.g., by the volume available within the glume and lemmas, and/or by the maximum dimensions of the grain attained following rapid expansion with water, and/or by the maximum size and number of endosperm cells); there is a real sense in which the sink size may present a physical limitation on yield. A more common reason for thinking of the sink as determining yield is that, because the developing ears (and then grain) compete for assimilate from other organs and because of hormonal and other factors, increasing the sink size increases the efficiency with which assimilate is partitioned to the ear and grain. Some take this approach a step further, supporting the idea that sink size affects resource capture and use-efficiency; e.g., increasing grain numbers set may lead to increased photosynthetic rates. Those supporting the idea that yield is sink limited point to evidence that variation in grain yield per unit area (particularly when comparing sites and seasons, varieties, and different levels of soil fertility) is much more closely correlated with grain numbers per unit area than with mean grain weight. Other common additional evidence comes from degraining experiments; e.g., removing half of the spikelets on an ear does not double mean grain weight, even though it would seem that there should be twice as much assimilate available per grain. It might also be expected that crops are often sink limited because breeders reject lines that have shriveled grain at harvest because this is unacceptable from a quality perspective; i.e., breeders often select lines for which the sink (grain) is full by harvest (R. SylvesterBradley, personal communication). These and other observations do not in themselves prove the hypothesis of sink limitation but are nonetheless consistent with it and emphasize that grain yield is mostly a function of grain numbers. The second approach is not counter to the first but focuses on resource capture, utilization, and partitioning. That is, biomass is a function of the capture of a resource and the efficiency with which this resource leads to the production of biomass. Grain yield is then a function of biomass and the efficiency with which this biomass is partitioned to the grain, i.e., the ratio of grain yield to biomass yield, or harvest index (HI). The resource most commonly used to express yield in this way is light, or more precisely, photosynthetically active radiation (PAR). Other common resources dealt with in this, or similar, ways are water (e.g., Araus et al 2002) and nitrogen (e.g., Kindred and Gooding 2004). Research and agronomy aimed at influencing resource capture are often thought of as concentrating on the source, i.e., the ability of the environment to provide and the crop to capture and assimilate resources for yield. With regard to light, the ratio of biomass produced per unit of PAR intercepted is the radiation use efficiency (RUEPAR), so yield can be expressed: Grain yield/m 2 = PAR interception/m 2 × RUEPAR × HI

(3)

That is, except in conditions of drought and/or heat stress, the production of biomass by a wheat crop during periods of leaf production is largely a linear function of light interception. An example of this type of analysis is given in Fig. 2.5A–E. Further expansion is again possible. PAR interception depends on the PAR available and the proportion intercepted by the crop. PAR

interception can be increased, therefore, by synchronizing highproportionate interception with times of the year receiving high radiation. The proportion of PAR that is intercepted depends on canopy size, canopy architecture, and canopy longevity. Canopy size is usually expressed in terms of leaf area index (LAI, the ratio of the upper surface of leaf area to the ground area) or, more usefully for cereals, the green area index (GAI, the ratio of leaf + stem + ear green area to the ground area). The relationship between GAI and the proportion of light intercepted at a particular time (LP) depends on canopy architecture and its influence on the extinction coefficient (k) in Beer’s Law:

LP = 1 – e–k·GAI

(4)

For wheat, the extinction coefficient may commonly vary from 0.4 to 0.6, but adopting a value of 0.5 implies that a canopy of GAI = 6 would be required to intercept 95% of available light (Sylvester-Bradley et al 1997). With regard to RUE, as for many crop species (Evans 1993), a net gain of about 2.8–3.0 g of dry matter is made for every megajoule of PAR intercepted (or about 1.5 g/MJ of sunlight). For the whole cropping period, the value may drop to 2–2.5 (Fig. 2.5D) because 1) biomass is lost in leaf drop; 2) later in the season, new leaves are not produced and photosynthetic efficiency declines as leaves age; and 3) respiration per unit of assimilation increases after anthesis (Gallagher and Biscoe 1978). For autumn-sown winter wheats, whole-season RUE may be significantly less, as canopy growth can be more limited by low temperatures than by light during the early stages of development. The yield component and resource capture methods of understanding yield are complementary because often grain numbers, and possibly also potential grain size, are in balance with the resources captured. The number of grain set often reflects the amount of resources assimilated (Sinclair and Jamieson 2006); i.e., in production fields, a balance exists between sink and source. It is also obvious that all dry matter production is dependent on photosynthesis and so is ultimately source limited. There are, however, times when the balance between source and sink is disrupted; reports mostly involve artificial disruption by crop physiologists, but imbalances between source and sink in commercial production are sometimes evident. For example, it is possible occasionally to extend canopy life beyond the end of grain filling (Pepler et al 2005). Also, harsh but transient climatic conditions at anthesis can reduce grain set and yield, despite more clement conditions, adequate canopies, and high light interception being achieved during grain filling (Ferris et al 1998). It is also possible that the balance that the crop reaches between source and sink is conservative; i.e., the crop may always set fewer grains than could be potentially filled (M. P. Reynolds, personal communication). Such a strategy may have had some selective advantage for the wild progenitors of wheat, whose grain development occurred in harsh and variable conditions.

WHEAT IMPROVEMENT As we have seen, selection pressures during the process of utilization by humans and then domestication brought about very major shifts in the wheat phenotype. The wheat plant has

The Wheat Crop  continued to change via both conscious and unconscious intervention by humans throughout millennia. Such selection led to wheats becoming adapted to local environments and farming systems so that, before the advent of formal breeding, wheats from different areas could be recognized as distinct “land races.” Land races (with putative area of origin) that have been described and named, partly because of their use in subsequent breeding programs (Swaminathan 2006), include Yaroslav Emmer (former USSR), Turkey (Crimea), Fife (Eastern Europe), Daruma (Korea), Rieti (Italy), Zeeuwse White (Holland), Hard Red Calcutta (India), Etewah (India), Indian G (India), Alfredo Chaves (Brazil), and Polyssu (Brazil). Selection has been not only for adaptation to local environments and cropping systems, but also in response to demand for end-use products. Sinclair (1998) reviews evidence that grain yields in certain areas close to the Euphrates could reach 2 t/ha as early as 4,400 years ago but concludes that the average yields throughout the Fertile Crescent, then more widely through Roman times and in Europe in the Middle Ages, probably averaged less than 0.5 t/ha. This compares poorly with 0.5–0.8 t/ha in dense wild stands of cereals in Israel and suggests that actual yields of wheat remained relatively stable for centuries (Evans 1993). This does not necessarily reflect the potential of the available wheat populations or systems to produce higher grain yields. Sinclair (1998) argues that grain yields were low partly because demand for straw was great, so crops were necessarily tall, with less biomass partitioned to the grain. Demand for straw was driven by its many potential uses, mostly as bedding and feed for livestock and as thatch for roofing. More debatable is Sinclair’s contention that taller crops were better suited to situations in which nitrogen fertility was low, although such conditions did prevail during the Roman era, when much of the manure produced was used on olive trees and in vineyards rather than in cereal fields.

The Decline of Stature Wheat cropping systems and selection pressures appeared to change significantly in Europe during the 1800s. The advent of multiple-year rotations incorporating fodder crops for livestock had major effects. The systems not only helped to disrupt life cycles of weeds, pests, and diseases, but they also increased the availability of manures for wheat fields. The 1800s also saw more conscious selection, following careful observation and hybridization (Swaminathan 2006). Certainly, informal selection for disease resistance, as well as for adaptation and grain quality, were major factors in the spread of types derived from the Fife land race through North America in the nineteenth century. Lead producers of this era were keenly aware of the relative merits of different types of wheat available, and their shortcomings. Roberts (1847) recommended that on rich soils, where an abundance of straw is produced, short and stiff strawed wheat yields the best crop as the weak and long-strawed wheat is liable to be spoiled by being laid.

A Thomas Garnett wrote to the Manchester (U.K.) Guardian in 1852 (Garnett 1883) about the advantages of short-strawed wheat, suggesting that it would

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bear highly manuring without lodging, and with much less liability to mildew, than a long strawed wheat….The proportion of grain to straw is greater than in a long strawed wheat….As it very rarely lodges, it will be far better suited to the reapingmachine.

Such sentiment illustrates that input responsiveness, disease resistance, HI, and the relationship between these characters and crop height have been long appreciated by wheat farmers. Since the rediscovery of the Mendelian laws of genetics in 1900, breeding efforts to improve wheat have been more targeted and formal. Swaminathan (2006) recognizes an initial phase from 1900 to 1930 that involved selection from existing diversity and hybridization using land races. Particular targets were disease resistance (hence yield stability) and also grain quality improvement. Despite some improvement in yield stability, it appears that yield potential was not greatly increased. Indeed, until 1950, average yields in major wheat-growing areas such as the United States, India, China, and Canada remained stubbornly around or below 1 t/ha. In more temperate and moist areas, such as the United Kingdom and France, average yields were higher but relatively constant at 2.5 and 1.5 t/ha, respectively (Dixon et al 2006). Significant limitations to grain yield still appeared to be those related to excessive height, as articulated by Roberts and Garnett a century earlier. The quote from Garnett recognizes two important aspects for crop improvement related to height. The first is that of responsiveness to inputs (“bear highly manuring without lodging”), which in turn can now be expressed in terms of resource capture by the crop. As described previously, high yields are dependent on large canopies for light interception. A degree of direct proportionality between GAI and the amount of nitrogen in the canopy suggests that canopies sufficiently large to intercept 95% of available light also contain between 150 and 200 kg of N per hectare (Sylvester-Bradley et al 1997, Lemaire et al 2007). However, crops supplied with this amount of nitrogen are at greater risk of lodging. That is, nitrogen encourages the production of more, often weaker, stems, which are taller and carry heavier ears. If lodging occurs during grain filling, yields are significantly depressed, but lodging after the end of grain filling still causes major losses through reduced harvesting efficiency and impaired crop quality. Lodging susceptibility is a complex character, but a major risk factor, as noted by our nineteenth century farmers, is crop height (Berry et al 2007). Hence tall varieties have poor yields partly because lodging risk prevents the economic use of sufficient quantities of nitrogen to produce canopies large enough to intercept the majority of available light during yield formation. The question of HI is also strongly linked to crop height. Garnett’s desire for a short wheat because “the proportion of grain to straw is greater than in a long strawed wheat” counters more recent commentary that modern wheats were bred to be shorter only to prevent lodging under fertile situations and that improvements in HI have been an unforeseen benefit. The HIs of varieties released before 1900 commonly were less than 0.35, although rapid improvements from 1950 to the mid-1980s saw values approach 0.55 in U.K. wheats (Austin et al 1989). The same trend is reported for many areas of the world and in studies

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also including diploid and tetraploid wheats (as listed by Evans 1993). At a given level of nitrogen fertilization, when disease and weeds are controlled with agrochemicals and when lodging is prevented mechanically, aboveground biomass production is often similar for varieties of contrasting ages (Feil 1992); i.e., grain yield is closely, and negatively, associated with straw dry weight (Austin et al 1980). Improvements in HI associated with reduced height can, therefore, account for more than 80% of the improvement in yield potential of wheat varieties within some twentieth century breeding programs. Height is a polygenic character, but a number of major reduced height (Rht) genes have been identified. Historically these have been listed as Rht1 through Rht21, but more modern notation recognizes that several of these are different alleles at the same locus and that some correspond to more than one allele (McIntosh et al 2003). Semidwarf and dwarf denote wheat genotypes with genes that cause moderate or severe stem shortening, respectively (Fig. 2.8). Only a few of the major Rht genes have been used in world agriculture because most are associated with yield reductions; i.e., any improvements in HI or lodging resistance are insufficient to compensate for reduced biomass production. However, those Rht genes that have effected an increase in HI, mostly through improved spikelet fertility while maintaining sufficient total biomass production (Fig. 2.8), have had a hugely significant impact on worldwide wheat production. Genes that confer reduced sensitivity to gibberellic acid (GA), located on the long arms of 4B and 4D, have been particularly important. Rht-B1b (formerly Rht1) and Rht-D1b (formerly Rht2) can each reduce final crop height by about 15% (Fig. 2.8) by reducing internode length. Both of these genes derived from the Japanese variety Norin 10, which itself was a descendant of the Daruma (semidwarf) and Turkey land races. Swaminathan (2006) recounts how Norin 10 was brought from Japan by S. D. Salmon and used by O. Vogel to produce semidwarf wheats for the United States in the 1950s. Vogel supplied F2 seeds of Norin 10 × Brevor to N. Borlaug in Mexico, who, in turn, developed widely adapted, photoperiod-insensitive, rust-resistant semidwarfs that could be used in breeding programs in developing countries (Reynolds and Borlaug 2006a). The use and release of semidwarf wheat and rice underpinned the “Green Revolution,” a term coined by W. Gaud in 1968 to describe the rapid increases in yield per unit area in South Asia during the 1960s. This revolution occurred simultaneously with improved availability of and technology for crop nutrition, irrigation, and protection. It should be noted, however, that the semidwarf wheat varieties are often superior for yield compared with older, taller varieties, even in more extensive systems, because of their improved HIs (Gooding et al 1999a, Reynolds et al 1999). The Norin 10 dwarfing genes are now present in more than 90% of the world’s semidwarf wheat production (Worland et al 1998). More semidwarfing genes are found in other Japanese varieties and, although not as prevalent as those from Norin 10, possibly predate them with regard to use in Western breeding programs. A further allele on 4B, Rht-B1d (formerly Rht1S), derived from the Japanese variety Saitama 27 but causing less reduced sensitivity to GA than Rht-B1b, was incorporated into the Italian variety Orlandi in 1947 and is still found in many southern European wheats. Additional dwarfing genes have been identi-

fied as originating from a third Japanese variety, Akakomugi, introduced into European wheats by the Italian breeder Strampelli in the 1930s. Akakomugi contains the photoperiod-insensitivity allele Ppd-D1a, which shortens height by reducing life cycle, but it also contains Rht8c on 2D, an allele conferring reduced stature without reducing sensitivity to GA. Since the widespread adoption of semidwarf wheats, the rate of improvement in wheat yield has declined (Slafer et al 2001). This is reflected in the fitting of a logistic curve to the wheat (and rice) yield in Fig. 2.2B. Some caution is required, however, before concluding that yield limits are being approached. First, so-called limits have often been “identified” in the past, only to be exceeded with the adoption of new varieties or technology (Evans 1993). Sylvester-Bradley et al (2005) fitted three logistic curves to the U.K. wheat yields, each with zero mean residual for 1900–2005 yet with projected asymptotes varying

Fig. 2.8. The effect of major dwarfing genes in near-isogenic lines on the grain yield, harvest index, and aboveground crop biomass of field-grown wheat. Points are genotype means, adjusted to remove effects of site × variety background. Backgrounds are Maris Huntsman (1), Maris Widgeon (p,P), Bersee (i), April Bearded (t), Maringa (+), Nainari (x), and Mercia (s,S). Closed symbols are grown according to common commercial practice; open symbols are grown organically. t = tall (rht); s = semidwarf (Rht1-B1b, Rht1-D1b, or Rht8c); b = double semidwarf (Rht1-B1b + Rht1-D1b); d = dwarf (Rht1-B1c, Rht-D1c, or Rht12; alone or in combination with Rht1-D1b). (Data from Flintham et al 1997a, 1,p,i,t; Rajaram and van Ginkel 1996, +,x; Gooding et al 1999a, P; Addisu-Delelle et al 2007, s,S)

The Wheat Crop  between about 10 t/ha (25% above current levels) and 20 t/ha. Second, the current slowdown in yield per unit area is at least partly due to economic, environmental, and political influences associated with the intensification of wheat production. These issues, together with the increasing profitability or suitability of other crops, must also be responsible for the current reduction in wheat area (Fig. 2.2C). Large increases in wheat yield have been associated with reductions in wheat prices. If these reductions are not matched by reductions in input prices, or by increases in input responsiveness, economically optimal input use places a downward pressure on wheat yield. In some developed countries where, in the past, price support has led to production greatly exceeding demand, not only have subsidies been removed from production, but they have instead been used to favor less-intensive systems. Third, the world average wheat yield does not approach the 10 t/ha already common in commercial production under moist, temperate, long-season conditions; i.e., yield limitation has as much to do with technological factors, such as the availability and use of irrigation and nutrients, and with the duration of the wheat cropping season as with the innate potential of existing germplasm. Sylvester-Bradley et al (2005) make the point that previous yield increases have been higher in Western Europe than in the United States, Canada, and Australia because yields were more constrained by light interception in the former but more by water availability in the latter. As already described, where water is not a major limitation, light interception can be increased by increasing the availability of nitrogen, subject to varieties being lodging-resistant and crop protection measures being sufficient. One of the major factors leading to large yield increases in India and China was the extension of lands under irrigation. Notwithstanding the above arguments, many commentators have suggested that the more gradual recent improvement in wheat yield potential is at least partly responsible for the current slowdown in the rate of yield increase. Advances in yield potential are unlikely to derive from further reductions in crop stature and associated increases in HI (Austin 1999). Flintham et al (1997a) compared near-isogenic lines with and without different combinations of alleles conferring varying insensitivity to GA and concluded that, under U.K. conditions, optimum height was likely to be around 0.8 m (Fig. 2.8), with an HI of 50–60%. Austin et al (1980) calculated a theoretical maximum HI of 60%. The use of dwarfing genes (as opposed to semidwarfing genes), such as Rht-B1c (previously Rht3), has not been successful (Swaminathan 2006). As plants become shorter and thus canopy density increases, it has been anticipated that efficiency of light interception and use is likely to decline, as would resistance to some leaf diseases. Partitioning efficiency and HI also decline as dwarfism becomes extreme, particularly if late competition from weeds hinders grain filling (Fig. 2.8). Shorter varieties and isogenic lines containing dwarfing genes are often less tolerant of weeds than are taller genotypes (Cosser et al 1996; Fig. 2.9). Shorter varieties with reduced GA sensitivity can also have reduced biomass and nitrogen accumulation before anthesis (Austin et al 1980, Feil 1992). Reduced GA sensitivity is associated with reduced coleoptile length and hence impaired ability to establish from depth, a character particularly relevant to deep sowing in drier areas (Rebetzke et al 2007).

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The Increase of Biomass The preceding discussion highlights the need to identify mechanisms by which yields of wheat might be increased in ways unrelated to reduced height and increased HI (Reynolds et al 1999). As already discussed, yield can be increased by increasing crop biomass as long as there are not commensurate reductions in HI. Even accepting a value of 80% (Austin et al 1989) for the contribution of HI increases to yield improvements, the implication is that 20% is due to increased aboveground biomass. This increase in biomass has been demonstrated within a number of breeding programs (e.g., Reynolds et al 1999), and, within some of these, the contribution of increased biomass to yield improvement is significantly greater than 20% (see Evans 1993 for examples). INCREASED LIGHT INTERCEPTION

Increased biomass production must necessarily derive either from increased light interception and/or increased RUE. We have already seen that modern varieties are capable with sufficient resources, particularly of nitrogen and water, to grow canopies that can intercept near-asymptotic proportions of available light without lodging. To maximize production, canopy size is especially important when photosynthesis is not constrained by cold temperatures, drought, or heat stress. It has been further argued that canopies that maximize light interception should be in place at least by anthesis, and then be maintained subsequently during grain growth (Sylvester-Bradley et al 1997). It is possible to increase light interception by having earlier canopy formation or longer-lasting canopies. The variation among genotypes in rates of canopy formation before anthesis is often associated with speed of emergence and seedling vigor. There remains a challenge, however, to increase early-season growth and biomass before anthesis while maintaining lodging resistance and HI. Additionally, excessive early canopy growth may result in wastage if lower leaves and tillers are shaded and shed. Prolific early vegetation may also increase infestation and infection pressures from pests and diseases. Also, early light interception can already be attained by agronomic manipulation of, for example, seeding rate, sowing date, and fertilization.

Fig. 2.9. Prevalence of weeds in near-isogenic lines of cv. Mercia differing in dwarfing genes following a three-year clover-rich grassland with no added fertilizer or crop-protection chemicals. 0, 1, 2, 3, 8, 10, and 12 correspond to rht, Rht1-B1b, Rht1-D1b, Rht1-B1c, Rht8c, RhtD1c, and Rht12, respectively.

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With regard to delaying canopy senescence, there are climatic and rotational limits to how long crops can be kept green. In the United Kingdom, the use of long-lived wheats, together with robust fungicide programs, has seen widespread application of nonselective late-season herbicides to desiccate crops and hence curtail canopy life. It also appears possible to delay senescence beyond the end of grain filling (Pepler et al 2005). Thus, without genetic advances to prolong grain filling, extra canopy longevity is of little benefit. Overall, there appear to be some possibilities for increasing wheat yields by extending the period of canopy closure and hence increase light interception and therefore biomass yields. It would seem, however, that the approach is likely to derive benefits through fine-tuning canopy development and senescence to particular production systems and environments rather than through large single steps in yield potential. Sylvester-Bradley et al (2005) give more details of opportunities and constraints for this approach and a possibly more optimistic view, but LAI and light extinction coefficient are not often closely correlated with progress in yield potential in wheat (Calderini et al 1997, Shearman et al 2005). INCREASED RADIATION USE EFFICIENCY

The second way of improving biomass yields is to increase RUE. Modification of canopy architecture offers some potential for achieving this. For example, in dense canopies under high irradiance, there can be some benefit in upper leaves being more erectophile, such that they are less likely to become light saturated while shading lower leaves. Feil (1992) reviews comparisons between old and modern cereal varieties and summarizes numerous changes to the canopies over time, including increased leaf erectness, increased size of upper leaves, and altered shape of leaves. Such changes were not, however, universal. Trends in changes in canopy architecture in different countries were often contradictory and were not conclusively associated with improved biomass production. Another approach to increasing RUE has centered around the argument as to whether RUE is in some way dependent on demand for assimilate, particularly by the developing florets and grain after anthesis. For example, is it possible, by increasing grain numbers, to maintain RUE closer to 2.8 g/MJ rather than, as observed (Gallagher and Biscoe 1978, Calderini et al 1997), having it decline after ear emergence? Of some promise have been associations with the leaf rust resistance gene (Lr19), transferred to wheat as part of a chromosome segment from Thinopyrum ponticum (syn. Agropyron elongatum) following irradiation with X-rays and thermal neutrons in the 1960s. The translocation, denoted 7DL.7Ag, also appears to confer improved yield potential, associated with increased postanthesis biomass production, photosynthetic rates in the flag leaf, and grain numbers (Singh et al 1998, Reynolds et al 2001). As already discussed, it has long been recognized that grain yields are much more closely related to grain number than to mean grain weight (Peltonen-Sainio et al 2007). Slafer et al (2001) reflect that grain numbers are often most closely related to assimilate supply between terminal spikelet initiation and anthesis, i.e., during stem elongation. As we have seen, however, it is

difficult to increase light interception during this period because it can already be high. Increases in RUE may accrue from improved canopy architecture, but again RUE is usually relatively high during this period. Alternatively, Slafer et al (2001) suggest that the duration of stem elongation could be extended. There are, however, further difficulties with this approach. In certain environments, earlier times of terminal spikelet formation may increase damage from frosts or reduce tillering, while later anthesis could reduce grain filling durations, as many crops mature in a terminal drought. Austin (1999) observes that earlier, not later, flowering contributed to yield gains in modern U.K. wheats because it was associated with prolonged grain filling. Genotypes do exist with supernumerary spikes with long or branched ears, but these have as yet generally failed to produce the anticipated yield improvements because the high grain numbers set per spike have been associated with reduced tiller numbers and/or mean grain weights (Swaminathan 2006, Gaju 2007). Sinclair and Jamieson (2006) argue that grain numbers are a consequence of resource availability, rather than a determinant of yield; i.e., the plant forms a grain population as a consequence of resources available and accumulated. There are numerous examples in which spike or grain removal has reduced photosynthetic rates, but Sinclair and Jamieson (2006) claim that the opposite has not been proven, i.e., that increases in grain number result in improved RUE. Nonetheless, for whatever reason, it does appear that modern varieties of wheat can have higher photosynthetic rates in flag leaves at the end of the season compared with their predecessors, and this remains a target for some potential yield improvement (Calderini et al 1997, Austin 1999, Reynolds et al 2001, Sylvester-Bradley et al 2005). Given that major advances in yield potential by increasing HI, light interception, canopy architecture, and/or grain numbers are difficult to envisage, some commentators have claimed that real breakthroughs in yield potential can arise only from increasing the efficiency of photosynthesis at the molecular level. Parry et al (2007) review the opportunities in wheat for increasing the efficiency of ribulose-1,5,-bisphosphate carboxylase/ oxygenase (RUBISCO). RUBISCO catalyzes the assimilation of CO2 into organic compounds via carboxylase activity, but it is deemed inefficient because the rate of assimilation is low and also because oxygenase activity leads to photorespiration and, depending on conditions, the loss of about one third of the carbon fixed. Parry et al (2007) claim that RUE could be improved by increasing the abundance of the enzyme; by improving the catalytic rate of RUBISCO; by increasing the enzyme’s ability to discriminate between CO2 and O2 in favor of the former (i.e., increasing the “specificity factor”); and/or by increasing the concentrations of CO2 and ribulose-1,5,-bisphosphate at the active site of the enzyme. There appears to be some natural variation in some of these characteristics that might be exploited in breeding programs and, despite regulation being complex and polygenic, there could eventually be some benefit from approaches using genetic transformation. HYBRIDS AND HETEROSIS

Commercial varieties of wheat are generally inbred lines and, as such, yield benefits may accrue through hybrid vigor, or het-

The Wheat Crop  erosis, when two parents are crossed. Such heterosis is widely used in commercial production of maize and also in certain areas of rice production but has been slow to be exploited in wheat. A number of commentators have mentioned the potential of heterosis to achieve future yield gains in wheat (Austin 1999, Rajaram 2001, Swaminathan 2006). Heterosis for grain yield can certainly occur in F1 hybrids of wheat and be exhibited as an increase in biomass (through combining high light interception and RUE) and, more rarely, an increase in HI (Kindred and Gooding 2005). However, although wheat hybrids have been commercially available in certain countries since the mid-1970s, until recently, hybrid wheat never occupied more than 3% of any national wheat area, a comparative failure that reflects a number of difficulties. Commercial hybrid wheat systems require that self-fertilization in one of the parents be prevented, either by exploiting inherited male sterility (cytoplasmic or nuclear) or by spraying chemical hybridizing agents (CHAs). Unfortunately, wheat is a poor pollinator for cross-fertilization, and seed set can be heavily dependent on weather conditions and variety. Selecting parents with good potential for cross-fertilization can severely limit the number of candidate varieties, while combining parents for greatest heterotic effect on yield often compromises end-use quality. In addition, heterosis in wheat appears to be relatively low, averaging only 5%, and may be even less with crosses involving the highest-yielding parents (Morgan et al 1989, Kindred and Gooding 2005). An explanation for low levels of heterosis is that much of the effect derives from dominance, rather than over-dominance, i.e., by the masking of unfavorable genes in heterozygotes, rather than by the presence of more than one allele at a locus. As wheat is an allopolyploid, it should exhibit some intragenomic heterosis; i.e., wheat could be regarded as a “fixed heterozygote.” It is, for example, possible to develop inbred lines with yield equal or superior to that of the F1 hybrids from which they were selected. The heterotic effect of some hybrids may, therefore, represent only a temporary improvement over the best available inbred lines, and this is further compromised if the choice of parents is constrained by considerations of crossfertilizing potential. Despite these difficulties, some evidence of over-dominance and/or favored intermediate phenotypes that cannot be achieved in inbred lines has been demonstrated for some wheat hybrids (e.g., Flintham et al 1997b). Occasionally, even by using two high-yielding parents, useful levels of heterosis can be achieved (Morgan et al 1989). Additionally, the development of improved CHAs in the 1990s has led to some local successes; for example, hybrid wheats in France contributed an estimated 5% of total wheat production in 1998.

Resistance to Disease Oerke (2006) estimates that fungal pathogens of wheat cause about 10% yield loss worldwide, a figure that would rise to 15% without targeted crop protection measures. Foliar diseases of particular note in breeding programs, and of importance in the major wheat-growing areas, include powdery mildew (Erysiphe graminis DC. f. sp. tritici E. Marchel), Septoria tritici Rob. in Desm. (perfect stage, Mycosphaerella graminicola), Septoria nodorum Berk. (perfect stage, Phaeosphaeria [syn. Leptosphaera] nodorum), rusts (Puccinia spp.), and tan spot (syn. yellow leaf spot, Pyrenophora

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tritici-repentis; asexual stage, Drechslera ­tritici-repentis). These fungal pathogens are particularly prevalent in high-yielding areas (Oerke 2006) with increased intensity of production. The need to develop resistant strains has therefore been a requirement parallel to that of increasing yield potential and responsiveness to inputs. Much early work identified major genes for resistance to the obligate pathogens (powdery mildew and rusts). Unfortunately, these diseases evolve rapidly, such that single-gene, race-specific resistance may typically last for only three to five years (Reynolds and Borlaug 2006a). Longer-lasting resistance can be achieved by combining genes that each confer only partial resistance and then by combining partial-resistance genes with major genes. A potentially important source of resistance genes is wild relatives or closely related species of wheat. Unfortunately, due to the transfer of undesirable characters, only a few of these genes have been used in the development of commercial varieties. Exceptionally, wide crosses for improved disease resistance have sometimes also appeared to increase yield potential, so germplasm of related species has become more prevalent in world wheat production. Of particular importance were crosses made in Germany between wheat and a variety of rye (Pektus) in the 1930s (Schlegel and Korzun 1997). This program generated a chromosome composed of the long arm of 1B from wheat and the short arm of the homeologous chromosome from rye, the so-called 1BL.1RS translocation. 1RS from Pektus carries race-specific resistance genes Lr26, Sr31, Yr9, and Pm8 against leaf rust (P. recondita), stem rust (P. graminis), yellow rust (P. striiformis), and powdery mildew, respectively. Other reported effects, depending on wheat background, include stem shortening, increased aboveground biomass, an increase in ear population, increased drought tolerance, better phosphorus extraction, heavier mean grain weights, improved spikelet fertility, and delayed heading and maturity (Villareal et al 1998, Rajaram 2001). Germplasm containing the 1BL.1RS translocation has been used in wheat breeding programs throughout the world, and, by 1998, it was estimated that more than 5 million hectares were cultivated with wheats with 1RS. The greater use of this source, however, may be limited. The resistance genes on 1RS are race-specific and hence liable to break down, as has already occurred in a number of areas (Villareal et al 1998). Additionally, the presence of 1RS is often associated with poor breadmaking quality.

Tolerance of Abiotic Stresses Wheat is subject to major abiotic stresses in significant areas of production; these include acidity, salinity, drought, heat stress, and nutrient deficiencies. There is also interest in overcoming stresses in nontraditional wheat-growing areas, where demand for wheat-based products has increased. This is not the place to review breeding strategies to overcome all stresses, but a notable success that can be mentioned is tolerance of acid soils. Soil acidity (pH < 5.5) reduces yield, partly due to the release of toxic concentrations of aluminum. Major genes have been identified that are associated with both exclusion of Al from the plant and tolerance of internal concentrations of Al. The relatively simple inheritance of Al tolerance has, therefore, facilitated the selection of high-yielding wheats for acid soils in, for example, Brazil (Reynolds and Borlaug 2006a).

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Breeding for drought tolerance or escape are genetically more complex. Desired traits are likely to be polygenic. The health of root systems becomes more important with drought severity; thus, selection for tolerance to water shortage can be complicated by genotypic variations in resistance to soilborne pests and diseases. The timing and severity of droughts vary greatly, as does the interaction with tolerance to heat stress. The severest type of drought is when rainfall during the growing season is negligible; i.e., the crop has access to residual water available only at emergence, such that water stress increases through time as the soil water is depleted. This situation is representative of major wheat-growing areas in India and Australia, where average yields of wheat are only 1 t/ha. In contrast, a typical Mediterranean environment may have significant rainfall until the start of stem extension, after which lower water receipts and increased temperatures generate significant soil moisture deficits and a postanthesis moisture stress environment. Richards (2006) lists a number of physiological traits that could form the basis of selection to enhance wheat yields through either improved water capture, use-efficiency, or escape. Selection could 1) modify the crop’s growing duration and its development to better match rainfall, increase root growth, and better exploit comparatively drought-resistant growth stages; 2) reduce the use of water in the early growth stages, for example, by selecting for narrow xylem vessels in the seminal roots, such that more stored water remains during grain filling; 3) improve transpiration efficiency (TE), i.e., photosynthesis per unit of transpiration, using carbon-isotope discrimination (plants discriminate against 13C, but less so when TE is high); or 4) improve osmoregulation, i.e., the ability of cells to maintain turgidity and function when leaf water potential is low. Additionally, Olivares-Villegas et al (2007) report significant negative correlations between canopy temperature and yield in the progeny of a wheat cross under drought, and they presumed this resulted from association with improved ability to extract water from the soil.

Modern Breeding Methods Traditional wheat improvement relies on pedigree breeding, whereby initial hand-crossing produces heterozygotes, which are then selfed for up to eight generations to increase homozygosity and make selections. Selection, however, is often less efficient in the early generations because heterozygosity complicates identification of suitable genotypes. Selection is generally based on morphology, disease reaction, and yield components, although some biochemical selection (e.g., for HMW-glutenin subunit composition as separated on electrophoretic gel systems) has been adopted in several commercial breeding programs. A more recent possibility is selection for DNA markers, linked or tagged, to single-gene traits (such as major disease resistance genes) or polygenic traits identified in chromosomal maps as QTLs. Marker technology is progressing rapidly, and longer-standing techniques such as restriction fragment length polymorphisms, polymerase chain reaction-based markers, and sequence tagged microsatellite markers are being significantly augmented with single nucleotide polymorphisms (Koebner and Summers 2003). The last decade has seen increased use of techniques to produce double haploids, usually from the first generation of

a cross (F1). This alleviates the complication of heterozygosity in the early generations of traditional systems. The most popular method is to fertilize the F1 plants with maize pollen, after which the maize chromosomes are eliminated to produce a haploid wheat embryo. Auxin treatment and then plating media are used to maintain embryo development, then plantlet development, until chromosome numbers are later doubled with colchicine treatment. This procedure allows the production of homozygous lines from the F1 and thus has the potential to increase the rate of variety selection and release. The technique also increases the efficiency of selection based on molecular markers, facilitates wide crossing between different species, and assists in improvements based on genetic transformation (Mujeeb-Kazi et al 2006a). With regard to wide crosses, intergeneric hybidization of wheat with related species has been increasing used. In particular, different accessions of Ae. tauschii have been combined with T. turgidum to produce synthetic hexaploids. This increases the genetic base for hexaploid wheat breeding because, although more than 250 accessions of Ae. tauschii are known, it is thought that very few have been involved in the evolution of common wheat through donation of the D genome. A wide range of resistances are attributed to Ae. tauschii, possibly deriving from the diverse habitats from which the numerous accessions of Ae. tauschii originate. These resistances can be transferred to synthetic hexaploids. They include resistance to disease (e.g., Mujeeb-Kazi et al 2006b) and to abiotic factors (cold, drought, salinity, and waterlogging; e.g., Reynolds et al 2005). The technique also has the benefit that the addition of the alternative D genomes leaves the tetraploid wheat genome intact, so there should be less disruption of nontarget traits than with crosses involving wild species of higher ploidy. There is also potential for intergenomic heterosis if novel alleles introgressed into the D genome interact positively with alleles at equivalent loci in the A and B genomes (Gill and Raupp 1987). The genetic transformation of wheat has lagged behind that of other important crops, and it was not until 1992 that the first reliable production of fertile transgenic wheat was reported (Vasil et al 1992). This transformation relied on the bombardment of cells derived from immature embryos with tungsten or gold particles coated with DNA to confer herbicide resistance. Further transformations have been achieved with particle bombardment, but Agrobacterium-mediated systems have also been developed (Hu et al 2003), and these have contributed to the development of wheat plants tolerant to herbicide. At the time of writing, release and use of genetically transformed wheat remains a politically sensitive issue in many parts of the world. However, Reynolds and Borlaug (2006b) review the potential of this technology for human benefit. In addition to herbicide tolerance, wheats with transformations for modified storage proteins, increased disease resistance, improved water-use efficiency, and drought tolerance are at various states of readiness for commercial exploitation should the political environment change.

AGRONOMY OF WHEAT This chapter does not attempt to give a full analysis and justification of all agronomic inputs to wheat, but aspects par-

The Wheat Crop  ticularly relevant to issues already raised in the analysis of yield and wheat improvement are worthy of mention. Discussion is restricted to the establishment, nutrition, and control of foliar fungal pathogens.

Sowing SEEDBED AND SOWING METHOD

Wheat can be established in soil prepared in diverse ways. More aggressive systems involve full soil inversion with moldboard plows or heavy disking, followed by secondary cultivation with various types of tines, harrows, and/or disks. At the other extreme are direct-drilling (zero-tillage) methods that place the seed in a slot formed in the soil, which is otherwise undisturbed since the harvest of the previous crop. The method of land preparation depends on soil type; timing and time available; rainfall; cost and availability of equipment, labor, and fuel; soil erosion risk; and the control of weeds, pests, and diseases. Plowing has traditionally been employed for weed control and trash burial and to render the soil suitable for the use of other implements to produce a suitable tillage (tilth). Ideally, the tillage should be fine enough to give intimate contact between the seed and soil to facilitate water imbibition for germination and to assist in the accurate placement of seed at optimum depth. Plowing and secondary cultivation relieves surface compaction, and the associated disturbance contributes to soil aeration. As well as aiding root growth, soil movement and ventilation may result in a flush of nutrients, particularly nitrate, because of increased microbial activity. Seedbeds with soil aggregates 2–3 and 1–2 mm in loam and clay soils, respectively, have been quoted as optimal, but establishment from seedbeds that are much more lumpy (cloddy) is possible, and often preferable, if there is a risk of the soil forming a crust or eroding. Despite the long-assumed benefits of plow-based systems, in many wheat-producing areas there has been a shift toward more minimal cultivation techniques, particularly as machinery has developed to allow savings of labor and time. Such techniques have also facilitated wheat production on soils too wet, heavy, or shallow to permit annual plowing. To varying degrees, minimal cultivation reduces the depth of cultivation and the degree of soil disturbance, the amount of nitrate released and at risk from leaching, the amount of organic matter breakdown and carbon dioxide released to the atmosphere, the evaporation of moisture, and soil erosion. Reduced cultivation systems, however, also favor grass, weeds, and slugs and therefore often increase reliance on graminicides and molluscicides. Sowing depths for wheat commonly range between 25 and 100 mm. The optimum depth for temperate and moist conditions is about 30 mm, but deeper planting is favored in drier conditions or where predation pressure is high or to avoid phytotoxic effects of some residual herbicides (Rebetzke et al 2007). Wheat can be sown through drills of various types or by broadcasting (through equipment ranging from hand-held seed spreaders to large pneumatic fertilizer spreaders) followed by light harrowing. The former gives greater control of depth and spacing between rows (commonly 100–200 mm apart), but the latter is more rapid and allows sowing on wetter soils or on rough terrain.

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SEED TREATMENTS

Before sowing, wheat seed is commonly treated with fungicide and/or insecticide. These are usually formulated as flowable concentrates or solutions and sprayed on or mixed with the seed. Cheaper, protectant fungicides are commonly used to “disinfect” the surface of the seed of peripheral pathogens such as bunt (Tilletia tritici) and Fusarium spp. Fungicides showing varying degrees of systemicity can control these pathogens, as well as loose smut (Ustilago nuda), and/or can protect the young seedling from foliar pathogens such as Septoria spp. and powdery mildew. Modern fungicides in this category come from the carboxamide, triazole, cyanopyrole, and guanidine groups. The development of a benzamide fungicide (silthofam) has allowed useful control of take-all (Gaeumannomyces graminis var. tritici) when applied as a seed treatment. The use of a neonicotinoid insecticide (imidacloprid) as a seed treatment can reduce damage from wireworms (e.g., Agriotes spp. and Cternicera spp.) and virus transmission by aphids. SEED RATES

The amount of seed sown per unit area is governed by several factors. Agronomic advice often revolves around the establishment of a target number of plants. The weight of grain needed to do this depends on the mean grain weight and the proportion of seeds expected to produce mature plants. Seed rates should therefore be increased as the expected establishment rate declines. Higher seed rates are required for poorer seedbeds and for grain of reduced germination capacity and vigor. Field emergence is always lower than germination in laboratory tests, and this difference is amplified as laboratory germination decreases or as field conditions deteriorate (Khah et al 1986). Seed rates (kg/ ha) should also increase with mean grain weight, although many small grains may also reflect poor seedling vigor. Seed size is often positively correlated with early seedling development and biomass. In good growing conditions, the benefits of increased seed size are comparatively small and disappear as development progresses. Rapid establishment and early-season vigor, however, are useful traits for improving yields in more marginal and drought-stressed environments or where weed pressure is high. Establishing higher numbers of plants increases the amount of light interception (Fig. 2.5E) and crop competitiveness, particularly in the early growth stages of the crop. Negative relationships between sowing rate and subsequent weed infestations are common (Fig. 2.10). However, there are a number of attendant risks. Dense crops often have increased susceptibility to diseases such as powdery mildew and Septoria spp. Interplant competition may also lead to weaker, taller stems susceptible to lodging. When moisture resources are scarce, drought problems may be exacerbated as the increased leaf area leads to greater evapotranspiration. Evidence is contradictory as to whether wheat must be sown thinly under semiarid conditions to avoid subsequent crop failure (Gooding and Davies 1997), but target populations are less when yield is limited by the availability of resources such as moisture and/or nitrogen (Fig. 2.11). Increasing seed rate has financial implications, but commentators also stress the relative cheapness of seed when the grower is faced with potentially higher costs of poor establishment in

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adverse circumstances resulting from false seed economies. Lowering seed rates places greater reliance on using compensatory mechanisms, mostly by increasing the production and survival of tillers (Fig. 2.5F) to form ears, less consistently by increasing the number of grains per ear, and, more rarely still, by increasing mean grain weight (Gooding et al 2002). Plants with a greater chance to tiller can therefore be sown at reduced seed rates. Earlysown winter crops can be sown at lower seed rates than later sowings. Similarly, spring wheats are often sown at higher rates than autumn wheats. Excessive reliance on compensatory growth can lead to several problems. The importance of the survival of individual plants is increased, such that pest attacks in the seedling stage that kill the growing points of only a relatively few plants could leave large gaps, resulting in poor resource utilization. Prodigious tillering resulting from reduced seed rates may also be the cause of variable and delayed maturation, making the crop uneven and difficult to manage and harvest.

Reductions in biomass per plant as plant population density increases result in a progressive decline in the dry matter yield response to seed rate. Typically, therefore, for wheat in fertile and moist conditions, when there is good weed and disease control and minimal lodging, biomass yield per unit area increases asymptotically with increase in plant population density (Ellis et al 1999), such that:

Yb =

ρ

ab + bb ρ



(5)

where Yb is biomass yield (g/m2), ρ is plant population density (plants per square meter), and ab and bb are constants. This was first derived mathematically by Shinozaki and Kira (1956) and experimentally by Holliday (1960) to describe yield of crop biomass (Fig. 2.5C). Given the relationship between biomass yield and PAR interception, it is not unreasonable to expect that a similar model should be adequate to describe radiation interception (eq. 6; Fig. 2.5E).

Ypar =

ρ

 apar + bpar ρ

(6)

When grain yield (Yg) declines at high densities, for example, if HI also declines (Fig. 2.5B) or lodging and disease pressures increase, the response can follow a parabola (Fig. 2.5A), as described by Bleasdale (1984), thus: 1

Yg = ρ

 1 θ ag + bg ρ 

(7)

SOWING DATE

Fig. 2.10. Effect of sowing density of Malacca winter wheat on aboveground weed biomass at anthesis. The crop was fertilized with N (100 kg/ha) at stem extension but received no herbicide. The fitted curve is exponential. The vertical bar is standard error of difference. DM = dry matter.

Fig. 2.11. Effect of sowing density and nitrogen on yield of Hereward winter wheat. ! = no nitrogen applied, 1 = average of plots receiving N (200 and 350 kg/ha) at the start of stem extension. Fitted with eq. 7 (see text). (Data from Gooding et al 2002)

The sowing date is largely governed by climate and the requirements of a rotation. In general, the higher the latitude, the cooler the summer temperatures, the longer the potential cropping period, the earlier the drilling of autumn-sown crops. In areas of Canada and in northern regions of Europe, the United States, Russia, and Japan, much winter wheat is sown in August and September and spring wheat in April and May. In Western Europe, winter wheat is mostly sown in late September and Oc­ tober and harvested the following July and August. In hotter climates, cropping is restricted to the cooler winter months. In such circumstances, sowing date is critical to making sure that the reproductive phase coincides with the coldest part of the season and the total growing season is restricted to just 90–100 days. Similarly, in South and East Asia, the sowing of wheat is often within rice rotations, such that wheat can be planted in the cooler, drier season between November and March, when land would otherwise be fallow from rice.

Nutrition Wheat growth requires at least 17 elements, which are classed as either macro- (major) or micro- (trace) nutrients. Crop demands for macronutrients are on the order of kilograms per tonne, compared with grams per tonne for micronutrients. Carbon (C), hydrogen (H), and oxygen (O) are macronutrients obtained from the atmosphere and water. Crop requirements most frequently met by fertilizer applications are for nitrogen (N),

The Wheat Crop  phosphorus (P), potassium (K), and sulfur (S). Other macronutrients include calcium (Ca) and magnesium (Mg). Calcium is not normally considered to be a nutrient that requires application because large amounts are often applied in liming material to correct for soil acidity. Micronutrients include copper (Cu), manganese (Mn), iron (Fe), boron (B), sodium (Na), chlorine (Cl), molybdenum (Mo),

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and zinc (Zn). Several of these are toxic at higher rates. Most growers rely on natural nutrient cycling from organic and rock material to satisfy micronutrient demand, but deficiencies of one or several are more likely on specific soil types under certain conditions, which may warrant application. As with macronutrients, deficiencies are more likely to occur with the adoption of high-yielding varieties and intensive growing systems due to the higher rates of removal from the soil over successive seasons and increased demands at peak growth periods. As a result, productivity that is more disappointing than predicted has often occurred in connection with long-term depletion of essential nutrients (e.g., Nambiar 1991). NITROGEN

Fig. 2.12. Effect of nitrogen applied at the start of stem extension on light interception, grain numbers, grain yield, and protein concentration of rain-fed Hereward winter wheat grown on sandy loam soil in the United Kingdom. PAR = photosynthetically active radiation. DM = dry matter. (Adapted from Clarke 2002)

Nitrogen, mostly applied as urea (CO(NH2)2) or ammonium nitrate (NH4NO3), is usually the most important fertilizer element determining the productivity of wheat. Nitrogen is a major component of proteins and therefore of enzymes and nucleic acids. In particular, during leaf production, more than 50% of the nitrogen in the plant can be in RUBISCO. Making more nitrogen available to deficient plants increases chlorophyll contents and leaf greenness (particularly of the older leaves), increases leaf size, delays senescence, stimulates tillering, increases height, and boosts grain site formation. Nitrogen is therefore a key determinant of canopy size, and thus light capture, as well as of the numbers of grain set per unit area (Fig. 2.12). As previously discussed, much of the grain yield response to nitrogen can be explained on the basis of effects on light interception. Hence, the asymptotic response of light interception to canopy size (eq. 4) and a near-linear relationship between canopy size and canopy nitrogen (Lemaire et al 2007) would contribute to an asymptotic effect of nitrogen availability on grain yield. This is often approximately the case (Fig. 2.12), but the response of yield to nitrogen is further modified by other aspects of light interception, such as changes in k and light availability during the season, effects on HI, nitrogen uptake as it is influenced particularly by soil and climatic conditions and also as efficiency of uptake is reduced as nitrogen availability increases, and associated negative effects of nitrogen such as heightened risks of frost damage, lodging, foliar disease, and delayed crop maturation (Fig. 2.13). Asymptotic

Fig. 2.13. Effect of nitrogen fertilizer applied at the start of stem extension on the grain yield, lodging, and disease of Hereward winter wheat (i, semidwarf variety [Rht1-D1b] of Triticum aestivum subsp. aestivum), Maris Widgeon (p, tall variety of T. aestivum subsp. aestivum), and Hercule spelt (1, T. aestivum subsp. spelta). (Adapted from Gandee 2001)

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models are therefore generally inadequate for describing the full response to nitrogen availability as levels pass from deficient to excessive for optimizing yield, or more importantly, financial margin. Alternatively, parsimonious empirical approaches have often found linear plus exponential (Figs. 2.12 and 2.13; Foulkes et al 1998) or polynomial models (Fig. 2.14; Holbrook et al 1983) sufficient to describe grain yield responses to nitrogen fertilizer applications. Understanding and predicting the amount of nitrogen fertilizer to apply to crops is complicated, not only because of the difficulty in predicting the positive and negative influences described above, but also in accounting for nitrogen made available from other sources, such as organic matter and previous cropping; nitrogen losses through immobilization, denitrification of N2, N2O, NO, or NO2 gas, volatilization of ammonia, and leaching of nitrate; effects of nitrogen on crop quality, particularly grain protein concentration (Fig. 2.12); limitations to responses due to other deficiencies, particularly water (Fig. 2.14); the health and activity of the root system and soil environment; and the price ratio of nitrogen fertilizer to the value of grain. Previous cropping, manure applications, and tillage systems can all have significant impacts upon soil organic matter and nitrogen release (Johnston and Poulton 2005). They also have additional effects on nitrogen availability and uptake through direct influences on mineral N contents, soil structure, and crop health. For example, preceding a wheat crop with a legume with symbiotic nitrogen fixation can decrease the economically optimal nitrogen fertilizer requirement for the wheat. Similar effects can be achieved with non-legume break crops, such as oilseed rape (Brassica napus), sunflower (Helianthus annuus), and potatoes (Solanum tuberosum L.), which are less exhaustive than wheat and may therefore leave a greater amount of applied and/

Fig. 2.14. Effect of nitrogen fertilizer applied at the start of stem extension on yield of rain-fed winter wheat on shallow soil over limestone in a cool, wet summer (1, May to July rainfall = 195 mm, mean temperature = 12.9°C) and a warm, dry summer (p, rainfall = 95 mm, mean temperature = 15.8°C). DM = dry matter. (Adapted from Gooding and Davies 1997)

or mineralized nitrogen in the soil for use by the following crop, as well as improving soil structure and breaking disease cycles (Gooding and Davies 1997). Agronomic advice with regard to nitrogen fertilizer applications to wheat is often informed by variety, previous cropping, manure applications, soil type, and rainfall (e.g., DEFRA 2000) because these are key determinants of nitrogen availability and wheat responsiveness to application. Some improvement to precision can be achieved by accounting for available nitrogen before application (Lobell et al 2004). In arid environments, and/ or for spring wheat, soil analysis of mineral nitrogen at sowing can be useful, as subsequent leaching may be negligible. For autumn-sown wheat, more-relevant soil analysis can be made in the spring, just before the period of rapid growth, when available N is the soil mineral nitrogen plus the crop nitrogen at the time of measurement. Further strategies have relied on periodic tests of sap nitrogen or leaf color, an evaluation of canopy size or light interception, comparison with test strips of crop given a surfeit of nitrogen, and/or previous experience of yield potential and grain protein concentration (Sylvester-Bradley et al 1997, Gooding et al 1999b, Ortiz-Monasterio and Raun 2007). The risks of nitrogen loss are reduced if timing of fertilizer application coincides with the onset of high crop demand, i.e., the start of stem elongation through to flag leaf emergence. Dressings before the reproductive stage can be taken up by the crop but are at risk of stimulating excessive tillering in autumn-sown wheats. Relatively small amounts of N (20–40 kg/ ha) can, however, stimulate tillering of poorly established and/ or backward crops. For spring wheat, or short-season wheat in hot environments, most, if not all, nitrogen can be applied to the seedbed or during the vegetative phase because crop development is rapid; long periods of slow growth are less likely; and leaching risk is diminished. Even for winter wheat, applications in the autumn may be suitable if the total crop requirement is low and leaching risk is small due to aridity. Sander et al (1987), for instance, cited work indicating that autumn applications of nitrogen were generally as effective at improving yield as spring applications to winter wheat grown in the central United States. In addition to yield, nitrogen applications commonly increase the grain protein concentration. Indeed, grain protein concentration continues to increase at fertilizer rates that are above optimal for yield (Fig. 2.12), but the response of protein concentration still becomes asymptotic at high application rates (Gooding 2005). Delaying nitrogen application beyond the second node detectable growth stage often reduces the yield response, but it can increase the response of grain protein concentration (Finney et al 1957). The precise timing for optimum effect on protein concentration, however, has varied with different systems. There is some evidence that, when water availability is low at and after anthesis, or the crop senesces early for other reasons, the greatest protein responses are from applications at anthesis (Finney et al 1957). When nutrient uptake occurs later into the season due to extended leaf and root life with sufficient soil moisture, however, applications as late as the milky ripe stage can be particularly beneficial (Gooding and Davies 1992). At timings as late as this, however, root activity might be expected to be impaired, and greater nitrogen uptake may be achieved through application

The Wheat Crop  to the foliage as a solution of urea (Gooding and Davies 1992). Typical amounts of nitrogen supplied as late-season foliar urea, specifically for increasing grain protein concentration, vary between 30 and 50 kg/ha. It should be remembered, however, that roots in some systems continue to grow during grain filling (Ford et al 2006). Also, applications of nitrogen late in the season can impair nitrogen redistribution within the plant and also the uptake of soil nitrogen (Gooding et al 2007), to the significant detriment of nitrogen use efficiency. This is particularly true when previous nitrogen availability has been high and/or when nitrogen quantity approaches 1 mg per grain (Gooding 2005, Gooding et al 2007). SULfUR

Sulfur is an important component of plant proteins and some oils. Sulfur-containing amino acids, such as cysteine and methionine, are responsible for forming sulfhydryl (SH) and disulfide (S-S) bonds among proteins and hence contributing to their three-dimensional configurations and subunit associations. Although the control of S uptake is closely linked to that of nitrogen uptake, remobilization of sulfur from senescing tissues is less efficient (Zhao et al 1999). Hence, while HIs for nitrogen may commonly reach 80% (Gooding et al 2005b), values for sulfur are more likely to be 50–60%. Leaf symptoms indicating poor availability are very similar to those of nitrogen, although chlorosis is more pronounced in younger, rather than older, leaves, i.e., the opposite of nitrogen deficiency, presumably because sulfur supply to the younger tissue is more dependent on concurrent uptake, rather than on redistribution from older leaves. Similarly, sulfur supply to the grain is also more reliant on uptake after anthesis. Sulfur deficiency in grain is deemed to have occurred if the N-S ratio is higher than 17:1 and the grain sulfur concentration is less than 0.12% (Wrigley et al 1984). In many parts of the world, sulfur deficiency in wheat is increasingly common (e.g., Haneklaus et al 1992, McGrath et al 1993) as a result of reduced industrial emissions, the reduced use of S-containing fertilizers, the increased demand from higher-yielding crops, and the decline in soil organic matter (Zhao et al 1999). As well as reducing yields, sulfur deficiency has a major impact on baking performance. In particular, sulfur has important influences on the viscoelasticity of doughs because S-containing amino acids provide the interchain S-S bonds that help maintain the polymer network of storage proteins (Shewry and Tatham 1997); S availability influences the relative proportions of different storage proteins that are synthesized (Zhao et al 1999); and/ or glutathione, an S-containing tripeptide, can modify rheology (Chen and Schofield 1996). Consequently, pan bread quality can be more closely associated with S concentration than with N concentration (Zhao et al 1997). Deficiency in sulfur can be alleviated by application of sulfurcontaining fertilizers such as elemental sulfur, potassium sulfate, and magnesium sulfate, applied either to the seedbed, if leaching is not a risk, or at the start of stem extension. Application of sulfur can be based on risks of deficiency as indicated by crop demand, atmospheric deposition maps, soil type, rainfall, and/ or plant tissue analysis. Nitrogen fertilizers applied late in the season to boost grain protein concentration are often combined

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with a source of sulfate to prevent excessive N-S ratios developing in the grain. POTASSIUM

Potassium plays critical roles in maintaining electrolyte and osmotic balances within plant cells and, therefore, also in controlling cell turgor. When plants are severely deficient, the tips of the older leaves become bleached or die back. Increasing K availability to deficient plants can improve tolerance to frost and drought; resistance to a number of diseases, including powdery mildew; and straw strength. Potassium is absorbed by the plant as K+ ions, which, being positively charged, are readily held on the surface of clay and humus in the soil or, more tightly, between clay plates within soil aggregates. The ion is thus less prone to leaching, so the timing of application is less significant, particularly on soils rich in clay. Clayey soils also tend to have a reserve of potassium that can offset the requirement for fertilizer applications. The need for potassium additions is often informed by soil analysis and is more likely on sandy soils, for high-yielding crops, and when straw is removed from the field. Potassium is regularly applied as potassium chloride (KCl; 60% K 2O) or potassium sulfate (K 2SO4; 48–50% K 2O). Potassium sulfate is more expensive per unit of potassium than the chloride salt and is therefore less frequently used. It does, though, also supply sulfur and hence can give additional benefits for yield and quality, as described above. PHOSPHORUS

Phosphorus, a component of cell membranes, is important for energy storage and transfer within cells. In particular, inorganic phosphate is essential for photosynthesis. Wheat plants deficient in phosphorus keep their green coloring, mature late, and have increased susceptibility to a number of diseases. In extreme cases, leaves have a dull, bluish green color with a purple or bronze hue and die back from the tips. Alleviating deficiency can aid establishment, increase growth rate of roots and seedlings, and encourage tillering. Phosphorus can be applied in many different forms, but once in the soil, it is comparatively immobile, only becoming available to the plant slowly as H2PO4– in weak soil solution. A low supply is more likely in acidic (pH 500 nm). Acid fuchsin stains protein red, while β -glucans present in cell walls show blue fluorescence with Calcofluor. Unstained starch appears black. B, stained for protein and starch with light green and iodine (observed using bright-field microscopy). Light green stains protein green, while iodine stains the amylose component of starch blue and amylopectin brown. (Courtesy VTT Technical Research Centre of Finland)

Fig. 3.36 (left). Transmission electron micrograph of a developing wheat aleurone cell, showing portion of partially filled aleurone grain (AG) containing a globoid (G) and storage protein (SP). Lipid droplets (L) lack typical membrane. Bar represents 0.5 µm. Fig. 3.37 (right). Transmission electron micrograph of a developing wheat endosperm eight days after flowering, showing small vacuoles (arrows) in the aleurone cells (A) and large vacuoles (V) in the subaleurone region (SB). S = starch grains in amyloplasts. Bar represents 10 µm.

Fig. 3.38. Transmission electron micrograph of a wheat endosperm cell undergoing cell division eight days after flowering. Note telephase nuclei (Nu) and the phragmoplast (arrows). Bar represents 4 µm.

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junction with subaleurone cells toward the center of the cheeks or, in the middle of the dorsal side, toward the top of the crease. Prismatic cells are 128–200 µm long by 40–60 µm wide (extremes from Bradbury et al 1956b). Typical subaleurone cells are not present next to the modified aleurone cells at the innermost extremity of the crease; neither do prismatic cells radiate from this region. Central cells occur in the center of the cheeks and have a length of 72–144 µm and a width of 69–l20 µm (extremes

from Bradbury et al 1956b). Although they vary considerably in size, they are similar in shape, being rounded or polygonal. Cell Walls. Cell walls are thin in relation to cell contents. Those around the peripheral cells are thickest, being up to 7 µm thick in the crease region and up to 4 µm elsewhere. The central cell walls are 2.6 µm thick, with a negative relationship between cell wall thickness and milling score (Larkin et al 1952). The cell walls of wheat starchy endosperm comprise about 15% protein and 75% polysaccharide (Mares and Stone 1973), the latter comprising about 70% arabinoxylans, 20% (1→3,1→4)-­β-­d-­glucan, 7% β -­glucomannan, and 2% cellulose (Bacic and Stone 1980) (see also Chapter 9). Wood et al (1983) confirmed the presence of ferulic acid in subaleurone cell walls by its autofluorescence, and the mixed-­linkage β-­d­glucan was also detected by staining with fluorochromes Congo red and Calcofluor. Some cellulose was also detected. The application of immunocytochemical and spectroscopic techniques has also allowed the distribution and properties of the major cell wall polymers to be studied in the developing and mature grain. Guillon et al (2004) used antibodies against arabinoxylan (AX) and (1→3,1→4)-­β-­d-­glucan to study mature wheat grain (Fig. 3.41). Two antibodies that recognize specific structural features of AX showed differences in their binding to the cell walls of aleurone, subaleurone, and central starchy endosperm cells, showing fine differences in cell wall structure, while the antibody to (1→3,1→4)-­β-­d-­glucans bound most strongly to aleurone and subaleurone cell walls. Further studies of developing grain (Philippe et al 2006b) showed a clear time sequence in the deposition

Fig. 3.39. Secondary ion mass spectroscopy of the aleurone and subaleurone cells of wheat, showing the distribution of ions as bright areas. C2– and CN– show the distributions of carbohydrates and proteins, respectively. O–, PO2–, Na+, K+, Ca+, and Mg+ are all clearly concentrated in aleurone grains. (Reprinted from Heard et al 2002, with permission from Blackwell Publishing)

Fig. 3.40. Transmission electron micrograph of mature aleurone cells, showing lipid droplets (arrows) surrounding aleurone grains (AG) and lining thick cell walls (CW). Most globoids (G) that have been removed during tissue preparation are surrounded by storage protein (SP). Bar represents 3 µm.

Development, Structure, and Mechanical Properties of the Wheat Grain  of cell wall polymers. Thus, in the early cellularization stage, the endosperm cell walls contained only (1→3)-­β-­d-­glucans, with (1→3,1→4)-­β-­d-­glucans appearing at the differentiation stage. Labeling with antibody to AX was also observed at the differentiation stage, with the intensity increasing through maturation. Labeling of Golgi stacks and of vesicles merging with the plasma membrane was also observed with the AX antibodies, demonstrating that synthesis occurred in the Golgi. Similar studies using Fourier transform infrared (FT-­IR) microscopy showed the presence of (1→3,1→4)-­β-­d-­glucans and AX at the end of the cellularization stage, with AX appearing and becoming dominant during cell differentiation (Philippe et al 2006a). Differences in AX structure were also reported within the grain, with the AX in the central cells being less substituted and the degree of AX substitution decreasing during development. FT-­IR microscopy was also used by Toole et al (2007) to compare the cell walls of developing endosperms of two U.K. wheat cultivars grown under cool/wet (23°C day temperature, watered to field capacity) and hot/dry (28°C, watered to 10% field capacity) conditions (Fig. 3.42). Their results also indicated that the structure of the AX in the starchy endosperm cell walls changed from a highly branched form to a less highly branched form during development and that the rate at which this transition occurred differed between the two genotypes and was greater in the material grown under hot/dry conditions. Barron et al (2005) also used FT-­IR microscopy to show differences in cell walls of four cultivars, two each with hard and soft endosperms. The cell walls of the soft cultivars showed greater heterogeneity, with walls of peripheral cells showing spectral similarities to smaller, more water-­soluble AX. Differences in

Fig. 3.41. Double labeling of arabinoxylan (AX) and β -glucans in aleurone cell walls from wheat grain. A polyclonal anti-xylan antibody and a monoclonal anti-β -glucan antibody were used with a second-stage goat anti-rabbit (orange-red fluorescence) and a secondstage goat anti-mouse (green fluorescence) antibody, respectively. Yellow fluorescence indicates the presence of both β -glucans and AX. (Reprinted, with permission, from Saulnier et al 2007)

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spectral properties of cell walls from both hard and soft wheat cultivars also indicated that changes in composition occurred with increasing maturation. Cell Contents. The starchy endosperm of mature wheat is dead at maturity but still possesses many of the structural features of live tissue. The principal features of endosperm cells are the two main storage reserves, starch and protein. The starch granules are surrounded by the matrix protein, with the cytoplasmic remnants being pressed into irregularly shaped regions. Within the cytoplasmic remnants are ribosomes and rough endoplasmic reticulum (RER) that contain medium-­dense material (Simmonds 1972, Gaines et al 1985). The protein in mature cells appears as a continuous matrix rather than as the series of individual bodies in which it develops. The proportional contributions of starch and matrix proteins also vary according to cell position. The peripheral cells have the lowest starch content (Fig. 3.35B) and, since all cells contain approximately the same mass of protein (Evers 1970), the protein percentage is highest in these cells. Values as high as 54% protein have been found (Kent 1966) in subaleurone cells in a flour of 12.5% protein. Large cores of protein can be seen in peripheral cells under the microscope and, even to the naked eye, a greater degree of vitreousness can often be detected in the outer layers. The increasing starch content found toward the center of the cheeks causes progressive dilution of other components as well as protein. Grain Texture. Grain of all classes of wheats can differ in appearance, having a floury (mealy) or vitreous (steely) appearance. This difference in texture probably reflects the number of microscopic air cavities within the protein matrix of the endosperm cell contents, with floury endosperm being characterized by such discontinuities. The latter create a chalky appearance by scattering light, and they contribute to mechanical weakness as foci for crack propagation. Conversely, vitreous bread wheats (and

Fig. 3.42. Spectroscopic Fourier transform near-infrared image overlaid onto a light microscope image of a transverse section of a grain of wheat cv. Spark at 30 days after flowering. The grain section has been treated to remove the cell contents, allowing the spectra of the cell walls to be determined. The distributions of highly branched and lessbranched forms of arabinoxylans are shown in blue and green, respectively, with residual starch shown in white and holes in black. (Courtesy Geraldine Toole, Institute of Food Research, Norwich, UK)

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ultrahard durum wheats) usually have no cavities and therefore have a vitreous appearance. In bread wheat, vitreousness can be influenced by environmental factors, with vitreous grain being favored by high temperature and high nitrogen availability (i.e., high protein). However, there is strong genetic control of hardness; a major locus (Hardness, Ha) located on chromosome 5D is the major determinant of whether grains are hard (ha) or soft (Ha) (Law et al 1978). This locus is not present in durum wheat, being absent from the A and B genomes of this species. However, minor loci contributing to texture have also been mapped (Turner et al 2004, Wilkinson et al 2008). Primarily, hardness results from strong starch-­protein interactions (Barlow et al 1973). This is illustrated by freeze-­fracture studies in which the protein matrix separates cleanly from the starch granule surface in soft cultivars such as the U.K. variety Riband (Fig. 3.43A) but not in hard cultivars such as the U.K. variety Mercia (Fig. 3.43B). Fragments of protein are clearly present on the starch granule surface in the latter, and the higher energy input required for milling leads to the damage of some granules. Softness is related to the presence of protein(s) (seen as a band of apparent molecular mass about 15 kDa by SDS-­PAGE) in fractions extracted from the surface of water-­washed starch granules. This band was present in large, small, and zero amounts on starch granules from soft, hard, and durum wheats, respectively (Greenwell and Schofield 1986, Schofield and Greenwell 1987). It was therefore termed “friabilin” because of its close correlation with soft (i.e., crumbly or “friable”) endosperm, and friabilin band intensity was shown to be linked to the Ha locus (Greenwell and Schofield 1989). On this basis, the “friabilin hypothesis” was postulated, that this protein material is both the product of the Ha locus and also the cause of softness because it reduces the strength of adhesion at the granule-­protein interface. Friabilin from soft wheat starch was further characterized as containing two major components, both of which are very basic, but it soon became apparent that these were identical to two proteins that had been independently isolated in 1991 on the basis of their hydrophobic, lipid-­binding character. A comprehensive study of the molecular genetics, protein structure, and lipid­binding properties of the two proteins (reviewed by Douliez

et al 2000) revealed that they are closely related in sequence, highly surface-­active, and have a strong affinity for phospholipids and other polar lipids. They are cysteine-­rich proteins of the “prolamin superfamily,” and they contain a loop region that is rich in the aromatic amino acid tryptophan and has an indole side chain (see Chapter 8). To reflect this fact, these proteins from wheat (Greek puros) were named puroindoline-­a (PIN-­a) and puroindoline-­b (PIN-­b), and the term friabilin is now used only for the puroindolines that are bound to the starch granule surfaces. The structural genes for puroindolines a and b have since been located at the Ha locus, as discussed in Chapter 8. Polar glycolipids and phospholipids show patterns of distribution on starch granules similar to those of friabilin (Greenblatt et al 1995), which is consistent with the puroindolines binding lipids at the starch granule surface. Subsequent detailed studies (reviewed by Morris 2002 and discussed in more detail in Chapter 8) have essentially confirmed the “friabilin hypothesis,” that puroindolines are products of the Ha locus and that specific allelic forms are able to bind to the surface of starch granules to cause softness by reducing the strength of adhesion between the starch granules and the protein matrix. This “nonstick” property of puroindoline-­lipid complexes may have two effects: first, they allow some of the shrinking protein matrix to pull away from the granules to form cavities during final maturation and desiccation of the grain, and second, they form a plane of weakness at the remaining matrix-­granule interfaces. The extent of these effects presumably depends on the ability of the puroindolines to saturate the available binding sites on the granule surfaces within the endosperm, as dictated by their amount and their affinity for the binding sites. As yet, the detailed molecular mechanisms that determine these physical differences are not known. Differences in endosperm texture (hard or soft) are apparently present throughout endosperm development in air-­dried wheat. Thus, hard wheats were hard and soft wheats were soft as early as they could be analyzed (15 DAA) with the single-­kernel characterization system, even though storage proteins were not present in sufficient amounts to surround the starch granules

Fig. 3.43. Differences in endosperm texture revealed by freeze-fracture of mature grains of the soft wheat cultivar Riband (A) and the hard wheat cultivar Mercia (B). In both cases, the fracture occurs within the cells of the starchy endosperm. In the soft wheat, the matrix proteins separate cleanly from the surface of the starch granule. In contrast, in the hard wheat, some protein remains attached to the starch granule surface, and damage to the granules may also occur. Bar represents 10 µm. (Courtesy Paola Tasi, Rothamsted Research, U.K.)

Development, Structure, and Mechanical Properties of the Wheat Grain  (Bechtel et al 1996, Turnbull et al 2003). Further studies using TEM related the structure of freeze-­dried or air-­dried wheat endosperm harvested at various stages of maturity to the development of endosperm texture (Bechtel and Wilson 1997). The most pronounced change upon air-­drying of these samples was the disappearance of individual protein bodies and a corresponding conversion of the protein bodies and cytoplasm into a matrixlike material similar in appearance to that of the storage protein matrix found in mature wheat endosperm. This suggests that the surface of the starch granule can bind to a number of endosperm components formed during senescence of preripe wheat to develop specific textural characteristics. Therefore, the surface properties of the starch granule may be important in determining hardness, as well as the presence of specific components that bind to the starch granule surface (Bechtel and Wilson 1997). Interactions of the starch granule surface and surrounding cellular components also apparently alter the starch granule size and shape. Starch granules isolated from hard and soft red winter wheats were separated into separate classes in the two types, and the basic morphometric features were determined by digital image analysis, even though some of the wheats had similar hardness values by near-­infrared (NIR) spectroscopy (Bechtel et al 1993, Zayas et al 1994). Effects of Environmental Factors. The effects of environmental and genetic factors on wheat flour quality have been studied extensively (Lukow and McVetty 1991, Huebner et al 1997, Bergman et al 1998, Ames et al 1999). While the genetic background of commercial wheats has been improved through breeding programs, environmental influences still continue to adversely affect wheat quality. Several studies on the effects of high temperature during grain filling and drying showed that flour from wheat harvested before maturity had quality traits superior to those of flour from wheat allowed to mature in the field (Finney 1954, Finney and Fryer 1957, Finney et al 1962). Finney (1954) found that wheat that was harvested 10–14 days before maturity exhibited optimum loaf potential as well as excellent crumb structure. The dough mixing requirement and mixing tolerance of flour from preripe wheat were also generally superior to, but never worse than, those of dough from wheat harvested at field maturity. The maximum loaf volume potential was apparently related developmentally to the time that the storage protein bodies fused to form the protein matrix present in mature wheat endosperm (Bechtel et al 1982b). The structural changes that occurred in the endosperm during the artificial drying of maturing wheat caryopses were investigated using TEM (Bechtel and Wilson 2005). Laboratory-­induced senescence caused the endosperm tissue to undergo several changes that resulted in the appearance of the tissue being similar to that of wheat that was not prematurely harvested. That suggests that the wheat plant has great capacity to develop normally even when subjected to environmental stresses. The molecular and biochemical changes accompanying the structural changes during senescence have not yet been investigated. Storage Protein Deposition. The starchy endosperm contains the major gluten storage proteins (gliadins and glutenins) and smaller amounts of globulin (triticin) storage proteins, as discussed in Chapter 8. The deposition of these storage proteins in the endosperm cells has been intensively studied. Graham et

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al (1962) showed that the earliest-­deposited endosperm storage protein was a single body enclosed by a membrane. Later in development, four or more bodies were present within the vacuole. The proteins were deposited into vacuoles from the RER via an unspecified mechanism called “internal secretion.” These results were confirmed by Buttrose (1963a), who suggested that the Golgi apparatus was associated with protein deposition and had a “condensing” function. Jennings et al (1963) described the effect of various fixatives on protein body appearance in wheat endosperm. They concluded that protein bodies occurred singly and had a tightly appressed membrane, rather than several protein bodies being present in vacuoles surrounded by a single membrane. The occurrence of single and multiple protein granules within vacuoles of the early-­developing wheat endosperm was observed by Barlow et al (1974) and Harvey et al (1974). During late stages of development, however, protein bodies appeared to be formed via a different mechanism (Barlow et al 1974). Subsequently, Simmonds (1978) hypothesized that the protein was synthesized in the cytoplasm and transported to vacuoles either through the lumen of the RER or by a process similar to pinocytosis. Briarty et al (1979) conducted an extensive stereological analysis on developing wheat endosperm. They concluded that the route followed by storage proteins to the vacuoles was unclear but that the Golgi apparatus was not involved because it was not detected at 12 DAA (Briarty 1978, Briarty et al 1979). Miflin and co-­workers suggested that prolamin-­containing bodies are not vacuolar in origin (Miflin et al 1980, 1981, 1983; Miflin and Burgess 1982) but that prolamins were synthesized on the RER and passed into the lumen, where they aggregated. Eventually the aggregates disrupted the RER membrane. Campbell et al (1981), in contrast, found that all protein bodies were surrounded by a single membrane but suggested that the origin may vary, some arising from RER distensions and others from vacuoles or the Golgi apparatus. Convincing microscopic evidence for the vacuolar location of protein bodies as well as the involvement of the Golgi apparatus was reported by Parker (Parker 1980, 1981, 1982; Parker and Hawes 1982), who showed that the Golgi apparatus was probably involved in the transport of the storage proteins from the cisternal RER to the vacuoles, at least during early stages of development (Parker 1982, Parker and Hawes 1982). Later in development, proteins accumulated in dilated regions of the RER and then apparently passed to the vacuoles (Parker and Hawes 1982). The early stages of protein deposition were described in a TEM/cytochemical study by Bechtel et al (1989), which provided explanations for several apparently conflicting observations. Vacuoles in wheat starchy endosperm cells were shown to originate via a process called “autophagy,” in which endoplasmic reticulum (ER) surrounded portions of the starchy endosperm cell cytoplasm that were devoid of organelles. The ER membranes then fused with one another, isolating the cytoplasm within the ER membranes (Bechtel et al 1989). The enclosed cytoplasm was then digested. The autophagic process was confirmed using cytochemical localization of acid phosphatase in both the RER and the autophagic vacuoles (Bechtel et al 1989). The process of autophagy helped explain why several investigators had

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­ bserved the presence of cytoplasm and cytoplasmic remnants o in vacuoles. Despite the observations of Briarty (Briarty 1978, Briarty et al 1979), it is clear that the Golgi apparatus is present during protein body formation in a variety of cereals, including wheat. Furthermore, the presence of proteins in dense-­cored Golgi vesicles was demonstrated by digestion with protease enzymes (Bechtel and Gaines 1982). In addition, Bechtel et al (1982b) identified protein bodies that were enzymatically digested by proteases. Those protein bodies were initiated at about 6–7 DAA of a 35-­DAA growing season and were observed near the Golgi apparatus (Bechtel et al 1982a). Direct connections between RER and protein bodies were not observed, suggesting a role for the Golgi apparatus in the initiation of protein bodies. A freeze-­fracture study on developing wheat endosperm that had not been chemically fixed yielded results that were in general agreement with those obtained from thin-­sectioned tissues

(Bechtel and Barnett 1986a,b). Protein bodies were observed in the starchy endosperm as discrete membrane-­bounded structures (Figs. 3.44 and 3.45) that fused with vacuoles and deposited the storage protein inside. The ER was present as large sheets during early development but was later converted into small cisternal elements interconnected by tubular ER. Freeze-­fracture revealed that vesicles were produced by the ER throughout development, something not previously shown by thin-­sectioned material. Freeze-­fracture also revealed direct connections between the ER and the Golgi apparatus (Fig. 3.46), disproving the previous suggestion that a soluble mode of protein secretion contributed to protein body formation (Bechtel et al 1982a). The pinocytotic vesicles observed in thin sections were found in freeze-­fractured material also, but they were not thought to be directly involved in protein body formation. Instead, Bechtel and Barnett (1986b) hypothesized that the pinocytotic vesicles were probably involved in recycling or sequestering of membrane. The initial formation of protein bodies is followed by a second stage in which the protein bodies fuse and form a matrix, with the protein body membranes being disrupted. These processes have been followed by isolating protein bodies using isopycic sucrose gradient centrifugation (Pernollet and Camilleri 1983) and by TEM and enzymatic digestion of thin sections (Bechtel et al 1982b). The latter study showed that protein bodies formed in the cytoplasm were transported to the central vacuole(s), where the protein body membrane and tonoplast fused and deposited the granules of protein into the vacuole (Fig. 3.40). The granules of protein fused with one another rapidly during grain filling and resulted in the conversion of the spherical protein granules into irregularly shaped protein masses that eventually became the matrix protein. Enzymatic digestion of thin sections with protease VI and Fig. 3.44. Transmission electron micrograph of endosperm cell with pepsin also confirmed that the vacuolar protein granules storage protein granules (SP) in vacuoles (V). Note how some granules were mostly proteinaceous. The only undigested regions were are fusing (arrows). Nu = nucleus, S = starch granules in amyloplasts. Bar represents 5 µm. peripheral electron-­dense inclusions, which were proposed to correspond to the last-­added protein. It is clear from the studies discussed above that prolamin-­containing protein granules occur both within the lumen of the ER and the vacuole, with the latter arising from transport via the Golgi apparatus. Based on such observations, Galili and co­workers (Levanony et al 1992, Galili et al 1993, Galili 1997) have proposed that two routes of protein body formation occur, with the populations of ER-­derived and vacuole-­derived bodies subsequently fusing (summarized in Fig. 3.47). The mechanism of this fusion is not fully understood, but Galili (1997) has proposed that the ER­derived protein bodies become engulfed by vesicles and “internalized” into vacuoles. It is not clear whether both routes opFig. 3.45 (left). Freeze-fracture electron micrograph of portion of vacuole in starchy enerate throughout grain development or dosperm cell. Vacuole V1 is fusing with vacuole V2 (arrow). The membranes of both vacuoles probably already have fused (arrow). Note how storage protein granules (SP) have fused. Bar whether individual gluten proteins take simrepresents 0.5 µm. ilar or different routes. However, Tosi and co-­workers (P. Tosi, M. Parker, C. Gritsch, Fig. 3.46 (right). Freeze-fracture micrograph, showing sheetlike rough endoplasmic reticuR. D’Ovidio, and P. R. Shewry, unpublished lum (ER) interconnected with a Golgi body (G). Bar represents 0.5 µm.

Development, Structure, and Mechanical Properties of the Wheat Grain  results) have recently expressed an epitope-­tagged low molecular weight (LMW) subunit in transgenic wheat and used a specific antibody to show that the same protein can be observed in Golgi­associated vesicles, vacuolar protein deposits (as shown in Fig. 3.48), and accumulating within the lumen of the ER. Hence, it is clear that any segregation of individual proteins into ER or vacuolar deposits is certainly not complete. Similarly, both Parker and Hawes (1982) and Levanony et al (1992) have suggested that the ER route becomes dominant during the later stages of protein deposition, although this may also reflect the relative ease with which the subcellular compartments can be identified at different stages of development. The location and distribution of gliadins and prolamins in the protein bodies and storage protein matrix has been investigated using immunocytochemical methods coupled with TEM. Prolamins in wheat and the related wild grass species Haynaldia villosa (Dasypyrum villosum) were localized in endosperm protein bodies (Kim et al 1988, Krishnan et al 1988). Bechtel et al (1991) used a variety of antisera raised against cereal storage proteins in an immunocytochemical study of wheat endosperm pro-

Fig. 3.47. Schematic summary of the two routes of gluten protein trafficking and deposition within the starchy endosperm cells of wheat grain. ER = endoplasmic reticulum.

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tein bodies. Antisera raised against α-­gliadin, γ-­gliadin, a high molecular weight (HMW) glutenin subunit, barley C hordein (the homologue of wheat ω -­gliadins), and the 12S “legumin-­like” globulin of oats all labeled the matrix of the wheat endosperm protein bodies, but they failed to label the electron-­dense inclusions present within these bodies (Bechtel 1990, Bechtel et al 1991). Similarly, Stenram et al (1991) also failed to show any differentiation between the contents of protein bodies using a range of monoclonal and polyclonal antibodies to gluten proteins. However, a minor class of storage proteins with legumin-­like properties has been identified in wheat endosperm (Singh and Shepherd 1985, 1987) (see also Chapter 8). The proteins, originally called “triplet proteins” but now referred to as “triticin,” are synthesized between 8 and 21 DAA (Singh and Shepherd 1987) and are concentrated in isolated wheat protein bodies (Payne et al 1986, Singh and Shepherd 1987). Immunocytochemical TEM localization using antibodies against triticin labeled both the matrix protein and the dense inclusions of protein bodies, with the latter being more heavily labeled. It was therefore concluded that the triticins are located in the inclusions, with the labeling of the matrix resulting from cross-­reaction of the antibody. It is also probable that the labeling of the matrix with the anti-­oat 12S globulin antiserum (Bechtel 1990, Bechtel et al 1991) resulted from cross-­reaction with prolamins. It is not known whether the triticin inclusions result from fusion of separate triticin­containing bodies with protein bodies containing prolamins or from phase separation of the two types of the protein after deposition in the same bodies. The pathway of deposition of triticins is not known but may be via the Golgi as it is for related 11S/12S globulins in legume and other dicotyledonous seeds and for the related glutelin storage proteins of rice (Kermode and Bewley 1999, Takaiwa et al 1999). The formation of large numbers of protein bodies, coupled with starch granule enlargement, caused the cytoplasm to be isolated into small regions of the cell. As deposition of reserves continued, the protein and cytoplasm became irregular. The irregularly shaped deposits became condensed to form the matrix protein during late stages of development, which was associated with the loss of water and the breakdown of integrity to form a

Fig. 3.48. Localization of an epitope-tagged, low molecular weight subunit within the Golgi apparatus (A) and vacuole (B) of a starchy endosperm cell of transgenic wheat cv. Cadenza at about 8–10 days after anthesis. The c-myc tag at the C-terminal end of the protein is detected by a specific monoclonal antibody with a second antibody linked to gold particles. (Courtesy P. Tosi, Rothamsted Research, U.K.)

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continuous proteinaceous matrix (Bechtel et al 1982b). Similar changes were also observed when developing wheat caryopses were artificially dried (Bechtel and Wilson 2005). Starch. Starch is the principal carbohydrate stored in the wheat caryopsis. Mature starchy endosperm of wheat contains approximately 63–72% starch by mass (Pomeranz and MacMasters 1968), making it the most prominent storage component. The starch granules in wheat, as in barley, rye, and triticale, make up more than one size class (Bechtel et al 1990, Langeveld et al 2000, Shewry and Bechtel 2001). Wheat starch possesses unique properties that do not allow it to be replaced by starches from corn, rice, or oats or by noncereal starches to yield a satisfactory baked product (Hoseney et al 1971). The size of wheat starch granules influences rheological properties (Kulp 1973, Rasper and Deman 1980, Casey et al 1997), baking characteristics (D’Appolonia and Gilles 1971), compositional differences (Meredith 1981), and processing properties (Stoddard 1999), as well as the exploitation of starch in nonfood uses (Langeveld et al 2000).

All plant starch granules are synthesized in a double­ embrane-­bound organelle called the plastid. Starch generally m occurs in two types of plastids: the chloroplast, where photosynthesis occurs and in which starch granules are variable in number and transient in occurrence, and the amyloplast, where starch is synthesized and stored for periods of time (Kirk and Tilney-­Bassett 1978). The development of starch granules in the starchy endosperm of wheat has been studied using light microscopy (Sandstedt 1946), TEM (Buttrose 1963a), and SEM (Evers 1971). The general scheme for the development of starch granules was formulated by Evers (1971). The large A-­t ype granules are formed first from a nucleus that is progressively surrounded by further starch deposits (Fig. 3.49). The deposits are preferentially added in the equatorial plane, and growth is from one side, which results in early granules having an irregular appearance. Continued growth around this plane results in a lens-­shaped granule with an equatorial groove. The final number of A-­t ype granules is determined by the number of plastids present when cell division ceases (Briarty et al 1979). The final size of these A-­t ype granules varies from about 30 to 50 µm (Sandstedt 1946, Evers and Lindley 1977, Dengate and Meredith 1984) and may be influenced by cultivar (Dengate and Meredith 1984) and environment (Baruch et al 1979). The number of size classes has been debated. May and Buttrose (1959) described two size classes of barley starch, the large A-­t ype and the smaller B-­t ype, a terminology for starch size classes that is now universally applied to wheat and other members of the Poaceae Fig. 3.49. Developmental sequence of type A starch granules of wheat endosperm. The nucleus (1) is progressively surrounded by starch deposits, which family that possess multiple starch size classes. The build up preferentially in the equatorial plane. Growth is asymmetrical, so early A-­t ype granules are formed first and are observed in forms (2–4) are only partially surrounded by liplike structures. The groove the cytoplasm after cellularization of the endosperm between the lips is present all around the circumference. Further deposits of (Buttrose 1960, 1963a). The possibility of more than two starch are added so that the groove decreases as thickness and diameter of the starch size populations was first reported for starch isogranule increase (5,6). (Reprinted, with permission, from Evers 1971) lated from New Zealand wheats (Meredith 1981, Baruch et al 1983) and later confirmed in U.S. hard red winter wheat (Bechtel et al 1990). Using starch isolated from developing wheat, Bechtel et al (1990) showed that there were three bursts of starch granule synthesis. The A-­t ype granules formed during the first week after anthesis, followed by smaller B-­t ype granules during the second week, while the C-­t ype granules developed during the third week after anthesis. Although data have shown that there are three distinct size classes of starch granules, most investigators have not investigated the C-­t ype granule class. The A-­ and B-­t ype classes of starch granules are generally considered to be synthesized within distinct compartments of the same amyloplasts (May and Buttrose 1959, Parker 1985). Parker (1985) studied the development of starch granules in wheats by TEM and found that the B-­t ype granules were not initiated until after cell division stopped. These granules were found often in association with the equatorial (peripheral) groove and tubuli of A-­t ype granules or in extensions of the A-­t ype plastids (Fig. 3.50). It was suggested that only one type of plastid, the complex A-­t ype, was present in wheat starchy endosperm. More reFig. 3.50. Formation of B-type starch granules in developing starchy cently, confocal laser scanning microscopy was used to examine endosperm cells of wheat. Two sites of granule initiation in a protruwheat endosperm tissue that had been transformed to express sion from an A-type amyloplast (A), resulting in a series of B-type a fluorescent protein within the amyloplast, revealing extensive granules (B). The scale bar is 1 µm in both figures. (Reprinted from interconnections between amyloplasts via tubular protrusions Parker 1985, with permission from Elsevier)

Development, Structure, and Mechanical Properties of the Wheat Grain  (Langeveld et al 2000). TEM of serial thin sections of endosperm tissue showed small B-­t ype starch granules within the protrusions emanating from the A-­t ype amyloplasts (Langeveld et al 2000). A TEM study of starch formation in developing wheat from the day of flowering through grain maturation showed that plastids in the coenocytic endosperm of young wheat caryopses were mostly in the form of proplastids, with a few containing small starch granules (Bechtel and Wilson 2003). During the first week of endosperm development, newly divided cells had plastids that were pleomorphic in shape, while subaleurone cells interior to the meristematic region contained amyloplasts that contained a single size class of starch granules (incipient A-­t ype). The pleomorphic plastids exhibited tubular protrusions that extended a considerable distance through the cytoplasm. Both subaleurone and central endosperm cells had amyloplasts that exhibited protrusions at 10–12 DAA, and some of the protrusions contained small starch granules (incipient B-­t ype). Protrusions were not observed in endosperm amyloplasts at 14 DAA, but incipient large A-­ and smaller B-­t ype starch granules were present. Amyloplast protrusions were numerous again at 17 DAA in both subaleurone and central endosperm cells, and by 21 DAA, a third size class of small C-­t ype starch granules was observed in the cytoplasm. Amyloplasts in the endosperm of wheat apparently divided and increased in number via protrusions, since the binary fission typical of plastid division was never observed (Bechtel and Wilson 2003). Those results suggest that wheat has three size classes of starch granules produced at specific times during wheat endosperm development. Isolated starch from mature endosperm has been investigated to determine the ratio of A-­t ype to B-­t ype granules. Stamberg (1939) used the data of Grewe and Bailey (1927) to calculate the distribution of granule sizes. He found that the small granules (14.8 µm) were only 12.5% by number but 93.0% by weight and 76.4% by surface area. The medium granules (7.4–14.8 µm) were a minor component in all three measurements. A number of methods have been used to measure size distributions of wheat starch, including microsieving, electrical-­sensing zone techniques, light scattering, and quantitative image analyses (Brocklehurst and Evers 1977; Evers and Lindley 1977; Baruch et al 1979, 1983; Karlsson et al 1983; Soulaka and Morrison 1985; Morrison and Scott 1986; Bechtel et al 1990; Raeker et al 1998; Peng et al 1999; Stoddard 1999). Evers and Lindley (1977) used microsieving, Coulter counter, and quantitative image analyses to study the distribution of starch granules in 12 wheats. All three techniques revealed that starch granules below 10 µm in diameter accounted for approximately one third to one half of the total weight of endosperm starch. Each analysis method has advantages and disadvantages (Langton and Hermansson 1993). While digital image analysis coupled with light microscopy offers the ability to record the physical parameters of each individual particle and to distinguish between individual granules, agglomerated granules, and nonstarch particles, it is extremely slow for routine analysis of many samples and has problems associated with particles touching the perimeter of the field of

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view. Light scattering instrumentation offers speed and reproducibility but yields erroneous data for nonspherical particles. Wilson et al (2006) developed a reference digital image analysis method and compared it to light scattering instruments for analyzing isolated wheat starch granules. This study verified errors associated with digital image analysis with respect to the perimeter of granules touching the edge of the field of view but found that the errors could be corrected. Starch granules from four wheat cultivars representing hard red winter, hard red spring, durum, and spelt wheat classes were analyzed by image analysis and light scattering. Correction of the perimeter error was done manually. The corrected image analysis data for starch distribution were then compared to light scattering data for identical starch samples (Wilson et al 2006). Light scattering underestimated peak diameters by 40–50% when compared with image analysis. An adjustment factor was developed from the image analysis data and applied to light scattering, which shifted the major peaks of the A and B granule populations to more accurately represent granule diameter. The adjusted data were then validated within each of the four wheat classes. A single method for routinely measuring wheat starch size distributions is not yet available without comparison to a reference method. Lipids. Lipid storage bodies do not commonly occur in the starchy endosperm. In fact, Seckinger and Wolf (1967) were unable to locate specific oil bodies and concluded that free (i.e., non­membrane-­associated) lipids were evenly distributed. However, oil bodies have now been described in the developing starchy endosperm (Bechtel et al 1982a) and in mature tissue (Hargin et al 1980). These bodies were observed in mature endosperm by fluorescence microscopy and were distributed throughout the tissue. The subaleurone region had the highest concentration of oil bodies, and the central region had the lowest (Hargin et al 1980).

Embryo The embryo (called “germ” by millers) lies on the lower dorsal side of the caryopsis. It comprises two major components, the embryonic axis and the scutellum. Both Percival (1921) and Bradbury et al (1956c) have studied the structure of the embryo with light microscopy. These in-­depth accounts have given us a clear picture of how the embryo is, in reality, a miniature living plant. EMBRYONIC AXIS

The mature embryonic axis can be divided into three regions: the shoot or epicotyl, the mesocotyl, and the radicle. A true hypocotyl region does not exist in the wheat embryo. The mesocotyl region is the axis between the scutellum, coleoptile, and radicle (Fig. 3.51). Within the epicotyl are the apical meristem (meristematic cells of the shoot tip) and the plumule (primary leaves). Surrounding the shoot is the coleoptile, a foliarlike structure that probably functions in a protective role for the shoot during germination as well as being a storage organ for reserves used during germination and a photosynthetic structure upon emergence. Provascular bundles are prominent in cross sections through the plumule and coleoptile. The number of bundles in the shoot and coleoptile is variable, depending upon the species (Percival 1921). The first foliar leaf of bread wheat typically has

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11 bundles, while the second leaf has seven (Percival 1921). The midvein of the first leaf is derived from a vascular trace of the scutellar bundle that passes down the scutellum into the mesocotyl and back upward into the vascular tissues of the epicotyl (Bradbury et al 1956c). Bradbury et al (1956c) found that at least two of the lateral bundles of the first leaf also had their origins from traces in the scutellum. The coleoptile possesses two vascular bundles located laterally on opposite sides (Percival 1921). The coleorhiza surrounds the radicle in much the same fashion as the coleoptile covers the shoot (Fig. 3.51). This structure is continuous with the suspensor in young developing embryos (Esau 1965). The suspensor persists as the coleorhizal papilla (Symons et al 1984) or embryonic appendage. The coleorhiza lacks a vascular system. Projecting upward from the outer dorsal surface is a small flap of tissue called the epiblast. The radicle or primary root forms just below the mesocotyl and is a well-

Fig. 3.51. Light micrograph of mature wheat embryo, showing major parts of the wheat embryo. SC = scutellum, R = radicle, M = mesocotyl, RC = root cap, E = epiblast, C = coleoptile, CR = coleorhiza, VT = vascular tissue, CP = coleorhiza papilla, SE = scutellar epithelium, EN = endosperm, F = fibrous region, CC = caryopsis coats, PL = primary leaves, SA = region where shoot apex is located, arrow = modified aleurone. Bar represents 100 µm.

­ ifferentiated system consisting of two pairs of lateral roots as d well as a vascular cylinder (Fig. 3.52). The radicle terminates at the apical meristem and the root cap. Bradbury et al (1956c) found that the epidermis, cortex, and central vascular cylinder could be distinguished in both longitudinal and transverse sections even though the tissues had not completely differentiated. The cortex consisted of four to six layers of parenchyma cells that contained small intercellular spaces. Both the endodermis and the pericycle were identified interior to the cortex. The embryo develops from the fusion of the egg nucleus and the second sperm nucleus. This fertilized egg divides after a few hours by forming a transverse cell wall. The basal cell develops little, but the apical cell continues to divide and ultimately forms the embryo (Percival 1921). Little differentiation occurs during the first week following anthesis; therefore, the embryo at this stage of development is club-­shaped. The first sign of differentiation is a small cleft that arises from the dorsal side of the bulbous portion of the embryo (Fig. 3.53). Percival (1921) conducted an investigation into embryo development and made drawings of the various stages of development. The cleft that develops shortly after the first week of development is actually the start of the formation of the shoot. The cleft separates the incipient coleoptile from the apical meristem. As the coleoptile enlarges, the first leaf primordium forms between the shoot apex and the coleoptile. Simultaneously, the primary root, the radicle, is initiated in the mass of tissue destined to differentiate into the coleorhiza. The radicle manifests itself as a line between the incipient coleorhiza and the mesocotyl, but it rapidly differentiates a root cap and root apex, as well as a protoderm, procambium, and cortex. The coleorhiza develops from tissues of the lower portion of the embryonic axis and is connected to the suspensor during the early stages. The embryo is essentially complete structurally by the start of the grain-­filling stage but before most of the storage reserves have been deposited. In Percival’s study (1921), lipid droplets were common in the embryonic tissues, as were irregularly shaped plastids containing one to several Fig. 3.52 (top). Light micrograph of a rounded starch granules. The cytoplasm cross section through the coleorhiza (CR) of most cells was also in an undifferentiand radicle (R) at the region where the latated state but one that was indicative of eral roots (LR) are located. Bar represents much metabolic activity. Large amounts 200 µm. of RER, Golgi bodies, mitochondria, and Fig. 3.53 (bottom). Light micrograph of polysomes characterized the cytoplasm longitudinal section through the embryo, of the embryonic cells. showing cleft region (arrow) that will deStorage reserves in the embryo begin velop into the epicotyl. Note that the scutelto be deposited toward the end of the enlum (SC), radicle (R), coleorhiza (CR), and dosperm cell division phase. The primary mesocotyl (M) have also been initiated alreserves are lipid droplets and protein bodready. Bar represents 100 µm.

Development, Structure, and Mechanical Properties of the Wheat Grain  ies. Plastids are present as proplastids; starch is only occasionally found in the mature embryo. Lipid droplets (oil bodies) are found in the embryo tissues early in development, but at low frequency. These become much more numerous during grain filling and continue to increase in number during embryo maturation. Protein bodies form in small vacuoles after the major structures of the embryo have formed. The storage proteins appear to be related to the 7S globulins of legumes, as discussed above for aleurone cells. SCUTELLUM

The scutellum is a storage organ that is considered to be a single cotyledon. It is bordered on the dorsal side by the embryonic axis and on the ventral side by the starchy endosperm (Fig. 3.51). The mature scutellum is a major store for protein, phytin, and lipid droplets (Swift and O’Brien 1972), with the lipid bodies encircling the protein bodies. Also present in the cytoplasm of the scutellar cells are mitochondria, plastids, RER, and free ribosomes (Swift and O’Brien 1972). Golgi bodies are not observed in mature dry tissue. Swift and Buttrose (1972) used freeze-­fracture techniques to study the dry state of the wheat scutellum. They found that water penetrates the scutellum within seconds of contact and causes modifications of cell organelles. The vascular tissues of the scutellum before germination are entirely provascular, even though sieve and tracheary elements appear mature (Swift and O’Brien 1971). Scutellar tissues develop from the ventral and apical portions of the young embryo (Fig. 3.53). The scutellum grows downward as endosperm cell division ceases, forming a projection extending into the endosperm at the level of the coleorhiza. The scutellum possesses an epithelium at the starchy endosperm boundary. This single-­cell layer of tissue is composed of columnar cells that are thought to function in a secretory and absorptive role during germination. A layer of crushed cells (fibrous region) separates the scutellar epithelium from the starchy endosperm (Fig. 3.51). These crushed cells are extremely important, for it is at this junction that the embryo is separated from the rest of the grain during milling. Crushed cells are formed from starchy endosperm cells that fail to develop normally. Instead, the cytoplasm of these cells degenerates, leaving the cell walls intact (Fig. 3.54). As the embryo and endosperm enlarge during devel-

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opment, the cell walls become crushed and are compressed to form this specialized layer.

MOLECULAR AND BIOCHEMICAL STUDIES OF GRAIN DEVELOPMENT A range of new molecular, biochemical, microscopy, and imaging methods have revolutionized developmental biology over the past two decades. Although applied initially to animal and model plant systems, these methods are increasingly being used to study wheat development. These developments may not be familiar to cereal scientists; hence, the principles are briefly described as well as the applications.

Gene Expression Analysis

The patterns of gene expression can be studied using several approaches, the most widely used being transcriptome analysis. In this approach, a total mRNA fraction is prepared from the tissue of interest and analyzed to determine the identities and abundances of the individual transcripts. Most studies of this type have used array systems in which transcripts are identified and quantified by hybridization to nucleotide sequences displayed on microarrays. Two types of array are in use. cDNA arrays use cloned sequences, which are usually derived from the species of interest. For example, a U.K. wheat transcriptomic resource comprises about 9,000 unique cDNA sequences derived from 35 cDNA libraries, including libraries from various tissues and stages of developing grain (Wilson et al 2004). Similarly, Leader (2005) used an array of approximately 10,000 unique cDNAs prepared for the biotech company Syngenta. cDNA-­based arrays have several limitations that make comparisons between and within samples difficult, as the sequences often differ in length, with cross-­hybridization occurring between related sequences (discussed by Leader 2005). These shortcomings are largely eliminated by using oligonucleotide probes, which also allow entire genome sequences to be exploited. Commercial systems have been developed that use a number of short oligonucelotides specific for each gene with some 55,000 genes being included on a widely used wheat array. This may represent up to half of the total expressed genes in wheat. Several studies have used array technology to determine transcriptome profiles of developing wheat grain. Leader (2005) and Laudencia-­Chingcuano (2006, 2007) used cDNA arrays comprising up to 10,000 elements to profile caryopsis development, while Wilson et al (2005) used a cDNA array of about 9,000 elements (Wilson et al 2004) to study embryo development and germination. More recently, Wan et al (2008) have reported an analysis of caryopsis development using a commercial array system. These studies showed that changes in patterns of gene expression were associated with the phases of grain development identified by classical studies, with distinct profiles associated with the Fig. 3.54. Light micrograph of a sagittal section through young wheat embryo pericarp, embryo, and starchy endosperm. It was also (EB), showing endosperm cells that will form the fibrous region (F) between possible to identify transcription factors that showed embryo and starchy endosperm (EN). Bar represents 100 µm.

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Fig. 3.55. Gene expression patterns in the central endosperm and coordinated expression patterns in maternal and endosperm tissues. A, α-thionin (ID 701996413) transcript is present throughout the central endosperm at 9 and 13 days after anthesis (DAA); B, gliadin (ID 701994563) transcript is present in the central endosperm but is reduced in the peripheral endosperm and aleurone layers; C, pyruvate orthophosphate dikinase (ID 701993242) is expressed in outer layers of the endosperm at 6 and 9 DAA and then is restricted to the aleurone layers at 13 DAA; D, β -expansin (ID 702008339) transcript is present in the modified aleurone layer, reaching a peak at 6 DAA; E, a pectinesterase (ID 702008330) transcript is present throughout the nucellus at 3 DAA and is maintained in the abaxial nucellar projection from 6 to 9 DAA; F, a serine carboxypeptidase (ID 702014362) transcript accumulates in both the modified aleurone and the nucellar projection from 6 DAA. The main tissue types are annotated as follows: crease region (cr), nucellar projection (np), integuments (in), central endosperm (cen), peripheral endosperm (pen), pericarp (p), modified aleurone (ma), and endosperm (e). Bar = 0.3 µm. (Reprinted, with permission, from Drea et al 2005)

Development, Structure, and Mechanical Properties of the Wheat Grain  expression profiles similar to those of specific gene families (e.g., gluten protein genes), implying that they play a role in the developmental programming of gene expression. A disadvantage of array technology is that it provides little or no information on spatial differences in gene expression patterns within cells and organs, as it is difficult to prepare sufficient quantities of homogeneous tissues or cell types for extraction of ribonucleic acid. To provide such information, Drea et al (2005) established a high-­t hroughput in situ hybridization system and used this to characterize the expression patterns of 888 genes during the first 13 days of grain development. They were able to identify classes of gene that were specific to individual tissues (maternal and seed) and stages of development (see examples in Fig. 3.55). Furthermore, their data indicated the importance of the crease region in establishing the developmental patterning, as the transition from cell proliferation to differentiation was initiated in this region and then spread to the rest of the endosperm. Array technology has also been used for wider comparisons between lines and treatments. Thus, Lu et al (2005) used the 9,155 cDNA array of Wilson et al (2004) to determine the expression profiles of developing endosperms at 14 DAA, comparing plants grown in the field with various fertilizer treatments, including farmyard manure (FYM). The transcriptome data formed three clusters corresponding to plants grown with low N, high N, and FYM (± additional N); the material grown with FYM differed most from the other samples. Several of the transcripts that were differentially regulated in these plants corresponded to enzymes or transporters involved in N metabolism, but a small number of unidentified genes were also shown to be up-­regulated in the FYM plots and in other plants grown organically in the United Kingdom and China. They were therefore considered to be diagnostic for organically grown grain. Arrays have also been used to compare the transcriptomes of near-­isogenic and transgenic lines. Thus, Gregerson et al (2005) and Baudo et al (2006) have described the use of the cDNA arrays described by Wilson et al (2004) to compare the substantial equivalence of transgenic wheat expressing a fungal phytase gene or additional HMW subunit genes, respectively. Neither of these studies showed substantial effects of the transgenes on the expression of unrelated genes, and it was concluded that the genetically modified (GM) and non-­GM lines were substantially equivalent. Similarly, Clarke and Rahman (2005) used cDNA microarrays comprising approximately 6,000 and 22,000 (16,000 unique) sequences to compare the expression profiles of near­isogenic lines differing in grain texture. Recently Kawaura et al (2005) have used expression profiling to compare the expression of genes encoding α/β -­gliadins and LMW subunits in developing grain. They initially analyzed more than 360,000 sequences from expressed sequence tag (EST)1 databases and assembled 35 contigs (continuous sequences) homologous to α/β -­gliadins and 15 homologous to LMW subunits. Comparison with previously determined gene copy numbers 1

ESTs are expressed sequence tags that correspond to mRNAs. EST libraries are generated by making cDNA copies of mRNA fractions from specific tissues, followed by partial sequencing. Thus, the abundance of ESTs in libraries should reflect the abundance of the corresponding mRNAs.

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(Sabelli and Shewry 1991, Cassidy et al 1998) suggested that these sequences corresponded to about 60% and half of the genomic sequences encoding α/β -­gliadins and LMW subunits, respectively, in hexaploid bread wheat. The chromosomal locations of the genes were determined by allele-­specific polymerase chain reaction, and their expression patterns were compared by counting the numbers of the corresponding ESTs in 32 cDNA libraries prepared from grain and other tissues (Ogihara et al 2003; Mochida et al 2006). This showed interesting and previously unrevealed differences in expression. The genes encoding LMW subunits showed similar patterns of expression, peaking at about 10 DAA and then declining. The ESTs for these genes corresponded to about 2.9, 0.8, and 0.4% of all ESTs at 10, 20, and 30 DAA, respectively. In contrast, the α/β -­gliadin genes from the B and D genomes were most highly expressed at 10 DAA and those from the A genome at 20 DAA. Thus, 4.2, 4.2, and 0.6% of all ESTs at 10, 20, and 30 DAA, respectively, corresponded to α/β -­gliadin genes. This striking difference in expression had not been revealed by previous studies, in which protein accumulation or hybridization of mRNA was used (e.g., Mecham et al 1981, Johansen et al 1994, Grimwade et al 1996, Altenbach et al 2002), emphasizing the power of modern EST and array technologies. HOW MANY GENES ARE EXPRESSED IN THE DEVELOPING WHEAT GRAIN?

Clarke et al (2000) suggested that between 4,500 and 8,000 genes are expressed in the developing wheat endosperm, based on sequencing 4,319 transcripts expressed between 8 and 12 DAA and mathematical modeling of the proportions of “singletons” (i.e., unique sequences) and gene families. Array technology cannot at present be used to provide data on all of the expressed genes in wheat, as the currently available systems do not provide whole-­genome coverage. However, analysis of rice using serial analysis of gene expression showed 3,080 unique tags in immature seed (7–14 DAA) (Gibbings et al 2003), while more­detailed studies of barley showed 14,548 unique tags in a whole­caryopsis library and 14,092 in an embryo library (Ibrahim et al 2005). Although a widely used commercial array for wheat includes 55,000 transcripts, it does not give full genome coverage. Nevertheless, preliminary studies have shown that at least 12,000 genes are expressed in developing caryopses of wheat cv. Hereward between 10 and 42 DAA (Wan et al 2007). Since some homoeologous transcripts would bind to the same array elements, this value is almost certainly an underestimate for hexaploid bread wheat.

Proteomic Studies Clarke et al (2000) also attempted to estimate the number of genes expressed in the developing wheat grain by separating and counting the gene products (i.e., individual proteins). They identified a total of almost 1,700 proteins by two-­dimensional electrophoretic analysis of a total protein fraction from endosperms at 10 DAA. This corresponds to 40% or less of the number of expressed genes calculated from analysis of the mRNA fractions from grain at 8–12 DAA (calculated as between about 4,500 and 8,000, see above).

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Several detailed proteomic studies of wheat grain development have been reported, comparing the patterns at different developmental stages and the impact of environmental factors on them. Skylas et al (2000) resolved about 1,300 individual components in total protein fractions from endosperm tissue of cv. Wyuna at about 17 DAA. A total of 321 proteins were transferred to membrane for determination of N-­terminal sequences by classical Edman degradation. A total of 177 proteins were identified by database matching, with the most abundant being seed storage proteins and inhibitors of proteinases or amylases. A further abundant protein was protein disulfide isomerase (PDI), with two forms being present. This is consistent with the role of PDI in catalyzing the formation of inter-­ and intrachain disulfide bonds in the newly synthesized protein (Boston et al 1996). About 17% of the proteins could not be identified based on sequences in databases, while 28% of the proteins analyzed did not give any N-­terminal sequence data. Comparison of the fractions from 17 DAA and mature (45 DAA) endosperms showed slightly fewer proteins in the latter, 1,125 compared with 1,300, with some proteins being especially down-­regulated. For example, PDI and the 60S acidic ribosomal proteins involved in protein synthesis were all down-­regulated at 45 DAA compared with 17 DAA. Vensel et al (2005) also compared the wheat endosperm proteome at two stages, 10 and 36 DAA, focusing on the salt-­soluble

proteins in order to exclude the major storage proteins. More than 250 components were identified by mass spectrometry and classified based on their putative biological roles (Fig. 3.56). The authors noted that the numbers and proportions of proteins involved in some processes, such as cell division, cytoskeleton, lipid metabolism, and signal transduction, were higher at 10 DAA (before grain filling) than at 36 DAA. In contrast, proteins involved in carbohydrate metabolism and protein synthesis were abundant at both stages, while storage proteins and proteins involved in stress and defense were dominant at 36 DAA. Skylas et al (2002) and Majoul et al (2004) focused on the effects of heat stress on the wheat proteome. Skylas et al (2002) compared heat-­tolerant (cv. Fang) and heat-­susceptible (cv. Wyuna) cultivars, analyzing developing (17 DAA) endosperms grown at day temperatures of 24 and 40°C. The heat-­tolerant cultivar, Fang, exhibited a stronger response to heat, and a total of 48 differentially expressed spots were characterized by mass spectrometry. Similarly, Majoul et al (2004) identified 43 proteins that were differentially expressed in mature grain when cv. Thésée was grown at 18 and 34°C during the day. In both cases, some of the up-­regulated proteins corresponded to previously characterized, small heat-­shock proteins, which are thought to prevent cellular damage. These small heat-­shock proteins accounted for most of the up-­regulated proteins in the study of Skylas et al (2002), but several of them were also differentially regulated between the cultivars. In addition, the heat-­stressed endosperms of cv. Fang expressed seven novel proteins of unknown function that were not detected in cv. Wyuna and could possibly be used as markers for thermal tolerance. In contrast, only five of the 24 up-­regulated proteins identified by Majoul et al (2004) corresponded to small heat-­shock proteins; the others included enzymes of starch synthesis, carbohydrate metabolism, and protein synthesis (initiation and elongation factors). These differences between the results of the two studies could relate to the fact that immature and mature grain were studied as well as differences in the temperatures used and in the tolerance and response of the individual genotypes.

STRUCTURE AND GRAIN MECHANICAL PROPERTIES The aim of milling is to isolate the starchy endosperm without contamination by the peripheral layers of the grain. This process depends on differences in mechanical properties among the various grain components. In fact, the rheological properties of the peripheral layers determine the degree of their incorporation into bran, while the mechanical properties of the starchy endosperm determine its fracture mechanics and the energy required to reduce it to flour. The differences in the mechanical properties of the endosperm and peripheral layers of the grain must be optimized by tempering to allow better separation of the tissues.

Fig. 3.56. Proteomic analysis of the timing of biochemical processes of wheat endosperm during grain development. A, profiles based on protein number. B, profiles based on normalized spot volume. Open columns are 10 days past anthesis (DPA); solid columns are 36 DPA. (Reprinted, with permission, from Vensel et al 2005)

Whole-­Grain Mechanical Properties

The mechanical properties of wheat grains are usually assessed on the basis of the grain hardness tests described by Pomeranz and Williams (1990). These assessments are generally performed on test samples containing a few grams of wheat

Development, Structure, and Mechanical Properties of the Wheat Grain  grains. They involve direct or indirect measurements of the particle size of the powder obtained after grinding under standard conditions using the particle size index or analysis of infrared spectra (by NIR spectroscopy). Energy consumption during milling reflects the mechanical resistance of the grains being processed. Milling energy is also often assessed indirectly by energy consumption measurements. Kilborn et al (1982) found that total milling energy ranged from 46 kJ/kg of grain for a soft wheat type to 124 kJ/kg for a durum wheat. Pujol et al (2000) described a micromill that directly measures the resistive mechanical torque on rolls of wheat during milling. They proposed a milling index based on energy consumption and on particle size reduction. This index varies between 100 kJ/kg of flour produced for soft wheat and 600 kJ/kg for durum wheat. A commercial “single kernel characterization system” (SKCS) apparatus has been described by Martin et al (1993). This instrument determines the force required to crush a grain between a rotating cylindrical roll and a smooth, fixed crescent-­shaped plate, the crushing force being measured with a strain gauge. The SKCS system is very convenient for rapid measurement of the mechanical properties of a single grain. However, the resulting data are processed to obtain values correlated with NIR hardness and do not provide a measure of the actual mechanical properties of the grains tested (Gaines et al 1996). Indeed, the complex geometric shape of the grain and the presence of a deep crease have complicated the study of their mechanical properties. Schoplanskaya (1952) used Hertz’s theory to describe the mechanical properties of wheat grain without taking into account the shape. This model was refined by Arnold and Roberts (1965) and by Shelef and Moshenin (1967), who determined a more rigorous value for the modulus of elasticity, which was in the range of 1.1–5.7 GPa for wheat grain of 10% moisture content. Although not devoid of interest, none of these global methods can discriminate between the physical properties and the effects of the morphology of the grain. Indeed, the crease depth constitutes a weakness point in the mode of rupture of the grains, and this characteristic is highly variable. Moreover, depending on the grain morphology (local curvature), the local stress and the contact surface at the interface between the kernel and the clamps may also vary widely. All this variability limits the establishment of a general relationship between the structure of the grain and its mechanical properties. Because the propensity of wheat to fracture results especially from the differences between the rheological properties of component tissues of the grain, research is now focusing on the measurement of the individual properties of each grain layer combined with the use of new tools for numerical simulation in order to reconstruct the properties of the whole caryopses.

Properties of the Outer Layers The separation between the flour and bran during milling is facilitated by the fact that the peripheral tissues form fragments that are larger than the particles originating from the endosperm. Determination of the mechanical properties of the outer layers of wheat is crucial in order to evaluate the ability of the raw material to be processed as well as to understand the underlying physicochemical basis for this ability.

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The main difficulty in determining the mechanical properties of the outer layers is in obtaining test samples from the peripheral layers of the grain. This was pioneered by Glenn and Johnston (1992a) and developed further by Peyron et al (2000) and Mabille et al (2001). In general, the embryo and brush are removed from the wheat grains by cutting transversely with a razor blade. The grains are then soaked overnight in distilled water. A crease incision is then made, and the endosperm is scraped from the outer layers, using a scalpel to obtain strips (10 × 2 mm) for rheological tests. Wheat bran is a composite material formed from different histological layers, and three different strips can be obtained from the soaked outer layers (Antoine et al 2003). The outer strip corresponds to outer pericarp (epidermis and hypodermis); the inner one corresponds to the aleurone layer; and the intermediate one remains a composite of several tissues (inner pericarp, testa, and nucellar tissue). All the strips can be obtained in a radial or a longitudinal orientation according to the long axis of the grain. Then strips are equilibrated at 17% moisture content in a saturated NaCl solution at 25°C before rheological measurements are made. Mechanical tests are generally performed under controlled temperature (30°C) and controlled relative humidity (75%) so as to maintain a moisture content of 17% during the experiment. After equilibration, the strips are subjected to a uniaxial tension test at a very low speed rate (90 Any Grain mixture, % 90% of all carotenoids in wheat (Kruger and Reed 1988, Chung and Ohm 2000). Dark green vegetables, such as cooked spinach and broccoli (62.6 and 16 µg/g wb, respectively), are considered the richest sources of lutein, followed by many yellow-colored fruits and other foods (e.g., egg yolk) (O’Neill et al 2001). According to the USDA National Nutrient Database, soft and hard white wheats, as well as hard red wheat, contain 2.2 µg (wb) of lutein and zeaxanthin per gram (USDA 2005). Among cereal grains, yellow corn is the richest source of lutein, containing 13.6 µg/g wb (USDA 2005). At the time of the third national health and nutrition examination survey (1988–1994), the median daily intake of lutein in the United States was about 1.7 mg (Institute of Medicine 2001). In five European countries, the median intakes range from 1.56 to 3.25 mg (O’Neill et al 2001). In two studies conducted in Finland in the 1990s, cereals were shown to be important contributors of dietary lutein intake. In one study, 430 µg of the 900-µg daily lutein intake was derived from cereals (Heinonen 1991), while in the other, 375 µg of the daily lutein intake of men (1,172 ± 471 µg) and 268 µg of that of women (1,046 ± 482 µg) was derived from cereals (Järvinen 1995). In a Spanish study, grains, corn, and peas accounted for only 2.8% of the daily lutein intake of 770 ± 780 µg (García-Closas et al 2004), while green leafy vegetables were the major source of lutein, with a share of 64.5%. The importance of wheat as a source of carotenoids is thus highly dependent on the overall diet. Even by increasing the consumption of wheat, it is not possible to reach the high lutein intake levels (e.g., 7.3 mg daily) suggested for optimal health and decreased risk of eye disease (Alves-Rodrigues and Shao 2004).

Carotenoids in Wheat Grains Data on carotenoid content in wheat, even in the most recent reviews, were derived mainly from the 1940s to the 1970s and indicated that the carotenoid content in wheat grains is in the range of 1.8–5.8 µg/g wb (Kruger and Reed 1988). Further, comparing carotenoid compositions of wheat samples from different studies is difficult because of the diversity in the analyti-

cal methods used. The data on carotenoids need to be updated. In the past, carotenoid content was measured spectroscopically from grain extracts and usually expressed as β -carotene equivalents. Nowadays, carotenoid composition can be obtained using more-specific HPLC techniques. Significant improvements have been gained in the separation of carotenoids and their geometrical isomers, as well as in specificity by using liquid chromatography-mass spectrometry. To measure total carotenoid content in wheat, modern techniques such as reflectance spectrometry (CIA L*a*b*) and front-surface absorbance spectra have been used as fast alternatives. Using these methods, tedious sample preparation procedures are simplified (Zandomeneghi et al 2000, Humphries et al 2004, Konopka et al 2004, Fratianni et al 2005). Since the year 2000, much new data have been published on the search for high-carotenoid varieties of wheat that could be exploited by plant-breeding programs and on wheat as a source of natural antioxidants and other biologically active compounds. Although carotenoid content in wheat is only moderate, high consumption levels make breeding programs worth the effort. The lutein and zeaxanthin contents of two commercial bread wheat cultivars grown in the United States differed markedly and the lutein contents were much lower than found in other studies (Table 7.5). Only 0.03 and 0.22 µg (wb) of lutein and 0.03 and 0.29 µg (wb) of zeaxanthin per gram of wheat were present in the two varieties (Humphries and Khachik 2003). Soft wheat grain samples from the state of Maryland contained more carotenoids than the previous samples. Lutein was the major carotenoid, contributing to 65–77% of total carotenoids. Its contents were between 0.82 and 1.14 µg/g wb, those of zeaxanthin between 0.20 and 0.39 µg/g wb, and those of β-carotene between 0.10 and 0.21 µg/g wb (Moore et al 2005). A green-harvested Australian cultivar contained much more carotenoids; the lutein content was 0.79 µg/g wb and that of zeaxanthin, 0.32 µg/g wb. All-trans isomers contributed to 79–89 and 61–77% of the total lutein and zeaxanthin contents, respectively (Humphries and Khachik 2003). Polish winter wheat cultivars contained lutein at levels of 1.48–1.80 µg/g db (Konopka et al 2004). In another Polish study, spring wheat cultivars contained even more carotenoids than the winter wheat varieties (Konopka et al 2006). The average carotenoid content of spring cultivars was 3.52 µg/g db and that of winter cultivars was 2.42 µg/g db, but the variation within each group was large (Table 7.5). Lutein accounted for 71–83% of all carotenoids, followed by zeaxanthin, β -carotene, and α-carotene. Lutein contents were higher than in the previous Polish study (Konopka et al 2004), which could be explained by the heat and light stress that the plants were subjected to in this study (Konopka et al 2006). Moreover, the number of carotenoids identified and measured was greater in this study, which partly accounted for the higher total carotenoid contents. Comparison of carotenoid contents (expressed as β -carotene equivalents) of grains from six durum wheat cultivars from three growing locations in Southern Italy over two years revealed that cultivar made the major contribution to the variation. In addition, a year × genotype interaction was evident. In all environments, the rank of each cultivar in terms of carotenoid content remained the same (Borrelli et al 1999). When grown under similar conditions, Italian durum wheat had a higher carotenoid

Micronutrients and Phytochemicals in Wheat Grain  content than bread wheats. Lutein contents in durum and bread wheat cultivars ranged from 1.69 to 2.75 µg/g wb and from 0.61 to 1.90 µg/g wb, respectively (Zandomeneghi et al 2000). In another study, lutein contents of Italian durum and soft wheat grains were 2.65 ± 0.65 and 1.31 ± 0.10 µg/g db, respectively (Panfili et al 2004). A similar trend was found in commercial Canadian wheats (Abdel-Aal et al 2002) but an opposite one in German wheats (Jahn-Deesbach et al 2004). When one durum wheat variety was grown in four locations in Italy, its lutein contents ranged from 1.63 to 3.23 µg/g db in 2001 and from 1.75 to 2.09 µg/g db in 2002. Thus, the variation within the same variety was twofold in one year and only 1.2-fold in the other, which means that the carotenoid contents are subject to great variation due to environmental conditions (Fratianni et al 2005). In a comparison of lutein contents of durum wheat samples grown in different locations in Italy in two different years, five samples showed higher lutein contents in the second year and two samples in the first year (Fratianni et al 2005). A study on several antioxidants performed on 11 bread wheat and durum wheat cultivars and lines in the United States showed that the variation was greatest in carotenoid levels (Table 7.5). Lutein was the predominant carotenoid, and its levels ranged from 0.26 to 1.43 µg/g wb. Zeaxanthin contents were in the range of 0.01–0.027 µg/g wb and β -cryptoxanthin contents in the range of 0.01–0.13 µg/g wb. The cultivars had fivefold, threefold, and 12fold differences in lutein, zeaxanthin, and β-cryptozanthin contents, respectively (Adom et al 2003). The variation among the

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wheat cultivars was great and could be useful in plant-breeding research. Moreover, durum wheat varieties did not show higher carotenoid levels than bread wheat varieties, which is an atypical result (e.g., Chung and Ohm 2000). In search of new sources of natural bioactive compounds, carotenoid compositions of alternative wheats such as einkorn and spelt grown in Canada were studied (Table 7.5) (Abdel-Aal et al 2002). Except for one einkorn cultivar, they all contained lutein levels of at least 7.29 µg/g db, which was much more than in the common soft white and red hard cultivars used as reference (2.39 and 2.09 µg/g db, respectively). Durum wheat also contained more lutein (5.93 µg/g db) than the reference wheats, while spelt had less (1.03–2.58 µg/g db). A diploid variety of wheat grown in Germany contained more lutein (3.95 µg/g wb) than two durum wheat cultivars (0.63–1.90 µg/g wb) and three spring and winter wheat cultivars (2.75–3.55 µg/g wb). Lutein contributed to 73–78% of the total carotenoids measured, the others being α- and β -carotene, violaxanthin, and neoxanthin (JahnDeesbach et al 2004). In a screening study conducted in Italy, 54 einkorn accessions contained, on average, 7.69 µg/g db of lutein and 8.41 µg/g db of total carotenoids (Hidalgo et al 2006). Also in this study, the levels for other wheat types (i.e., bread wheat, durum, spelt, and tetraploid emmer cultivars) were always lower, ranging from 1.40 to 4.79 µg/g db of lutein. In another study, 53 accessions of tritordeum, grown under greenhouse conditions in Spain, contained 2.6–9.4 µg/g wb total carotenoids that consisted of 99% lutein. The mean total carotenoid content (6.2 µg/g

TABLE 7.5 Lutein and Total Carotenoid Contents (µg/g) of Wheat Grains from Different Sources Sample Description

Lutein

1)b

Commercial bread wheat cultivar, Catoctin, from USA (n = Commercial bread wheat cultivar, Pioneer, from USA (n = 1) Green-harvested wheat, Freekeh, from Australia (n = 1) Soft wheat cultivars from Maryland, USA (n = 8) Winter wheat from Poland (n = 6) Winter wheat from Poland (n = 6) Spring wheat from Poland (n = 5) Bread wheat cultivars from Italy (n = 3) Durum wheat cultivars from Italy (n = 5) Bread and durum wheat cultivars from USA (n = 11) Commercial hard red spring wheat from Canada (n = 1) Commercial soft white wheat from Canada (n = 1) Commercial durum wheat from Canada (n = 1) Winter and summer wheats from Germany (n = 3) Durum wheat from Germany (n = 2) Genotypes of einkorn from Canada (n = 3) Breeding lines of einkorn from Canada (n = 12) Winter and spring spelts from Canada (n = 4) Diploid species of wheat from Germany (n = 1) Tritordeum accessions from Spain (n = 53) Durum wheat from Spain (n = 6) Einkorn accessions from Italy (n = 54) a References:  1

x 

0.03 0.22 0.79 0.82–1.1 1.5–1.8c 1.2–3.0c 2.0–3.8c 0.61–1.9 1.7–2.8 0.26–1.4 2.1c 2.4 c 5.9c 2.8–3.6 0.63–1.9 1.8–8.2c 7.7–8.7c 1.0–2.6 c 4.0

4.1–12.6 c

Total Carotenoids

1.3–1.7 1.7–2.2c 1.6–3.6 c 2.5–4.8c 0.77–2.2 2.1–3.4

3.1–4.1 0.83–2.3

5.1 2.6–9.4 0.7–1.8 5.3–13.6 c

Ref.a

1 1 1 2 3 4 4 5 5 6 7 7 7 8 8 7 7 7 8 9 9 10

= Humphries and Khachik (2003), 2 = Moore et al (2005), 3 = Konopka et al (2004), 4 = Konopka et al (2006), 5 = Zandomeneghi et al (2000), 6 = Adom et al (2003), 7 = Abdel-Aal et al (2002), 8 = JahnDeesbach et al (2004), 9 = Atienza et al (2007), 10 = Hidalgo et al (2006). bn  = number of samples. c Data  given on dry matter basis.

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wb) was 5.2 times higher than that in the control durum wheat samples (mean of 1.2 µg/g wb, n = 6) (Atienza et al 2007). These findings indicate that early cultivated and wild wheat genotypes could be potentially good sources of carotenoids.

Carotenoids in Wheat Milling Fractions Not much data are available on the distribution of carotenoids in the wheat kernel. Studies from the 1940s and the 1950s indicate that the color of wheat flour is constant up to an extraction rate of 72% (but if more outer layers of the grains are included, the flour becomes more yellow) and that whole-grain wheat contains ~15% more carotenoids than the endosperm and only 25% as many as the embryo (Kruger and Reed 1988). Lutein and β -carotene contents in commercial bread wheat flours (0.7–1.4% ash) and wheat bran consumed in Finland varied between 1.9 and 2.4 µg/g wb and between 70%). In all of the transformation events studied, suppression of BEIIa also led to loss of expression of BEIIb. Analysis of expression at the mRNA level indicated that, in plants containing the construct targeting BEIIa, the mRNA for BEIIa was strongly reduced; however, expression of BEIIb was not affected, indicating that the suppression of BEIIb activity was via a posttranscription mechanism (Regina et al 2006). A provisional conclusion from research on the linkage between the expression of BEIIa and BEIIb, and the phenotypes resulting when each gene is suppressed, is that BEIIa and BEIIb may have highly overlapping functions in starch biosynthesis, differing in their roles only by virtue of their differential expression patterns in different cereals. The properties of the high-­amylose wheat generated by RNAi are described in a later section. Debranching Enzymes: Essential for Normal Starch Synthesis in Cereals

Two classes of debranching enzymes (direct and indirect) are found in nature. Indirect debranching enzymes, found in fungi and animals, are bifunctional enzymes carrying both α-­ 1,4-­glucanotransferase and α-­1,6-­glucosidase active sites. Such indirect-­acting debranching enzymes act on glycogen to release d-­glucose and a modified polysaccharide having longer external chains. Direct-­acting debranching enzymes are found in bacteria and higher plants and contain only an α-­1,6-­glucosidase active site. Direct debranching enzymes cleave α-­1,6 linkages, resulting in the release of linear maltooligosaccharides. In plants, two classes of direct-­acting debranching enzymes have been described, isoamylase-­t ype enzymes that act on glycogen and amylopectin but not pullulan, and pullulanases, which can cleave the α-­1,6 linkages of pullulan. The recognition that isoamylase-­t ype debranching enzymes were required for normal starch synthesis came through the work on the sugary-­1 mutation in maize (James et al 1995) A transposon-­tagging approach was used to demonstrate the linkage between a mutation in the isoamylase gene in maize and the sugary phenotype. In the sugary phenotype, amylopectin synthesis is reduced and replaced to some extent by the presence of a noncrystalline “phytoglycogen.” The necessity for isoamylase for normal starch synthesis has been confirmed in a wide range of species, including Chlamydomonas (Mouille et al 1996), rice (Kubo et al 1999, Fujita et al 2003), Arabidopsis (Zeeman et al 1998), and barley (Burton et al 2002a). The availability of whole genome sequences has led to the identification in Arabidopsis of three isoamylase-­t ype genes and one pullulanase (also known as limit dextrinase) type (AtISA1, AtISA2, AtISA3, and AtPU1, respectively) (Delatte et al 2005, Wattebled et al 2005). Elimination of either AtISA1, AtISA2, or both AtISA1 and AtISA2 results in a low-­starch, phytoglycogen-­accumulating phenotype (Delatte et al 2005, Wattebled et al 2005), consistent with the view first proposed on the basis of research in potato that both gene products are subunits of a complex isoamylase heteromultimer (Hussain et al 2003). On the basis of an excess­starch phenotype, the AtISA3 gene appears to be involved in the catabolism of starch rather than in the synthesis of starch

Carbohydrates  (Wattebled et al 2005). Down-­regulation of pullulanases in wild­t ype backgrounds leads to subtle changes in starch structure only, whereas in backgrounds deficient in isoamylase-­1 or -­2, a marked additional phenotype is observed (Dinges et al 2003, Wattebled et al 2005). The roles of isoamylases in starch synthesis have been a controversial topic, with at least three roles proposed for isoamylase. One model proposes that isoamylase acts on “preamylopectin” by editing or shaping the polysaccharide such that crystallization into amylopectin lamellae can occur (Ball et al 1996, Myers et al 2000). A second view suggests that isoamylase is required to clear water-­soluble polysaccharides from the stroma of the amyloplast that might compete with starch synthesis (Zeeman et al 1998). A third role proposed is for isoamylase to be involved in the initiation of starch granules (Zeeman et al 1998, Burton et al 2002a, Bustos et al 2004). Further research is required to fully define the role of isoamylases in starch granule synthesis; however, the alterations in the fine structure of amylopectin in isoamylase-­deficient starches (Dinges et al 2001, Kubo et al 2005, Wattebled et al 2005) argue for a direct role in amylopectin synthesis rather than an indirect role or a role confined to granule initiation alone. Isoamylase genes in wheat have been described (Genschel et al 2002, Rahman et al 2003). In wheat, no isoamylase mutants or down-­regulation events have been reported; however, given the strength of observations in other plant systems, notably barley (Burton et al 2002a), it would be highly unlikely if suppression of the activity of isoamylase-­1 or -­2 did not result in a sugary phenotype. The strength of this prediction is increased by work showing that the rice sugary-­1 mutation could be complemented by transformation with the wheat isoamylase-­1 gene (Kubo et al 2005). The Roles of Other Genes in Starch Biosynthesis

Several other classes of genes have been suggested to be involved in starch synthesis on the basis of evidence from other systems. Disproportionating enzymes (D-­enzymes) transfer α -­1,4-­g lucans among oligosaccharides, with the concomitant release of glucose. Mutations eliminating D-­enzyme in Chlamydomonas reinhardtii caused a major reduction in starch synthesis (Colleoni et al 1999a,b), although the reason for this reduction is unclear. In Arabidopsis, mutation of D-­enzyme activity in the leaves has no impact on starch synthesis, suggesting that this enzyme has no role in starch synthesis in Arabidopsis leaves (Critchley et al 2001). A D-­enzyme activity from wheat endosperm has been described; however, its role in starch synthesis, if any, is yet to be defined (Bresolin et al 2005). Further research is required to define the role of this enzyme, if any, in starch biosynthetic processes in higher plants. Another enzyme with potential roles in starch synthesis and degradation is starch phosphorylase. A major role in starch synthesis is not indicated by the major impact on starch content of mutations affecting ADPG PPase; however, a minor role is possible. Two cytosolic forms of phosphorylase and a plastidic form have been described in wheat (Schupp and Ziegler 2004). The cytosolic phosphorylase is expressed during germination of the seed, suggesting a role in starch degradation. The plastidic form

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is expressed in endosperm, but no clear role for this enzyme in synthesis or degradation has been defined. The molecular events responsible for starch granule initiation have been of interest not only because of the intrinsic scientific interest but because of the potential to manipulate starch granule number and size. An autocatalytic, self-­g lucosylating protein covalently attached to glycogen, named glycogenin, was first described by Whelan (1985). In yeast (Cheng et al 1995) and mammals (Hurley et al 2005), there is strong evidence for the involvement of glycogenins in glycogen biosynthesis. There is no evidence for any requirement for a glycogenin-­like protein in bacterial glycogen synthesis. Plants contain diverse glycogenin­like genes, which may be involved in the synthesis of a range of polysaccharides; identifying those that may be involved in starch synthesis in plants has proved challenging. There is one report of glycogenin being required for starch synthesis in Arabidopsis (Chatterjee et al 2005), but further confirmation of this phenomenon is required. The recent report of a potential role for SSIV in granule initiation in Arabidopsis leaves (Roldan et al 2007) suggests that plants may employ a mechanism for granule initiation that does not involve a glycogenin-­like precursor. The glucan water dikinase (GWD) family of proteins (also known as R1) has been demonstrated to be responsible for the phosphorylation of starch in leaves and tubers (Ritte et al 2002, Mikkelsen et al 2004), where it is thought to form a part of the starch degradation system. Antisense inhibition of GWD leads to a starch-­excess, low-­starch-­phosphorylation phenotype (Lorberth et al 1998). A phosphoglucan water dikinase (PWD) encoded by gene At5g26570 has been identified in Arabidopsis (Bárcenas Baunsgaard et al 2005, Kotting et al 2005). This enzyme preferentially introduces phosphate groups at the C3 position of starch, but only after phosphorylation of starch by GWD. Interruption of the PWD results in a starch-­excess phenotype and the loss of phosphorylation of starch at the C3 position. The presence and role of these enzymes in the developing cereal endosperm is undefined. Regulation of Starch Biosynthesis

Research over the past decade has generated considerable evidence that starch biosynthesis is regulated at several levels and that this regulation is important in determining both the amount and structure of starch produced in any one tissue. Five regulatory processes have been identified. Gene Duplication and Transcriptional Control of Gene Expression. One basic mechanism for controlling starch synthesis in various tissues is the duplication of genes for enzymes, accompanied by the differential expression of members of the duplicated families in different tissues. Examples of this type of regulation are seen for ADPG PPase (Burton et al 2002b), GBSS (Vrinten and Nakamura 2000), SSIIa (Jiang et al 2004), SSIII (Dian et al 2005), and BEIIa/BEIIb (Yamanouchi and Nakamura 1992, Gao et al 1997, Sun et al 1998). Two rounds of duplication appear to have occurred for cereal ADPG PPases, an initial ancient duplication leading to the generation of the large and small subunits and a presumably more recent duplication leading to the generation of genes that lead to the plastidic and cytosolic forms of the enzyme. Within a particular tissue, evidence from a range of species indicates that enzymes involved in starch

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s­ ynthesis are under circadian control (Wang et al 2001, Dian et al 2003, Tenorio et al 2003). Microarrays were used to investigate oscillations in starch gene expression in Arabidopsis leaves; the study concluded that GBSSI and SSII oscillated on a diurnal light-­dark cycle (Smith et al 2004). Allosteric Regulation. The well-­characterized sensitivity of ADPG PPase to activation by 3-­phosphoglycerate (3PGA) and inhibition by inorganic phosphate (Pi) (reviewed in Ballicora et al [2004]) modulates ADPG PPase activity levels in response to photosynthetic activity in chloroplasts. In sink tissues such as cereal endosperm, the cytosolic form of ADPG PPase is clearly much less sensitive to 3PGA activation, while retaining sensitivity to Pi inhibition (Gomez-­Casati and Iglesias 2002). Given the absence of direct photosynthetic activity producing 3PGA in this tissue (and yet the need to modulate activity in response to energetic status, as indicated by Pi pools), this pattern of allosteric regulation is in keeping with the metabolism and functions of these tissues. Redox Regulation. ADPG PPase from a number of species has been shown to be regulated by redox mechanisms (Fu et al 1998, Hendriks et al 2003, Tiessen et al 2003). Preliminary reports suggest that ADPG PPase from barley and wheat endosperms is also regulated by redox control (Tetlow et al 2004), and recently, a number of targets of redox regulation have been identified in the cereal endosperm, including ADPG PPase subunits, the brittle-­1 ADPG transporter, starch phosphorylase, and BEIIa (Balmer et al 2006). In Arabidopsis leaves, a redox-­regulated phosphatase has been demonstrated to bind to starch in the light and dissociate at night; disruption of the gene encoding this enzyme leads to a high-­starch phenotype (Sokolov et al 2006). Phosphorylation. In extracts of wheat endosperm amyloplast, a large number of proteins have been shown to be phosphorylated when amyloplasts are incubated with 32P-­ATP (Tetlow et al 2004). Among those phosphorylated are BEI, BEIIa, BEIIb, and plastidic phosphorylase; dephosphorylation of these enzymes resulted in a reduction in enzyme activity (Tetlow et al 2004). The role of protein phosphorylation in regulating starch synthesis requires further research. Complex Formation. Immunoprecipitation experiments have been used to show that complexes of starch biosynthetic enzymes exist in the wheat endosperm, including a complex containing BEI, BEIIb, and starch phosphorylase (Tetlow et al 2004). As noted previously, both isoamylases and ADPG PPase exist in heteromultimeric forms in plants, suggesting that the protein-­protein interactions formed through protein aggregation into complexes may be a general feature of starch biosynthesis. However, further work is required to define the number, composition, and function and the formation/disintegration behavior of such complexes. Degradation of starch

The degradation of starch during germination has been extensively studied in malting barley, and the reader is referred to the extensive literature on this subject for information that is likely to be of strong relevance to the events occurring during germination in wheat. There is limited information on starch degradation events occurring during starch deposition

in the developing endosperm, although a number of enzymes associated with starch degradation, including the α-­amylases, β -­a mylases, α-­glucosidases, starch phosphorylase, and disproportionating enzyme, are present in the developing endosperm. Several extensive reviews of starch degradation in plants have been published (Manners 1985, Lloyd et al 2005, Smith et al 2005, Zeeman et al 2007).

Relationships Between Starch Synthesis, Structure, and Functionality The range of uses for starch, and indeed wheat starch, is extremely broad, ranging from feedstocks for industrial processes such as ethanol production and industrial materials such as sizing and adhesives through to the traditional food uses in bakery products, noodles, beverages, pastas, and many more. The degree to which starch structure and functionality impact these myriad end uses is an area of research that is in many ways in its infancy, largely because the range of genetic variation in starch properties in wheat has been hitherto extremely limited compared to the range of variation possible in protein properties. However, the tools for generating new wheats based on both conventional breeding and transgenic technologies are developing rapidly, and it is anticipated there will be an exponential growth in the amount of genetically defined diversity available; the challenge will be to define the key relationships that deliver utility and value. Four general mechanisms for increasing the genetic diversity in wheat starch properties are available: 1) screening for natural variation, 2) mutagenesis, 3) use of down-­regulation technologies via genetic engineering, 4) use of genetic engineering to express foreign genes or over-­express endogenous genes. The remainder of this section focuses on the use of natural diversity and mutagenesis screens to identify and combine altered or null alleles of starch biosynthetic genes as a powerful technology for developing new wheats. The identification and exploitation of genetic diversity at the GBSS loci serve as an excellent case study for generating new opportunities in wheat starch diversity and wheat end use. The exploitation of genetic diversity at the GBSS locus was made possible by the observation that the products of the three GBSS loci could be separated under carefully defined conditions on sodium dodecyl sulfate–polyacrylamide gel electrophoresis gels (Yamamori et al 1992). Given the existence of engineered lines with highly defined chromosomes, it was a straightforward matter to assign each of the bands to a specific chromosome and to begin a search for “null” alleles for GBSS (Yamamori et al 1992). This search was rewarded by the identification, in natural populations, of mutations eliminating GBSS expression from each of the 4A, 7A, and 7D GBSS loci (Nakamura et al 1995, Yamamori et al 1995). While alleles for 4A and 7A are reasonably abundant, the 7D allele was rare and has been used from a limited number of primary sources. Identifying this set of null alleles has facilitated the development of four types of products based on GBSS alleles: 1) triple-­null “waxy” wheats, 2) single-­null lines, 3) double-­null lines, and 4) partial­waxy lines generated through mutagenesis of double-­null lines, yielding lines with a single partially active GBSS allele. At the molecular level, the structural basis of several null loci used in

Carbohydrates  germplasm development has been characterized (Vrinten et al 1999; Yanagisawa et al 2001, 2003). The structural and processing functionality of “triple-­null” waxy wheats has been extensively analyzed. The most obvious feature of such lines is an amylose content of less than 1% (Nakamura et al 1995, Yasui et al 1996, Demeke et al 1999), typical of waxy wheats across many species. The size distribution of starch granules in waxy wheats is reported to be very similar to that in nonwaxy wheats (Fujita et al 1998). The structure of the amylopectin of waxy wheats differs in its overall molecular weight distribution, having a lower molecular weight distribution with a more compact branching structure, i.e., a decrease in very long chains within the amylopectin fraction (Yoo and Jane 2002b). The chain length distribution of amylopectin between DP 6 and 50 remains unaltered (Yasui et al 1996, Yoo and Jane 2002b). DSC analysis has demonstrated that waxy wheat starch has higher gelatinization onset temperatures and gelatinization enthalpy and the absence of an amylose-­lipid complex compared with nonwaxy wheat (Yasui et al 1996, Fujita et al 1998, Demeke et al 1999, Lee et al 2001, Abdel-­Aal et al 2002). X-­ray diffraction studies show that waxy wheat retains the classical A crystal packing unit of the cereals and has a higher degree of crystallinity than nonwaxy starches (Fujita et al 1998, Demeke et al 1999, Kim et al 2003), consistent with the increase observed in gelatinization onset temperature. RVA analysis of waxy lines has shown higher peak viscosity, lower peak temperature, and shorter peak time than for other nonwaxy and partial-­waxy genotypes (Abdel-­Aal et al 2002, Kim et al 2003). Properties of partial-­waxy (low-­amylose) starches in yellow alkaline noodles (Tanaka et al 2006) and starch gels (Sasaki et al 2007) have been investigated. Starch swelling has been examined in durum, where waxy starch had four times more swelling power than nonwaxy durum starches (Grant et al 2001). Lipid contents (0.12–0.29 g/100 g) are much lower than for nonwaxy lines (1.05–1.17 g/100 g) (Yasui et al 1996). No amylose-­lipid peak is detectable in DSC analysis (Abdel-­Aal et al 2002, Yoo and Jane 2002b). Waxy wheat has now been examined for suitability in a range of end products. In milling tests, waxy wheat starch granules are considerably less resistant to mechanical damage than normal starch granules (Bettge et al 2000). Despite starch granule size and protein contents being consistent with those of nonwaxy wheats, water absorption values are greatly increased (Lee et al 2001, Abdel-­Aal et al 2002, Baik and Lee 2003, Kim et al 2003). Baking tests with waxy wheats have used both straight waxy flour and blends with nonwaxy flours. The results to date suggest that unblended waxy wheats are not suited for use in baking applications (Kim et al 2003); however, the addition of up to 20% waxy wheat had interesting potential in terms of reducing crumb firmness (Lee et al 2001) and extending shelf life through reduced retrogradation (Bhattacharya et al 2002, Hayakawa et al 2004). Several studies have examined the utility of waxy wheats for production of white salted noodles, concluding that full waxy noodles are softer, thicker, less adhesive, less chewy, and more cohesive and springy than nonwaxy lines (Epstein et al 2002, Baik and Lee 2003). Instant noodles prepared from waxy wheat flours yielded thicker strands and higher free-­lipid contents than nonwaxy wheat flours, with numerous surface bubbles, and the

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waxy instant noodles stuck together during frying. Waxy instant noodles were softer and more cohesive than nonwaxy lines (Park and Baik 2004). Pasta produced from waxy semolina had decreased firmness and increased stickiness; thus, waxy starch is not favorable for pasta quality (Grant et al 2001, 2004; Gianibelli et al 2005). End users of wheat may favor particular sources of grain because of price, ability to meet technical specifications, or reliability of supply. In one case study, consumers of a specific end product drove the selection of a specific wheat that is now understood to derive from a specific alteration in starch synthesis, linking soft wheat properties to consumer preferences for Japanese white salted noodles (Nagao et al 1977). Endo et al (1989) recognized that Japanese consumers preferred wheats from Western Australia and suggested that this was due to the particular starch properties of lines derived from that source. Further investigations of this starch type (Toyokawa et al 1989, Crosbie 1991, Miura et al 1994) demonstrated that low amylose content, high starch paste viscosity, and higher swelling properties underpinned the desired characteristics. These characteristics are associated with the loss of the GBSS protein encoded by the GBSS locus on chromosome 4A (Zhao et al 1998). The association between that locus and a range of starch properties has been confirmed in a wide range of association and mapping studies (Batey et al 2001). This observation led to the development of high-­t hroughput methods designed to assist breeders to routinely deliver wheats for this market (Crosbie et al 1992, Zhao and Sharp 1996, Batey et al 1997, Briney et al 1998, Graybosch et al 1998, McLauchlan et al 2001, Shariflou et al 2001, Gale et al 2004). This example is powerful in demonstrating that very subtle changes in starch structure can translate into significant differences in starch functionality that, in turn, result in changes in the consumer appeal of high-­value end products. Two further categories of variation in GBSS alleles have been explored. One is the use of double-­null lines (lines lacking either the 4A and 7A, 4A and 7B, or 7A and 7D GBSS alleles) (Zhao and Sharp 1998, Baik et al 2003, Kim et al 2003, Park and Baik 2004), and the second is the exploitation of mutations that modify the activity of a single GBSS allele induced through the mutagenesis of double-­mutant lines (Yanagisawa et al 2001, Yasui et al 2002). TILLING methods demonstrate that it is now possible to generate a nearly inexhaustible allelic series of mutants that can be used to fully explore the impact and utility of double-­null waxy germplasm carrying mutations that modify the activity of the third, partially active GBSS allele (Slade et al 2005). The identification of the sgp-­1 proteins from the wheat starch granule as the products of a homeologous series of genes (Denyer et al 1995, Yamamori and Endo 1996) led to the development of wheat lines lacking each of the homeoforms of sgp-­1 (Yamamori 1998, Yamamori et al 2000). The sgp-­1-­deficient wheat has an elevated amylose content (30.8–37.4%), deformed starch granules, and a decrease in amylopectin chains from DP 11 to 25. X-­ray crystallography showed a reduced level of starch crystallinity, and DSC showed a reduced gelatinization onset temperature. The properties of flours from sgp-­1 lines have been investigated in breadmaking (Morita et al 2002), where such flours were shown to have high water absorption and showed weaker, less­stable properties compared with the control. Loaf volume was

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reduced. The properties of wheat flours containing waxy and elevated-­amylose starches have recently been reviewed (Hung et al 2006). A combined Sgp-­1/Waxy wheat line has been generated (Nakamura et al 2006) and shows a dramatically shrunken phenotype and a high sugar level.

Starch and the Staling of Bread The staling of bread is a significant phenomenon restricting the shelf life and acceptability of bread. “Staling” is defined as a term which indicates decreasing consumer acceptance of bakery products caused by changes in crumb other than those resulting from the action of spoilage organisms

(Bechtel et al 1953). The subject has been reviewed over many years (see, for example, Herz 1965, Zobel 1973, Knightly 1977, and most recently, Gray and BeMiller 2003). The overall conclusion of these reviewers is that there is still no comprehensive understanding of the factors that lead to staling, although retrogradation processes involving starch are highly correlated with crumb firming and decreases in sensory appeal. Amylose retrogradation is thought to happen rapidly after baking, whereas staling occurs after extended storage, leading to the hypothesis that amylopectin retrogradation, which occurs on the same time scale as staling, is responsible (Gray and BeMiller 2003). However, Gray and BeMiller (2003) advise caution on the basis that this is a correlation and not a demonstrated cause-­a nd-­effect relationship A range of other phenomena have been suggested to be important in staling. The redistribution of water within the loaf (Rasmussen and Hansen 2001, Ribotta et al 2004, Ribotta and LeBail 2007) and protein-­starch interactions (Gerrard et al 2001) have also been postulated to contribute to staling. Mechanisms to prevent staling have fallen into four general classifications: 1) addition of polymers such as hydroxymethyl cellulose (Bárcenas and Rossell 2005) or cereal pentosans (Denli and Ercan 200l); 2) addition of emulsifiers (see Gray and BeMiller [2003] for a review); 3) addition of enzymes such as α-­a mylase (Blaszczak et al 2004), xylanase, cellulase, and β -­g lucanase (Caballero et al 2007); and 4) use of modified starches, either chemically modified (e.g., hydroxypropylated [Miyazaki et al 2006]) or genetically modified (e.g., waxy wheat starch [Hung et al 2007]). Modes of action of these enzymes or additives in retarding staling are suggested to include either the prevention of retrogradation through starch degradation or inhibition of retrogradation through hindrance of crystallization processes. Further research is clearly required to fully define the mechanism(s) underpinning staling. The generation of a wide range of genetically modified starches, such as waxy wheats with a demonstrated staling-­retardation effect (Hung et al 2007), provides the prospect of finding bread formulations that contain starches or combinations of starches that are inherently less prone to crumb firming on storage and thus less prone to staling.

Starch and Nutrition Linkages between starch properties and human gastrointestinal physiology are subjects of emerging importance, not least because of the increasing incidences of diseases such as type II

diabetes and increased obesity in many countries. Starch structure and functionality can impact gastrointestinal functionality in two main ways. First, they can affect the rate of digestion through the gastrointestinal tract to the point of glucose uptake in the small intestine, and second, they can influence the extent of digestion to that point, determining the amount of starch and starch digestion products passing into the large bowel. The rate of digestion influences the glycemic response of a food, while the extent of digestion controls the “resistant starch” content of a food. One challenge for researchers in this field is that neither glycemic index nor resistant starch represents a chemical entity that can be simply assayed by an analytical procedure; both are measures of physiological processes occurring in the human body and are strongly influenced not only by the properties of the raw material, but also by the nature of the processing that has occurred in the formulation of the food. A number of reviews of glycemic index and resistant starch have been published (Brown 1994, 1996; Topping and Clifton 2001; Jenkins et al 2002; Topping et al 2003a; Kendall et al 2004), and several recent publications have explored aspects of granule structure and in vitro digestibility in cereals (Stevnebo et al 2006; Zhang et al 2006a,b; Benmoussa et al 2007) Resistant starches have been traditionally classified into four groups: RS1, physically inaccessible starch; RS2, starch granules resistant to digestion; RS3, retrograded starch; and RS4, chemically modified starch. High-­amylose maize starches are known to provide one source of resistant starch, delivering a combination of RS2 and RS3 resistant starches (Brown 1994; Muir et al 1995; Topping et al 1997; Brown et al 1998, 2000; Wang et al 1999). Studies using a high-­amylose wheat starch fed to rats (Regina et al 2006) indicate that wheats with an amylose content of 70% can also deliver benefits such as increased digesta weight, reduced luminal pH, and increased short-­chain fatty acid production, all typical of other sources of resistant starch. Mutations in the SSIIa gene can also lead to grains and flours with altered nutritional characteristics. For example, the high-­amylose (70% amylose) sex6 mutant of barley shows impacts on gastrointestinal performance consistent with the presence of high levels of resistant starch (Topping et al 2003b; Bird et al 2004a,b) Products made from sgp-­1-­deficient wheats with amylose contents of 35–40% contain slightly increased levels of resistant starch. Use of an in vitro enzyme hydrolysis method to predict resistant starch levels showed that the levels of RS increased in breads with 10, 20, and 50% substitutions of sgp-­1 null wheat to 1.6, 2.6, and 3.0%, respectively, compared to a baseline level of 0.9% for wheat with a standard flour (Hung et al 2005). Further increases in resistant­starch levels, as predicted, were observed on storage (Hung et al 2005).

Future Directions The past decade has seen an explosion of knowledge about the synthesis, structure, and functionality of starches. While model systems such as pea, Arabidopsis, Chlamydomonas, and rice have been fundamental tools in developing our understanding, substantial progress has been made in key delivery systems such as bread wheat and durum. A number of areas of wheat starch research are in need of further investment. In this section,

Carbohydrates  selected areas are discussed, but clearly this is not intended to be an exhaustive list. Many of the fundamental questions in starch synthesis are most likely to be addressed through model systems, although some groundbreaking work has occurred in wheat, such as the identification of complex posttranslational regulation of starch synthesis enzymes involving phosphorylation and formation of complexes (Tetlow et al 2004). Two aspects of starch synthesis research have specific relevance for wheat. First, the relationship between the enzymatic machinery of starch synthesis and the biology of the amyloplast and cell requires further attention. The mechanisms for initiating B granules clearly implicate the evaginations of the amyloplast membrane, and researchers as long ago as 1960 (Buttrose 1960) suggested that the tubuli, invaginations of the amyloplast membrane, are involved in directing the supply of substrate to starch granules, thus potentially influencing the highly spatially directed growth of granules. Second, the regulation of starch synthesis is an area for further research. Several mechanisms (transcriptional control, allosteric regulation, redox mechanisms, phosphorylation, and complex formation) appear to be important in regulation and have species-­ or tissue-­specific elements that need to be understood in various crop species. The promise of transgenic manipulation of plant productivity, composition, and functionality has been delayed because of real or perceived political, consumer, or regulatory issues surrounding the use of transgenic technologies. This has been particularly the case in wheat, where international trade issues have mitigated against commercial deployment of the technology. Recent studies (Meyer et al 2004, Regina et al 2006) demonstrate that transgenic technology in wheat is a powerful research tool, facilitating proof-­of-­concept studies on genetic mechanisms. One consequence of the issues surrounding deployment of transgenic technology has been the spawning of a plethora of technologies for the rapid and high-­t hroughput identification of specific mutations in the plant genome, and, in wheat, the use of techniques such as TILLING technology (Slade et al 2005) exemplifies this trend. Undoubtedly, the next decade will see a further explosion in knowledge driven by the ability to generate and analyze defined diversity through the application of both transgenic and mutagenic technologies. The availability of vastly greater genetic diversity will sharpen the need to be able to rapidly and rigorously phenotype populations in order to derive both scientific and commercial value. Two trends have become evident. The first is the need to define functionality in terms of specific end-­product categories because of the individual needs of many classes of products, from bakery products to noodles, pasta, etc. The second is the need to consider functionality in the human gastrointestinal tract as a key phenotype worthy of ever-­increasing attention because of the growing pace of development of lifestyle diseases such as type II diabetes, bowel health problems, cardiovascular disease, and obesity in many developed and developing countries. The knowledge and tools available to cereal scientists now open the door to the exploration of the impact of carbohydrates on cereal products and their health outcomes, leading to a new era of research delivering a combination of scientific knowledge, commercial opportunity, and benefits for consumers.

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CELL WALL POLYSACCHARIDES Occurrence, Composition, and Organization in Walls of Cell Types in Grain Cereal and grass species belong to families of the Poaceae among the Poales group of commelinid monocotyledons (Harris 2005, Trethewey et al 2005). In their grains, the unlignified (primary) walls of cells in the starchy endosperm, aleurone, scutellum, and embryonic axis are characteristically rich in (glucurono)arabinoxylans and, in the case of starchy endosperm and aleurone, (1→3,1→4)-­β-­d-­g lucans (Table 9.5). Smaller amounts of cellulose and glucomannans are also present, but xyloglucans and pectins, the typically abundant components of noncommelinid monocotyledon and dicotyledon species, are minor components, if present at all. The lignified secondary walls of cells in the pericarp­seed coat of the grain are rich in cellulose (60%) and (glucurono)arabinoxylans (30%), with smaller amounts (1.2%) of (1→3,1→4)-­β-­d-­g lucans (Harris et al 2005). In addition to lignin (Bunzel et al 2004), other nonpolysaccharide components may be present, e.g., suberin in the nucellus, cutin in the walls of the seed coat, and cutin and silica in the walls of epidermal cells of the pericarp (Chapter 3). Structural and enzymic (glyco)proteins are probably present in walls of all cell types (Jose-­Estanyol and Puigdomenech 2000, Rhodes and Stone 2002). There are relatively few compositional studies on walls isolated from specific cell types in the grain; more often, studies are of milling fractions, which invariably contain more than one cell type. Thus, wheat flour, although composed largely of starchy endosperm cells, includes more or less bran (aleurone + pericarp-­seed coat) depending on the cultivar and the milling regime. This is shown dramatically in the broad range of compositional values of 20 French wheat flours (Ordaz-­Ortiz and Saulnier 2005). Bran, another easily accessible milling fraction, has been extensively characterized, but its tissue heterogeneity and variability in composition, e.g., the amount of associated starchy endosperm, must be borne in mind when evaluating compositional data and functional attributes (Saadi et al 1998). Cellulose Occurrence and Structure

Cellulose, a homopolymer composed of (1→4)-­linked β-­d­glucopyranose units (Fig. 9.8A), is found in walls of all cells of

grain tissues. The total content in grain is 2% (dw). In flour, the cellulose content is as low as 0.3% (Fraser and Holmes 1959). In the primary walls of cells of the starchy endosperm and aleurone, it makes up ~2% (w/w) (Table 9.5). The highest concentrations, 30% (w/w), are in the secondary walls of pericarp-­seed coat tissues and intermediate layers (Table 9.5). The long, unbranched molecular chains of cellulose (6,000 glucose units in primary walls and up to 14,000 in secondary walls) have an extended, ribbonlike conformation that allows parallel packing of the chains into three-­dimensional micro­ fibrillar aggregates stabilized by extensive intermolecular hydrogen bonding and van der Waals interactions (Fig. 9.8B). The

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microfibrils may reach diameters of ~3 nm in primary walls and 5–10 nm in secondary walls. In the microfibrils, individual cellulose molecules are mostly packed into highly ordered crystalline arrays, although there are less ordered regions, particularly on

the surfaces, where the molecular conformation and hydrogen bonding arrangement of the chains may be different. The noncellulosic matrix polysaccharides of the wall—the arabinoxylans, glucomannans, and (1→3,1→4)-­β-­d-­glucans—are believed

TABLE 9.5 Compositions of Cell Wall Types in Wheat Grain Tissues (% dry weight) Ref.a

Cellulose

Gluco­ mannan

Starchy endosperm Aleurone

A B

 2  2

7 2

Aleurone cv. Barouder Aleurone cv. Scipion Intermediate layerc cv. Barouder

C C C

Intermediate layerc cv. Scipion Bran (pericarp, seed coat, aleurone)

Origin of Walls

Bran

(1→3,1→4)­ β-D-Glucan

Heteroxylan

Feruloyl Ester

20 29

70 65

 4  3 25

34 42  8

62 54 67

1.8 1 in 70 AHPb 7.10 mg/mg 6.28 mg/mg 5.01 mg/mg

C

20

11

69

5.00 mg/mg

D

29

 6

64

E–G

7.1 mg/g (E) 10.3 µg/mg (G)

Beeswing bran (outer pericarp)

H

30

Pericarp-rich fraction Pericarp cv. Barouder Pericarp cv. Scipion Beeswing bran, mol% total carbohydrate

E C C H

30 33 30 19e

79f A/X g 1.12

4.9 mg/g 3.06 mg/mg 4.36 mg/mg 1 µg/mg (high DDFAg)

Cross cells, mol% total carbohydrate

I

20e

74f A/X 1.06

1 µg/mg (low DDFA)

Testa + nucellar epidermis, mol% total carbohydrate

I

  6e

91f A/X 0.13

5 µg/m (low DDFA)

Aleurone, mol% total carbohydrate

I

15e

80f A/X 0.37

9 µg/mg (low DDFA)

Starchy endosperm, mg/100 mg tissue

J

1.7/1.5h A/X 0.81/0.87

Scutellum, mg/100 mg tissue

J

7.9/8.1h A/X 1.31/1.39

Embryonic axis, mg/100 mg tissue

J

14.8/14.9h A/X 1.35/1.35

Aleurone layer, mg/100 mg tissue

J

28.3/20.5h A/X 0.47/0.41

Hyaline layer, mg/100 mg tissue

J

66.8/53.1h A/X 0.10/0.13

Intermediate layer, mg/100 mg tissue

J

39.4/38.3h A/X 0.42/0.43

Outer pericarp, mg/100 mg tissue

J

47.8/46.2h A/X 1.19/1.13

0.05/0.04 µg/mg tissue 7354/6076i 3.48/3.32 µg/mg tissue 271/331i 0.31/0.57 µg/mg tissue 2053/1860i 8.14/7.98 µg/mg tissue 1299/1195i 9.84/10.83 µg/mg tissue 2417/3003i 4.67/4.96 µg/mg tissue 1359/1306i 2.04/4.50 µg/mg tissue 318/240i

a Reference

60   1.2  4 12

66.7 63 57

Acetyl Ester

1 in 3 AHP

Lignin

Protein

Nil Nil

0.5 (B) 1

  8.3

9.2d

12

6d

0.4 g/10 g (F) 5.2 µg/mg (G)

letters apply to the whole line, except where additional references are given in parentheses. A = Mares and Stone (1973a); B = Bacic and Stone (1980), Rhodes and Stone (2002), Rhodes et al (2002); C = Antoine et al (2003); D = Selvendran et al (1980), Ring and Selvendran (1980); E = Harris et al (2005); F = Kabel et al (2002); G = Mandalari et al (2005); H = Du Pont and Selvendran (1987); I = Parker et al (2005); J = Barron et al (2007). b AHP = anhydropentose. c Intermediate layer comprises inner pericarp, seed coat, and nucellar remnants. d Values for protein may include cellular protein. e Includes noncellulosic glucose. f  Sum of Xyl, Ara, and uronic acid. g A/X = Ara-Xyl ratio, DDFA = dehydrodiferulic acid. h Cultivars Caphorn and Crousty. i Molar ratio Xyl/total DDFA.

Carbohydrates  to associate noncovalently with microfibrillar surfaces through the cellulose-­like regions of their molecules, thus contributing to cohesive forces in the wall structure. After exhaustive extraction of walls of starchy endosperm (Mares and Stone 1973a) or aleurone (Bacic and Stone 1981a) with alkaline reagents, cellulose remains as an insoluble residue in the form of narrow microfibrils (2–3 µm). In the primary walls of the starchy endosperm and aleurone, the microfibrils are oriented randomly (Mares and Stone 1973a, Bacic and Stone 1981a), whereas in secondary walls, such as those of cells of the pericarp-­seed coat tissues, which are rich in cellulose (Table 9.5), the microfibrils may be organized more regularly. In wheat bran, cellulose is found predominantly in walls of pericarp-­seed coat cells and is of moderate crystallinity, as judged by solid state 13C nuclear magnetic resonance (NMR) (Ha et al 1997). Lignin and cutin are also detected, the latter being more prominent. Cellulose is insoluble in water, swells but does not dissolve in concentrated 18–22% sodium hydroxide, and is soluble only in powerful hydrogen-­bond-­breaking reagents such as LiCl in dimethylacetamide or N-­methylmorpholino-­N-­oxide. Alkaline peroxide treatment of wheat bran leaves secondary wall residues enriched in cellulose and lignin (Maes and Delcour 2001). Cellulose from similarly derived residues from maize hulls, after removal of lignin by shearing, can be converted to a gel for use as a dietary fiber source (Inglett and Carriere 2001). Analysis

Cellulose can be measured gravimetrically using the acetic acid-­nitric acid reagent (Updegraff 1969), which dissolves non-

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321

cellulosic polysaccharides and lignin, leaving the cellulose. The fluorochromes Calcofluor White and Congo Red both fluoresce when associated with cellulose, but the interaction is not specific for cellulose, since (1→3,1→4)-­β-­d-­glucans and other noncellulosic β-­d-­glucans also induce fluorescence (see section on (1→3,1→4)-­β-­d-­glucan). Biosynthesis

A putative gene (BAD06322.1) for a cellulose synthase (EC 2.4.1.12) belonging to the glycosyltransferase family GT2 (http:// afmb.cnrs-­mrs.fr/CAZY/) has been identified in developing wheat grain. In rice and barley, families of cellulose synthase (CesA) genes (Burton et al 2004), whose protein products associate to form large complexes (rosettes) located in the plasma membrane, are responsible for the formation of microfibrils. In cellularizing barley endosperm, cellulose is present in the first formed walls 3–4 dpa at the time that the (1→3)-­β-­d-­glucan callose is also deposited (Burton et al 2006).

Glucomannans Occurrence and Structure

Glucomannans are minor components of walls of cells of the starchy endosperm and aleurone (Table 9.5). Their presence is inferred from the appearance of mannose and glucose in acid hydrolysates of polysaccharides extracted by concentrated alkali, containing borate from alkali-­unextractable residues of the walls (Mares and Stone 1973a, Gruppen et al 1992a).

Fig. 9.8. Cellulose structure. A, portion of a cellulose molecule. B, crystal structure of cellulose. Part i, projection down the fiber axis showing the hydrogen bond layers (ac planes) and the absence of interplane hydrogen bonding. Part ii, view of the origin layer approximately perpendicular to the ac plane, showing two intermolecular hydrogen bonds in the fiber axis direction and the interchain hydrogen bonds. (B, reprinted with permission, from Kroon-Batenberg and Kroon 1995)

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The structures of glucomannans from wheat and other cereal grains have not been examined in detail. Those from other cereal and grass sources are linear copolymers of (1→4)-­linked β-­ d-­glucopyranose and β-­d-­mannopyranose in the proportion of 3:7 (Bacic et al 1988). Their degrees of polymerization range from less than 100 to several thousand. In some glucomannans, α-­d­galactopyranosyl residues are attached at position 6 of mannosyl and glucosyl residues in the linear backbone (Fig. 9.9), and they may also be acetylated. The conformation of the glucomannan backbone is similar to that of cellulose (Marchessault et al 1981), enabling the glucomannans to associate strongly with the surfaces of cellulose microfibrils (Chanzy et al 1982). Glucomannans are also present in the secondary walls of the pericarp-­seed coat (Mandalari et al 2005). Biosynthesis

A CslA gene encoding a (1→4)-­β-­d-­mannan synthase that belongs to the GT2 family of cellulose synthaselike (Csl) enzymes (http://afmb.cnrs-­mrs.fr/CAZY/) has been cloned from guar (Cyamopsis tetragonoloba) seeds and is involved in synthesis of the mannan backbone of guar galactomannans (Dhugga et al 2004). It is not clear whether the same gene is involved in glucomannan biosynthesis in cereals, but CslA family members from rice encode (1→4)-­β-­d-­mannan synthases (Leipman et al 2005). (Gluco)mannans are detectable in walls and in the Golgi and associated vesicles of cellularizing barley endosperm at 5–6 dpa (Wilson et al 2006) when a mannan-­specific monoclonal antibody is used (Pettolino et al 2001).

Arabinoxylans Occurrence and structure

The arabinoxylans (heteroxylans, pentosans) are quantitatively the major noncellulosic polysaccharides of primary and secondary cell walls in cereals and grasses, whether from the endosperm, aleurone, scutellum, embryonic axis, pericarp-­seed coat, or vegetative parts of the plant (Table 9.5). Arabinoxylans (AX) have a basic backbone chain of β-­d­x ylopyranosyl (Xylp) residues linked through (1→4)-­glycosidic linkages (Fig. 9.10). Some of the Xylp residues are substituted for by α-­l-­arabinofuranosyl (Araf ) residues at position 3, or both positions 2 and 3 of the same Xylp residues (Fig. 9.10) (Perlin 1951, Renard et al 1990, Hoffmann et al 1991a, Izydorczyk and Biliarderis 1994). Additionally, Araf at position 2 has been found on AX from beeswing bran (Brillouet and Joseleau 1987), wheat endosperm (Gruppen et al 1992c, Izydorczyk and Biliaderis 1994), and wheat bran (Shiiba et al 1993, Schooneveld-­Bergmans

et al 1999b). Characteristically, the arabinofuranoside substituents are readily removed by dilute acid hydrolysis, although the Araf at position 2 on disubstituted Xylp residues appears more resistant to trifluoracetic acid cleavage than that at position 3 (Schooneveld-­Bergmans et al 1999b). The AX from the secondary walls in the pericarp-­seed coat tissues of bran carry 4-­O-­methyl α-­d-­glucuronic acid (Fig. 9.10) as an additional substituent at position 2 of Xylp units (Schooneveld-­Bergmans et al 1999b). These glucuronoarabinoxylans amount to 2.6 mol% of the AX in barium hydroxide extracts of pericarp-­seed coat walls and as high as 7.7 mol% in a fraction soluble in 80% ethanol (Schooneveld-­Bergmans et al 1999b). Terminal Xylp residues (Gruppen et al 1992a; Schooneveld­Bergmans et al 1999a,b) and short arabinose side chains occur in endosperm and bran AX (Brillouet and Joseleau 1987, Hoffmann et al 1991b, Gruppen et al 1992b, Izydorczyk and Biliaderis 1993, Shiiba et al 1993). More complex disaccharide substituents are found in minor amounts on AX from wheat bran and other cereals (Brillouet and Joseleau 1987, Hoffmann et al 1991b, Gruppen et al 1992b, Izydorczyk and Biliaderis 1993, Shiiba et al 1993, Schooneveld-­Bergmans et al 1999b). The arabinoxylans in beeswing bran (outer pericarp) and in the cross cells, intermediate layer, scutellum, and embryonic axis are relatively highly substitued with arabinose (Ara/Xyl > 1), whereas those of aleurone (Ara/Xyl < 0.5) and especially those of the seed coat (testa)/nucellar epidermis/hyaline layer are poorly substituted (Ara/Xyl < 0.2) (Parker et al 2005, Barron et al 2007) (Table 9.5). Frequency of Substitution. Although the chemical structures of water-­soluble and water-­unextractable (alkali-­extractable) AX from wheat endosperm are basically the same (Mares and Stone 1973a,b; Gruppen et al 1992a–c), the AX show considerable heterogeneity with respect to degree of arabinosylation (Table 9.6). This heterogeneity is evident from the Ara-­Xyl ratios of the water-­soluble polysaccharide fractions from whole-­grain flour eluted from diethylaminoethyl-­Sepharose with water and with 2.5 mM and 25 mM sodium borate, which had Ara-­Xyl ratios of 0.45, 0.42, and 0.76, respectively (Girhammar and Nair 1992a). The frequency of Araf substitution on AX depends on the wall type, and large natural variations occur, even within a single wall type. Thus, unfractionated AX from endosperm walls have Ara­Xyl ratios typically in the range of 0.5–0.7, but values as low as 0.3 have been reported (Cleemput et al 1995, Dervilly et al 2000, Dervilly-­Pinel et al 2001a). Ara-­Xyl ratios are cultivar dependent, as shown by the range of Ara-­Xyl ratios in water-­extractable (0.47–0.58) and water-­unextractable (0.51–0.67) AX fractions of flours from 20 French wheats (Ordaz-­Ortiz and Saulnier 2005). AX obtained by fractional precipitation of water-­soluble endosperm AX with ammonium sulfate (Izydorczyk and Biliaderis

Fig. 9.9. Structural representation of portion of a glucomannan chain, a copolymer of (1→4)-linked β -d-glucopyranosyl and β -dmannopyranosyl residues. A few backbone residues may be replaced at position 6 by single α-d-galactopyranosyl residues.

Carbohydrates  1993, 1994), water-­unextractable (alkali-­extractable) AX with ethanol (Gruppen et al 1992b, Dervilly et al 2000, Dervilly-­Pinel et al 2001b), and ethanol fractions of water-­soluble durum AX (Roels et al 1999) show increasing Ara-­Xyl ratios as the con-

Fig. 9.10. Substituents on (1→4)-β -d-xylan chain residues. α-lArabinofuranose may substitute at position 3, position 2, or both on one xylopyranosyl residue. Some arabinofuranosyl residues may carry an esterified hydroxycinnamic acid at position 5, which may be either p-coumaric acid (R = H) or ferulic acid (R = OCH3). 4-O-Methyl α-dglucopyranuronic acid (4MeGA) is also a substituent at position 2 of the xylopyranosyl residues, and acetyl esterification may occur at position 2 or position 3.

T. aestivum 20 French cultivars Alkali-extractable fractions

Water-extractable fractions

Water-extractable Water-extractable fractions

T. durum Water-extractable Water-extractable fractions

Percent Doubly Substituted Xylosyl Residues (mol%) References

Unfractionated, 0.47–0.58 Xylanase-extractable, 0.51–0.67

Ordaz-Ortiz and Saulnier (2005)

20% ethanol ppt., 0.36 30% ethanol ppt., 0.46 40% ethanol ppt., 0.55 50% ethanol ppt., 0.68 60% ethanol ppt., 0.80

  4.8   8.3 10.8 14.9 17.6

Gruppen et al (1992a)

55% (NH4)2SO4, 0.50 60% (NH4)2SO4 , 0.67 70% (NH4)2SO4 , 0.80 80% (NH4)2SO4 , 0.88 100% (NH4)2SO4 , 0.91 Unfractionated, 0.55

  9.8 12.8 12.9 14.0 16.4

Izydorczyk and Biladeris (1993, 1994)

Dervilly et al (2000), Dervilly-Pinel et al (2001a)

30% ethanol ppt., 0.39 40% ethanol ppt., 0.58 50% ethanol ppt., 0.67 60% ethanol ppt., 0.82 Unfractionated, 0.62 0–20% (NH4)2SO4, 0.42 20–30% (NH4)2SO4 , 0.48 30–40% (NH4)2SO4 , 0.61 40–50% (NH4)2SO4 , 0.70 50–65% (NH4)2SO4 , 0.80

323

centration of precipitant increases; i.e., the higher the ratio, the more soluble the AX becomes (Table 9.6). The proportion of doubly substituted xylosyl residues in the fractions also increases as the concentration of precipitant increases (Gruppen et al 1992b; Izydorczyk and Biliaderis 1993, 1994). The acidic AX in alkali-­soluble fractions from undelignified secondary walls of cells from beeswing bran (outer pericarp— cuticle, epidermis, and hypodermis) are also mixtures composed of highly branched AX, slightly branched AX, and AX in close association with xyloglucan (Ring and Selvendran 1980). Distribution of Substituents. Perlin and colleagues (Perlin 1951, Ewald and Perlin 1959, Goldschmid and Perlin 1963) showed that the distribution of the Araf on the xylan backbone is not regular. Later, Gruppen et al (1993) showed that endosperm AX consists mostly of highly branched regions in which mono-­ or disubstituted Xyl residues are separated by single unsubstituted residues or by domains of at least two to five (and possibly more) unsubstituted Xyl residues. Based on these data, two structural models were generated (Fig. 9.11A). Izydorczyk and Biliaderis (1994) also identified three major substitution patterns in different regions of the backbone of a single wheat AX fraction (Fig. 9.11B). In region I, isolated unsubstituted Xylp units are separated by one or two mono-­ or disubstituted units; in region II, high proportions of Araf units

TABLE 9.6 Comparative Ara-Xyl Ratios of Endosperm Arabinoxylans and Their Fractions Ara-Xyl Ratios

x 

Roels et al (1999)

324 

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Wheat: Chemistry and Technology, 4th ed.

are linked at position 3; and in region III, high proportions of contiguous (up to six and possibly more) unsubstituted residues are present. Dervilly-­Pinel et al (2004) compared a simulated random distribution of Araf units on the xylose backbone with experimentally derived distributions. The results suggest, in agreement with Gruppen et al (1993) and Izydorczyk and Biliaderis (1994), that the distribution of the type of substitution (mono-­ or di-­) on the backbone is not random. However, when only the distribution of substituted (irrespective of the substitution type) and unsubstituted residues are considered, the results are consistent with a random distribution. Dervilly-­Pinel et al (2004) also found that some runs of up to six contiguous substituted Xyl residues may be present. Among 20 French wheat flours (Ordaz-­Ortiz and Saulnier 2005), the proportions of disubstituted Xyl residues in water­extractable and in xylanase-­extractable AX (a fraction similar to water-­unextractable AX) were not significantly different, but the xylanase-­extractable AX had more monosubstituted residues. There was a strong correlation between the Ara-­Xyl ratio and the proportion of doubly substituted xylosyl residues in both fractions. This was also observed in the ethanol and ammonium sulfate subfractions of both water-­extractable and alkali-­extractable AX (Table 9.6). Esterified Substituents. Characteristically, cereal AX are esterified with both hydroxycinnamic acids (HCA) and acetic

acid. Both esters are partly or wholly lost when AX are extracted from grain or grain fractions using alkaline reagents (Mandalari et al 2005). The hydroxycinnamic acids ferulic (FA) and p-­coumaric (pCA) acid are esterified at position 5 of Araf substituents located at position 3 of the xylose residues (Smith and Hartley 1983) (Fig. 9.10). The FA-­pCA ratio is typically about 9:1. Small amounts of sinapic acid are present in aleurone layer preparations (Barron et al 2007), but their origin has not been determined. The extent of feruloylation of AX in cell walls of grain tissues is variable, as shown in Table 9.5. AX in the walls of the starchy endosperm are very lightly esterified with FA, whereas the AX in the walls of aleurone and scutellar cells are highly esterified. In the latter, pCA is in low abundance (Barron et al 2007). Notably, the AX in the walls of the starchy endosperm, aleurone, cross cells, and testa/nucellar epidermis have low amounts of esterified dehydrodiferulic acid (DDFA), whereas the pericarp-­seed coat fractions (beeswing bran) and scutellar fractions are extensively cross-­linked by DDFA (see section on molecular associations of AX in cell walls of grain tissues). The AX in the aleurone and pericarp-­seed coat walls are also acetylated (Fig. 9.10) (Ha et al 1997, Kabel et al 2002, Rhodes and Stone 2002, Mandalari et al 2005) (Table 9.5). The location(s) of the acetyl esters has not been defined. They may be either on the xylosyl residues, as in glucuronoxylans from wood (Kabel et al 2002), or on the arabinofuranosyl residues, as in cell walls from bamboo shoots (Ishii 1991). A low degree of acetylation of polysaccharides enhances their water solubility (Katz 1965) and may prevent their association with one another and other polysaccharides in walls. The acetyl substituents on the wheat bran xylan are more labile than the HCA esters (Mandalari et al 2005). No detectable bound acetate remained on wheat bran after extraction with 1M KOH. Chromatographic fractionation of water-­soluble polysaccharides from wheat flour (Meuser and Suckow 1986) shows some protein eluting with water­extractable AX; however, there is no definitive evidence that the association is covalent. Molecular size

Fig. 9.11. Models of the distribution of arabinosyl substituents on arabinoxylan fractions from starchy endosperm of wheat, showing regions with different substitution patterns: Gruppen models (A) and Izydorczyk and Biliaderis models (B). Symbols 1 and ! represent Xylp residues; S represents Araf residues. (Reprinted, with permission from Elsevier, from Gruppen et al 1993 [A] and Izydorczyk and Biliaderis 1994 [B])

The recorded molecular weights for water-­soluble AX are quite variable and dependent on extraction and determination procedures. They may be as low as 65,000 for an AX fraction measured by equilibrium sedimentation or may range between 800,000 and 5,000,000 (DP 5,000–38,000) measured by gel filtration, viscosity, and osmometry (Table 9.7). The high molecular weights may reflect molecular aggregation.

Carbohydrates 

x 

325

TABLE 9.7 Molecular Weight Values of Wheat Arabinoxylan Preparations Source

Method

Mol. Wt.

Axial Ratio

References

Whole grain (water extract) Flour (water-soluble fraction) Endosperm cell wall (water extract)

Sedimentation equilibrium Sedimentation equilibrium Gel filtration

255,000 ± 300   65,000

   4.2 ± 0.1 140 ± 10

Girhammar and Nair (1992b) Andrewartha et al (1979) Mares and Stone (1973b)

Flour (purified water extract) Ethanol fractions Flour (purified water extract)

Gel filtration

Ethanol fractions Flour (purified water extract) Flour (total water extract) Bran glucuronoarabinoxylan fraction (waterunextractable residue extracted with H2O2) Bran glucuronoarabinoxylan fraction (Ba(OH)2 extract)

800,000–5,000,000a 280,000 200,000–285,000

Dervilly-Pinel et al (2001b) Dervilly et al (2000)

Gel filtration Gel filtration

300,000 b 307,000–408,000 134,000–201,000   55,000–85,000

Gel filtration

100,000–110, 000

Hollman and Lindhauer (2005)

Viscometry

150,000

Schooneveld-Bergmans et al (1999b)

Gel filtration

Rattan et al (1994) Cleemput et al (1995)

a Aggregation.

b Pronase-treated.

AX are polydisperse with respect to molecular weight (Table 9.7), with the ratio of the weight­average molecular weight (Mw) to the number­average molecular weight (Mn) ranging from 1.3 to 2.5 for alkali-­soluble AX (Gruppen et al 1992a,b) and 4.2 for water-­extractable AX (Girhammar and Nair 1992b). Solid state properties The β-­d-­x ylopyranosyl (Xylp) residues in

the AX backbone are in the 4C1 chair conformation (Nieduszynski and Marchessault 1972, Winterburn 1974, Atkins 1992). The unsubstituted (1→4)-­β-­d-­x ylan chains adopt a twofold helical conformation (i.e., two Xylp residues per turn), with only one hydrogen bond (O5–H–O3') between adjacent Xylp units (Nieduszynski and Marchessault 1972, Winterburn 1974). This conformation is more flexible than the twofold helix of the homomorphous (1→4)-­β-­d-­glucan (cellulose), which has two hydrogen bonds between adjacent glucosyl units (see section on cellulose above). The absence of the two cooperative inter-­residue associations allows xylan molecules to be conformationally more versatile. However, in the solid state, an unsubstituted (1→4)-­β-­d-­x ylan chain preferenFig. 9.12. Arabinoxylan conformations. Projections: perpendicular (top) and partially exists in the fully extended, threefold, leftallel (bottom) of (1→4)-β -d-xylan backbone (A), backbone with single-arabino­ ­handed helix (Fig. 9.12). Araf substitution does not furanosyl side groups (B), and backbone with double-arabinofuranosyl side groups substantially change this basic conformation. In (C). Hydrogen bonds are shown by dotted lines. In each case, the backbone is a leftfact, substituted xylans cannot assume the twohanded, threefold helix. (Reprinted, with permission from Elsevier, from Atkins fold helical conformation due to significant steric 1992) interactions between the Araf units and the xylan backbone (Atkins 1992) (Fig. 9.12) and preferentially assume a cellulose microfibrils and may do so in the twofold helical conthreefold helix. formation. With increasing degree of substitution, the affinity for In cell walls, AX with low degrees of substitution may incellulose decreases, presumably due to the increased incidence teract reversibly through hydrogen bonding with the surfaces of of the threefold conformation, which would be unfavorable for

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association with cellulose due to the steric hindrance to interaction imposed by the substituents themselves.

tion of the residual AX with low Araf substitution (Andrewartha et al 1979).

Solution properties

Gel formation

AX molecules with xylan backbones of DP 5,000–10,000 may have extended lengths of 2.5–5 µm (Xylp = 0.5 nm) and may behave in solution as partially stiff (semiflexible), wormlike, cylindrical molecules whose flexibility is largely unaffected by the Ara-­Xyl ratios in the range of 0.39–0.82 (Izydorczyk and Biliaderis 1995, Dervilly-­Pinel et al 2001b, Picout and Ross­Murphy 2002). The α-­l-­Araf residues, either linked singly at position 3 or doubly substituting internal Xylp residues, have considerable conformational freedom. They may be either of the twist type or the envelope type, with equal amounts of the two types at conformational equilibrium (Hoffmann et al 1992). Direct imaging using atomic force microscopy (Adams et al 2005) of populations of water-­soluble FA-­A X preparations containing protein shows the absorbed molecules as extended structures. Their estimated Kuhn statistical extended lengths (a statistical length that divides the end-­to-­end length [contour length] of a polymer chain into freely jointed segments) are 128 nm, which contrasts with the moderately extended, sinuous (reptate) solution conformation, which has Kuhn lengths of only 16 nm. This suggests that, on absorption, the molecules adopt a more ordered and extended structure (see the section on solid state properties). A proportion (15%) of the molecules exhibit branches that are believed to be (1→4)-­β-­d-­x ylan chains attached through phenolic linkages (see the section on gel formation) to the backbone (Adams et al 2005) since treatment with a feruloyl esterase leads to reduction of the density of the branches. The intrinsic viscosities of AX solutions are influenced not only by the overall asymmetry of the molecules and their DP, but also by the specific arrangement of the Araf units along the backbone (Dervilly-­Pinel et al 2001b, Picout and Ross-­Murphy 2002). Intrinsic viscosities recorded for starchy endosperm AX are 0.8–5.5 (Izydorczyk and Biliaderis 1995), 3.2 (Dervilly-­Pinel et al 2001b), and 1.7 (Girhammar and Nair 1992b), and for bran glucuronosyl AX in 0.1M KCl, the value is 2.5 (Schooneveld­Bergmans et al 1999a). The viscosities of solutions of AX with different Ara-­Xyl ratios are strongly dependent on concentration. At low concentrations (0.27–1.05 g/dL) (Izydorczyk and Biliaderis 1992a). This behavior is typical of populations of polymers with extended, rodlike conformations (Izydorczyk and Biliaderis 1992a, 1995), which, through associations over short sequences, lead to formation of shear-­dissociable aggregates at high concentrations. In dilute solutions, the “zero” shear-­rate specific viscosity (ηsp)0 increases linearly with increasing AX concentration (Fig. 9.13). However, above certain concentrations, there is an abrupt increase in the concentration dependence of (ηsp)0, which corresponds with the onset of coil overlap among polymer chains. The critical concentration c* depends on the hydrodynamic volume. Removal of Araf units by dilute acid treatment or by α-­l-­ arabinofuranosidase action leads to aggregation and precipita-

Covalently Stabilized Gels. As first shown by Durham (1925) and later investigated by Geissmann and Neukom (1971) and Markwalder and Neukom (1976), stable AX gels are formed by cross-­linking (dimerization) of feruloyl units on neighboring AX chains by oxidative (radical) coupling reactions (Izydorczyk et al 1990, Ralph et al 1994). Covalently cross-­linked AX gels may hold up to 100 g of water per gram of polysaccharide. The oxidative cross-­linking reaction is catalyzed by enzymes and other reagents, as listed in Table 9.8. Oxidants such as iodate, bromate, or ascorbate are not effective (Crowe and Rasper 1988). Gamma-­irradiation, a possible generator of free radicals, does not induce gelation of FA-­A X but causes depolymerization, with concomitant loss in viscosity (Micard et al 2003). Laccase­generated radicals cause xylan depolymerization and slowly

Fig. 9.13. Concentration dependence of “zero” shear-rate specific viscosity (ηsp)0 for aqueous solutions of wheat AX fractions with different Ara-Xyl ratios. Inset: “zero” shear-rate specific viscosity as a function of the coil overlap parameter (c[η]). (Reprinted, with permission from Elsevier, from Izydorczyk and Biliaderis 1995)

Carbohydrates 

x 

327

TABLE 9.8 Agents for Oxidative Dimerization of Feruloylated Arabinoxylans Oxidant

References

Horseradish peroxidase (EC 1.11.1.7)/hydrogen peroxide

Ciacco and D’Appolonia (1982a,b), Crowe and Rasper (1988), Durham (1925), Fausch et al (1963), Garcia et al (2002), Geissmann and Neukom (1971, 1973a,b), Girhammar and Nair (1992b), Hoseney and Faubion (1981), Lempereur et al (1997, 1998), Moore et al (1990) Garcia et al (2002) Schooneveld-Bergmans et al (1999c) Carvajal-Millan et al (2005a), Figueroa-Espinoza and Rouau (1998) Figueroa-Espinoza et al (1999b), Labat et al (2001) Neukom and Markwalder (1978) but see also Crowe and Rasper (1988) Izydorczyk and Biliaderis (1995), Schooneveld-Bergmans et al (1999a) Izydorczyk and Biliaderis (1995) Crowe and Rasper (1988)

Wheat germ peroxidase Peroxidase/glucose/glucose oxidase Laccase (p-diphenol: oxygen oxidoreductase) (EC 1.10.3.2) Manganese-dependent peroxidase (EC 1.11.1.13) Linoleic acid-lipoxygenase (EC 1.13.11.12) Ammonium persulfate Potassium periodate Ferric chloride and other metal chlorides, chlorine, and chlorine dioxide Sodium chlorite/peroxidase

Crowe and Rasper (1988)

weaken the gel, but this is prevented by inactivating the laccase (Figueroa-­Espinoza and Rouau 1998). The development of three-­dimensional networks of solutions of AX undergoing gelation is effectively followed by small­amplitude oscillatory measurement of shear stress. The viscous aqueous solutions of AX show mechanical spectra with G" (a measure of a sample’s ability to dissipate energy) higher than G' (a measure of a sample’s ability to store energy) (Fig. 9.14, inset A). At high concentrations (2.5% w/v), high molecular mass AX fractions with intrinsic viscosities 8.5 and 6.2 dL/g form thermoreversible gels, stabilized by noncovalent, chain-­chain associations and chain entanglement, which exhibit elastic properties. On the other hand, after oxidative gelation, the storage modulus, G', predominates over the loss modulus, G", across the frequency range measured (Fig. 9.14, inset B) (Izydorczyk et al 1990; Izydorczyk and Biliaderis 1992b, 1995). The kinetics of development of the storage modulus (G') of an arabinoxylan solution during radical coupling induced by horseradish peroxidase in the presence of peroxide shows an initial rapid rise followed by a plateau region (Fig. 9.14). The rate of peroxidase-­catalyzed gelation depends on temperature, pH, and the concentration of oxidizing agent (Izydorczyk and Biliaderis 1995). As gelation proceeds, the peaks at λmax 320 (375 nm) in the UV spectrum decrease, due to esterified FA on the AX, since the DDFA formed have lower absorptions than FA (Kundig et al 1961, Izydorczyk and Biliaderis 1995). The kinetics of gel formation for peroxidase and laccase are similar initially, but when the H2O2 generated is exhausted, the peroxidase reaction stops (Figueroa-­Espinoza and Rouau 1998). Well-­developed, three-­dimensional gel networks are obtained with AX with high FA substitution, high molecular masses, and relatively unsubstituted xylan backbones (Izydorczyk and Biliaderis 1992b). The greater flexibility of the unsubstituted chain backbone facilitates the initial approach between reacting feruloyl groups on neighboring AX chains. There are varietal differences in the oxidative gelling capacity of AX (Izydorczyk et al 1990). Several isomeric DDFA, 8-­5', 8-­O-­4', 8-­8, and 5-­5' (Fig. 9.15), are formed in vitro by peroxidase-­hydrogen peroxide coupling in

Fig. 9.14. Kinetics of the development of the storage modulus, G', derived from small-amplitude shear oscillatory measurements, for a water-extractable wheat endosperm arabinoxylan (2.5%, w/v) treated with horseradish peroxidase and peroxide. Inset A, mechani­cal spectrum (small-amplitude shear oscillatory measurements) showing typical behavior of a viscous AX solution before addition of oxidant. The viscous component (the loss modulus, G") predominates over the storage modulus, G', over the frequency range tested. Inset B, mechanical spectrum after addition of oxidant (horse­radish peroxidase/ H2O2). The storage modulus, G', predominates over the loss modulus, G". n' = the non-Newtonian index of the power law model, η = k γ n–1. (Reprinted, with permission from Elsevier, from Izydorczyk and Biliaderis 1995)

the proportion 5:3:1:1 (Schooneveld-­Bergmans et al 1999c), indicating that both the aromatic ring and the propenoic side chains are involved in dimerization. Laccase also oxidizes phenols to free radicals, which couple through nonenzymatic reactions, ­forming,

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principally, the isomeric DDFA 8-­5' and 8-­O-­4' (Figueroa­Espinoza et al 1998, Vansteenkiste et al 2004). The 8-­8 dimer was not detected, but very small amounts of a trimer, (5-­5')-­(8-­O-­4') (Fig. 9.15), are formed (Rouau and Moreau 1993, Antoine et al 2003, Bunzel et al 2003, Carvajal-­Millan et al 2005a). Extensive investigations (Figueroa-­Espinoza and Rouau 1999) of the cross-­linking of feruloylated AX (FA-­A X) by laccase in the presence of high molecular weight glutenin subunits, bovine serum albumin, and small molecules such as lysine, tyrosine, cysteine, glutathione, cysteinylcaffeic acid, caffeic acid, and L-­dihydroxyphenylalanine produced no evidence for covalent coupling of the proteins or the small molecules to the FA­A X, either through sulfhydryl groups, as suggested by Hoseney and Faubion (1981), or through tyrosine residues (Fig. 9.16). The small molecules did, however, inhibit gelation by lowering the concentration of laccase-­induced FA–semiquinone intermediates (Figueroa-­Espinoza and Rouau 1998, 1999; Figueroa­Espinoza et al 1998, 1999a,b, 2002). On the other hand, gelling of FA-­A X with manganese-­dependent peroxidase as the cross­linking catalyst was slowed in the presence of cysteine. Tyrosine accelerated the disappearance of FA, suggesting that it linked to FA esters or to DDFA diesters and possibly to tyrosine in proteins (Figueroa-­Espinoza et al 1999b). This was confirmed by Schooneveld-­Bergmans et al (1999c), who showed that tyrosines in proteins in AX preparations can act as partners with FA to form mixed dimers. Additionally, Oudgenoeg et al (2001) dem-

onstrated the oxidative coupling of a tyrosine-­containing peptide to free FA. Preparations of hot-­water-­extractable nonstarchy polysaccharides (AX with a low degree of arabinosylation and (1→3,1→4)-­β-­d-­glucan) from wheat bran exhibited shear thinning behavior at low concentrations (0.5%, 25°C) and formed a thermally reversible gel on cooling (Cui et al 1999). Extraction of AX from cell walls and grain

Water-­soluble AX amount to 0.3–0.8% (dw) of wheat grain, and a further 3.7–5.2% is water unextractable. Variable proportions of AX are extractable by water from the walls of endosperm cells or from wheat flours at 40°C and higher temperatures, but a portion remains unextractable. Other solvents are required to extract the residual AX. Although 0.05M Na 2CO3, 8M urea, and dimethylsulfoxide extract only a small part of the water-­u nextractable AX, most can be extracted from walls of endosperm cells with 1M NaOH, 1M hydroxylamine hydrochloride (at pH 7) (Mares and Stone 1973b), saturated Ba(OH)2 (Gruppen et al 1991), or N-­methylmorpholino-­N-­oxide (Joseleau et al 1981). Of these, saturated Ba(OH)2 containing NaBH4 is the most successful, although its use results in loss of acetyl and HCA esters. The water-­u nextractable AX once brought into solution with alkali are then water soluble (Mares and Stone 1973a). The amount of AX extracted from wheat flours increases

Fig. 9.15. Feruloyl units on neighboring AX chains in solution or in cell walls may be cross-linked by radical coupling into ferulate dehydrodimers (structures 1–5) or dehydrotrimers (structure 6). Dotted arrows indicate potential sites for radical coupling with hydroxycinnamoyl alcohols or lignin oligomers in lignifying cell walls, resulting in cross-linking of arabinoxylans to lignin. “Ara” is an arabinofuranosyl residue on an arabinoxylan. (Reprinted, with permission from Elsevier, from Grabber et al 1995)

Carbohydrates  as the alkali concentration increases, but the alkali-­extractable polysaccharides represent only ~80% of the water-­unextractable polysaccharide (Michniewicz et al 1990). The unextractable polysaccharide may be AX in complex with protein (see next section). Molecular associations of AX in cell walls of grain tissues

The integrity of the walls of mature starchy endosperm and aleurone cells is dependent on noncovalent interactions between individual AX chains, between AX and the surfaces of the cellulosic microfibrils, and, putatively, between the AX and the (1→3,1→4)-­β-­d-­glucan components (Izydorczyk and MacGregor 2000). Potentially, the polysaccharide associations in these walls could be stabilized by covalent cross-­linking of AX through DDFA; however, such bridges do not appear to be important in aleurone walls, since, notwithstanding the high (1.8%) concentration of esterified FA (Table 9.5), only small amounts of DDFA are released on saponification of walls from aleurone cells (Rhodes et al 2002, Antoine et al 2003, Parker et al 2005). However, in aleurone walls, a fraction that resists digestion by

x 

329

hydrolases for AX and (1→3,1→4)-­β-­d-­glucans contains a highly branched AX that appears to be linked to a protein, possibly through an FA-­t yrosine bridge (Rhodes et al 2002). Confocal Raman spec­troscopy (Piot et al 2001) applied to walls of starchy endosperm and aleurone cells in wheat grain sections identified AX, esterified FA, and small amounts of protein and phospholipid. The possibility of tyrosine-­FA, cysteine-­FA, phospholipid linkages was suggested. During grain development, there is a decrease in the protein-­A X ratio in the endosperm wall and a simultaneous increase in the FA-­A X ratio, which may relate to changes in cohesiveness of the wall matrix. The noncovalent associations between the hydrated matrix­phase polysaccharides in walls of starchy endosperm and aleurone cells are presumably sufficiently numerous and intensive to form a gel, reinforced by the cellulosic microfibrillar phase (Antoine et al 2003, 2004a,b). These interactions would be sufficient to maintain the integrity of these walls during endosperm development. However, these walls, unlike those of the pericarp-­seed coat, have a minimal role in protection or maintenance of tissue structure in the mature grain and are destined for enzymatic deconstruction during germination to allow access of the aleurone-­derived hydrolases to their starchy-­endosperm-­located substrates. The hydrated noncellulosic components in this simple wall structure are readily accessible to hydrolase attack. This was demonstrated by the use of active and inactive xylanases as molecular probes (Beaugrand et al 2005), which showed that the walls of aleurone cells are rapidly degraded. In contrast, lignified walls of the pericarp­seed coat are not penetrated by active xylanases, and the inactive xylanase probe fails to bind. The exclusion of xylanases from their substrates in the pericarp-­seed coat walls is presumably due to the quantitatively important covalent cross-­linking of AX with one another through DDFA bridges (Table 9.5) involving a number of isomeric bridging dimers (Fig. 9.15) (Antoine et al 2003, Parker et al 2005, Barron et al 2007). Moreover, AX are also linked to lignin through both direct covalent interactions and indirect covalent bridges through ester-­ether bridges involving ferulic acid and DDFA (Fig. 9.16) (Iiyama et al 1990, 1994b). The covalently cross-­linked AX and AX­lignin overlies the cellulose in the walls of pericarp-­seed coat tissues, providing a robust and refractory protective and structural complex. Thus, extraction of AX from walls of the pericarp-­seed coat in wheat bran first requires cleavage of the lignin inter-­residue linkages and lignin-­polysaccharide complexes by treatment with alkali (Chanliaud et al 1995) or alkaline H2O2 (Maes and Delcour 2001) or by oxidative degradation with chlorite (Whistler and BeMiller 1963). Biosynthesis

Fig. 9.16. Three modes of covalent cross-linking between wall polymers. A, a diferulate cross-link between two arabinoxylan chains. B, a tyrosyl-ferulate cross-link between a protein and a feruloylated arabinoxylan. C, a dityrosyl cross-link between two protein chains. (Adapted from Geissmann and Neukom 1973a,b)

A membrane-­bound (1→4)-­β-­xylosyltransferase from wheat seedlings uses uridine-­5'-­diphosphate (UDP)-­[14C]­d-­x ylose as substrate to produce a polymeric xylan (Porchia and Scheller 2000), and a similar enzyme has been described in developing barley endosperm (Urahara et al 2004). The genes and the encoded enzymes involved

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in assembly of the (1→4)-­β-­d-­x ylan backbone of AX have not been identified, although the enzymes presumably belong to the cellulose synthaselike (Csl) family of GT2 glycosyltransferases. The enzyme responsible for Araf substitution of the xylan backbone has not been described, but the donor molecule is likely to be UDP-­β -­l-­Ara, whose biosynthesis from UDP-­α-­d­Xylp is catalyzed by a 4-­epimerase (EC 5.1.3.5) (Pauly et al 2000). Arabinosyltransferases using this substrate are known for other polysaccharides (Reiter and Vanzin 2001, Seifert 2004). The heterogeneous (nonrandom) pattern of Araf substitution favoring disubstitution and contiguous substitution of xylosyl residues on AX (see the section on AX structure) is presumed to be a manifestation of the specificity of the arabinosyltransferase-­directed step (Dervilly-­Pinel et al 2004). AX have been located immunocytochemically in Golgi or Golgi-­derived vesicles in the developing endosperm of both wheat (Philippe et al 2006c) and barley (Wilson et al 2006). In both cereals, AX deposition commences at the beginning of differentiation. Perlin and co-­workers (Perlin 1951, Ewald and Perlin 1959, Goldschmid and Perlin 1963) proposed that the pattern of Araf substitution on AX could be explained by concerted de­arabinosylation of a first-­formed heavily substituted AX. Some support for this view is provided by the presence, in subfractions, of endosperm AX with Ara-­Xyl ratios as high as 0.91 (Izydorczyk and Biliaderis 1993, 1994) (Table 9.6) and also from the changes in the composition of tissues from developing outer layers (pericarp, nucellus, and aleurone) of grain from 11 dpa to maturity (Beaugrand et al 2004). At 11 dpa, the AX content was low and the Ara-­Xyl ratio (0.82) was at its highest. The concentration of AX reached a maximum at 20 dpa, and the Ara-­Xyl ratio had fallen to 0.58 and thereafter did not change significantly. Thus, the already deposited AX may be enzymatically de-­arabinosylated during endosperm development, or the subsequently deposited AX may have a lower degree of arabinosylation. Such remodeling has been demonstrated in walls of developing coleoptiles of barley (Gibeaut et al 2005). α-­l-­Arabinofuranosidases (EC 3.2.1.55) are present in germinating wheat (see Chapter 11) but have not been described in developing grain. The esterification of Araf residues by FA is intraprotoplasmic (Brett et al 1999, Fry et al 2000) and takes place in the Golgi complex, as indicated by the immunochemical study of Philippe et al (2007), but the enzymatic mechanism has not been elucidated. There are two candidate donors: feruloyl CoA and feruloyl 1-­O-­β -­glucose. The former appears to be involved in feruloylation of proteins but not of AX (Kohler and Kauss 1997). Kinetic experiments with suspension-­cultured wheat cells support the view that FA is incorporated intracellularly from feruloylglucose (Obel et al 2003). O-­Acetylation of the AX is presumed to be catalyzed by an O-­acetyltransferase (EC 2.3.1.-­) with acetyl CoA as the donor substrate, as found for other polysaccharides—e.g., pectin (Pauly and Scheller 2000)—and probably also occurs within the Golgi compartment. Deposition in developing cell walls

Chemical and cytochemical analysis and xylanase degradability have been used to monitor the development of external

layers of wheat grain (Beaugrand et al 2004). At all stages of development, xylanase treatment degraded the aleurone and nucellar layer but the pericarp remained intact. This is likely to result from the poor penetration of the xylanase probe into the pericarp walls, which have a low porosity, and is limited to molecules of below 20-­ to 50-­nm diameter (Chesson 1988). Lignin was present in the late stages of development, while esterified HCA, mostly FA, reached highest levels at 20 dpa and then decreased, possibly due to increasing dimerization to DDFA and cross-­linking to lignin by etherification (Fig. 9.17). A mass balance of the different types of HCA present was not possible (an unknown proportion of the etherified HCA is not released by current analytical procedures), but it is likely that DDFA are responsible for cross-­linking AX in the pericarp walls before lignification and later in formation of lignin-­polysaccharide cross­links (Fig. 9.17). Immunolabeling of nucellar walls with an antibody specific for AX with a low degree of Araf substitution shows that the AX is deposited in three layers (Beaugrand et al 2005). In contrast, aleurone walls are labeled uniformly by this antibody, but inactivated xylanase gave intense labeling only at the anticlinal­periclinal junctions, indicating heterogeneity in the type of AX deposited. Spectral differences in structures of AX between cell walls of the peripheral and central endosperm (Barron et al 2005) also suggest modulation of biosynthetic events during endosperm development. Fourier transform-­infrared (FT-­IR) microspectroscopy of walls in developing starchy endosperm of wheat showed that AX was deposited only after cellularization and that, during differentiation, AX features dominated (Philippe et al 2006b). At the beginning of differentiation, AX was more substituted and, moreover, the AX in the central cells was less substituted than in peripheral cells at the differentiation stage. Raman spectrometry (Philippe et al 2006a) also showed that the AX in walls of endosperm cells were more substituted in the early stages of differentiation than in later stages. Toole et al (2007), using FT-­IR, found that AX in endosperm walls changes from a highly substituted form to a less substituted form during development. The restructuring occurs at higher temperatures with restricted water availability from 14 dpa, and there were differences in the rate of restructuring between two wheat cultivars. Immunocytochemistry (Philippe et al 2007) showed that FA­A X is first deposited in the walls of the aleurone and the starchy endosperm of wheat grain at 13 dpa and continues to accumulate until the aleurone cells are differentiated at 19 dpa. The concentration of FA-­A X was high in both the peri-­and anticlinal aleurone walls, with highest concentrations at the cell junctions of the seed coat. The amount of FA-­A X in the starchy endosperm cell walls was low at all stages of development. Genetics

There is a significant genetic influence on the water-­soluble AX content of bread wheat cultivars (Lempereur et al 1997; Martinant et al 1998, 1999). The heritability coefficient, the proportion of the phenotypic response due to genetic factors, for water-­extractable AX content is high, and the values of the broad-­sense heritability parameter h2 (the ratio of genotypic

Carbohydrates  variance to phenotypic variance) for Ara-­Xyl ratio and flour extract viscosity are strong (Martinant et al 1999). The content of AX and arabinogalactan-­peptide in flours has been investigated in 1B/1R wheat translocations in which the short arm of chromosome 1B of wheat is replaced by that of chromosome 1R of rye (Biliaderis et al 1992). The mean values of the total and water-­soluble AX content were only slightly higher in the normal line, and the molar ratios of the component monosaccharides and FA contents were similar. However, the intrinsic viscosities of the AX from the translocation lines were 1.69 and 3.18 dL/g, and there was a correspondingly lower proportion of high molecular weight AX in the translocation line with the higher intrinsic viscosity, suggesting a genetic effect on AX molecular weight. In wheat-­r ye addition lines (Cyran et al 1996), the double addition of rye chromosomes led to important transgression effects on the content and composition of nonstarch polysaccharides. Chromosomes 2R and 5R increased soluble AX content above the rye level, whereas the 3RS arm decreased soluble nonstarchy polysaccharides below the wheat

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level. Selanere and Andersson (2002) also found that 1B/1R translocation wheat lines had ~20% more water-­extractable polysaccharides and significantly more water-­extractable xylose and water-­unextractable mannose residues than standard wheat AX. The molecular weight of the water-­extractable AX from the translocation wheat was lower than that of the standard wheat AX and resembled the xylanase-­resistant AX from standard wheat (i.e., the Ara-­Xyl ratio was 0.80 compared with 1.04 for rye). NMR showed that fewer than one in every second xylose residue was disubstituted, whereas in the rye xylanase-­resistant AX, on average, every second xylose residue was disubstituted. A quantitative trait locus (QTL) accounting for ~35% of the variation in both Ara-­Xyl ratio and extract viscosity is present on the long arm of chromosome 1B of bread wheat (Martinant et al 1998). Genes located at this QTL controlled relative viscosity by modifying the Ara-­Xyl ratio. There was no apparent change in AX concentrations in the wheat 1BL/1RS translocation lines, suggesting that the genes involved are located outside the segment of the chromosome that was introgressed from rye. QTLs affecting monosaccharide composition of cell walls of maize pericarp have been mapped, potentially allowing identification of genes involved in cereal cell wall biosynthesis (Hazen et al 2003) (see also Chapter 12). Analysis and detection

Fig. 9.17. Schematic diagram showing possible covalent cross-links between polysaccharides and lignin in cell walls. ! = p-coumaryl, 1 = feruloyl, 1–1 = dehydrodiferuloyl residues. a = direct ester linkage, b = direct ether linkage, c = ferulic acid esterified to poly­ saccharide, d = p-coumaric acid esterified to lignin, e = hydroxycinnamic acid etherified to lignin, f = ferulic acid ester-ether bridge, g = dehydrodiferulic acid diester bridge, h = dehydrodiferulic acid ester-ether bridge. (Reprinted, with permission, from Iiyama et al 1994b)

AX determination in grain and grain fractions relies on the colorimetric phloroglucinol-­HCl (Douglas 1981) or orcinol-­HCl (Hashimoto et al 1987) reactions, which show a high, but not absolute, specificity for pentoses. A rapid, semiautomatic method based on the phloroglucinol­HCl reaction has been developed for determining total AX and water-­unextractable AX in wheat flours (Rouau and Surget 1994). Uronic acids are determined colorimetrically using the m-­hydroxydiphenyl reagent (Filisetti-­Cozzi and Carpita 1991). Near-­infrared (NIR) spectroscopy measures AX content (Hong et al 1989, Kacurakova et al 1994). NIR peak intensity ratios at 1,164 and 990 cm–1 allow estimation of the degree of Araf substitution (Kacurakova et al 1994). FT-­IR and FT-­Raman spectroscopies can be applied in complementary ways to determine structural features of AX (Kacuráková et al 1999, Philippe et al 2006a). Spatially resolved FT-­IR microbeam spectroscopy (Wetzel and Reffner 1993) successfully mapped local differences in composition of walls of cells in wheat grain tissues. On the basis of the characteristic IR spectrum of AX, FT-­IR microspectroscopy with principal component analysis discriminates between AX with different degrees of arabinosylation in the walls of the starchy

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endosperm and aleurone (Barron et al 2005, Robert et al 2005, Toole et al 2007). Raman spectroscopy (Piot et al 2001, Barron et al 2006, Philippe et al 2006b) also allows determination of structural heterogeneity and relative concentrations of AX as well as HCA esterification and protein ratios in the walls of grain sections. 1H-­NMR allows the determination of mono-­ and disubstituted Xylp residues (Kormelink et al 1993, Hollmann and Lindhauer 2005). HCA monomers and dimers may be analyzed directly by HPLC after saponification (Waldron et al 1996) or by gas chromatography as their trimethylsilyl derivatives (Lam et al 1990). The degree of acetylation may be measured by HPLC (Voragen et al 1986) or by NMR (Iiyama et al 1994a). Esterified HCA on AX are sensitively detected in tissue sections by their bright blue, UV-­induced fluorescence (excitation λmax 310 nm, emission λmax 415–445 nm) (Harris and Hartley 1976), and this technique has been applied in UV absorption microspectrophotometry (Atkin 1995). Lignins also fluoresce under UV irradiation and may be differentiated from ferulate esters by examining sections at high pH, where lignins continue to fluoresce blue but ferulate esters fluoresce green (Fulcher et al 1972, Harris and Hartley 1976, Beaugrand et al 2005). The FA content of cell walls can be estimated using the 1,600 cm–1/1,094 cm–1 intensity ratio in Raman spectra (Philippe et al 2006b). A number of polyclonal and monoclonal antibodies raised against AX with both high and low substitution allow their location and differentiation to be determined in cereal tissues by light and electron microscopy (Barry et al 1991; Migne et al 1994, 1996, 1998, 1999; Ordaz-­Ortiz et al 2004; McCartney et al 2005). Both active and inactive xylanases (Adams et al 2004, Beaugrand et al 2005) and xylan-­specific carbohydrate-­binding domains (McCartney et al 2006) have also been used in a similar way. Antibodies specific for Araf (Kaku et al 1986, Smallwood et al 1996) and 5-­O-­(trans-­feruloyl)-­l-­arabinose (Philippe et al 2007) are also available.

(1→3,1→4)-­β-­d -­Glucans Occurrence and Structure

Among higher plants, the (1→3,1→4)-­β-­d-­glucans are found exclusively in the graminoid (Poaceae, Poales) group of commelinid monocotyledons, to which grasses, cereals, and related families belong (Harris 2005, Trethewey et al 2005). In wheat grain, (1→3,1→4)-­β-­d-­glucans constitute 20% (w/w) of the walls of the starchy endosperm and 29% (w/w) of the aleurone walls (Bacic and Stone 1981a,b) (Table 9.5). Endosperm cell wall (1→3,1→4)-­β-­d-­glucans from flours of a range of Triticum species are not extractable by water at 65°C (Beresford and Stone 1983). The content in the flours from 29 cultivars of T. aestivum ranged from 0.52 to 0.99% (w/w) (Beresford and Stone 1983). (1→3,1→4)-­β-­d-­Glucans are also found in the walls of coleoptiles (Nevins et al 1977) and are minor components in walls of cells in beeswing bran (outer pericarp) (Harris et al 2005), stems, and leaves (Wilkie 1979). The (1→3,1→4)-­β-­d-­g lucans are linear, unbranched polymers in which the β-­d-­glucopyranosyl residues are joined by both (1→3) and (1→4) glucosidic linkages. Single (1→3) linkages are separated by two or more (1→4) linkages (Fig.

9.18A and B) and regions of two or three adjacent (1→4) linkages predominate. The (1→3,1→4)-­β-­d-­glucan extracted from wheat bran with dilute alkali, and almost certainly arising from walls of aleurone cells (Table 9.5), when treated with a specific (1→3,1→4)-­β-­d-­g lucan hydrolase (Fig. 9.18B) released 3-­O-­β -­cellobiosyl-­and 3-­O-­β -­cellotriosyl-­d-­glucose and longer oligosaccharides (Lazaridou et al 2004, Li et al 2006) (Table 9.9). The trisaccharide-­tetrasaccharide ratio is significantly higher than for barley and oat (1→3,1→4)-­β-­d-­g lucans. The high proportion of trisaccharide in the wheat (1→3,1→4)-­β-­d-­g lucan resembles lichenin from the lichen Cetraria islandica (78.1 mol%); however, lichenin has a much higher trisaccharide­tetrasaccharide ratio (Table 9.9). Molecular size A (1→3,1→4)-­β-­d-­glucan purified from wheat bran had an

average molecular weight (Mw) of 487,000 and a polydispersity (Mw/Mn) of 1.65 (Li et al 2006). Fractionation of the purified polysaccharide using ammonium sulfate gradient precipitation gave six fractions with Mw ranging from 43,000 to 758,000, each with narrow Mw distributions and low polydispersities (Mw/ Mn between 1.03 and 1.26) compared to the parent preparation. There were no significant structural differences between the six fractions or between the fractions and the original sample. Physical properties

The cereal (1→3,1→4)-­β-­d-­glucans are generally soluble in water due to their extended, flexible (reptate) chain conformation (Buliga et al 1986) (Fig. 9.18C), and this may be compared to the linear, extended conformation of water-­insoluble cellulose, the all-­(1→4)-­linked β-­d-­glucan (Fig. 9.8A). The insertion of (1→3)-­glucosidic linkages interrupts the extended conformation of the otherwise (1→4)-­linked β-­d-­glucan and is responsible for the chain flexibility (Buliga et al 1986). The chain conformation of (1→3,1→4)-­β-­d-­g lucans (Fig. 9.18Ciii) leads to solutions with high intrinsic viscosities (Table 9.9) due to their occupancy of high volumes of solution (high hydrodynamic volume). At low concentrations, the viscosity is independent of shear rate, but as the concentration increases, the individual chains interpenetrate and the viscosity diminishes with increasing shear rate (i.e., shear thinning occurs) (Izydorczyk and Biliaderis 2000). At relatively high concentrations, 4–10% or more, (1→3,1→4)-­ β-­d-­glucans form thermoreversible elastic gel networks that exhibit broad melting transitions and show syneresis (Bohm and Kulicke 1999, Izydorczyk and Biliaderis 2000). The melting temperatures of wheat and lichenin (1→3,1→4)-­β-­d-­glucan gels are similar but higher than those from barley and oats (Table 9.9). This is proposed to be due to the formation of junction zones between pairs of consecutive cellotriosyl units (Bohm and Kulicke 1999, Cui 2001), which predominate in wheat and lichenin (1→3,1→4)-­β-­d-­glucans but are less frequent in those from barley and oats. The gelation rate and the melting enthalpy (∆H) of wheat (1→3,1→4)-­β-­d-­glucan, measured by dynamic rheometry and scanning calorimetry, respectively, increase as Mw decreases

Carbohydrates  (Li et al 2006). This is interpreted as indicating that wheat (1→3,1→4)-­β-­d-­glucan molecules (above a minimum size) form junction zones more easily than their barley and oat counterparts and establish stronger three-­dimensional networks due to their high mobility and structural regularity (high trisaccharide-

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t­ etrasaccharide ratio). Moreover, the melting temperature of wheat (1→3,1→4)-­β-­d-­g lucan gels increases with increasing molecular weight, again suggesting that high-­Mw wheat (1→3,1→4)-­β-­d-­glucans form stable structural networks. Solutions of low-­Mw barley (1→3,1→4)-­β-­d-­g lucan prepared by

Fig. 9.18. A, portion of a (1→3,1→4)-β -d-glucan molecule. B, distribution of linkages in a (1→3,1→4)-β -d-glucan; G = β -glucosyl unit, 3 = (1→3) linkage, 4 = (1→4) linkage, red = reducing end, arrows = sites of hydrolysis by (1→3,1→4)-β -d-glucan endohydrolase (EC 3.1.2.73). C, perspective drawings of computer-generated, instantaneous conformations of β -d-glucans; (i) = (1→4)-β -d-glucan (cellulose), (ii) = (1→3)-β -dglucan (callose), (iii) = (1→3,1→4)-β -d-glucan; ! = (1→4)-linked residues, 1 = (1→3)-linked residues. (Reprinted, with permission, from Fincher and Stone 1986) TABLE 9.9 Comparative Properties of Cereal (1→3,1→4)-β-d -Glucansa Source

Wheat Bran

Barley Flour

Oat Flour

Lichenin (Cetraria islandica)

Trisaccharide-tetrasaccharide ratio

  4.2–4.5b, 3.7c

  2.7–3.0d, 2.8c, 3.0c

  2.2–2.4 d, 2.1c

18.6 e, 24.5c

Percent trisaccharide + tetrasaccharide Percent penta-nonasaccharide

93.3b, 91.3c

91.0–92.1d, 90.9c, 91.2c

92.4–94.0d, 90.3c, 90.5c

77.5c Not available

Percent penta-tetradecasaccharide

  8.7c  

0.49 b,

Polydispersity (Mw/Mn) Intrinsic viscosity (dL/g)

 

1.65f

 

4.96f

Gelation melting transition (°C)

72c

Average Mw ×

a Asterisks

105

 

6.7b

 

7.8b

 

  9.1c, 8.8c 2.09c*

 

1.26–2.39d *,

 

1.3–1.9e

 

4.6–6.9e

  9.7c, 9.5c 2.13c*,

1.07c*

65e, 67.7d, 69.3d

indicate the peak fraction of the main peak in the HPLC chromatogram. et al (2000). c Lazaridou et al (2004). d Papageorgiou et al (2005). e Bohm and Kulicke (1999). f Li et al (2006). b Cui

8.1b

 

0.44–1.10d *,

 

1.3–1.5e

 

2.0–9.6 e

62e

22.5c 2.03c*,

1.05c*

  0.55e, 1.06 c*   1.8e Not available 73e, ~89c

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partial acid hydrolysis undergo a sol-­to-­gel transition on storage (Vaikousi et al 2004). This was especially true of a low-­Mw fraction with a high trisaccharide-­tetrasaccharide ratio, again implying that these structural features are important in the formation of ordered structures. Gelation of (1→3,1→4)-­β-­d-­glucans may be induced by disc centrifugation (Letters et al 1985) and by freezing and thawing (Morgan and Offman 1998, Lazaridou and Biliaderis 2004, Vaikousi and Biliaderis 2005). Under certain circumstances, barley (1→3,1→4)-­β-­d-­glucan and lichenin can be induced to form structures that give weak X-­ray diffraction patterns (Tvaroska et al 1983), indicating a relatively high degree of structural regu­ larity, which might be expected to be even higher for wheat. Freeze-­drying wheat (1→3,1→4)-­β-­d-­glucan solutions induces chain-­chain interactions, and consequently the product redissolves with difficulty, but drying by solvent exchange yields a more easily solubilized product (Cui 2001). Biosynthesis

The enzymes synthesizing (1→3,1→4)-­β-­d-­glucan are found in microsomal membrane fractions from ryegrass (Lolium multiflorum) endosperm (Smith and Stone 1973, Henry and Stone 1982, Henry et al 1983, Meikle et al 1991), barley (Hordeum vulgare) coleoptiles (Becker et al 1995) and endosperm (Tsuchiya et al 2005), maize (Zea mays) (Buckeridge et al 1999), and other cereals (Stone and Clarke 1992). The synthase complex is presumed to reside in the plasma membrane since, in immunochemical studies of developing endosperm and other tissues in wheat (Philippe et al 2006c) and barley (Wilson et al 2006), there was no evidence that the (1→3,1→4)-­β-­d-­glucan co-­locates with Golgi membranes or vesicles. The enzyme system involved in the biosynthesis of the (1→3,1→4)-­β-­d-­glucan homopolymer, with its unique organization of the two linkage types in the chain, involves at least one cellulose synthase-­like (Csl) enzyme, encoded by a CslF gene, one of two members of the Csl group found only in graminoid monocotyledons that encode GT2 glycosyltransferases (Burton et al 2006). In rice, there are six CslF genes on chromosome 7 and others on chromosomes 8 and 10. In barley, at least two CslF genes are present on chromosome 2H. The involvement of other enzymes, cofactors, or protein carriers in directing the synthesis of the (1→3,1→4)-­β-­d-­g lucan remains to be determined. The second member of the Csl group, CslH, found only in graminoid monocotyledons, may also be involved in (1→3,1→4)-­β-­d-­g lucan biosynthesis, but definitive proof is not available. In the developing wheat grain, (1→3,1→4)-­β-­d-­glucans are deposited in the walls of the starchy endosperm cells during cellularization and later in the walls of the aleurone (Philippe et al 2006c). Some modification of the endosperm wall (1→3,1→4)-­β-­d-­glucans may occur during development (Philippe et al 2006c). depolymerization during germination (malting)

During germination, the walls of the endosperm and aleurone cells are degraded (Fincher and Stone 1974, Joyner 1985) by

hydrolases (see Chapter 11) that are synthesized and secreted by the aleurone protoplast (Corder and Henry 1989). Genetics

The concentration of (1→3,1→4)-­β-­d-­g lucan in cereal grains is influenced by both genotype and environment. Regions of the barley genome that control (1→3,1→4)-­β-­d-­g lucan concentrations have been identified by QTL mapping and placed on high-­density maps (Han et al 1997). Since there is a high degree of gene order (synteny) between wheat and barley, the barley maps are highly relevant to determining the chromosomal positions of (1→3,1→4)-­β-­d-­g lucan QTLs in wheat (Burton et al 2006). In wheat-­r ye addition lines, chromosomes 2R and 5R notably increased the proportion of soluble noncellulosic glucose in the nonstarchy polysaccharide fraction (Cyran et al 1996). Analysis and detection (1→3,1→4)-­β-­d-­Glucans are estimated with high specificity using the (1→3,1→4)-­β-­d-­glucan endohydrolase (EC 3.2.1.73)

from Bacillus amyloliquefaciens to cleave the (1→4) linkage in the sequence -­G4G3G↓4G-­, releasing oligosaccharides (Fig. 9.18B) that are further hydrolyzed to glucose using a β -­glucosidase. The glucose released is estimated using a glucose oxidase–peroxidase procedure (AACC 2000). The enzymatically released oligosaccharides may be separated by size exclusion chromatography (Woodward et al 1983, Wood et al 1991b) and their structural features determined by 13C NMR (Wood et al 1994) or matrix­assisted laser-­desorption ionization–mass spectroscopy (Jiang and Vasanthan 2000). (1→3,1→4)-­β-­d-­Glucans in cell walls of grain sections, in milling fractions, or in solution may be detected by the fluorescence induced by complexing them with the diaminostilbene sulfonate fluorochrome Calcofluor or with the diphenyldiazo dye Congo Red to give a bright blue fluorescence (excitation at λmax 350 nm, emission at λmax 420 and 440 nm) or a bright red fluorescence (excitation λmax 470 nm, emission λmax 588 nm) (Wood et al 1983). Scanning microspectrofluorimetry of cell-­wall-­bound Calcofluor has been used to compare the distribution of (1→3,1→4)-­β-­d-­glucan in the grains of cultivars of barley and oats (Miller and Fulcher 1994). Calcofluor binding is also applicable to measurement of solution concentrations of (1→3,1→4)-­β-­d-­glucans above 30,000 kDa (Wood et al 1991a). It should be noted, however, that the induced fluorescence is not specific for (1→3,1→4)-­β-­d-­glucans (see section on cellulose). In light and electron microscopy, a monoclonal antibody used with a gold-­labeled, second-­stage antibody detects (1→3,1→4)-­β-­d-­glucans in cell walls of cereal grains with high selectivity and sensitivity (Meikle et al 1994, Philippe et al 2006c, Wilson et al 2006). NIR spectra of (1→3,1→4)-­β-­d-­glucans are complex, making their interpretation difficult, but principal component analysis of spectra of different polysaccharides has enabled characteristic NIR wavelengths to be discerned and applied to quantitation of (1→3,1→4)-­β-­d-­glucan in barley varieties (Munck et al 2004, Jacobsen et al 2005). NIR has been evaluated for the determina-

Carbohydrates  tion of both (1→3,1→4)-­β-­d-­glucans and arabinoxylans in cereal grains (Blakeney and Flinn 2005).

Arabinogalactan-­Peptides Occurrence and Structure

Arabinogalactan-­proteins and -­peptides are ubiquitous components of plant tissues (Fincher et al 1983, Johnson et al 2003). The arabinogalactan-­peptide (AG-­peptide) from wheat flour (Fincher et al 1974, Neukom and Markwalder 1975, Loosveld et al 1997) consists of a 15-­residue peptide backbone (molecular mass 7,800 Da) with three hydroxyproline residues (Van den Bulck et al 2002). Each residue is glycosidically linked (McNamara and Stone 1981, Strahm et al 1981) to a branch-­on­branch (1→3)-­ and (1→6)-­l inked β-­d-­galactopyranan (~80%, 3,6-­l inked Galp; 6.0–11.5%, 3-­l inked Galp; 5.1–11.8%, 6-­l inked Galp; trace terminal Galp). All cereal AG-­peptides have a 6-­l inked β -­Galp backbone substituted at position 3 with either single α-­l-­A raf residues or, to a lesser degree, with single 3-­l inked β -­Galp residues, which are mostly substituted at position 3 with single α-­l-­A raf residues (Strahm et al 1981, Loosveld et al 1998, Van den Bulck et al 2005) (Fig. 9.19). The Ara-­Gal ratio is in the range of 0.63–0.72:1 (Loosveld et al 1997). AG-­peptides with structures very similar to that of wheat AG­peptide have been found in durum wheat, spelt, rye, barley, and triticale (Van den Bulck et al 2005). No acetyl or HCA esterification of AG-­peptides has been reported. The wheat AG-­peptide is very soluble in water, giving solutions with low intrinsic viscosities (0.045–0.062 dL/g), and is not precipitated at saturating ammonium sulfate concentrations that allow its separation from AX (Fincher et al 1974). The concentration of the wheat AG-­peptide in the grain ranges from 0.27 to 0.38% (dw) (Ingelbrecht et al 2001). In wheat milling fractions, the concentration is higher in the second and third reduction fractions than in the three break and first reduction fractions (Loosveld et al 1997, 1998). Among eight European cultivars, the water-­extractable AG-­peptide content varied between 0.24 and 0.33% and the Ara-­Gal ratio varied between 0.66 and 0.73 (Loosveld et al 1998). In eight Canadian wheat cultivars, the peptide of the AG-­peptide molecule constituted 6.5– 14.3% (cf. 4.7–7.0%, Van den Bulck et al 2005), reflecting differences in the size of the AG component. Among eight Canadian wheats, Ara-­Gal ratios ranged from 0.66:1 to 0.72:1 (Izydorczyk et al 1991). The viscosities of AG-­peptide solutions do not change in oxidative reactions catalyzed by peroxidase/hydrogen peroxide, consistent with the absence of esterified ferulic acid (Meuser and Suckow 1986).

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Biosynthesis

Membrane preparations from suspension-­cultured ryegrass (Lolium multiflorum) endosperm cells incorporate Gal residues from UDPGal into presumptive AG molecules, identified by their interaction with the (1→6)-­β-­d-­galactan-­specific myeloma protein, J539 (Mascara and Fincher 1982, Cohen et al 1983). Sub­ cellular fractions enriched in Golgi vesicles had the highest Gal transferase activity (Schibeci et al 1984). A number of noncereal AG-­peptides have been described whose short (10–13) amino acid peptide backbones are encoded by dedicated genes; however, genes encoding the peptide of cereal AG-­peptides have not been found in the cereal expressed­sequence-­tag database (Schultz et al 2004). Significantly, sequences homologous to the wheat and other cereal peptide sequences have been identified near the NH2 terminus of the 15-­k Da grain softness protein, GSP-­1, found on the surface of wheat starch granules (Van den Bulck et al 2002, 2005). This pre­pro-­protein consists of an N-­terminal signal peptide followed by about 20 residues before the first amino acid of the mature protein, both of which are cleaved in the processing steps. Thus, the peptide backbone of the AG-­peptide may arise by excision of a processing product of the GSP-­1 protein. Presumably, glycosylation follows the excision step. AG-­peptides are one of the main polysaccharides detected by FT-­IR in association with walls of starchy endosperm cells of wheat at the end of cellularization (Philippe et al 2006b). In barley endosperm, AG-­peptides were first detected immunologically at 6 dpa but at very low levels and in a patchy distribution (Wilson et al 2006). By 7 dpa, labeling could be seen at the

Physiological role

The physiological role of the cereal AG-­peptides is not understood, although there is increasing evidence that their AG­protein counterparts are involved in events in cell proliferation, cell elongation, and embryo development (Majewska-­Sawka and Nothnagel 2000, Showalter 2001). An AG-­protein from barley may have a role in gibberellin-­induced α-­amylase production in the aleurone of germinating grain (Suzuki et al 2002).

Fig. 9.19. Structure of wheat arabinogalactan-peptide (AG-peptide) based on the data of Fincher et al (1974), Strahm et al (1981), Loosveld et al (1998), and Van den Bulck et al (2005). ! = (1→6)-linked-β -dgalactopyranosyl unit, 1 = (1→3)-linked-β -d-galactopyranosyl unit, p = terminal α-l-arabinofuranosyl unit.

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plasma membrane–cell wall interface along most walls of the starchy endosperm. Genetics

Soluble fractions of flours from 1B/1R wheat translocation lines (Biliaderis et al 1992) showed substantial amounts of galactose arising from the AG-­peptide, as shown by the gel filtration profile, indicating that the 1R chromosome of rye does not contribute to AG-­peptide formation. Walls from normal and 1B/1R translocation grains had comparable galactose levels, but galactose was not detected in the rye parent (Selanere and Andersson 2002). The grain softness protein gene sequences containing the AG-­peptide sequence are found on the A, B, and D genomes of Triticum (Van den Bulck et al 2005).

Radioautography using tritiated myo-­inositol (Tanaka et al 1974) has been used to follow the accumulation of phytate in the globoids. The content of phytate in the phytin globoids depends on the phosphate concentration in the soil in which the wheat plant is grown. The protein bodies in plants grown under low­phosphate conditions have globoids that are small and numerous, in contrast to control plants in which the globoids are large (Batten and Lott 1986). Williams (1970) recorded phytate deposition at 18 dpa, which continued rapidly between 25 and 30 dpa and slowly thereafter up to ~35 dpa. Maximal rates were reached at 28 dpa. In the first days of germination, the inositol phosphate ester groups are cleaved by the aleurone-­located phytase (EC 3.1.3.36), a phosphomonoesterase, to provide phosphate for the developing seedling. Biosynthesis

Detection

The AG-­peptides, unlike their AG-­protein counterparts, do not bind to the glycosylphenylazo Yariv reagents (Yariv et al 1967). However, (1→6)-­β-­d-­galactan epitopes on AG-­peptides (Andrew and Stone 1983) and -­proteins are recognized by the myeloma protein J539 (Glaudemans 1975) and by the LM2 monoclonal antibody (Smallwood et al 1996, Wilson et al 2006). The immobilized J539 myeloma protein has been used in affinity purification of AG-­peptides and -­proteins (Andrew and Stone 1983).

Carbohydrates in Aleurone Protein Body Inclusions—Phytin Globoids and Niacytin Granules Cells of the aleurone and scutellar parenchyma of wheat and other cereals are characterized by the presence in their cytoplasm of special storage vacuoles containing albumins and globulins, termed “protein bodies” (3–6 µm in diameter) (Fulcher et al 1981) (Chapter 3). In the aleurone, these membrane-­enclosed organelles carry two types of inclusions (Fulcher et al 1981) (see also Chapter 3, Fig. 3.40). The first type, the phytin globoids, are of two sizes (large, 1–1.5 µm in diameter, and small, 0.1–0.2 µm in diameter; Batten et al 1994) and are themselves bounded by a unit membrane (Jiang et al 2001). These inclusions are the storage forms of inositol and phosphate. The second type, the electron­dense niacytin granules (2 µm in diameter), are storage forms of niacin and aromatic amines (Fulcher et al 1972, 1981). In the scutellum, protein bodies in the epithelial and parenchymatous cells contain a single type of inclusion body, identified as phytate deposits (Swift and O’Brien 1971, Fulcher et al 1981).

Phytin Globoids The phytin globoid inclusions in the aleurone protein bodies are rich in phytate, the hexaphosphate derivative of the hexahydric cyclic alcohol myo-­inositol (myo-­inositol-­1,2,3,4,5,6­hexakisphosphate, or IP6) (Fig. 9.20). In the aleurone, phytate occurs as mixed salts of cations such as K, Mg, Fe, Zn, Mn, and Ca at levels of 0.5–2% of aleurone by weight. Phytate inclusions are electron dense (Batten et al 1994) and birefringent, stain red with Toluidine Blue, and are soluble in dilute acid (Fulcher et al 1981).

The molecular basis of phytate synthesis is poorly defined but may involve either phospholipid-­dependent or -­independent pathways (Raboy et al 2002). Inositol polyphosphate kinases participate in both pathways, and the disruption of their encoding genes in Arabidopsis leads to phytate-­free seeds (Stevenson­Paulik et al 2005). Nonlethal, recessive mutations in cereals with low phytate (lpa) have been isolated (Raboy et al 2001). Two phenotypically distinct lpa mutant types are found. The lpa-­1-­like mutants show seed phytate reductions between 50 and 95%, with molar equivalent increases in inorganic phosphate. Mutations in the lpa-­2-­like gene, which in maize encodes a variant of the inositol kinase gene family, lead to a 50–75% reduction in phytate, with corresponding increases in inorganic phosphate and in myo-­inositol phosphates containing five or fewer phosphates. A heritable wheat lpa-­1-­like mutant has been described (Guttieri et al 2004) in which the phytate concentration in the bran is 45% of normal and inorganic phosphate increases fourfold. In wheat, two or more genes may be involved; however, the genotypes derived from the wheat mutations are agronomically unacceptable and have greatly reduced grain yield. Analysis

Phytate may be directly determined by a colorimetric method based on the reaction between ferric ion and sulfosalicylic acid (Latta and Eskin 1980) or by separation and independent measurement of inorganic and phytate-­bound phosphorus (Batten et

Fig. 9.20. Two conformational isomers of phytic acid (myo-inositol hexaphosphate). 1 = phosphorus atom. (Reprinted, with permission from Springer Science and Business Media, from Stone 1996)

Carbohydrates  al 1994). The cationic composition of phytate may be determined semiquantitatively in situ by energy-­dispersive X-­ray analysis (Batten and Lott 1986) or quantitatively by inductively coupled plasma atomic emission analysis (Batten et al 1994).

Niacytin Granules The niacytin granules in the aleurone protein bodies contain niacin (nicotinic acid and its amide) covalently complexed with saccharides and/or polypeptides that, as yet, are inadequately described. Bound forms of niacin are found in several wheat bran fractions: a chloroform-­methanol extract, a 60% ethanol extract of the lipid-­free (chloroform-­methanol extracted) residue, and the insoluble residue after ethanol extraction (Kodicek and Wilson 1960, Mason and Kodicek 1973, Mason et al 1973, Koetz and Neukom 1977, Koetz et al 1979). The 60% ethanol extract contains complexes with molecular masses ranging from 1,500 to 17,000 Da and composed of 75.5% carbohydrate, 15% protein, 1.0% nicotinic acid (representing 18.4% of the total bran nicotinic acid), and 0.9% ash. Cinnamic acid esters and aminophenols are also part of this complex (Koetz and Neukom 1977, Koetz et al 1979). Separation of an AG-­peptide from the nondialyzable nitrogen fraction by saturation with ammonium sulfate gave an insoluble fraction composed of protein and carbohydrate, yielding arabinose, xylose, and glucose on complete acid hydrolysis and 3-­O-­nicotinoyl-­d-­glucose on partial acid hydrolysis (Koetz and Neukom 1977, Koetz et al 1979). Niacin in cereal fractions may be assayed using the cy­ anogen bromide–amine reagent colorimetric reaction (AACC 1975) or chromatographically after saponification (Chase et al 1993). Niacytin granules are detected histochemically using the periodate-­Schiff reagent for carbohydrate (Morrison et al 1975) and the cyanogen bromide–amine reagent for niacin (Fulcher et al 1972, 1981).

Impact and Applications of Cell Wall PolysaccHarides in Grain Utilization, End-­Use Quality, and Nutrition The chemistry of the cell wall polysaccharide components— in particular, the arabinoxylans and their organization in wheat grain tissues—has important impacts on the utilization of the grain, especially in milling and bread manufacture. These components also impact human and monogastric digestion and nutrition. These aspects are discussed in the following sections.

Milling and Conditioning The pattern of grain breakage during milling is dependent on the compactness, thickness, composition, and resilience of the outer (bran) layers and on the hardness of the starchy endosperm (Fincher and Stone 1986, Bass 1988) (see also Chapter 5). In roller milling, the aim is to produce an endosperm flour with the minimum amount of bran particles. In the first break rolls, the bran separates from the starchy endosperm, and the success of this step is governed by the characteristics of the bran layers. The aim is to have minimal loss of endosperm with the bran and minimum breakage of the bran into particles that con-

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taminate the endosperm flour. The reduction milling steps that follow reduce the size of the endosperm particles. Bran separation is controlled by adding water to the grain in the conditioning (tempering) step, rendering the bran more plastic while maintaining endosperm hardness. Endosperm compressive strength in hard wheat is more sensitive to moisture content than in soft wheat, and this difference in moisture response accounts for the difference in the endosperm strengths of hard and soft wheats below 22% moisture content (Glenn and Johnston 1992). Conditioning times and rates of water penetration are cultivar-­dependent and influenced by the amount, chemical composition, and physical organization of the outer layers of the grain (Lee and Stenvert 1973), which strongly influence the friability of the cell walls of bran. Differences in Ara­Xyl ratio were found in bran layers of three hard and three soft wheats, being lower in soft (0.76) than hard (0.86) wheats (Lee and Stenvert 1973), with cultivars with highest Ara-­Xyl ratio having the most rapid water penetration during conditioning (Stenvert and Kingswood 1976). The efficiency of separation of bran and endosperm in experimental roller milling of durum wheat has been followed using phenolic markers specific for starchy endosperm, aleurone layer, and pericarp (Peyron et al 2002a,b). The critical step is the separation of the aleurone from the endosperm; there is considerable variation between cultivars in the extent of contamination of the coarsely ground flour (semolina) with aleurone. This was attributable to differences in the plasticity of the bran, which, in turn, is dependent on the moisture distribution (Crewe and Jones 1951, Glenn and Johnston 1992) and the chemistry of the bran layers. The profile of the aleurone-­subaleurone interface (Crewe and Jones 1951) and the thickness of the walls of the subaleurone endosperm (Larkin et al 1951) may also govern this behavior. The mechanical and chemical properties of brans from hard and soft bread wheats are correlated (Antoine et al 2003, 2004a). Strips of aleurone, intermediate layers (inner pericarp, seed coat, and nucellar remnants), and outer pericarp were dissected from wheat grain and bran and their mechanical properties compared with their chemical composition and tissue microstructure. AX cross-­linking is high in the outer pericarp and low in inner layers and aleurone. (1→3,1→4)-­β-­d-­Glucan content is high in aleurone, but lower in other layers; lignin content is high in the outer pericarp and absent in the aleurone; and the cellulose content is high in outer pericarp and low in aleurone (Table 9.5). Antoine et al (2003, 2004b) propose a structural model of a bran composite, with the three layers exhibiting differing mechanical behaviors to account for the facile detachment of the outer pericarp when the bran undergoes mechanical stress. Aleurone cell contents, together with unresolved factors involved in tissue plasticity and the cohesiveness between, for instance, aleurone and nucellar remnants, also influence milling behavior (Piot et al 2000). Intrinsic differences are found in the breakage patterns of the endosperms of hard and soft wheats (Fincher and Stone 1986) (see also Chapter 5). Hardness influences the mode of fracture and the mechanical properties of the whole grain and endosperm (Greffeuille et al 2006b). In milling, hard wheat produces less coarse bran than soft wheat and, as a consequence of the greater breakage of the aleurone, more aleurone cell contents are released into the flour at the reduction step from hard than

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from soft wheat (Greffeuille et al 2005). Again, these differences may reflect underlying differences in the mechanical properties and chemistry of the outer layers of the two kinds of wheat. However, attempts to find correlations between the content of water-­extractable AX in hard, soft, and club wheats using NIR analysis failed to verify that AX was the component correlating with hardness (Hong et al 1989). Moreover, FT-­IR spectral features of walls in entire sections of wheat endosperm do not discriminate between hard and soft wheats (Barron et al 2005), as is also shown in chemical studies on AX from walls of endosperm cells (Mares and Stone 1973a). Nor are there marked differences in the chemical compositions of the outer layers of hard and soft wheats (Antoine et al 2003, Greffeuille et al 2006a). Nonetheless, when the location of AX in the grain is considered, hard wheat is clearly distinguishable from soft wheat (Barron et al 2005). These regional differences in composition may contribute to the differences in mechanical properties of the walls. Cell walls in the peripheral layer in soft wheat are characterized by a higher level of water-­extractable AX than are those of hard wheat. Differences in the walls of central cells are more complex. The differences between the walls of endosperm cells of hard and soft wheats become apparent at about 15 dpa at the stage when the hard/soft characteristic is manifest (Bechtel et al 1996). In later stages, changes in wall composition also occur, as shown in the immunochemical studies of Philippe et al (2006c).

Bread and Other Baked Products There is abundant evidence that wall polysaccharides, especially AX, have a significant role in the various steps in the breadmaking process and in the storage and organoleptic characteristics of the final product (Courtin and Delcour 2002). Water Absorption and Mixing

In bread manufacture, the first step is the formation of a dough by the mixing of wheat flour with a predetermined amount of water, yeast, salt, and supplements such as emulsifiers, enzymes, and oxidizing and reducing agents. A wheat flour dough is thus a heterogeneous system composed of thermodynamically incompatible polymers (Fessas and Schiraldi 2001b) and consists of separate phases, each with a predominant specific polymer— namely, a gluten-­r ich phase, a starch-­r ich phase, and an AX/ soluble protein-­rich phase (Larsson and Eliasson 1996, Grinberg and Tolstoguzov 1997). Water exchanges between these phases occur during dough mixing and baking (Larsson and Eliasson 1996), and, although the partitioning is incomplete because of limited molecular mobility and the changing conditions of the polymers in the phases as the process proceeds, these lead to changes in water affinity as the molecular structures of the gluten and the AX are modified (Fessas and Schiraldi 2001b). Water added to the dough mixture is absorbed to an extent that depends primarily on the protein content of the flour and the degree of damage the starch grains have suffered during milling (which is higher in hard wheats). In addition, although their concentration is low (~2%), the highly hydratable nonstarch polysaccharides (in particular, the water-­soluble and the water-­unextractable AX) are also important contributors to

water absorption. Water is absorbed by the starchy endosperm fragments in the flour and additionally is entrapped inside the macromolecular complexes formed by the swollen endosperm cell contents, with the following resultant distribution: granular starch, 25–30%; damaged starch, 25–30%; gluten, 20%, and AX, 22% (Bushuk 1966). Thus AX have an impact on the water absorption properties of the flour that is disproportionately higher than their absolute content. Added water-­soluble and water-­unextractable wheat AX markedly increase farinograph water absorption (Michniewicz et al 1990, Vanhamel et al 1993, Biliaderis et al 1995, Denli and Ercan 2001), and their hydration capacity is further increased by oxidative gelation (Izydorczyk et al 1990). Dough Rheological Properties

Water Binding. The extent to which water in dough is bound to the dough polymers or is free influences the physicochemical behavior of doughs. The proportion of frozen (bound) water and unfrozen (free) water in doughs has been evaluated using differential scanning calorimetry (DSC) (Roman-­Gutierrez et al 2002). The free water contents of dough components were gluten, 38–47%; starch, 38–42%; damaged starch, 37–40%; water­soluble AX, 51%; and water-­unextractable AX (containing 52% starch), 40–44%. Bound water is interpreted as the amount of water necessary to fully hydrate and plasticize the flour components, whereas free water is partly responsible for the flow and mobility of dough (Fessas and Schiraldi 2001b, 2005; Schiraldi and Fessas 2003). Water-­soluble AX increases the viscosity of the dough water (Udy 1956), and the viscosity is further increased by oxidative gelation reactions that cross-­link FA-­substituted xylan chains and that possibly cross-­link FA on AX and tyrosine on flour proteins (Udy 1957) (Fig. 9.16). The addition of water-­soluble AX to doughs results in an increase in bound water and concomitantly in the resistance of dough to extension and a decrease in dough extensibility (Pence et al 1950; Cawley 1964; Jelaca and Hlynka 1971, 1972). Conversely, addition of xylanase-­containing enzyme preparations releases AX-­bound water (probably from the AX gel) and decreases the resistance to extension, giving a slacker dough (Cawley 1964, Tracey 1964). The involvement of AX in moderating dough behavior is clearly demonstrated by the action of endogenous and added xylanases (Rouau 1993, Rouau and Moreau 1993, Rouau et al 1994, Petit-­Benvegnen et al 1998). At optimal levels, xylanase addition improved dough properties, leading to greater uniformity in quality characteristics and a higher level of quality for all samples. When an excess of enzyme was added, the dough characteristics deteriorated due to loss of water-­holding capacity. During breadmaking, a continuous change occurs in the molecular mass of the AX in the dough through the action of endogenous flour xylanases (Rouau et al 1994, Cleemput et al 1997). In “native” doughs, part of the water-­unextractable arabinoxylan becomes extractable during processing, but solubilization occurs to a greater degree when xylanase is added, with a concomitant increase in the specific viscosities of water extracts of doughs (and the dough liquor) as water-­soluble AX is released. The effectiveness of the enzyme preparations appears to be re-

Carbohydrates  lated to the amount and size of the extractable AX released from the water-­unextractable AX. Enzymes in sourdough may also alter the degree of arabinosylation of AX during breadmaking (Martinez-­Anaya and Devesa 2000). Endogenous xylanases in wheat flour are accompanied by endogenous proteinaceous xylanase inhibitors (Chapter 11) so that the activity of the xylanases during breadmaking depends on the relative amounts of the enzymes and their inhibitors (Gebruers et al 2001, Courtin and Delcour 2002). Dough syruping, encountered after refrigeration of doughs, is another manifestation of loss in water-­holding capacity, resulting from enzymatic degradation of AX by endogenous and exogenous xylanases, the latter contributed by microorganisms on the bran (Gys et al 2004). Gluten Aggregation-­Disaggregation. The functional properties of gluten in bread doughs are affected by the presence of both water-­extractable AX and water-­unextractable AX (Jelaca and Hlynka 1971, 1972; Kim and D’Appolonia 1977; Michniewicz et al 1990, 1991; Courtin et al 1999; Wang et al 2003a,b, 2004a,b, 2005). Water-­soluble AX affects gluten development by competing for water, and, in addition, FA on water-­extractable AX may interact covalently with gluten, leading to a less-­extensible dough (Wang et al 2002). On the other hand, water-­unextractable AX negatively affects the development of gluten in doughs. Wang et al (2004a,b, 2005) have explored the rheological behavior of glutens from wheats with varying amounts of water-­unextractable AX and also the role of xylanases in modifying these effects. Models for the involvement of AX in the dough properties at various stages of dough development have been proposed (Han and McDonald 1998; Wang et al 2004a,b, 2005). Generation of DDFA cross-­linked AX in situ in doughs during mixing led to only minor changes in mixing behavior, mainly involving dough consistency (Labat et al 2001). No covalent interaction between AX and the dough proteins was observed, suggesting that the gluten protein and AX formed separate networks (Labat et al 2002). The aggregation-­disaggregation of the gluten proteins during dough development is affected by the water absorption and water-­holding capacity of AX (Michniewicz et al 1991, 1992). Added water-­soluble and water-­u nextractable AX both have marked effects on farinograph properties; both water absorption and dough development time increase. There are also effects on gluten yield, which depends on the characteristics of the base flour and the type and amount of AX added. In general, the yield of wet gluten decreases, particularly at low AX supplementation levels (1 and 2%). Added water-­unextractable AX had a significant effect on the extractability of proteins from the dough and gluten of a Canadian western red spring wheat (Katepwa) by acetic acid (AA) and acetic acid–urea (AA-­U) solvents. Added water-­unextractable AX decreased the amount of protein solubilized from the dough samples in AA and increased the amount in AA-­U; opposite trends were observed on gluten yield. Both types of AX affect the aggregation-­disaggregation process of the high molecular weight gluten proteins but produce only minor changes in the electrophoretic patterns of the AA-­ and AA-­U­soluble proteins. The interactions of AX with gluten in the dough may be direct or take place through their water-­binding effects. Fessas and

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Schiraldi (2001a, 2005) have made a thermogravimetric study of the state of water in a wheat flour dough at mixing and at rest after mixing and have followed the effect of adding globular proteins and water-­soluble wheat flour AX. At the concentration of the nonstarch polysaccharides used (1% with respect to flour mass), the state of water in the nongluten phase was un­ affected, but the water-­soluble AX were almost completely associated with the gluten-­rich phase of the dough, from which the moisture is not released during thermogravimetric analysis at room temperature. This behavior is interpreted on the basis of the general thermodynamic incompatibility between proteins and polysaccharides (Grinberg and Tolstoguzov 1997), and, in the case of the gluten and water-­soluble AX, the extent of the effects is related to the molecular mass of the AX. Bread Volume and Loaf Properties

AX have been shown to affect loaf volume and the crumb and crust characteristics of breads. Loaf volume is enhanced by the addition of water-­soluble AX to doughs (Delcour et al 1991, Michniewicz et al 1992, Biliaderis et al 1995). However, there is an optimum addition due to the increased viscosity of the dough liquor (Delcour et al 1991), which is dependent on the molecular mass of the added AX (Biliaderis et al 1995). Whereas addition of water-­soluble AX (at 2%, w/w) (Delcour et al 1991, Michniewicz et al 1992) increased specific loaf volume, water-­unextractable AX did not significantly affect this parameter. However, Kulp and Bechtel (1963) reported that water-­u nextractable AX depresses loaf volume. Xylanase addition has a positive effect on loaf volume (Debyser et al 1999) through the conversion of water­unextractable AX to water-­soluble AX (McCleary 1986, Biliaderis et al 1995, Petit-­Benvegnen et al 1998, Courtin et al 1999, Denli and Ercan 2001). Courtin and Delcour (2002) have provided a model for the role of AX and AX hydrolases in breadmaking. Westerlund et al (1989a) followed the fate of low molecular weight flour carbohydrates and AX in dough, crumb, and the inner and outer crust portions of white bread during baking. Analysis of 80% ethanolic extracts from the bread fractions showed that Maillard reactions had occurred, new gluco­saccharides with β -­1,6-­anhydroglucopyranose end units that resist amylolytic cleavage were formed, and maltulose (α-­d-­glucopyranosyl-­(1→4)-­d-­fructose) was generated by epi­ merization of maltose. Compared with flour and dough, water­extractable AX in bread had a higher degree of arabinosylation. Heat treatment during baking rendered some of the water­unextractable AX soluble, but there was little effect on arabinogalactan (Westerlund et al 1989a,b, 1990). Dough Fermentation—Gas Cell Stabilization and Gas Retention

Gas cell stabilization and gas retention in the fermenting dough is necessary to produce a loaf with a light and even texture (Gan et al 1995). Gas cells have liquid film “walls” whose mechanical strength is important for cell stability. AX protects the gas cells against thermal disruption, enhancing the strength and elasticity of the surrounding gluten protein films and slowing the rate of diffusion of CO2. AX with high intrinsic viscosity is

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effective in stabilizing aqueous protein foam during thermal expansion (Izydorczyk et al 1991). The emulsion-­stabilizing activity of AX is due to increases in viscosity of the continuous phase by the various AX fractions (Schooneveld-­Bergmans et al 1999a). Among the various AX fractions, an AX having an Ara-­Xyl ratio of 0.94 and an intrinsic viscosity of 2.4 dL/g showed the highest emulsion-­stabilizing activity at concentrations as low as 0.05 g/dL of aqueous phase. The AX in protein foams not only stabilizes them by increasing the viscosity of the interlamellar liquid but may also enhance foam stability by formation of cross-­links between AX and adsorbed protein (Sarker et al 1998). Bread Staling and Crumb Properties

Using the increase in bread crumb firmness as a measure of bread staling, Biliaderis et al (1995) showed that breads with added AX were consistently less firm than controls. This effect is attributed to the increased moisture content of the bread crumb, which plasticizes the starch-­gluten matrix, lowering the rigidity of this composite network. Breads supplemented with water­soluble AX showed higher amylopectin retrogradation rates, as measured by DSC, presumably due to their higher moisture content (Michniewicz et al 1992, Biliaderis et al 1995). Thus, starch retrogradation and firming occur concurrently on storage, both being influenced by the moisture content of the bread crumb. On the other hand, water-­soluble AX retarded the aggregation of amylose molecules, as shown by the amount and type of carbohydrates extractable by water from bread crumb. The finding that breads with added AX have lower firmness is supported by elastic modulus measurements on staling bread crumb (Fessas and Schiraldi 1998), which showed that doughs enriched by addition of water-­soluble AX remain softer than those from control doughs. Again, there was no evidence that starch retrogradation was involved in crumb properties. Image analysis of crumb texture (Fessas and Schiraldi 1998) showed that bread crumb with added soluble AX had a coarse alveolar structure compared with that of control breads and breads baked with added soluble protein. These results were interpreted in terms of the different effects of added AX and protein on the properties of the liquid film over the alveoli. Wheat Arabinogalactan-­Peptide In Doughs and pasta

The effect of addition of wheat AG-­peptide, larch wood arabinogalactan, and gum acacia, an AG-­protein, at 1 and 2% substitution levels, on the mixing characteristics of doughs from flours of two wheat varieties and the volumes of the resulting breads was tested, but it was unclear whether the effects were beneficial or unfavorable (Westerlund et al 1989a,b, 1990; Loosveld and Delcour 2000). No changes in AG-­peptide content or structure were observed during pasta processing, but AG-­peptide leached more rapidly than AX during cooking (Ingelbrecht et al 2001). Gluten and Starch Separation

Gluten products from a starch/gluten separation process are associated with AX, which represents ~0.4% of the weight of the gluten (Saulnier et al 1997). Some AG-­peptide is also pres-

ent. These polysaccharides are physically entrapped in the gluten network. Numerous factors affect the performance of industrial-­scale separation of wheat flour into gluten and starch fractions. Among these are the level and type of nonstarch polysaccharides, in particular AX (Van Der Borght et al 2005). For example, separation in the batter process, which relies on agglomeration of gluten proteins in a dilute salt solution, is hindered by the competition for water between gluten and the AX in solution (Roels et al 1993, 1998; Christopherson et al 1997; Frederix et al 2003; Wang et al 2003a,b, 2004a,b). Moreover, the presence of AX in the gluten increases the water content and hence the energy input required for drying (Roels et al 1993). Additionally, in the batter and dough-­batter processes, the viscosity imparted by the AX to the solution affects gluten aggregation by slowing the diffusion of the polymeric components (Weegels et al 1992, Christopherson et al 1997, Redgwell et al 2001, Frederix et al 2003). In the presence of AX, gluten yields are lowered, but both gluten and starch yields improve after the viscosity is reduced using endoxylanase (Weegels et al 1992). Covalent cross-­linking of the AX with one another and to gluten proteins may enhance the negative effect of the AX (Wang et al 2002, 2003a,b). The particulate, water­unextractable AX also interferes with the gluten agglomeration not only by its action as a physical barrier, but also through its water-­holding capacity and putatively through the formation of DDFA cross-­links with gluten (Frederix et al 2003, 2004).

Applications of Arabinoxylans Arabinoxylan Preparations

Alkaline extraction of wheat bran provides a good source of AX (Maes and Delcour 2002, Mandalari et al 2005) for use in food formulations as gelling agents, cryostabilizers, or a source of prebiotics and for nonfood uses such as film formation. Gel Formation

Hydrogels have been created by laccase-­catalyzed crossl­ inking of water-­soluble wheat AX in the presence and absence of bovine serum albumin (Vansteenkiste et al 2004). When the degree of FA substitution is varied, the resulting AX gels are able to include proteins with molecular masses from 43 kDa up to 669 kDa. Protein loadings ranged from 0.5 to 3 mg/mL of gel (Carvajal-­Millan et al 2005b). Their use in controlled delivery of protein, with the possibility of modulating protein release by controlled degradation, is proposed (Carvajal-­Millan et al 2005a,b). A mixed gel has been prepared by peroxidase/ peroxide cross-­linking of FA-­A X with feruloylated beet pectin (Schooneveld-­Bergmans et al 1999c). Film Formation

AX from maize bran, which is comparable in structure to wheat pericarp AX, has been cast into edible films that are soluble, form a continuous and cohesive matrix with neutral taste and odor, and have mechanical barrier properties similar to those of films of gluten or starch (Chanliaud et al 1995). Hydro­

Carbohydrates  phobic films have been produced by covalent binding of lauryamine chains to maize bran AX (Fredon et al 2002). Films prepared from emulsions of maize AX in hydrogenated palm kernel oil have enhanced functional properties (Phan The et al 2002). Edible maize AX-­based films with reduced water vapor permeability have been created by grafting hydrophobic stearyl acrylate and methacrylate subsituents, using oxygen plasma and electron beam irradiation (Peroval et al 2002, 2004). Acetyl esters (Fang et al 2000) and methyl ethers (Fang et al 2002) have been prepared from an acidic wheat straw arabinoxylan. An acetylated, highly branched, acidic AX from maize fiber produces optically clear films and forms polymer blends with cellulose acetate (Buchanan et al 2003). Cryostabilization

Water-­soluble AX are candidates for cryostabilizing agents in food and drug preparation since their aqueous solutions at 0.5% have a viscosity that hinders the growth of ice crystals on cooling. The state diagrams of wheat AX-­H2O binary mixtures have been determined (Fessas and Schiraldi 2001a) using two AX preparations with Ara-­Xyl ratios of 0.66 and 0.51, molecular weights of 235,000 and 650,000, and dispersity values of 4.2 and 1.6, respectively. The glass transition temperature, T'g, the lowest temperature at which liquid is still present, was higher for the higher molecular weight AX (–17°C) than for the lower molecular weight AX (–35°C), and the X'g, the molar fraction of the solute at T'g, was one order of magnitude lower for the lower molecular weight AX than for the higher molecular weight AX.

Nonstarch Polysaccharides in Human and Animal Nutrition The wall polysaccharides of cells of wheat grain tissues— cellulose, arabinoxylans, (1→3,1→4)-­β-­d-­g lucans, and glucomannans—and the soluble fructans are important components of fiber in diets for humans and monogastric animals. These species produce no autochthonic enzymes that degrade the wall polysaccharides or fructans, although they are to a greater or lesser extent fermented by the microbial population in the lower gut, with the production of volatile (short-­chain) fatty acids, which are in turn metabolized or absorbed by the mucosal cells. Thus, dietary fiber makes a small contribution to digestible energy. Wheat bran nonstarch polysaccharides have a digestible energy of 4.2 kJ/g and a net energy of 2.9 kJ/g (Livesey 1992). Human Nutrition

Fructans. Although mature wheat contains only low concentrations of fructans (1–2.5%) (Table 9.2), grain is the most important source of dietary fructans, since wheat products constitute a high proportion of human diets (Van Loo et al 1995). There are indications that fructans may have beneficial effects in the promotion and protection of human health (Nardi et al 2003). Thus, they are reported to have favorable effects on prebiotic activity (Gibson 1999, Niness 1999), on reducing the level of circulating lipids and glucose (Delzenne and Kok 1999, 2001), and on modu-

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lating the immune response (Schley and Field 2002, Merendino et al 2006). Fructans are not digested by acids at concentrations found in the monogastric stomach (Dahlquist and Nilsson 1984) or the small intestine and may be considered a component of dietary fiber (Roberfroid and Slavin 2000). They are readily fermented by microorganisms in the lower gut (Nilsson and Bjorck 1988). Developing T. durum grain, which is high in fructans, has been proposed as a source of fructose oligosaccharides for use in functional foods (Nardi et al 2003). Cereal fructans are hydrolyzed by bakers’ yeast during fermentation of doughs prepared from wheat flour (Nilsson et al 1987). After 1 h of fermentation, most of the tri-­, tetra-­, and pentasaccharides disappeared, amounting to half of the fructan initially present. Cell Walls and Access to Cellular Nutrients. Most cells of the starchy endosperm are broken in roller milling, so endosperm proteins are accessible for complete digestion. However, aleurone cells, with their thick, bilayered walls, may survive intact, so the valuable protein-­, lipid-­, and vitamin-­rich cell contents are not accessible until the unbroken cells reach the lower gut, where the cell wall polysaccharides are subject to fermentation. Depending on milling conditions, about 30% of bran protein, essentially aleurone protein, remains undegraded (reviewed by Fincher and Stone 1986). As discussed in the section on milling and conditioning, the extent of breakage of aleurone cells, with the release of their contents into the flour, depends on the type of wheat—hard vs. soft (Greffeuille et al 2005, 2006a,b). Ball milling of wheat bran particles cracks the aleurone walls, allowing extraction of the contents (Antoine et al 2004b). Up to 30% of the protein can be extracted by water from sheets of aleurone cells prepared from bran by hammer milling but with apparently undamaged walls (Stevens 1973). Soluble Cell Wall Polysaccharides and Their Effects in the Small Intestine. Soluble polysaccharide components of dietary fiber may enhance the viscosity of the digesta in the small intestine, delaying gastric emptying, inhibiting the dispersion of digesta along the intestine, and suppressing convectional stirring of the fluid layer adjacent to the mucosal surface, resulting in a slowing of the rate of absorption of glucose and so reducing the glycemic response (Johnson 2005). A consequence of this is the lowering of the postprandial glucose response (as measured by the glycemic index) and the concomitant insulin response (Chapter 7). Additionally, the reabsorption of cholesterol in the distal ileum may be reduced, leading to a reduction of serum cholesterol (Johnson 2005). For wheat grain, the so-­called “viscous fiber” is essentially the soluble arabinoxylan, the (1→3,1→4)-­β-­d-­glucan being essentially unextractable by water at body temperatures (Beresford and Stone 1983). Wheat arabinoxylan fiber preparations behave like the soluble fermentable fiber in the large bowel of rats (Lu et al 2000a,b). Dietary supplementation with arabinoxylan-­rich fiber reduces the postprandial glucose and insulin responses in normal human subjects (Lu et al 2000a,b; Zunft et al 2004) and improves metabolic control in people with type II diabetes (Lu et al 2004). No data are available comparing the effects of wheat arabinoxylan or (1→3,1→4)-­β-­d-­glucan with those established for other viscous fibers such as barley and oat (1→3,1→4)-­β-­d-­glucans, which have well-­documented ­beneficial

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effects (Brennan and Cleary 2005). Some lowering in the degree of arabinosylation of AX during residence in the stomach may occur. Thus, Zhang et al (2003) have reported that, after 3 h at 37°C at acidities comparable with those found in the human stomach (pH 1–3), up to 10% Araf residues on maize hull AX, larch arabinogalactan, and noncellulosic polysaccharides from banana peel are lost. Particulate (Insoluble) Fiber and Colonic Health. In the large intestine (colon and rectum), undigested cellulose and noncellulosic polysaccharides in the form of particulate cell wall residues and soluble fiber are fermented by commensal microorganisms (i.e., Gram-­negative anaerobes of the genus Bacteroides and Gram-­positive Bifidobacterium, Lactobacillus, and Clostridium spp.). This results in the production of the short­chain fatty acids butyrate, propionate, and acetate, which are used as energy sources, allowing recovery of some of the energy from the undigested polysaccharides. Butyrate is preferentially a source of energy for colonic mucosal cells, whereas propionate and acetate are absorbed and metabolized systemically. Butyrate has a number of effects in vitro (causing differentiation of tumor cells, suppression of cell division, and induction of apoptosis), but whether these effects are seen in vivo is not established (reviewed by Topping and Cobiac 2005). The digestibility of wheat bran and its fractions in the lower guts of monogastric animals has been followed in rats (Cheng et al 1987, Harris 2005) and pigs (Bach Knudsen and Hansen 1991, Bach Knudsen et al 1991) and in vitro by incubation with fecal bacteria (Stevens and Selvendran 1988, Stevens et al 1988), by digestion with mixtures of mammalian digestive enzymes (Amrein et al 2003), and by digestion with (1→4)-­β -­endoxylanase (Benamrouche et al 2002). With rats fed diets supplemented with pericarp-­seed coat, bran, aleurone, or aleurone-­rich fractions of wheat (Cheng et al 1987), the lignified and cutinized pericarp-­seed coat was only poorly digested. Separated aleurone tissue was extensively digested, but when associated with the pericarp-­seed coat, as in bran, digestion was less complete. These differences are reflected in the yield of the volatile fatty acids: aleurone > bran > pericarp­seed coat. Aleurone supported the highest fecal bacterial mass. Harris et al (2005) fed a pericarp-­rich fraction and an aleurone­seed coat fraction to rats and isolated cell walls from the feces. The aleurone walls were partially degraded, but those of the pericarp were not. Mongeau et al (1991) compared wheat bran in ileal digesta with that in fecal pellets, using fluorescence microscopy. The pericarp-­seed coat tissues remained essentially intact, but the aleurone was partially degraded. In an exhaustive study (Bach Knudsen and Hansen 1991, Bach Knudsen et al 1991), researchers compared the digestibility and bulking properties of polysaccharides and other constituents of wheat fractions (white flour, aleurone, pericarp-­seed coat, and bran) in the gastrointestinal tract of the pig. As found in rats, the order of nonstarch polysaccharide breakdown was endosperm > aleurone > pericarp-­seed coat, again emphasizing the differences in organization of the cell walls in these fractions. The stimulation of microbial activity by the fermentable carbohydrate leads to increased microbial biomass in fecal material and softer feces, and, although the pericarp-­seed coat is scarcely fermented, its

presence and water-­holding capacity increases fecal dry matter and bulk. In wheat bran incubated for 72 h in slurries of human feces, the arabinoxylans and the (1→3,1→4)-­β-­d-­glucans of the aleurone were preferentially degraded, but the glucuronoarabinoxylan cross-­linked by phenolic acids in lignified outer layers of the bran was very resistant to attack (Stevens and Selvendran 1988, Stevens et al 1988). Alkali treatment of the bran increased the extent of degradation threefold due to saponification of ester­linked phenolic acids and possibly of glucuronosyl ester linkages. The alkali-­soluble arabinoxylan was readily digested. Com­ parable results have been reported in a comparison of in vitro fecal digestibility of two differentially enriched aleurone preparations (Amrein et al 2003). Chemical and histological examination of wheat bran digested with (1→4)-­β -­endoxylanase (Benamrouche et al 2002) showed that 80 and 50% of the carbohydrate was removed from the aleurone layer and the inner bran (nucellus, seed coat, and tube and cross cells), respectively, but the outer bran (hypodermis and epidermis) was undigested. The hydrolysis of arabinoxylans and (1→3,1→4)-­β-­d-­glucans in wheat bran dietary fiber by enzymes of colonic bacteria produces oligosaccharides that can be considered prebiotics in that they support the growth of colonic organisms (Jaskari et al 1998, Van Laere et al 2000, Crittenden et al 2002). Binding of bile acids and mutagens to wheat bran dietary fiber has been suggested to provide a protective role against colorectal cancer. In particular, the lignin in the walls of the pericarp-­seed coat is good at absorbing dietary carcinogens, and the antioxidant capacity of ferulic acid and other phenolic components of the bran may be effective in inhibiting carcinogenesis (reviewed by Ferguson and Harris 1999). Additionally, the water-­holding capacity of soluble polysaccharides reduces the residence time of digesta in the lower bowel and, acting as a diluent, reduces exposure to potential mutagenic agents. In summary, the nonstarchy polysaccharides of the aleurone cell walls, but not those of the pericarp-­seed coat fraction of wheat bran, are important substrates for microbial fermentation in the large intestine, leading to increased growth of colonic microorganisms with consequential increases in fecal bulk and in the water-­holding capacity of the intestinal contents. Animal Nutrition

Antinutritional Effects of Cell Wall Polysaccharides. In monogastric animals, such as pigs and poultry, diets with high levels of cereal nonstarchy polysaccharides, which include arabinoxylan and (1→3,1→4)-­β-­d-­g lucans, have antinutritive effects (Austin and Chesson 1996, Austin et al 1999). Thus, when soluble wheat arabinoxylan is added to experimental diets for broiler chickens, the digestibility of ileal starch, protein, and lipid is reduced (Choct and Annison 1990). This is consistent with the fact that the apparent metabolizable energy of wheat fed to broiler chickens is negatively correlated with the content of water-­soluble, nonstarchy polysaccharide, largely arabinoxylan (Annison 1990, 1991; Choct and Annison 1992a,b). This effect may be explained by the increase in viscosity of the digesta in

Carbohydrates  the gut, which impedes the absorption of nutrients. The problem can be overcome by enzymatic pretreatments of the feed (Choct et al 1995). When wheat grain is fed to ruminants, energy is lost in the fermentative breakdown of starch, cellulose, and the noncellulosic polysaccharides; however, 80–90% of the energy released is available for the animal’s metabolic processes (Black 2004). Phytate. Phytate has antinutritional activities in human and monogastric animal diets through its strong chelation of Ca, Fe, and Zn to form insoluble complexes that are not absorbed and can contribute to deficiency of Fe and Zn (Raboy 2001). Moreover, poultry, pigs, and fish are unable to digest phytate, and feeding grain-­based diets leads to excretion of phytate, which contributes to the accumulation of soil and water phytate. Phytate phosphorus is poorly utilized by plants (Raboy 2001). On the other hand, phytate has a positive nutritional role as an antioxidant, through suppression of Fe-­mediated ·OH formation by Fe, which is complexed by phytate (Graf et al 1987), and as an anticancer agent (Graf and Eaton 1993) (see also Chapter 7). Niacin. Niacin in cereals is largely unavailable biologically since the ester linkage to carbohydrate is not extensively cleaved in the acidic regions of the upper alimentary tract. Pretreatment of cereal fractions with alkaline reagents, e.g., dilute NaOH (Kodicek et al 1956), NaHCO3 (Clegg 1963), or lime water, as used by Mexican Indians in preparation of masa from maize, liberates the niacin (Katz et al 1974). ACKNOWLEDGMENTS

The helpful comments of Marta Izydorczyk and Philip Harris were greatly valued, as was the assistance of Kathy Andrewartha with the references and Fung Lay with the illustrations. References

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CHAPTER  10

Wheat Lipids Okkyung Kim Chung (Retired) Grain Marketing and Production Research Center USDA, Agricultural Research Service Manhattan, Kansas Jae-­Bom Ohm Northern Crop Science Laboratory USDA, Agricultural Research Service Fargo, North Dakota

Lipids in wheat or wheat flour are a minor constituent, and yet they play major roles in wheat production, storage, processing, products, nutrition, and consumer acceptance of finished goods (Chung and Ohm 1997). The topics of quantification and composition of lipids in wheat kernels, their structural parts, and even the processed product, i.e., flour, have been the focus of many researchers, especially the role of lipids in wheat-­product quality. The studies between the mid-­1960s and the 1980s made extensive use of the techniques of defatting flour and reconstituting the flour by adding back the removed lipids. This research concentrated mainly on “free lipids” (FL), i.e., lipids easily extractable with relatively nonpolar solvents, which would not disrupt the binding between lipids and other constituents such as proteins or carbohydrates. Until the late 1970s and the beginning of the 1980s, most researchers referred to the wheat or flour lipids as “nonstarch lipids” (NSL). The presence of phospho­lipids (PL) in starch granules was suspected due to the presence of phosphorus in highly purified starch granules. However, starch lipids (SL) could not be extracted with any solvent system at ambient temperature. Therefore, the terminology of wheat or flour lipids is greatly dependent on the extraction conditions, including the extractants (solvents), extracting temperature, moisture contents, or extraction and quantification methodology. In addition, because lipids are unevenly distributed in wheat structural parts, lipid content and composition are also affected by milling practice, i.e., flour extraction rate, various milled streams, etc. Furthermore, the growing environments as well as the genetic backgrounds of the wheat result in variations in lipid content and composition. Many abbreviated terms are used in order to simplify a complex subject (Table 10.1). Wheat and flour lipids have been reviewed periodically (Lasztity 1966; Mecham 1971; Chung et al 1978; Morrison 1978,

M. S. Ram (Retired) Seok-­Ho Park Grain Marketing and Production Research Center USDA, Agricultural Research Service Manhattan, Kansas Crispin A. Howitt CSIRO Plant Industry Canberra ACT 2601, Australia

1979, 1989, 1995; Pomeranz and Chung 1978; Chung and Pomeranz 1981; Berger 1982, 1983; Pomeranz 1985, 1988b; Chung 1989, 1991; Chung and Ohm 1997, 2000; Marion and Clark 2000; Nebesny et al 2002; Day and Vu 2004). Especially, earlier research efforts were well summarized and reviewed by Mecham (1971) and Morrison (1988a) in the editions of Wheat: Chemistry and Technology edited by Pomeranz and Hlynka (1971) and Pomeranz (1988a), respectively, and also by Morrison (1978). Wheat lipid content and composition were compared among classes and also with other cereal lipids (Chung 1991, Chung and Ohm 2000), and cereal lipid compositions were compared with those of oilseeds (Day 2004). Numerous enzymatic studies on lipids have been reported; the effect of lipoxygenase on the mechanical development of wheat flour was reviewed by Daniels et al (1970) and nonamylolytic enzymes were extensively reviewed by Van Eijk and Hille (1996). The subject of enzymatic effects on lipids is discussed in Chapter 11 of this book. Our main effort focuses on newer reports since the last publication of this book in 1988. This chapter covers recent progress in lipid methodology, comprehensively reviews functional roles of wheat/flour lipids in relation to wheat product quality, and discusses genetic research on nutritional improvement by manipulating carotenoid, tocochromanol, and fatty acid (FA) contents in wheat seeds.

WHEAT LIPID CLASSIFICATION Lipids are generally classified as “simple lipids” and “complex lipids.” Simple lipids in wheat are compounds with two types of structural moiety, including hydrocarbons, wax esters, steryl esters (SE), the glycerol esters, and fatty acids (FA); they are also biochemically classified as nonpolar lipids (NL). Complex lipids are compounds with more than two types of

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structural moiety, including PL and glycolipids (GL); they are polar lipids (PoL). The NL and PoL are separated according to their structure and polarity. The NL fraction includes acylglycerols and the smaller amounts of SE and minor acylated GL.

Free fatty acids (FFA) are considered NL, even though they have a slightly polar nature. Wheat lipids are also grouped into saponifiable and nonsaponifiable lipids, as discussed below.

TABLE 10.1 Abbreviations and Definitions Regarding Lipids Grouping

Abbreviation Definitiona

Grouping

General terms

ASE DAD db ELSD FID GC HPLC HPTLC LV MS NIR PetE QTL RI SC SF SFC SFE SPE TLC WSB wt

Accelerated solvent extraction Diode array detector Dry basis Evaporative light-scattering detector Flame ionization detector Gas chromatography High-performance liquid chromatography High-performance TLC Loaf volume Mass spectrometry Near-infrared Petroleum ether Quantitative quality loci Refractive index Supercritical Supercritical fluid Supercritical fluid chromatography Supercritical fluid extraction Solid-phase extraction Thin-layer chromatography Water-saturated n-butanol Weight

Lipid classes Fatty acids

BL EFA FA FAME FFA FL GL HCBN HL LC-PUFA LPL NL NSL NSTL PL PoL PUFA SL SSL T T-3 TFL TL HRS HRW HWS HWW SRW SWS SWW

Bound lipids Essential fatty acids Fatty acids Fatty acid methyl esters Free fatty acids Free lipids Glycolipids Hydrocarbons Hydrolysate lipids Long-chain polyunsaturated fatty acids Lysophospholipids Nonpolar lipids Nonstarch lipids Nonstarch total lipids Phospholipids Polar lipids Polyunsaturated fatty acids Starch lipids Starch surface lipids Tocopherols Tocotrienols Total free lipids Total lipids Hard red spring Hard red winter Hard white spring Hard white winter Soft red winter Soft white spring Soft white winter

Lipids—general terms

Wheat classes

a Although 

Acylglycerols

Glycolipids

Phospholipids

Sterol lipids

Tocol derivatives

  Biosynthetic enzymes

Abbreviation Definitiona 14:0 16:0 16:1 18:0 18:1 18:2 18:3 MAG DAG TAG MGMG DGMG MGDG DGDG AMGMG AMGDG ADGDG PC PE PG PI PS DPG PA LPC LPE LPG LPI LPL LPS APE ALPE FS SE SG ASG α-T α-T-3 β-T β-T-3 γ-T γ-T-3 δ-T δ-T-3

DGAT DGD1 MGD1 PDS PSY Biosynthetic genes dgat1 dgd1 mgd1 vte

most of the acronyms are defined as plural, they can be read as singular when that is more appropriate.

Myristic acid Palmitic acid Pamitoleic acid Stearic acid Oleic acid Linoleic acid Linolenic acid Monoacylglycerols Diacylglycerols Triacylglycerols Monogalactosylmonoglycerides Digalactosylmonoglycerides Monogalactosyldiglycerides Digalactosyldiglycerides Acylmonogalactosylmonoglycerides Acylmonogalactosyldiglycerides Acyldigalactosyldiglycerides Phosphatidylcholines Phosphatidylethanolamines Phosphatidylglycerols Phosphatidylinositols Phosphatidylserines Diphosphatidylglycerols Phosphatidic acids Lysophosphatidylcholines Lysophosphatidylethanolamines Lysophosphatidylglycerols Lysophosphatidylinositols Lysophospholipids Lysophosphatidylserines N-Acylphosphatidylethanolamines N-Acyllysophosphatidylethanolamines Free sterols Steryl esters Steryl glycosides Acylated steryl glycosides α-Tocopherols α-Tocotrienols β -Tocopherols β -Tocotrienols γ-Tocopherols γ-Tocotrienols δ -Tocopherols δ -Tocotrienols Diacylglycerol transferase DGDG synthase 1 MGDG synthase 1 Phytoene desaturase Phytoene synthase Diacylglycerol transferase 1 DGDG synthase 1 MGDG synthase 1 Vitamin E pathway gene

Wheat Lipids 

Saponifiable Lipids Fatty Acids

FA are the simplest form of lipids. The most commonly found FA and their nomenclature in different forms are given in Table 10.2 and are named according to the International Union of Pure and Applied Chemistry’s Rules for the Nomenclature of Organic Chemistry (Nichols and Sanderson 2002). Long-­chain alcohols are designated by their systematic names. The nomenclature generally includes the number of carbon atoms, followed by a colon and then the number of double bonds and the position of the double bond counting from the COOH end of the molecule. In the “n =” or the ω system, the position of the double bond is counted from the CH3 end of the FA. The geometry about the double bond in unsaturated FA is designated according to the E-­Z system. The double bonds in unsaturated FA in all natural compounds have the “cis” or the “z” configuration, i.e., the two hydrogen atoms are on the same side of the double bond. Acylglycerols

In classical terms, acylglycerols contain triglycerides, diglycerides, and monoglycerides. The currently recommended and preferred terms are triacylglycerides or triacylglycerols (TAG), diacylglycerides or diacylglycerols (DAG), and monoacylglycerides or monoacylglycerols (MAG), respectively. Most of the FA in wheat are present as saponifiable esters of glycerol 1,2,3-­propane-­triol. In TAG, all three hydroxy positions are esterified with FA. The most common esters are the TAG. When R=R'=R" are stearoyl groups, it is called tristearoylglycerol or tri-­O-­stearoyl glycerol or glycerol tristearate, or glyceryl tristearate. Typically, unsaturated FA are predominantly located in the C2 (i.e., R') position in plant TAG. Wheat lipids consist of some DAG. For DAG with the same FA, three positional isomers, i.e., sn-­1, sn-­2; sn-­1, sn-­3; and sn­2, sn-­3, are possible, and, with two different FA, six isomers are possible. MAG are also FA esters of glycerol but have one FA unit less than the DAG per glycerol unit. For MAG, three positional isomers, sn-­1, sn-­2, and sn-­3, are possible, of which the sn-­2 isomer is nonchiral. MAG are considered to be nutritionally healthy because of their low FA content per mole. Glycolipids/Glycosylglycerides

The GL class consists of the glycosylglycerides and the sterylglycosides, but, in practice, the GL are synonymous with glyco-

Phospholipids/Phosphoglycerides

Phosphatidic acid is a derivative of a glycerol phosphate or glycerophosphate in which both remaining hydroxyl groups of glycerol are esterified with FA. If the phosphate group in phosphatidic acid is esterified with the amino alcohol choline, it is known as phosphatidylcholine (PC). The systematic name for this compound is 1,2-­d iacyl-­sn-­g lycero-­ 3-­phosphatidylcholine, and the common name is lecithin. If only one position is acylated and the other position remains free, it is one of the lysophospholipids, including lysophosphatidylcholine (LPC). The PL class in wheat or milled flour mainly consists of LPC but also contains PC, lysophosphatidylethanolamine, N-­acylphosphatidylethanolamines, and N-­acyllysophosphatidylethanolamines. Sterols

Sterols are any of a group of predominantly unsaturated solid alcohols of the steroid group. The principal sterols in wheat are the C29-­and C28-­desmethyl sterols, 5'-­sterols, 4-­methyl sterols, 4, 4'-­dimethyl sterols, and triterpenols. The more abundant sterols are the β -­sitosterol C29, the campesterol C28, and the stigmasterol C29. Stigmasterol has one CH2 group less at C24 than sitosterol, and campesterol has a double bond at C22. The sterol class is often esterified with FA or phenolic acids, such as ferulic acid or coumaric acid. Also, sterols form glycosides with sugars. However, Barnes (1983) included sterols, together with tocol derivatives and carotenoids, in the nonsaponifiable lipids, as the nonacyl parts of these lipids often occur in the unesterified form.

Nonsaponifiable Lipids

Structure

Systematic Namea

Common Namea

Palmit-

16:0

-[CH 2]14-

Hexadecano-

18:0

-[CH 2]16 -

Octadecano-

Stear-

18:1 ∆9

-[CH 2]7CH=CH[CH 2]7-

Z-9-Octadeceno-

Ole-

18:2 ∆9, 12

-[CH 2]3[CH 2CH=CH]2[CH 2]7-

Z-9, Z-12-Octadecadieno-

Linole-

-[CH 2CH=CH]3[CH 2]7-

Z-9, Z-12, Z-15-Octadecatrieno-

9,12,15-Linolen-

-[CH 2]3[CH 2CH=CH]3[CH 2]4-

6,9,12-Octadecatrieno-

6,9,12-Linolen-

18:3

∆ 9, 12, 15

18:3 ∆6, 9, 12 a Endings

365

sylglycerides. The term “glycolipid” designates any compound containing one or more monosaccharide residues linked by a glycosyl linkage to a lipid part. GL are generally named as glycosyl derivatives of the corresponding lipids, e.g., diacylgalactosylglycerol. Since wheat GL contain galactose as the main sugar moiety, wheat “glycolipids” can be generally termed wheat “galactolipids.” The main GL classes in wheat are digalactosyldiglycerides (DGDG) and monogalactosyldiglycerides (MGDG). Minor GL are digalactosylmonoglycerides, monogalactosylmonoglycerides, and acylated steryl glycosides (ASG). The nomenclature of GL was recommended by the International Union of Pure and Applied Chemistry and the International Union of Biochemistry (IUPAC-­IUB 1997).

TABLE 10.2 Nomenclature of Fatty Acids Commonly Found in Wheat Numerical

x 

in -ic, -ate, and –oyl are for acid, salt or ester, and acyl radical, respectively.

Two classes of lipids, the caretonoids and tocopherols, are nonsaponifiable and contain no FA. Carotenoids are polyisoprenoid compounds derived from large (35–40 carbon) polyene chains. Carotenoids have two major forms: an oxidized form, xanthophylls, including lutein and zeaxanthin, and an unoxidized form, carotenes, including α-­ carotene, β-­carotene, and lycopene. Carotenoid hydrocarbons are known as “carotenes”

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and are the biosynthetic precursors of the oxygenated derivatives called “xanthophylls.” The characteristics and properties of the carotenoids reflect the presence of the conjugated polyene chain, which accounts for the color and sensitivity to light, heat, oxygen, and acid (Barnes 1983). In a food system, carotenoids are important because they exhibit a range of different colors from yellow through bright orange to deep red, due to their different carbon double bond structures. Waxes are acyl derivatives of ethanediol and propanediol and have very-­long-­chain hydrocarbons and long-­chain (FA > C20) esters that are found essentially on the wheat pericarp. Wax is only a minor constituent of the wheat grain. For information on lipid nomenclature and chemical structures, several references are recommended (Morrison 1978, 1988a; Nawar 1996; Akoh and Min 2002; Christie 2003; Nichols and Sanderson 2003; Day 2004; Godber and Juliano 2004). The nomenclature used here for general lipids and GL is that recommended by IUPAC-­IUB (1976) and IUPAC-­IUB (1997), respectively.

Lipid Class by Location of Lipids in Wheat Structural Parts Wheat or flour lipids can be grouped or classified in various ways depending on the location of the lipid in the wheat or flour constituents or on extraction methods, including the solubility parameter of the extracting solvents, the apparatus, and the flour moisture content (Chung et al 1977a,b, 1980a, 1984) (Table 10.3). NSL or nonstarch total lipids (NSTL) are present in wheat fractions other than starch granules and are extractable at ambient temperature, while SL are associated with starch granules and extractable most efficiently by a mixture of 1-­propanol or 2-­propanol with water at an elevated temperature at which most of the starch granules are gelatinized (Morrison and Coventry 1985). NSTL contain all classes of lipids, such as acylglycerols (TAG, DAG, MAG), FFA, GL, and PL, whereas SL are mainly PoL, i.e., PL with a very high level of LPC (Fig. 10.1). A third category, starch surface lipids (SSL), was created by Morrison (1981, 1988a,b) for portions of NSL that become firmly absorbed onto or into starch granules during the formation of starch and contaminate internal SL. SSL composition differs from SL compo-

sition. SSL are monoacyl NSL, mainly FFA. However, Gailliard and Bowler (1987) contended that SSL should be considered SL.

Lipid Class by Extraction Method and Lipid Solubility There are several methods for extracting lipids, which include Soxhlet, Butt-­t ype extractor, and Weibull-­Stoldt acid hydrolysis followed by extraction. However, these methods do not always give consistent results. Differences in the lipid level obtained may be due to the extraction solvent. Solvents used may include petroleum ether (PetE) in a Soxhlet, a mixture of chloroform and methanol (2:1), water-­saturated n-­butanol (WSB) at room temperature or at 100°C, a mixture of n-­butanol and water (2:1), and acid hydrolysis. These methods extract different lipid classes, leading to different results. Another reason for the inconsistent results is that most of the analysis methods use a gravimetric method; i.e., the lipid material is extracted with an organic solvent. The solvent is evaporated, and the remaining residue is calculated as weight percent (% wt) lipids. It is extremely difficult to compare the lipid data reported by different researchers because there are large variations in lipid extraction, purification, and quantification, as well as in the reporting of results. Free Lipids

The lipids that are easily extractable from a dry material, usually flour or freeze-­dried dough, with a nonpolar solvent such as hexane, ether, or PetE, are called FL. The solvents of choice are the low-­boiling-­point petroleum hydrocarbon fractions or hexane, which are totally immiscible with water. Other solvents do not offer any advantages. The FL fraction in flour contains a proportion of every class of lipid, such as NL, GL, and PL of NSL, whereas the FL fraction of dough is essentially NL with low-­polarity classes, mainly TAG and SE. The yield and composition of flour FL are affected by flour moisture content, extraction method, and solvent temperature. If a solvent refluxing apparatus (e.g., Goldfisch, Soxhlet, or Butt-­type) is used, the extraction temperature will be near the boiling point of the solvent. A simple extraction procedure involves extracting a 200-­ to 500-­mg sample

TABLE 10.3 Grouping of Wheat Flour Lipidsa Group

Lipid Category

By location

Nonstarch lipids Starch surface lipids Starch lipids Free lipids Bound lipids Total nonstarch lipids Hydrolysate lipids Simple lipids (nonpolar lipids) Complex lipids Polar lipids Glycolipids Phospholipids

By extraction

By biochemistry

  a Modified

from Chung and Ohm (1997).

Fig. 10.1. The subdivision of wheat flour lipid classes. TAG = tri­ acylglycerides/triacylglycerols, DAG = diacylglycerides/diacylglycerols, MAG = monoacylglycerides/monoacylglycerols, FFA = free fatty acids, SE = steryl esters, HCBN = hydrocarbons, LPC = lysophosphatidylcholines, LPE = lysophosphatidylethanolamines.

Wheat Lipids  with 8 mL (20 volumes of solvent) of PetE in a 10-­mL screw cap vial shaken for 1 hr in a water bath with a controlled temperature of 20 ± 1°C in a hood. The definition of the term “free lipids” has been well accepted by cereal chemists. The FL content of wheat or wheat products is closest to the “crude fats” content. Bound Lipids

Strictly speaking, bound lipids (BL) should include not only NSL but also SL and SSL. However, “BL” refers to the NSL extracted with a very polar solvent such as WSB at ambient temperature from the defatted flour, from which FL were removed by PetE or hexane. When FL and BL are extracted from flour and its lyophilized dough meal, the FL fraction in dough is only one third the amount of flour FL, because two thirds of the flour FL content becomes bound, mainly to gluten, during dough mixing and is not extracted (Chung 1986, 1989). Daniels et al (1970) reported the effect of air on lipid binding. Nonstarch Total Lipids and Starch Lipids

NSTL are extracted from ground wheat meal, flour, or lyophilized dough or other wheat products with WSB at room temperature (Pomeranz et al 1966, Chung et al 1980a) and consist of both FL and BL. NSTL contain NL, GL, and PL (Fig. 10.1). SL are located in the starch granules, and often the SL are reported together with SSL. Unless starch granules are gelatinized, most of the lipid extractants used are not able to reach the inside starch granules at ambient temperature. Therefore, a hot solvent system such as WSB or propanol-­water mixture is required to extract SL from a purified starch fraction. Hydrolysate Lipids

The true total BL, including NSL and SL, bound to or complexed with proteins or polysaccharides, especially starch granules, are released only by acid hydrolysis. Acid hydrolysis facilitates the release of BL into easily extractable forms; typically, this involves refluxing with approximately 3M HCl for 1 hr before filtration. During this process, some tissue may be destroyed also, and some nonlipid materials may be extracted. Generally, SL are also extracted. Therefore, the hydrolysate lipids are closest to the true total lipids, including NSTL as well as SL.

Analytical MethodS Lipids are extracted from wheat, flour, or wheat products or from food systems by various methods. General lipid methodologies of extraction and fractionation in food systems were recently reviewed by Nawar (1996), Akoh and Min (2002), Nichols (2002), Christie (2003), and Day (2004). These references will be of great help to readers of this chapter.

Methods for Extraction of Lipids Soxhlet or Butt-­Tube Methods

Two official solvent extraction methods are used for the extraction of FL. Approved Method 30-­25 of AACC International

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367

(AACC 2000) is a Soxhlet extraction using PetE to extract lipids from cereal grains and products, and AOCS Official Method Aa­38 (AOCS 2000) is a modification of the procedure using a Butt­tube, which overcomes the drawback of the extraction temperature not being uniform in a Soxhlet method. The AOCS method is a continuous solvent extraction, and the AACC method is semicontinuous. Semicontinuous extraction facilitates contact between substrate and solvent (Schweizer et al 1974, Clements 1977). Selected data indicate that the Soxhlet and the Butt­tube procedures gave nearly identical results for hard red winter (HRW), hard red spring (HRS), and soft red winter (SRW) wheats and a commercial wheat blend (Hubbard et al 2004). Aqueous Alcohol Extraction Methods

For the extraction of BL, the method is the same as for the extraction of NSTL, except that FL-­defatted flours, meals, or lyophilized dough meals are used as the starting materials. Polar solvents such as mixtures of alcohol and water, including WSB, are used for stirring, shaking, or high-­speed blending methods. A mixture of chloroform and methanol (2:1) has also been used commonly, but the extraction efficiency was not as high as for WSB. One caution is that very polar solvents extract nonlipid materials in addition to lipids, and thus, a procedure to remove nonlipids, such as the Folch method (Pomeranz et al 1966, Mecham 1971), is needed. Traditionally, the WSB extracts were dried by a rotary evaporator, desiccated over P2O5, and then redissolved with a typical lipid solvent to obtain gravimetric results of lipids only (Pomeranz et al 1966). For the extraction of NSTL, wheat meals or flour samples are extracted with an aqueous alcohol mixture such as WSB at room temperature to obtain NSTL containing FL and BL. For the extraction of SL, which consist of >90% PoL, mainly lysophospholipids such as LPC (Fig. 10.1), and do not contain any hexose-­phosphate, an efficient extractant requires mixtures of hot aqueous alcohols in proportions optimized for controlled swelling of starch granules and solubilization of lipids. The rates of solubilization of SL and NSL in WSB are given in Figure 10.2 (Morrison 1978). Starch solubilization is very temperature­dependent. Because SL are the most difficult to extract, if hot solvents are used to extract the NSL and SL from flour together, substantial quantities of alcohol-­soluble proteins are also extracted with the lipids. The best solvents are propan-­1-­ol and propan-­2-­ol with water, 3:1 by volume; alternatively, butanol-­or methanol-­water mixtures can be used under nitrogen at 100°C. The butanol-­water system extracts smaller amounts of proteins than the propanol-­water system at either room temperature or elevated temperatures. For the most accurate quantification of SL, the use of highly purified starch samples, rather than wheat meal or flour samples, is recommended. Supercritical Fluid Extraction

Several reviews on supercritical fluid extraction (SFE) and supercritical fluid chromatography (SFC) that pertain to lipids are available (King and France 1992; King and Hopper 1992; Clifford and Walker 1996; King and List 1996; King et al 1997; Taylor 1997; Doane-­Weideman and Messe 1998; King 2003a,b,

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2004; Wang et al 2004). Carbon dioxide is the most widely used supercritical fluid (SF). The solubility of many compounds is low in liquid CO2. Increasing their density improves the solubility, but the density of liquids cannot be varied much by applying pressure only; a temperature increase is needed. An increase in temperature also means lowering the SF viscosity, increasing the diffusivity, and weakening the analyte-­matrix interactions such as those found in the “bound lipids.” It is often said that solubility in the SF does not always mean better extractability, but the matrix effects are critical. Sample Preparation and Other SF Solvents. The preparation of the sample before extraction is extremely important (Eller and King 2001). Usually, the material is ground because smaller particles increase surface area, which promotes more efficient extractions. For example, although the presence of water is useful during the extraction of caffeine, water tends to adversely affect the extraction of fat. A patent (U.S. patent 5151188, 1992) describes the use of pelletized diatomaceous earth as an extraction aid to dry and disperse samples before supercritical (SC)-­CO2 extraction (Hopper and King 1992). The use of the material was well demonstrated by the various dispersing agents sold by the major manufacturers of analytical SFE equipment. This dispersive aid also prevents certain materials from being compressed or pelletized under the initial flow of the SFE fluid. Inadequate sample preparation can lead to unreliable fractionation or quantification. In one application, SC-­CO2, when used with an extraction enhancer, formed an SFE system for extraction of pesticides and matrix components from fatty and nonfatty foods containing high levels of moisture (Hopper and King 1991). Instead of using large amounts of modifiers, trifluromethane or any other commercially available Freon can be used. Propane is another SF solvent with lipid selectivity and has been used to extract fat from moist turkey and chicken without removing moisture.

Use of Modifier Solvents. SC-­CO2 is generally considered a more selective solvent than hexane. In addition, it is possible to vary the selectivity slightly by proper control of the temperature and pressure. The fact that SC-­CO2 is a good solvent for nonpolar and moderately polar compounds makes it an excellent choice for extracting fats and oils without coextracting other, more-­polar nonfat components. In this manner, it is possible to extract TAG without the coextraction of PL, which are generally present in oilseeds or cereal grains. However, to extract PoL such as PL, some sort of modifier or cosolvent must be added to the SC-­CO2. Although there are many potential modifiers, including methanol (Via et al 1994), the most common modifier used is ethanol, which increases the polarity of the SC-­CO2 to allow for the extraction of more-­polar compounds not removed by SC­CO2 alone (Clifford and Walker 1996, Hubbard et al 2004, King 2004, Wang et al 2004). The solubility of compounds in SF solvents can also be increased by adding a small amount of a modifier or cosolvent, generally at less than 20% (v/v) of the SF solvent. Beyond this amount, one loses the advantages of SFE by reintroducing the sometimes hazardous or toxic waste collection and waste disposal and decreases the efficiency of the extraction. Fundamentally, a phase separation takes place. Thus, the “selectivity” factor introduced in SFE is completely lost. SFE Extraction of BL from Flour and Dough. The optimum conditions for NSTL extraction from wheat flour by the SFE system were determined in our laboratory in Manhattan, KS. Extraction with WSB was used as a reference method. The conditions were 10,000 psi at 130°C for the extraction chamber pressure and temperature, respectively, and 60 mL of extractant (a CO2 and ethanol mixture, 1:1, v/v) at 3 mL/min. Lipid binding during dough mixing is well recognized by cereal chemists; thus, the extraction of NSTL from dough also must be optimized. For extraction of NSTL from lyophilized flour-­water doughs, mixed to optimum, the optimum SFE condition was 10,000 psi and 140°C for the extraction chamber pressure and temperature, respectively, and 80-­mL extraction with a mixture of CO2 and ethanol (1:1, v/v) at 3 mL/min (unpublished data). Official SFE-­Based Test Methods. AOCS has an official method (Am 3-­96) for “oil in oilseeds” by the SFE method, which was reapproved in 1997 and revised in 2000 (AOCS 2000). The two-­part method involves extraction with SC-­CO2 alone or with SC-­CO2 plus 15% ethanol modifier. This method is applicable for determination of process-­scale extractable oil or determination of total oil. The SFE method is fast, requiring only a 30-­min extraction to determine process-­extractable oil or 60 min for total oil. The SFE method uses no hazardous solvents, so it is well suited for the process-­control environment. SFE instruments that extract samples in parallel rather than in sequence have become available. Although there is an SFE method of wheat lipid extraction (Hubbard et al 2004), as yet there is no official SFE-­based method for extraction of lipids from wheat and other similar cereal grains.

Fig. 10.2. Extraction of wheat flour nonstarch lipids (upper curve, at 20°C) and starch lipids (middle and lower curves) extracted by watersaturated n-butanol. (Reprinted, with permission, from Morrison 1978)

Accelerated Solvent Extraction

Accelerated solvent extraction (ASE) is known by different names depending on manufacturer. Dionex (Salt Lake City, UT)

Wheat Lipids  named it “ASE,” whereas ISCO (Lincoln, NE) calls it “enhanced solvent extraction.” It is also called “pressurized solvent extraction” and “pressurized fluid extraction.” In spite of the different names, all of them are the same method and belong to the general category of subcritical fluid extraction (King 2004). ASE is gaining in popularity, due in part to its appearance, which resembles high-­performance liquid chromatography (HPLC), and its use of regular solvents (King 2004). The solvents are heated to above their boiling point, and then sufficient pressure (1,500–2,500 psi) is applied to keep them liquid. Once the solvent is compressed to liquid, pressure does not have any effect on its density. With increased temperatures, solubility increases. Although this technique is similar to the classic extraction, the velocity with which the solvent flows through the sample is very high, and the extraction process is very efficient (Schafer 1998).

Fractionation and Characterization Open-­Column Chromatography

Traditionally, NL, GL, and PL fractions have been sequentially eluted by chloroform, acetone, and methanol, respectively, from extracted lipids using silicic acid open-­column chromatography (Fisher and Broughten 1960; Pomeranz et al 1966; Rouser et al 1967; Ponte and De Stefanis 1969; Chung et al 1980a, 1982; Bekes et al 1986). However, silicic acid open-­column chromatography requires much solvent and time to elute each lipid class based solely on gravity. Solid-­Phase Extraction System

The solid-­phase extraction (SPE) system with prepacked columns has some advantages over open-­column chromatography, including less solvent consumption and thus lower hazards, less use of glassware, shorter sample handling time, and higher analyte recoveries (Wachob 1991). Prieto et al (1992a) reported the use of an SPE cartridge for the fractionation of wheat flour lipids extracted by WSB. SPE systems were used for fractionation of flour FL and NSTL into NL, GL, and PL (Ohm and Chung 1999, 2000, 2002; Néron et al 2004). Ohm and Chung (1999) found that a chloroform and acetone mixture (4:1, v/v) was more effective at fractionating NL, including FFA, from FL than chloroform alone. This indicates that FFA were not completely eluted into the NL fraction using chloroform alone; thus, some residual portions were eluted into the GL fraction by acetone. However, Néron et al (2004) reported that chloroform alone could separate NL by using an SPE system that used a larger amount of chloroform. These results suggest that the optimum solvent composition to separate NL varies according to the condition of the SPE systems. Thin-­L ayer Chromatography

The thin-­layer chromatography (TLC) methods have been widely used for analysis of lipids because TLC is a simple and rapid procedure with high separation efficiency and sensitivity. Normal-­phase TLC has been used to separate complex wheat lipid classes, using a glass plate coated with silica gel (Pomeranz

x 

369

et al 1966, Graveland 1968, Prabhasankar et al 2000, Néron et al 2004). High-­performance TLC (HPTLC) techniques require less test volume and a shorter separation time and give better resolution, sensitivity, and accuracy due to the improvement in layer characteristics. They include thinner layers, with smaller size and tighter distribution of particles than those of conventional TLC. Separated lipid classes could be visualized by spraying a specific or universal coloring agent for lipid classes on the TLC plate, with or without heating, UV absorption, or fluorescence (Pomeranz et al 1966, Prabhasankar et al 2000, Néron et al 2004). Lipid classes separated by TLC have been quantified by determining the size of separated spots, by densitometry (i.e., measuring the intensity of spots; Pomeranz et al 1966), or by chemically analyzing the eluted lipid fractions from the TLC spots. For example, gas chromatography (GC) analysis can be used for FA (Graveland 1968) or phosphate analysis for PL (Morrison et al 1980). Flame ionization detection (FID) is another way to quantify lipids separated by TLC. In this system, lipids are separated on a quartz rod coated with silica gel and analyzed by FID with burning by hydrogen flame. A TLC-­FID system was used to analyze FFA and for a storage stability test of cereal grains (Nishiba et al 2000). High-­Performance Liquid Chromatography

This system has been used to analyze wheat flour lipid classes and their species. The HPLC determination of wheat flour lipids has been focused on the separation and quantification of GL classes because of their importance in breadmaking (Tweeten et al 1981, Christie and Morrison 1988, Walker 1988, Prieto et al 1992b). Tweeten et al (1981), using a reverse-­phase HPLC with a refractive index (RI) detector, quantified two main GL subclasses of wheat flour lipids, i.e., MGDG and DGDG, and found the relationship of equivalent carbon number to log K', as shown in Figure 10.3. Although the FA composition of the GL fraction can be determined by conversion to fatty acid methyl esters (FAME), it is not possible to determine the various DAG fractions from this information. Therefore, a procedure for characterizing the intact GL by equivalent carbon numbers is necessary at first to construct a plot of log K' vs. equivalent carbon number, such as in Figure 10.3, for the GL-­DAG of saturated FA. The K' is defined as (TR – T0)/T0, where TR and T0 are the retention times of lipid peak and solvent (void time), respectively. Marion et al (1988) reported the isocratic separation of wheat flour lipids into GL and PL classes using a normal mobile silica column. The GL fraction of wheat flour was analyzed by HPLC with a UV detector after being converted to benzoyl derivatives (Walker 1988, Prieto et al 1992b). Papantoniou et al (2001) developed a preparatory HPLC procedure that could separate lipid classes from 100 mg of FL injection. They reported that pure MGDG, DGDG, and phosphatidylcholines could be separated and used for study of their functionalities in baking. The use of RI and/or UV detectors has some disadvantages, including the sensitivity to temperature changes for the RI detector and the effect of FA saturation and limitation of choice in eluting solvent for the UV detector (Moreau 1994, Ohm and Chung 1999). An evaporative light-­scattering detector (ELSD) measures the degree of light scattering of the solute droplets after

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the evaporation of the mobile phase (Moreau 1994). The advantages of an ELSD include gradient elution, diverse use of organic solvents, and analysis at relatively high temperatures (Shulka 1988). The peak areas of ELSD indicated a linear response, with the mass of most lipid classes in the range of 10–200 µg (Moreau 1994). Christie and Morrison (1988) detected the wheat flour GL classes using an ELSD. Likewise, Conforti et al (1993a) separated and quantified the wheat flour lipid classes using an ELSD. PL fractions, obtained through the SPE system from lipid extracts of flour and dough, were analyzed and quantified by HPLC with ELSD (Néron et al 2004). However, the ELSD also has disadvantages. Moreau (1994) pointed out that an ELSD requires large amounts of gas for nebulization, while the response of the ELSD is not linear to the mass of the lipid. Also, the detection of a solute is difficult in cases where a solute is highly volatile. In addition to fixed or variable wavelength UV detectors, a diode array detector (DAD) has recently been used in HPLC. The development of a DAD makes it possible to scan a range of wavelengths at about 100-­µs intervals (Moreau 1994). Ohm and Chung (1999) found that quantification of MGDG and DGDG was possible using DAD with ELSD as a reference. The FA composition of MGDG or DGDG was not complex enough to interfere with their quantification by UV detector, as evidenced by significantly linear correlations between peak areas obtained by the ELSD and DAD within hard winter wheats grown in Kansas. Recently, the development of mass spectrometry (MS) has greatly improved lipid analysis. Complexity in the structure and types of lipids has been a big obstacle to the analysis of lipid mixtures. MS has been suggested as a great tool to overcome this problem because it simplifies the analysis procedure and increases detection sensitivity. An automated HPLC system coupled with electrospray ionization MS could characterize and

Fig. 10.3. Relationship of equivalent carbon number (C24 = di­ linolenic, C34 = palmitylstearyl) vs. log K' of monogalactosyldi­ glycerides (MGDG, 1) and digalactosyldiglycerides (DGDG, !) analyzed by high-performance liquid chromatography with a refractive index detector. K' = (TR–T0)/T0, where TR = retention time of peak (min) and T0 = void time (min). (Adapted from data of Tweeten et al 1981)

quantify more than 100 components of the PL class of about 35 samples in a day (Hermansson et al 2005). This result suggests that application of MS coupled with HPLC could be very helpful in allowing the relationship between quantitative and qualitative variations in wheat lipids and their quality characteristics to be studied in greater depth than before. Gas Chromatography

Gas chromatography (GC) techniques have been mainly used for FA analysis of wheat lipids or their classes separated by HPLC or TLC. FA of separated lipid fractions are usually saponified, converted to FAME, and analyzed by GC with FID. Hepta­ decanoic acid, which is rarely found in wheat lipids, is added as an internal standard in GC analysis of FA (Néron et al 2004). The GC system has also been applied to analyze sterols, using the following procedure: alkaline hydrolysis and saponification of free sterols and their conjugates, extraction and clean-­up of nonsaponifiable matters by SPE, derivatization to sterol trimethylsilyl ether, and separation and quantification by GC and MS or FID (Piironen et al 2000). When analyzing steryl glycosides (SG) by GC, acid hydrolysis is performed before alkaline saponification because of difficulty in the saponification of SG by alkaline hydrolysis (Toivo et al 2000). Near-­Infrared Spectroscopy

Near-­infrared (NIR) spectroscopy has been used to analyze the constituents of agricultural products, including lipids/fats/ oils, especially for corn, soybeans, etc. Law and Tkachuk (1977) reported the identification of bands in the NIR for wheat lipids and other contributing groups. However, the direct determination of wheat flour lipids or their classes, such as GL content, using NIR spectroscopy was not satisfactory, probably due to the low concentration of GL. To overcome this problem, Simmons (1985) tried a filter-­disk technique in which a paper disk was coated with the extracted GL. Ohm and Chung (2000) derived the prediction equations of FL, GL, and DGDG contents, calibrated using the NIR transmittance of FL solution in hexane, scanned over the wavelengths of 400–2,499 nm. Ohm and Chung (2000) reported that the prediction model of FL content was derived by modified partial least squares and showed an R 2 of 0.95 for the calibration set and 0.89 for the validation set. The prediction model developed for GL showed R 2 values of 0.87 and 0.89 for calibration and validation, respectively. For DGDG, the model developed using modified partial least squares showed R 2 values of 0.94 for the calibration and 0.88 for the validation sets. Thus, NIR transmission spectroscopy using FL in hexane solution is expected to improve flour lipid analysis so that this method can be used for flour quality evaluation in breeding programs with a significant decrease in the hazards involved with lipid analysis. Fractionation of Lipids Using SUPERCRITICAL FLUID extraction with a Modifier

The ratio of NL to PoL is important in clinical and nutritional chemistry as well as in breadmaking parameters. Determining the ratio of NL to PoL by sequential SFE would be convenient

Wheat Lipids  and accurate, as the manual sequential elution on an open column or solid-­phase extraction is labor-­intensive and tedious, with large consumption of solvents. SF-­CO2 is a useful system for extracting NL from wheat flour. However, a polar modifier, such as ethanol, is necessary to extract the PoL, and the amount of lipids extracted by an SFE system varies with the percent of modifier used (Fig. 10.4). FL extraction by an SFE system was successful at 7,500 psi and 80°C with 12% (v/v) ethanol as a modifier, and then the extracted FL were fractionated to NL, GL, and PL (Hubbard et al 2004). Ram and Chung (2004), using four HRW wheat flours, developed the sequential extraction method for flour FL using SFE; first, the NL fraction of flour FL was extracted by using SF-­CO2 only, and then the PoL fraction was extracted with the addition of the modifier ethanol to SF-­CO2. The amounts of lipids extracted by the first and second sequential extractions were in agreement with the amounts of NL and PoL fractionated from the FL by solid-­phase extraction, and the success of the sequential extraction was evidenced by TLC chromatograms (M. S. Ram and O. K. Chung, unpublished data). Montanari et al (1996) selectively extracted a PL mixture by SC-­CO2 and cosolvents. Some novel techniques are available for efficient extraction, recovery, separation, identification, and analysis of lipids. Even the most difficult extractions, such as of SL, can be performed with automated supercritical modified-­fluid extraction with minimal use of aqueous solvents. Systems that run parallel, instead of serial, extractions are available. The properties of SF can be fine-­tuned with adjustment of pressure and temperature. Supercritical Fluid Chromatography

SFC is applicable for mixtures of organic compounds that are thermally unstable, not volatile enough to easily pass through a GC, and too high in molecular weight to be well resolved by HPLC (King and France 1992, Thompson and Taylor 1994, Taylor 1997, King 2003b, Wang et al 2005).

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SF can be used as the mobile phase to separate analytes with solvating powers similar to those of organic solvents but with higher diffusivities, lower viscosity, and lower surface tension. The lower viscosity allows higher flow rates compared with liquid chromatography, and the solvating power can be adjusted by changing the pressure. SF can have liquidlike solubility of the substrates but can use gas-­phase detectors such as the FID. The column is usually a capillary GC column. SFC has been revived after demonstrating the use of a packed column for the separation of lipids. It is very much like HPLC, except that it is faster with far less solvent waste. For separation of PL, Eckard et al (1998) used a packed­column subcritical fluid chromatography to separate five PL on a Luna octyl column (25 cm × 4.6 mm i.d., 5 mm) employing an evaporative light-­scattering detector. The mobile-­phase modifier contained 0.10% (v/v) trifluoroacetic acid initially, for 2 min, increasing to 15% in 14 min and to 40% at 14.1 min, at a column temperature of 70°C. The SF outlet pressure was 2,000 psi, and the mobile-­phase flow rate was 2.0 mL/min. The addition of trifluoroacetic acid significantly influenced the sharp separation of PL into five classes.

LipidS IN Wheat Fractions AND WHEAT CLASSES Approximately 2–3.5% (w/w, db) of the whole grain consists of total lipids (TL), with those lipids unevenly distributed in various parts of wheat grain. A relatively small number of studies report lipid compositions in the various wheat fractions, and even the available data are not complete or vary greatly among reports. These variations likely result from the methods or techniques used during the analysis, as well as the use of samples of different varieties and growing conditions. Wheat FL in different parts of the kernel anatomy were reported by MacMasters et al (1971), who used hand-­d issected and divided kernels (Table 10.4). The endosperm fraction, which makes up about 75–87% (db) of total kernel weight, contains a rela­t ively low concentration of FL (0.8–2.2%, db), whereas the germ, which is 2–4% of kernel weight, contains a high concentration of FL (28.5%, db). Chung et al (2002) compared the FL content in straight-­g rade flour and air-­classified high-­protein-­f raction TABLE 10.4 Weight Distribution of Grain Fractions and Their Free Lipid Contentsa

Fig. 10.4. Effects of ethanol modifier concentration on amount of lipids extracted by supercritical fluid extraction from 10 g (db) of wheat flours: 1 = Cargill flour, ! = Karl 92 flour.

Fraction

Weight (%)

Whole kernel Bran Pericarp Testa (hyaline) Aleurone Endosperm Outer Inner Germ Embryonic axis Scutellum

100    3.8–4.2    5.0–8.9    0.2–1.1    4.6–8.9   74.9–86.5    –    –    2.0–3.9    1.0–1.6    1.1–2.0

a Data

from MacMasters et al (1971).

Free Lipid Content (% wt of fraction)

  1.8   5.1–5.8   0.7–1.0   0.2–0.5   6.0–9.9   0.8–2.2   2.2–2.4   1.2–1.6 28.5 10.0–16.3 12.6–32.1

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flour. The air-­classified high-­protein fraction contained approximately twice the amount of FL compared with the straight-­grade flour regardless of lipid classes, whereas the composition of NL was similar for both flours (Table 10.5). NSL from milling fractions including whole wheat, endosperm, germ, and bran fractions were reported (Table 10.6) by Zeringue and Feuge (1980). About 63% of NSL were distributed in the milled endosperm fraction even though the NSL content made up only 2.2% of this fraction. However, in this study, 92% of the milling product was the endosperm fraction, which is much higher than the usual milling flour extraction rate of 75%. Germ (4% of the milling product) contained about 34% of milled wheat NSL because of the high NSL content (27.8%) in the germ fraction. The composition of soft wheat NSL in different milling fractions, including straight-­run flour, coarse offal, fine offal, finished bran, and bran flour, was reported by Morrison and Hargin (1981). A more complete picture of lipid content and composition is given in Table 10.7, which was calculated by Chung and Ohm (2000), using the data reported by Morrison et al (1975) and Hargin and Morrison (1980). One whole kernel contained about 900–1,250 µg of TL, consisting of NSL (270–319 µg in germ, 220–387 µg in aleurone, and 259–387 µg in endosperm) and SL (139–256 µg). Of the TL from a single kernel, the biggest portion was NL (44–57%), followed by PL (31–42%) and GL (13–14%). TL extracted from milled flour also showed similar trends. NSL in the germ and aleurone layers had higher portions of NL and a smaller portion of GL, whereas NL, GL, and PL were evenly distributed in endosperm. On the other hand, lipids in starch, including SSL, showed a substantially different lipid composition compared with other fractions, showing that PL (90–94%) is the dominant lipid class, followed by NL (4–6%) and GL (1–6%). TL (NSL and SL) content falls in the range of 2.5–3.3% of wheat kernel weight (db) and 2.6–2.8% of milled flour weight. Milled flour contained 1.70–1.95% NSL, and the purified starch granules contained 0.8–1.2% SL. The germ and aleurone fractions are rich in TAG, whereas the endosperm is rich in GL and the germ fraction is poor in GL (Table 10.7). Lipid content and composition are influenced by genetic variations, including wheat class, cultivar, etc.; environmental TABLE 10.5 Free Lipids (FL) in Straight-Grade Flour (SF) and Air-Classified High-Protein Fractions (ACHPF)a SF Lipids

Lipid content (mg/10 g of flour, db) Total FL Nonpolar lipids Glycolipids Phospholipids Lipid composition (% FL)c Nonpolar lipids Glycolipids Phospholipids a Data b **

Range

Mean

92.8** 74.1** 12.8**   4.9**

82.0–101.3  61.3–83.3 10.1–17.2   3.9–6.1

178.5 141.9   20.9   12

76.1–83.4 12.2–17.3   4.1–6.8

  81.2   12    6.9

from Chung et al (2002). = Mean values significantly different from those of ACHPF at P