Guttation: Fundamentals and Applications 1108487025, 9781108487023

Table of contents :
Cover
Front Matter
Guttation Fundamentals and Applications
Copyright
Dedication
Contents
Figures
Tables
Foreword
Preface
Acknowledgments
Abbreviations
1 Phenomenon of Guttation and
Its Machinery
2 Principles of Guttation and
Its Quantification
3 Mechanism of Guttation
4 Regulation of Guttation
5 Chemistry of Guttation
6 Plant Microbiology and
Phytopathology of Guttation
7 Significance of Guttation
in Soil–Plant–Animal–
Environment Systems
8 Significance of Guttation,
Associated Structures,
and Root Secretion in the
Production of Pharmaceuticals
and Other Commercial
Products
9 General Conclusions and
Future Perspectives
Appendices
Bibliography
Index

Citation preview

Cambridge University Press 978-1-108-48702-3 — Guttation Sanjay Singh Frontmatter More Information

Guttation Guttation is the phenomenon of bleeding or oozing of exudates or fluids from plant organs through special structures called hydathodes or sometimes ‘water stomata’ or ‘water pores’, located on the tip, periphery, and surfaces of leaves. This text is an up-to-date review of the knowledge in the field and it discusses the principles, mechanisms, regulation, and applications of guttation. The book covers genetic, environmental, and edaphic factors that control and regulate the phenomenon of guttation. It comprehensively discusses the impact of guttation on important aspects including soil–plant–animal–environment systems, soil fertility and soil productivity, plant water balance, plant physiological research, ecosystem maintenance, and hydathode retrieval of water and solute. A separate chapter covers the applications of guttation in the production of recombinant proteins for commercial use, seed protein, alkaloids, pharmaceutical drugs, resins, gums, and rubber. Sanjay Singh is a UNDP Associate Professor of Plant Physiology in the Department of Plant Sciences at Mizan-Tepi University, Ethiopia. He has published more than 40 papers in national and international journals. He organized the ‘First World Congress on Guttation 2015’. His research interest includes plant stress physiology.

© in this web service Cambridge University Press

www.cambridge.org

Cambridge University Press 978-1-108-48702-3 — Guttation Sanjay Singh Frontmatter More Information

Guttation Fundamentals and Applications

Sanjay Singh

© in this web service Cambridge University Press

www.cambridge.org

Cambridge University Press 978-1-108-48702-3 — Guttation Sanjay Singh Frontmatter More Information

University Printing House, Cambridge CB2 8BS, United Kingdom One Liberty Plaza, 20th Floor, New York, NY 10006, USA 477 Williamstown Road, Port Melbourne, VIC 3207, Australia 314–321, 3rd Floor, Plot 3, Splendor Forum, Jasola District Centre, New Delhi – 110025, India 79 Anson Road, #06–04/06, Singapore 079906 Cambridge University Press is part of the University of Cambridge. It furthers the University’s mission by disseminating knowledge in the pursuit of education, learning and research at the highest international levels of excellence. www.cambridge.org Information on this title: www.cambridge.org/9781108487023 © Sanjay Singh 2020 This publication is in copyright. Subject to statutory exception and to the provisions of relevant collective licensing agreements, no reproduction of any part may take place without the written permission of Cambridge University Press. First published 2020 Printed in India A catalogue record for this publication is available from the British Library Library of Congress Cataloging-in-Publication Data Names: Singh, Sanjay (Associate professor of plant sciences), author. Title: Guttation : fundamentals and applications / Sanjay Singh. Description: First. | New York, NY : Cambridge University Press, 2020. | Includes bibliographical references and index. Identifiers: LCCN 2019058596 (print) | LCCN 2019058597 (ebook) | ISBN 9781108487023 (hardback) | ISBN 9781108487023 (pdf) Subjects: LCSH: Plants, Motion of fluids in. | Stomata. | Xylem. | Plant exudates. Classification: LCC QK871 .S47 2020 (print) | LCC QK871 (ebook) | DDC 582.1--dc23 LC record available at https://lccn.loc.gov/2019058596 LC ebook record available at https://lccn.loc.gov/2019058597 ISBN 978-1-108-48702-3 Hardback Cambridge University Press has no responsibility for the persistence or accuracy of URLs for external or third-party internet websites referred to in this publication, and does not guarantee that any content on such websites is, or will remain, accurate or appropriate.

© in this web service Cambridge University Press

www.cambridge.org

Cambridge University Press 978-1-108-48702-3 — Guttation Sanjay Singh Frontmatter More Information

To the loving memory of my mother, Mrs Sampatti Devi, who left for her heavenly abode on November 30, 2017 (1950–2017), who has been a rock of stability throughout my life, and whose loving spirit sustains me still. May her soul rest in peace.

© in this web service Cambridge University Press

www.cambridge.org

Cambridge University Press 978-1-108-48702-3 — Guttation Sanjay Singh Frontmatter More Information

Contents

List of Figures List of Tables Foreword Preface Acknowledgments List of Abbreviations 1.

Phenomenon of Guttation and Its Machinery 1.1 Introduction 1.2 Definition of guttation 1.3 Biographical sketch of Abraham Munting (1626–1683)—discoverer of guttation 1.4 Phenomena resembling guttation 1.4.1 Dew 1.4.2 Bleeding or oozing 1.4.3 Other plant secretions 1.5 Taxonomic distribution of guttation 1.5.1 Angiosperms and gymnosperms 1.5.2 Pteridophytes 1.5.3 Algae and fungi 1.6 Gateway of guttation: hydathodes as exit pores 1.6.1 Structural biology of hydathodes 1.6.1.1 Morphological and anatomical aspects 1.6.1.2 Ultrastructural and histological aspects 1.6.2 Induction of hydathodes by auxin 1.6.3 Genetic aspects of hydathodes

© in this web service Cambridge University Press

xiii xv xvii xix xxi xxiii 1 1 2 2 4 4 4 4 5 5 7 7 8 8 9 14 16 17

www.cambridge.org

Cambridge University Press 978-1-108-48702-3 — Guttation Sanjay Singh Frontmatter More Information

Contents

viii 2.

3.

Principles of Guttation and Its Quantification 2.1 Introduction 2.2 Natural guttation 2.3 Periodicity of guttation and bleeding 2.4 Induced guttation 2.4.1 Induction of guttation in intact plant without application of pneumatic pressure 2.4.2 Induction of guttation in intact plant by applying pneumatic pressure 2.4.3 Induction of guttation in intact plant in a plastic enclosure 2.4.4 Induction of guttation in intact plant inside bell jar 2.4.5 Induction of guttation in excised plant 2.5 Measurement of guttation 2.5.1 Qualitative assessment 2.5.1.1 Image analysis of guttation droplets 2.5.1.2 Measurement of shape of guttation droplets 2.5.1.3 Measurement of size of guttation droplets 2.5.2 Quantitative measurement 2.5.2.1 Measurement of guttation through mass collection of leaf drippings 2.5.2.2 Pedersen technique for measuring guttation 2.5.2.3 Komarnytsky technique for measuring guttation 2.5.2.4 Wagner technique for guttation measurement 2.5.2.5 Singh technique for measurement of guttation 2.6 Guttation intensity Mechanism of Guttation Introduction 3.2 Mode of guttation 3.3 Mechanism of guttation 3.3.1 Ascent of sap in plants: a key to exudation 3.3.1.1 Theories of ascent of sap 3.3.1.2 Magnitude of root pressure and exudation 3.3.1.3 Pressure in the shoot and leaf causing bleeding and guttation 3.3.2 Mechanism of root pressure 3.3.2.1 Osmometer model of root pressure 3.3.2.2 Metabolic model of root pressure 3.4 Integrated view of sap movement and guttation 3.4.1 Water forced upward-like mechanism of ascent of sap and guttation 3.4.2 Compensating pressure theory and guttation 3.4.3 Plant hearts theory of ascent of sap and guttation 3.4.4 Chemico-mechanosensory signal and guttation 3.4.5 Light signal and guttation 3.4.6 Chemical communication between opposite plant poles and guttation 3.4.7 Molecular aspect of guttation: role of contractile proteins and AQPs 3.4.8 Energy coupling in water and solute transfer during root pressure development resulting in guttation 3.4.9 Sum of the mechanism at a glance 3.4.10 The unknowns—a look at the future 3.1

© in this web service Cambridge University Press

19 19 19 21 21 22 22 22 23 24 24 25 25 25 25 25 26 26 26 26 27 28 30 30 30 31 31 31 33 35 35 36 38 43 45 46 46 46 47 47 49 51 54 54

www.cambridge.org

Cambridge University Press 978-1-108-48702-3 — Guttation Sanjay Singh Frontmatter More Information

Contents 4.

Regulation of Guttation Introduction 4.2 Internal factors 4.2.1 Genetic factors 4.2.1.1 Species variability 4.2.1.2 Genotypic variability 4.2.1.3 Phenological variability 4.2.1.4 Hormonal variability 4.2.1.5 Enzymatic variability 4.3 External factors 4.3.1 Environmental factors 4.3.1.1 Mechanical stimuli 4.3.1.2 Atmospheric temperature 4.3.1.3 Light 4.3.1.4 Atmospheric humidity 4.3.1.5 Wind 4.3.2 Edaphic factors 4.3.2.1 Soil and root temperature 4.3.2.2 Soil moisture 4.3.2.3 Soil nutrients 4.3.2.4 Soil aeration 4.3.2.5 Soil mycorrhizae 4.3.2.6 Soil salinity and pollutant 4.1

5.

Chemistry of Guttation 5.1 Introduction 5.2 Organic constituents of guttation fluids 5.2.1 Proteins and enzymes 5.2.1.1 Protein profile: new proteins 5.2.2 Nucleobases and RNAs 5.2.3 Amino acids and amides 5.2.4 Carbohydrates 5.2.5 Lipids, lipoides, alkaloids, glucosides, and toxins 5.2.6 Pesticide residues 5.3 Inorganic constituents of guttation fluids: cations, anions, and salts 5.3.1 Silica 5.3.2 Boron 5.3.3 Nickel, cobalt, manganese, zinc, and magnesium 5.3.4 Calcium 5.3.5 Arsenic 5.3.6 Aluminum 5.4 Hormones 5.4.1 Auxins 5.4.2 Abscisic acid and ethylene 5.4.3 Cytokinins 5.4.4 Gibberellins 5.5 Vitamins

© in this web service Cambridge University Press

ix 55 55 55 55 55 58 59 61 61 62 62 62 62 63 65 65 66 66 67 68 69 70 70 71 71 72 73 73 80 81 81 81 82 83 85 85 85 86 87 87 87 88 89 90 91 92

www.cambridge.org

Cambridge University Press 978-1-108-48702-3 — Guttation Sanjay Singh Frontmatter More Information

Contents

x 6.

Plant Microbiology and Phytopathology of Guttation Introduction 6.2 Guttation-induced mode of plant injury 6.2.1 Guttation-induced non-pathogenic abnormalities in plants 6.2.1.1 Non-pathogenic injury caused by chlorotic and necrotic lesions 6.2.1.2 Non-pathogenic injury caused by pesticide residues of guttation fluids 6.2.1.3 Non-pathogenic injury caused by loss of nutrients through guttation from leaves 6.2.2 Guttation-induced pathogenic abnormalities in plants 6.2.2.1 Salt incrust formation on leaf portions and pathogen infection 6.2.2.2 Pathogenicity and its link with parasitic diseases 6.3 Natural defense mechanisms induced by guttation and implications for phytopathology 6.3.1 Plant defense achieved through preformed chemicals 6.3.2 Plant defense achieved through infection-induced chemicals 6.3.3 Plant defense achieved by infection-induced physical factors 6.3.4 Guttation as a device for enhancing natural disease resistance in crop plants 6.3.5 Guttation as a device for producing natural anti-pathogenic peptides by plants 6.3.6 Guttation as a device for fungal taxonomy for developing disease-resistant plants 6.1

7.

Significance of Guttation in Soil–Plant–Animal–Environment Systems 7.1 Introduction 7.2 Soil-related implications of guttation 7.2.1 Impact of guttation on soil fertility and soil productivity 7.2.2 Impact of guttation on soil-moisture build-up 7.2.3 Role of guttation in phytoremediation of wasteland 7.3 Plant-related implications of guttation 7.3.1 Guttation as an indicator of root activity 7.3.2 Role of guttation in the maintenance of plant water balance 7.3.3 Guttation as a noninvasive assessment test for nutritional status of plants 7.3.4 Guttation as a new noninvasive screening tool for salt tolerance in crop plants 7.3.5 Guttation as a noninvasive assessment test for pesticide residues in plants 7.3.6 Role of guttation in plant growth, biomass build-up, and economic yield 7.3.7 Role of guttation in disease resistance of plants 7.3.8 Role of guttation in fungal classification 7.3.9 Role of guttation in plant injury 7.4 Benefits of guttation for animal systems 7.4.1 Significance of guttation for herbivores and insect-pest management 7.4.2 Significance of guttation for domestic animals and rangeland management 7.5 Benefits of guttation for environment systems 7.5.1 Role of guttation in ecological perspectives and ecosystem maintenance 7.5.2 Role of guttation in evolutionary perspectives 7.6 Implications of guttation in plant physiological research 7.6.1 Use of guttation as a research tool for hydathodal retrieval of water and solute 7.6.2 Use of guttation as a tool for studying plant hydraulic and nutritional traits 7.6.3 Use of guttation as a tool for investigating the physiology of host–parasite relationship

© in this web service Cambridge University Press

93 93 94 94 94 95 95 96 96 97 102 102 102 102 102 104 105 106 106 107 107 108 108 109 109 109 110 111 112 112 113 115 116 116 116 117 117 117 119 119 120 120 120

www.cambridge.org

Cambridge University Press 978-1-108-48702-3 — Guttation Sanjay Singh Frontmatter More Information

Contents 7.6.4 7.6.5 7.6.6 7.6.7 7.6.8 7.6.9

8.

9.

Use of guttation as a tool for studying nutrient loss from leaves Use of guttation as a tool for investigating the excretion mechanism of toxic elements, waste products, and other organic compounds Use of guttation as a tool for unravelling the mechanism of pesticide transport and their fate in plants Use of guttation as a noninvasive assessment tool for chemical health of plants Use of guttation as a new tool for understanding salt tolerance in plants Use of guttation for field screening of germplasm and varieties of crop plants for yield enhancement

Significance of Guttation, Associated Structures, and Root Secretion in the Production of Pharmaceuticals and Other Commercial Products 8.1 Introduction 8.2 Pharmaceutical implications 8.2.1 Guttation and production of drugs, human growth factors, hormones, toxins, alkaloids, etc., by transgenic plants 8.2.2 Rhizosecretion and production of drugs, human growth factors, hormones, toxins, alkaloids, etc., by transgenic plants 8.3 Industrial and commercial implications 8.3.1 Production of recombinant proteins at a commercial scale 8.3.2 Production of quality seed protein and human nutrition 8.3.3 Production of quality cotton and other cotton-like fibers 8.3.4 Production of medicinals, spice principals, cosmeceuticals, resins, gums, rubber, etc. 8.4 Progress and prospect of designing plants for future: plant bioreactor technology, gene mining, and molecular farming of guttation-, rhizosecretion-, and bleeding-efficient plants 8.4.1 Molecular farming 8.4.2 Trichome modification and metabolism in hydathode 8.4.3 Molecular cloning 8.4.4 ABA biosynthesis as a factor for increased guttation General Conclusions and Future Perspectives

Appendices Bibliography Index

© in this web service Cambridge University Press

xi 121 122 122 122 123 123

125 125 126 128 129 132 132 134 135 136

136 136 138 142 143 144 148 159 185

www.cambridge.org

Cambridge University Press 978-1-108-48702-3 — Guttation Sanjay Singh Frontmatter More Information

Figures

1.1 Guttation in the form of droplets in different plant species.

6

1.2 A striking example of ‘dewatering’ process, i.e. guttation having a number of polypores

exuding droplets of water during development of fungus Polyporus squamosus.

8

1.3 External and internal features of the laminar hydathodes in Ficus formosana Maxim

and Physocarpus opulifolius (L.) Maxim.

10

1.4 Magnification of a hydathode of Ficus formosana Maxim.

12

1.5 Ultrastructures of laminar hydathodes in Ficus formosana Maxim.

15

1.6 Early stages of hydathode differentiation in leaf primordia of Arabidopsis thaliana (L.)

Heynh., showing DR5::GUS gene expression in transformed plants, marking by the blue colour GUS staining the sites of high concentrations of the auxin hormone.

17

2.1 The root of a four-week-old maize plant was placed in a root-pressure chamber so that

the xylem pressure in the leaves could be changed to cause guttation by altering the pneumatic pressure in the root chamber. 2.2 White pine shoot showing guttation with a pressure of 100 kPa applied to the cut stem.

23 24

2.3 The relationship, for six rice cultivars, between the rate of guttation during pre-heading

stage and their panicle weights (the yields-sink potential).

27

3.1 Morphology and anatomy of root systems and water and solute collection and transport.

34

3.2 (a) Hypothetical interplay of membrane transporters in the plasma membrane of xylem

parenchyma cells for water secretion.

41

3.2 (b) Alternative model for water secretion that makes use of different water ion coupling

ratios in outward- and inward-rectifying K+ channels.

42

3.3 Photographs show the arrangement for investigating the effect of applied pneumatic

pressure on water transport in intact rice plant.

© in this web service Cambridge University Press

45

www.cambridge.org

Cambridge University Press 978-1-108-48702-3 — Guttation Sanjay Singh Frontmatter More Information

xiv

Figures 3.4 Singh model of guttation, proposed to account for the mechanism of guttation.

53

4.1 The rate of guttation as affected by plant growth stages of hybrid rice (cv. NDRH-2).

59

4.2 Effect of wind velocity on the rate of guttation over a period of 30 min at tillering stage

in hybrid rice (cv. NDRH-2).

65

4.3 Effect of water stress on the rate of guttation over a period of 20 min at anthesis stage

in hybrid rice (cv. NDRH-2).

68

5.1 Guttation fluid containing a number of organic and inorganic compounds including

metabolites, enzymes, hormones, vitamins, salts, ions, nutrients, pathogens, etc., impacting plant behaviour.

© in this web service Cambridge University Press

72

www.cambridge.org

Cambridge University Press 978-1-108-48702-3 — Guttation Sanjay Singh Frontmatter More Information

Tables

4.1 Estimates of flow velocities inside the xylem vessels, pressure gradients, root pressure,

and hydraulic conductance in S. emersum and L. dortmanna. Median values and ranges in parentheses are shown apart from the mean guttation rate.

57

4.2 Variations in guttation as revealed by different intact leaf portions of rice leaf at anthesis

during 30 min (cv. NDRH-2).

60

5.1 Organic and inorganic constituents commonly found in guttation fluids of different plant

species.

74

8.1 Important pharmaceutical proteins that have been produced in plants.

131

8.2 Plant-based vaccines, antibodies, and therapeutic proteins in clinical development or in

the market.

133

8.3 Selected transgenic and mutant lines with effects on N-transport, primary N-assimilating

genes, and secondary N-metabolism.

© in this web service Cambridge University Press

139

www.cambridge.org

Cambridge University Press 978-1-108-48702-3 — Guttation Sanjay Singh Frontmatter More Information

Foreword

Guttation is a prominent example of natural secretion, containing organic and inorganic solutes, by plant leaves. The oldest references related to guttation in this volume are the studies of de Saussure (1804) followed by the studies of Duchartre (1859) and Unger (1861) on the secretions of calcareous matters and other compounds and salts of leaves. The author of the book, Sanjay Singh, from the windows of his parents’ house saw guttation with its myriad brilliant drops glistering on the tips of leaves of rice plants in the early morning sunshine. He fell in love with guttation in his childhood. He followed it up throughout his professional academic life as a teacher, a researcher, and an author of a number of review articles. The present book is born out of this love. The book is unique because until now no such treatise on guttation existed. It takes the important phenomenon of guttation out of unjustified relative negligence. The book is a comprehensive source of references on guttation with a remarkable completeness in coverage of the literature. Beyond that, the author provides far-reaching excursions into the background of how guttation and related phenomena such as root exudation are embedded in general in the various fascinating features of plant life including water relations and transport, solute transport, regulation in response to external and internal signals, and ecology. From this, it unfolds guttation as innovative emergence in the true sense of the term. Doing this, the author with over 600 references covers an immense breadth of the literature going back to the beginning of the 19th century and up to the most recent works on implications of molecular biology. Through this monumental work, the author has created a history on guttation research, which forays into many outlooks on further work and progress to be anticipated. With techniques of sampling and quantification of guttation, inorganic and organic chemistry of guttation, biotic interactions with viruses, bacteria, fungi, and animals, and with pharmaceutical implications, the various chapters of the book, which can be taken as self-contained entities, evidently address a very broad audience interested in plant biology, ecology, agriculture, horticulture, animal husbandry, pharmacology, and medicine. Thus, the book is worth being kept on personal bookshelves by students, teachers, and researchers and being acquired by private and government institutions interested

© in this web service Cambridge University Press

www.cambridge.org

Cambridge University Press 978-1-108-48702-3 — Guttation Sanjay Singh Frontmatter More Information

xviii

Foreword

in policy advice, libraries of colleges, and universities for use in teaching and research. Repeatedly, in the book, the author’s sincere concern shines up striving to promote the understanding of guttation for serving human advantage in innovative supply and sustainable management of resources. Professor Ulrich Luettge Department of Biology Darmstadt University of Technology Schnittspahn Str. 3–5 64287 Darmstadt Germany

© in this web service Cambridge University Press

www.cambridge.org

Cambridge University Press 978-1-108-48702-3 — Guttation Sanjay Singh Frontmatter More Information

Preface

The childhood perception of multiple observations of a visible plant event turning into an academic pursuit has been a miraculous twist in my life, which made all the difference between what I am today and what I might have been in the past. The early morning sunlight reflected by diamond-like water drops oozing profusely from rice plants growing all around my house, officially provided to my father, Professor Tarak Nath Singh, by Narendra Deva University of Agriculture & Technology Crop Research Centre, Faizabad in India where I was born, raised, and educated, triggered my mind during school days and aroused curiosity and anxiety regarding this fascinating and intriguing phenomenon of plant oozing, that is, guttation. Driven by the desire to understand guttation, I had then decided to work on these so-called teardrops of plants to unravel the internals and externals of how, when, why, and what spectra of this phenomenon, setting aside the prospect of choosing medical or engineering streams of education for lucrative job opportunities. The curiosity that was aroused during my school days culminated in investigations on guttation, constituting a chapter on it in my doctoral thesis submitted to Dr R. M. L. Avadh University, Faizabad, UP, India. With this began the exploratory journey on guttation that has seen no end till date, resulting in several invited publications on this subject authored by me. My guttation journey was further boosted by organizing and hosting the ‘First World Congress on Guttation and Root Pressure’ from December 2, 2015 to December 5, 2015 at the College of Agriculture & Rural Transformation of the University of Gondar, Ethiopia, which was attended by overseas guttation specialists from the United States, Germany, the Netherlands, Israel, and Hong Kong, in addition to those from within Ethopia. As a matter of fact, guttation has never been in the mainstream of research because of the prevalent belief and opinion that it is of no use to plants and people, which, of course, was proved wrong as you will witness when you harbor through different chapters of this book. It is this wrong notion that actually stirred and teased me so much that I began digging the literature on this topic from the beginning of this millennium and accumulated a good amount of information, old and new, on different aspects of guttation since its discovery in 1672 by Abraham Munting about three-and-a half-centuries ago (to be exact 348 years) in the Netherlands (erstwhile Holland). However, it is by no means an exhaustive collection, and I may kindly be excused for having inadvertently missed referencing the contributions of

© in this web service Cambridge University Press

www.cambridge.org

Cambridge University Press 978-1-108-48702-3 — Guttation Sanjay Singh Frontmatter More Information

xx

Preface

some authors. Subsequent to preliminary topic-wise classification and synthesis, the idea crystallized for writing a book. So, the book, first and only one ever written on guttation, is in your hands. For the sake of clarity and maintaining the sequence of various aspects of guttation in order as far as possible, this book has been organized into 9 chapters. Chapter 1 deals with the nature of guttation, its machinery, and the biography of its discoverer. Chapter 2 describes the principles of guttation and its quantification, whereas Chapter 3 details the mode and mechanism of guttation, reflecting on the chain of events involved in this process. Chapter 4 reflects on the genetic, environmental, and edaphic factors that regulate the phenomenon of guttation. Going further, Chapter 5 gives readers a glimpse of its chemistry—both organic and inorganic aspects. Chapter 6, not behind its predecessor chapters in substance and material, goes on to presenting pathological aspects, pathogenic and non-pathogenic, including phycology, mycology, bacteriology, and virology of guttation. Matters of most significant interest to readers and scholars that now stand to dispute and set aside the previous negative belief and opinion about guttation have been highlighted in Chapter 7, which deals with its impact on soil– plant–animal–environment systems including biomass formation, agriculture, horticulture, forestry, soil, water, animal, ecosystem balance, etc. Chapter 8 sheds light on the latest discovery of secretion of biopharmaceuticals, paving the path for cheaper, safer, and faster production of drugs, vaccines, and a number of recombinant proteins by molecular farming for guttation and rhizosecretion, for animal and human well-being under changing environment and society. Finally, Chapter 9 draws integrated conclusions, apart from those indicated at various places in previous chapters as and when required, and underlines the future perspectives, pointing out gaps in our knowledge of guttation, which is expected to ignite the minds of students, teachers, and scholars to take up research into this so far least talked and written-about topic in plant biology with the objective of translating it into a subject to be extensively researched to explore the unresolved issues of guttation and root secretion to the advantage of mankind, as the biology of guttation is connected to our lives in many ways. Here, I would like to mention that the computer typing of the manuscript, draft after draft, was done by me alone, depriving my daughter Yashvi, son Reyansh, wife Sangita, and my respected mother late Mrs Sampatti Devi of my due love, affection, and care for them, taxing heavily on their social and family life. I highly appreciate their limitless patience during the write-up up to the final submission of the manuscript of the book after its various chapters had been reviewed by learned specialists of the world. In the end, I hope the materials presented and their arrangement in various chapters will make the book useful, accessible, and interesting to students, research scholars, teachers, and professional researchers. Although the various chapters have been reviewed by a panel of competent specialists, nonetheless, the final responsibility for what you read here remains mine, and you may confidently attribute to me any errors of omission or commission in these pages. To help me produce an even better text in the next edition, please send your comments and suggestions at [email protected].

© in this web service Cambridge University Press

www.cambridge.org

Cambridge University Press 978-1-108-48702-3 — Guttation Sanjay Singh Frontmatter More Information

Acknowledgments

I wish to acknowledge and place on record the contributions of several people whose cooperation and encouragement made this book possible. I owe my special debt of gratitude to the most senior and learned specialists, including my father Professor T. N. Singh, from whose insights and suggestions I have benefitted greatly and borrowed skills and expertise freely. Among overseas specialists are the services of Professor Roni Aloni, University of Tel Aviv, Israel, for reviewing Chapter 1; Professor J. T. M. Elzenga, Groningen University, the Netherlands, for particularly providing the biography of Abraham Munting as well as reviewing Chapters 2 and 9; Professor Lars H. Wegner, Karlsruhe Institute, Germany, for Chapters 3 and 4; Professor Slavko Komarnytsky, North Carolina State University, USA, for Chapters 5 and 8; Professor Dani Shtienberg, The Volcani Center, Israel, for Chapter 6; Professor Peter Brimblecombe, City University of Hong Kong, for Chapter 7: all of which are gratefully recognized and appreciated. I am heartily indebted to Professor Ulrich Luettge, Darmstadt University of Technology, Germany, and Professor Wolfgang Kundt, University of Bonn, Germany, for their invaluable contributions by way of critical comments, constructive suggestions, and technical editing of the entire manuscript of the book. These acts of selfless service by all the leading experts mentioned above as well as those anonymous reviewers appointed by the Cambridge University Press are once again highly appreciated and they are indeed unforgettable. I am also grateful to those authors and publishers who have very kindly consented and permitted the use of their copyrighted works and materials for inclusion in this book. I am indeed very thankful to Mr Haile Negash (Head, Department of Plant Science), Dr Desta Firdu (Dean, College of Agriculture and Natural Resources), Dr Getachew Mekonen (Postgraduate Coordinator), and other staff of the department. I also express my obligations and extend sincere thanks to Dr Mitiku Woldesenbet (Vice-President for Research and Community Development Support), Dr Ahmed Mustefa, Vice-President (Academic Affairs), and the most visionary person Dr Faris Delil, President of the Mizan-Tepi University, Ethiopia, for their moral support and encouragement during the writing of this book. My special recognition, high appreciation, and sincere thanks go to the publisher Cambridge University Press whose consistent hard work and careful editing contributed much to the clarity and timely publication of the book.

© in this web service Cambridge University Press

www.cambridge.org

Cambridge University Press 978-1-108-48702-3 — Guttation Sanjay Singh Frontmatter More Information

Abbreviations

AAO3 ABA ADP AQPs AtABA2 AtNCEDs ATP AtPUP1 BOR1 Bot1 CK CPMCW CTP DNP DPIC GSTs KCN MALDI-TOF MRI NDRH-2 PEG PUP PVC ROS SUTs TSP WT

Abscisic aldehyde oxidase 3 Abscisic acid Adenosine diphosphate Aquaporins Arabidopsis thaliana abscisic acid Arabidopsis thaliana 9-cis epoxycarotenoid dioxygenases Adenosine triphosphate Arabidopsis thaliana purine permease 1 Boron transporter Arabidopsis thaliana  Boron transporter barley Cytokinin Cytoskeleton–plasma membrane–cell wall Compensating tissue pressure 2,4-dinitrophenol Diphenylene iodonium chloride Glandular-secreting types Potassium cyanide Matrix-assisted laser desorption/ionization time-of-flight Magnetic resonance imaging  Narendra Deva Rice Hybrid-2 Polyethylene glycol Purine permease Polyvinyl chloride Reactive oxygen species Sucrose transporters Total soluble protein Wild-type

© in this web service Cambridge University Press

www.cambridge.org

C h a p t er

1

Phenomenon of Guttation and Its Machinery

1.1  Introduction Secretions in plants, animals, and humans constitute natural and fundamental metabolic processes vital for their life (Fahn 2000; Squires 2010; Vivanco and Baluska 2012; Wilson 2009). In plants, guttation is one of the conspicuous secretions, which has been known for about three-and-a-half centuries (precisely 348 years). Abraham Munting of Holland (now the Netherlands) was the first to discover this phenomenon way back in 1672, though the word ‘guttation’ was not in existence then. Moreover, the subject was not in the mainstream of research until about two to three decades ago, though the secretory tissues are present in most vascular plant species. The substances present in the secretions of hydathodes, secretory glands, and nectaries are mostly in unmodified or only slightly modified forms, and they are supplied directly or indirectly through vascular systems. Apart from these secreting organs, some other secreting tissues exist that secrete, for example, polysaccharides, proteins, and lipoids, and these substances are produced by them in their own cells. Secretory tissues usually contain numerous mitochondria; however, the distribution of other cell organelles varies in accordance with the materials they secrete. The side wall of the lowest stalk cell in the most glandular trichomes is completely cutinized, which prevents the flow of secreted materials back into the plant body. In this chapter, the occurrence and taxonomic distribution of guttation and morphological, anatomical, and ultrastructural aspects of hydathodes, that is, the mouths of guttation, have been described in some detail based on the current available information and discovery (Singh 2013, 2014a,b, 2016a,b).

2   Guttation

1.2  Definition of guttation The term ‘guttation’ (coined by Burgerstein in 1887) is derived from a Latin word gutta, which means ‘drop’. It is also referred to as the secondary compound exudated from certain trees, that is, trans-1,4 polyisoprene, which has a lower molecular weight than that of natural rubber. The phenomenon of guttation and its process have been known for 350 years (Ivanoff 1963); however, its significance in plant physiology was not known (Stocking 1956a). It is basically a biophysical and physiological occurrence in which fluid is expelled out through the leaves. Guttation has many implications and applications in agriculture, in balancing the ecosystem, as well as in pharmaceutical and industrial products (Singh et al. 2008, 2009a; Singh and Singh 2013; Singh 2014a,b, 2016a,b). Water loss in the form of liquid from the edges, surface, or tips of uninjured or undamaged leaves is called guttation. This is different from transpiration, in which water is lost in vapor form. Guttation occurs during minimal evaporation, with escalating water transport to the leaves. For example, guttation is observed in the early morning in case of grasses, where water droplets accumulate at the tip of the grass, which may be confused with dew. Exudation of water droplets or fluid occurs through specialized plant organs known as hydathodes (otherwise known as water stomata or water pores). These specialized structures are found at the tip of the leaf, its boundary or periphery, and on the leaf surface. Guttation assists the plant in getting rid of excess water along with certain dissolved materials, which is why it is also referred to as ‘teardrops’ of leaves. In case of land plants, the fluid is excreted intermingled xylem or phloem sap and is translucent. This is called ‘terrestrial guttation’ (Stocking 1956a). Guttation of submerged aquatic plants is called ‘aquatic guttation’ (Pedersen 1993, 1994, 1998). Moreover, space explorations have observed guttation in case of dwarf wheat plants, which is called ‘aero-guttation’ (Bugbee and Koerner 2002). The process is such that the more moisture the plant draws to itself, the more it throws off again (Kundt and Gruber 2006). Guttation builds up when the water transport exceeds the evaporation from leaves, which is usually the case during the night or early in the morning when the atmosphere is highly humid. In most environments, humidity is relatively low and usually the water of guttation evaporates as fast as it is exuded and therefore goes unnoticed. In other cases, guttation occurs on the leaf periphery (e.g. in case of lettuce) (Curtis 1943). This occurs because of the low surface tension of the guttate, which prevents water droplet formation. Before guttation, the formed water droplets gather in the leaves’ convolutions. However, surface guttation should not be confused with either transpiration or condensation of dew on horizontal leaves.

1.3  Biographical sketch of Abraham Munting (1626–1683)—

discoverer of guttation Ivanoff (1963) wrote that Munting’s name has not been mentioned in any of the history books of botany or plant physiology that he studied. This is adequate reason for developing a brief biographical sketch, from Munting’s work ‘Naauwkeuringe beschryving der aardgewassen’, of a researcher who discovered the process of guttation. The English translation of the work states: The discoverer Abraham Munting, Hendrikszoon [son of Hendrik], Arts en Meester [Doctor and Master], was born on the ‘19th day of the summer month’ [August?], 1626 in Groningen, Holland. Dr

Phenomenon of Guttation and Its Machinery    3 Hendrik Munting was his father, who was a Professor at Groningen Academy. He excelled in the fields of Botany and Chemistry. One of his most popular works for which he is known is referred to as the ‘Hörtus universae materiae medicae gazophylacium’, published in the year 1646. The work listed out large varieties of gardening specific plants, such as medicinal, tulips, pinks, water hyacinths, and so on, which were cultivated by him. This printed publication work can be considered as the first ever catalogue for gardeners. Munting’s maternal grandfather, Johan Reriaman, was treasurer of d’Ommelanden. His paternal grandfather, Joris Munting, was a gold and silver businessman.

At the age of 18 years, Munting started going to the college at Groningen. Later he lived in France for some time studying medicine and botany. He travelled extensively. Returning to Holland, he became a faculty member at the University of Groningen, where he worked for 24 years, earning eminence as a professor of botany and chemistry. With passing time, he advanced upon the work of his father. Between 1658 and 1683, he directed one of the best and most extensive botanical gardens of the time. Holland was already famous for its collection of plants. Building upon his father’s gardens Munting cultivated a large collection of different species, which he named ‘Paradise of Groningen’. He also grew many a rare species. During a sojourn, in 1658, Munting was conferred the title of Doctor of Medicine, in France (Angers). In his professional career of 24 years, he worked on some brilliant pieces, some of which were published posthumously after his death, due to ‘catarrhe suffoquant’, on January 31, 1683. In 1680, he published works like ‘Aleidarium’ or ‘History of the American Aloes’; in 1681, he published ‘De vera antiquirum Herba britanica’. Munting’s most famous work ‘Naauwkeurige beschryving der aardgewassen’ was published in 1696, 13 years after his death, which is an enlarged and revised version of his previous book ‘Waare oeffening der planten’, earlier published in 1672 and republished in 1682. The Latin editions of ‘Naauwkeurige’, which gathered much appreciation and recognition, were later republished in a series under the title ‘Phytographia Curiosa’, beginning in 1702. Plumier named a genus Muntingia in his honor. The genus consisted of a single variety of plant, which was then included under Rhamnus by Linnaeus and was named as Rhamnus mican, thus including Muntingia to the genus list of the Tiliaceae family. Why was Munting’s work not recognized by botanists? In this context, Ivanoff (1963) agrees with Michaud (1854) who stated that ‘The Munting generation, though has [having] contributed to [the] cultivation of some rare as well as wide range of plants, however, made least contribution to the field of botany as a whole.’ The process of guttation and its impact on a plant’s water economy was not well understood or even recognized by botanists at that time.* Moreover, categories of injuries linked to guttation became topics for research much later, and they still require considerable attention from botanists.

* An

explanatory note has been referenced over here that was presented by DeBary, the editor, who published it in Botanische Zeitung, which was followed by publication of a discussion on water pores. The discussion was held at Russian Botanical Society in Moscow, 1869. DeBary stated, ‘Wenn Man bei einem zweige rifler geeigneten Pflanze, z.B. Fuchsia globosa, Wasser durch den massigefi Druck rifler Quecksilberaule in das Holz einpresst, so treten aisbald Wassertropfen aus den grossen Stomata hervor.’ Here, no discoveries are mentioned; however, artificial guttation method was talked about, in which the cut part of stems or petioles of leaves showed artificial guttation under mercury pressure.

4   Guttation

1.4  Phenomena resembling guttation For the sake of clarity, it is pertinent to describe briefly a few other phenomena resembling guttation. 1.4.1  Dew Dew is the condensation of atmospheric water vapor on the leaves of plants, especially when the atmospheric humidity is high and the nights are cool. It is different, however, from guttation where water generally comes out from within the leaves through hydathodes. Dew is relatively pure water, which does not contain anything except some gases of the atmosphere dissolved in it, whereas guttation fluid, which is actually xylem and phloem saps intermingled together, consists of both organic and inorganic substances. 1.4.2  Bleeding

or oozing

In case of herbaceous/woody plants, sap exudation from a cut or injured stem is called bleeding or oozing. The bleeding rate can be either slow or fast depending on the root pressure in xylem (positive pressure), phloem pressure in sieve tubes, and the pressure around the injury. In a single day, 5–6 L of sap are exuded, and nearly 150 L of sap are exuded from the upper end of the trunk of a 30–40 feet high vigorous Indian date palm (Phoenix sylvestris), although the average yield is about 25–75 L of sap in a season (Bose 1923). The highest bleeding, about 50 L per day, is found in Caryota urens. The average sugar content of the sap is about 2–3 percent (Kramer 1949). The exudation of latex from rubber (Ficus elastica) is a relevant instance of bleeding. However, the mechanism behind bleeding is unclear. For exudation, many conditions are essential such as warm days and freezing nights during the winters and early springs when leaf expansions occur. During these freezing temperatures, the root systems are usually within the frozen soil (Johnson 1945). Hence, for the movement of sap, the plant roots must have moisture available, which favors bleeding. Exudation of water from detached pine needles was also considered to be due to processes distinct from those causing guttation (Kramer 1949). 1.4.3  Other

plant secretions

Secretion assists the plant in conducting various functions such as attracting pollinators, disposing solutes, and taking up nutrients. Moreover, it helps the plant in maintaining the functionality of the secretory organs like that of the nectaries, salt glands, and trichomes, which are mainly found in vascular plants; however, both the secretory and retrieval mechanisms are not well understood (Pilot et al. 2004). Some of these secretory organs exudate unmodified or moderately modified materials that are directly/indirectly supplied from vascular tissues. Secretory tissues seem to have developed from secretory idioblasts scattered among the cells of ordinary tissues during the course of evolution. Subsequently, ducts and cavities seem to have developed and given rise to secretory trichomes (Fahn 1979, 1988, 2000). Certain substances, such as polysaccharides, lipophilic materials, and proteins, are secreted in the cells of certain types of secreting tissues. However, plasmodesmata present in the nectariferous tissue provide passage for the prenectar products to the secretory cells. The vesicles of dictyosomal origin generally tend to eliminate nectar, which is of phloem origin, from the secretory

Phenomenon of Guttation and Its Machinery    5

cells. The nectar secretion, which in some cases involves both these organelles, may be achieved eccrinally as well (Fahn 2000; Vivanco and Baluska 2012). Dictyosomes are known to synthesize carbohydrate, mucilages, and gums. Chloroplasts (or plastids) synthesize lipophilic substances, which are secreted by almost every cell compartment. In some cases, these substances move toward the plasma lemma. Resin or gum secretion from a plant part can occur as a natural process of exudation or because of certain external stimuli (e.g. microbes and growth substances) (Telewski 2006). Among the external stimuli, ethylene is considered as the most effective (Baluska and Mancuso 2009). The secretory tissues consist of numerous mitochondria; however, the concentration of other cell organelles depends on the nature of secreted substances. The secretory glands, which are modified cells (or hair), are usually not as tightly bound with the xylem tissues as in the case of hydathodes. Some glands secrete dilute substances consisting of sugar and salts, whereas some of them release enzymes, volatile oils, nectar, and resins. Thus, such exudation of sap is commonly referred to as secretion. It is apparently caused by forces that develop within the gland, but the mechanism of force development is unclear (Vivanco and Baluska 2012).

1.5  Taxonomic distribution of guttation Guttation is observed in a wide range of angiosperms, gymnosperms, ferns, algae, and fungi. Early studies of hydathodes, the natural leaf pores through which guttation takes place, include those of Haberlandt (1914) and Lepeschkin (1923). Later studies reported this phenomenon and associated it to structures occurring in plants belonging to Crassulaceae, Moraceae, pteridophytes, and cereals (Maeda and Maeda 1987, 1988; Lersten and Peterson 1974; Rost 1969; Sperry 1983). A marked presence of hydathodes has been noted in many dicot families, which includes Salicaceae, Balsaminaceae, Rosaceae, Asteraceae, Begoniaceae, and Urticaceae (Brouillet et al. 1987; Chen and Chen 2005, 2006, 2007; Curtis and Lersten 1974; Elias and Gelband 1977; Lersten and Curtis 1982, 1985, 1991). Hydathodes are found on the entire leaf surface, tip, and edges (Singh et al. 2009a). Moreover, they exist on hypodermis of grape (Vitis vinifera) tendrils or submerged aquatic plants (Fahn 1979, 1988, 2000; Pedersen 1993, 1994, 1998; Pedersen et al. 1997; Tucker and Hoefert 1968). 1.5.1  Angiosperms

and gymnosperms

Guttation has been noted across a wide range of plants in the plant kingdom, including lower as well as higher plants (Figure 1.1). However, it is not widely observed among the taxons. Burgerstein (1920) and Frey-Wyssling (1941) worked on a list of plant species showing guttation (around 333 genera and 115 families as listed by Bergerstein; 43 sub-alpine and alpine genus, that is, Equisetum to Salix as reported by Frey-Wyssling), which has now gained a status of a common phenomenon in case of plants. Burgerstein (1920) also suggested the process of guttation in case of woody and berbaceous plants. Twelve genera were left out by Burgerstein as reported by Klepper and Kaufmann (1966). As per the previous research, only three dicot families have laminar hydathodes (Lersten and Curtis 1991). In case of Urticaceae, a clear linkage was observed among the minor vein junctions in all five tribes. This was seen in 43 species belonging to 30 genera of which just one species did not have hydathodes. Adaxial hydathodes were observed in 28 genera. In case of Elatostemeae tribe, laminar hydathodes were observed in Pilea and Pellionia species, which are abaxial, adaxial, or present

6   Guttation

(a) Horsetail (Equisetum arvense)

(b) Strawberry leaf (Fragaria ananassa)

(d) Annual bluegrass (Poa annua)

(f) Garden burnet (Sanguisorba minor)

(c) Wheat (Triticum aestivum)

(e) Tomato leaf (Solanum lycopersicum)

(g) Water lobelia (Lobelia dortmanna)

Figure 1.1  Guttation in the form of droplets in different plant species [Source: (a) https://commons. wikimedia.org/wiki/File:Dew_on_a_Equisetum_fluviatile_Luc_Viatour.jpg, (b) https://commons.wikimedia.org/ wiki/File:Guttation_ne.jpg, (c) https://commons.wikimedia.org/wiki/File:Guttation_on_Christmas_Wheat.JPG, (d) https://commons.wikimedia.org/wiki/File:Leaves_with_drops.jpg, (e) https://botweb.uwsp.edu/Anatomy/ images/hydathodes/pages/anat0976.htm, (f) https://commons.wikimedia.org/wiki/File:Sanguisorba_minor,_ Botanischen_Garten_zu_Berlin.jpg].

Phenomenon of Guttation and Its Machinery    7

on both sides of the leaf. As guttation was considered a widely occurring phenomenon by FreyWyssling (1941), he stated that the guttation process must be considered a crucial phenomenon in determining water and plant relationship. Pedersen (1993, 1994, 1998) has considered the guttation process as the sole method of transfer of water and nutrients throughout the plant, especially in case of aquatic and submerged plants, where transpiration is minimal. In case of terrestrial plants, guttation is observed in herbaceous as well as woody plants, and it depends on environmental factors as well. Some of the examples are garden Nasturtium, lotus, Colocasia, strawberry, Cucurbita, Abies balsamea, and gramineous family. Broad-leaved trees show the phenomenon of guttation; however, it has also been readily observed in low-growing herbaceous plants. However, plants with reduced leaves, such as in the case of conifers, showed a lack of guttation or root pressure in a study (Kramer 1949). Conversely, Daniel (1949) noted the occurrence of root exudation in 8-month cultured plantlets of plant species like Sequoia gigantea, redwood trees, pine trees, Monterey pine, and Port Orford cedar. Tall trees that grow in areas of high rainfall and humidity, such as tropical rainforest trees, show high guttation and low transpiration (Choat et al. 2012; McCulloh et al. 2011; Sperry 2011). It has been observed in trees like Amborella (a kind of shrub), Trimenia papuana, T. weinmannifolia, Saruma henryi, S. glabra, S. chinensis, Kadsura longipedunculata, K. japonica, and all Chloranthaceae genera; however, plant species having leaves with complete margins did not show guttation (Austrobaileya) (Feild et al. 2003; Feild and Arsens 2007). Furthermore, future studies are needed to determine the reasons for guttation in plants where root pressure is minimal or absent. The conditions favoring this process must be investigated. The effect of mycorrhizas on the guttation process is not well researched, neither has its role in water and nutrient uptake been stated variedly. 1.5.2  Pteridophytes The fronds of many ferns in Polypodiaceae and Cyatheaceae possess swollen vein endings associated with specialized adaxial epidermal cells (Ogura 1972). Their structure is similar in all ferns (von Guttenberg 1934), including Blechnum lehmannii Hieron (Sperry 1983). The endings of developing fronds secrete water when transpiration reduces or stops. In some species, the secreted water contains salts, which form ‘chalk scales’ after the water evaporates. Guttation in gametophytes has been observed on illumination in certain fern species such as common horsetail (Equisetum arvense) and maidenhair spleenwort (Asplenium trichomanes). These are some of the classic examples or models for studying the mechanism of guttation in detail. On illumination, changes in the value of membrane potential have been recorded in the presence of an ion channel and proton pump inhibitors, in order to reveal the response nature and its connection to guttation. A potential role of potassium and chloride ion channels in light-induced guttation has been implicated (Szarek and Trebacz 1999). 1.5.3  Algae

and fungi

The fungus Pilobolus has been studied as a model for guttation because of its fluid exudation capabilities (Tarakanova et al. 1985; Tarakanova and Zholkevich 1986) (Figure 1.2). Research was conducted on large Pilobolus cells that confirmed the link between a cell’s electrical polarization and water flow occurring in a single direction. A positive correlation was observed in the fungus,

8   Guttation

taking into consideration the rate or intensity of guttation and membrane potential gradient value. Gareis and Gareis (2007) observed guttation in eight different kinds of ochratoxigenic isolates (from 11 known isolates) of Penicillium nordicum and P. verrucosum. The fluid exudates contained ocratoxin A and B in high amounts (these are mycotoxins) when the isolates were grown on high concentrations of mycotoxins from Czapek yeast extract agar for about 1 to 2 weeks (10–14 days) at 25°C. The production of exudates differs significantly depending on the growth media. For example, parallel culture of one strain each of P. nordicum and P. verrucosum on malt extract agar demonstrated the efficacy of this agar by yielding higher volumes of exudates. Similarly, a number of polypores, for example, Polyporus squamosus, exude droplets of water during their development. This fungus is parasitic and saprobic in nature, and it feeds on living or dead tree trunks/logs. This kind of guttation is similar to the ‘dewatering’ phenomenon that occurs in higher plants, thus presenting a good example of the guttation process (Emberger 2008). Moreover, algae (also genetically modified ones) have shown fluid secretions (Daniell et al. 2002).

Figure 1.2  A striking example of ‘dewatering’ process, i.e. guttation having a number of polypores exuding droplets of water during development of fungus Polyporus squamosus [Source: Emberger 2008].

1.6  Gateway of guttation: hydathodes as exit pores 1.6.1  Structural

biology of hydathodes

In higher living organisms, both plants and animals, various physiological functions are based on the laws of division of labor because of the complexities of life activities. Individual functions are, therefore, performed by various separate sets of specialized tissues. Plants are known to possess specialized systems for various functions such as photosynthesis, transpiration, translocation of organic metabolites, solute and water absorption and transport, and the exit of guttation fluid from leaves. For example, transpiration, that is, the loss of water in the form of vapor, takes place through stomata, whereas guttation, again the loss of water but in the form of liquid, takes place through special structures called hydathodes. Thus, form and function are intimately linked to each other, they go hand in hand (Dodd and O’Sullivan 2012; Tyree and Zimmermann 2002). Below are, therefore, discussed the morphology, anatomy, and ultrastructures of hydathodes, that is, mouths without lips, for the exudation of liquid in guttation.

Phenomenon of Guttation and Its Machinery    9

1.6.1.1  Morphological and anatomical aspects During the 18th century, the guttation process was observed in case of barley, where water droplets were seen at its edges, especially during spring mornings. It was due to the positive root pressure. A similar phenomenon was observed in oat, wheat, and maize because of their large, conspicuous stomata at coleoptile apices that cause the water droplets to escape from these gaps when the atmosphere is moist. Currently, these pores are called water pores, and the discharge of water and solutes from the pores is termed guttation. As stated earlier, guttation is a widespread phenomenon, which is observable in many different families of plants. These plants are known to possess a specialized system for the exit of guttation fluid from leaves. Just as transpiration takes place through stomata, guttation occurs through special structures called hydathodes, which are also known as ‘water stomata’ or ‘water pores’. Hydathodes are present at the tips, margins, and adaxial and abaxial surfaces of leaves (Chen and Chen 2005; Drennan et al. 2009; Lersten and Curtis 1991; Singh et al. 2009a). The guttation fluid is usually exuded through these special structures known as hydathodes (Figure 1.3, a-h). In plants showing guttation and having these specialized structures, the hydathodes have less dense cytoplasm. Tracheids exist below the vascular bundles, which act as the passage for liquid flow with least resistance. In most of the angiosperms, the tracheids have been observed in association with parenchyma cells of the epithem, which has large intercellular spaces. The flow of water into the epidermis occurs through these intracellular spaces. Epidermal openings found above the epithem are usually incompletely differentiated stomata, which have lost the ability of opening or closing (Esau 2006). Some plant species lack epithem, whereas some plant species have a complicated association of the epithem with the secretory tissue (Aegthe 1951; Sperlich 1939). Sperlich (1939) presented a detailed study on hydathodes and water glands, and their structure and function, with respect to previous studies. To understand the path of guttation, it is necessary to review briefly the present knowledge on the structure, function, and mode of action of the various guttation apparati. Hydathodes form the natural openings on the surface of leaves, which provide the least resistant pathway for fluid transmission that occurs through apoplastic or intercellular spaces found in epithem or mesophyll cells (Stocking 1956a). However, guttation can also occur through the cuticle (Lausberg 1935) or stomata (Bald 1952). Morphologically, the laminar hydathodes of Ficus formosana are complex epithem-type which consist of structures such as water pores, tracheid ends, epithem cells, and a bounding sheath layer. Each water pore is made of two guard cells, and these pores are permanently open. Thus, in reality, hydathodes are the apparatus of guttation, which are made of stomata-like pores in the epidermis and epithem, having a large chamber composed of masses of thin-walled parenchyma cells and a sheath layer that surrounds its tissue (Dieffenbach et al. 1980a). Each hydathode is formed of colorless epidermal cells. Parenchymatous and loose tissue lies beneath the hydathodes, which are known as ‘epithem’. It is also known as ‘transfer tissue’. The cells of epithem are soft and made of loosely arranged thin-walled parenchyma cells and without chloroplast and are involved in absorption and secretion. Surrounding the epithem is a mass of chlorenchymatous tissue. In the anterior part of epithem, a cavity is present which is called ‘water cavity’. Each hydathode opens to the exterior by means of a pore in the leaf epidermis. Hydathodes always remain open but exert flow-controlling effect on the exudation. In fact, hydathodes are the structures that mediate guttation. Among dicots, they have been reported to occur in many families

10   Guttation

Figure 1.3  External and internal features of the laminar hydathodes in Ficus formosana Maxim (a, e–h) [Source: Chen and Chen 2005] and (b, c) Physocarpus opulifolius (L.) Maxim [Source: Lersten and Curtis 1982]. (a) Hydathodes on the adaxial surface of the leaf and scattered in a linear arrangement between the midrib and leaf margin. White points indicate hydathodes. (b) Magnification of hydathodes (circled). (c) Light micrographs of the longitudinal section of the leaf, showing a laminar hydathode. (d) Drawing of a longitudinal section of the hydathode [Source: http://www.plantscience4u.com]. (e) Resin-embedded cross-section of a hydathode. Outer surface consisting of epidermis and water pores; inside region included with the epithem consisting of a group of small lobed cells; two vascular bundles extending upward (arrowheads). (f) Oblique paradermal section of the hydathode through the epidermis level, showing the vicinity of a hydathode consisting of water pores and epidermis cells. (g) Paradermal section of a hydathode through the middle level of epithem, showing the epithem consisting of a group of small lobed parenchyma cells and surrounded by a sheath layer. (h) Paradermal section of a hydathode through the vascular bundle level, showing four vascular bundles and their tracheid element junction under the epithem. Scale bars = 50 mm. E, epithem cell; H, hydathode; PT, palisade tissue; ST, spongy tissue; VB, vascular bundle; and WP, water pore.

Phenomenon of Guttation and Its Machinery    11

(Lersten and Curtis 1982, 1985, 1986, 1991). Most hydathodes have been described similarly with respect to location and morphology. Typical hydathodes occur along the leaf margin, usually singly at the tip of some kind of dentations, and consist of permanently open stomata (water pores), which may vary from one to several in the epidermis adjacent to the epithem. Hydathodes are found at the tip, on the margin, and, in some cases, over the entire leaf surface. As stated earlier, in some plant species, hydathodes exist on leaf hypodermis as well (Fahn 2000). Still, in a few cases, their presence can be traced on other organs too such as the grape tendrils (V. vinifera) (Tucker and Hoefert 1968). On the basis of the structure and function, the organs causing guttation (hydathodes) have been divided into two categories by Haberlandt (1914) and Lepeschkin (1923): (a) ‘passive/epithemal hydathode’ (occurrence of guttation due to root pressure), and (b) ‘epidermal/active hydathode’. In the former, these organs are directly associated with the water-conduction system of plants. The hydathodes start functioning in the presence of root or hydrostatic pressure, which is also called ‘exudation pressure’. When the root pressure becomes highly intense due to reduced transpiration rate, it leads to guttation. In such cases, some of the plant species release water through stomata or water pores that are found on leaf margins. However, some of the plant species lack these structures. The name ‘epithemal hydathode’ is so because the major portion of hydathode constitutes the epithem tissue. Epithem consists of thin-walled parenchymatous cells that are colorless and have a large nuclei and abundant protoplasm. These cells are the association between the epidermis and tracheid’s terminal end. From this end, the tracheid terminal emerges out, which is a fingerlike projection. These projections are closely linked to epithem cells, and its ends direct into the intercellular spaces within epithem cells. These intercellular spaces found between the cells lead the communicative pathway to the chambers that are present just below the water pore. The water pore is generally surrounded by guard cells. Because of this feature, these stomata do not have the ability to open and close in the presence of a stimulus such as light, carbon dioxide, darkness, or any other external or internal factors as do the normal stomata. This special arrangement of waterconducting cells assists in forced flow of water from the tracheids under the effect of positive root pressure. This builds up a least resistant path for water flow through these pores, leaf boundary, as well as tip into the other environment. These kinds of hydathodes are referred to as passive hydathodes. This is because they are not involved directly in the fluid exudation process, rather they just act as a filtering apparatus. Laminal guttation or guttation occurring at the leaf lamina was observed and investigated by Ivanoff (1960) who conducted experimentation on cantaloupes as well as other plant species at different locations and times. On the basis of his observation, he stated that laminal guttation was more frequent. The surface area where guttation occurs can vary from microscopic to large surface areas, that is, some square centimeters to even the complete blade of the leaf. The leaf gets covered with a thin, glistening film of sticky material, which is formed by merging of the small water droplets. Under a microscope, the leaf hairs of cantaloupes showed a coating of solidified exudate that seemed to have exudated from trichome pits in acone-like formation (Figure 1.4). The results were similar to the phenomenon of guttation, which was closely related to guttation through epithemal hydathodes. However, another water egress channel was involved, which also needs to be focused on. Lepeschkin (1923) and Haberlandt (1914) showed concurrent findings stating that guttation can occur through a normal opening as well as water stomata, which are present along the leaf blades. According to Lepeschkin (1923), the force causing guttation was thought to be the sap pressure, and any kind of cellular involvement, far off or near the stomata, was not known. However, in case

12   Guttation

Figure 1.4  Magnification of a hydathode of Ficus formosana Maxim. (a) Hydathode (arrowhead) consists of a trichome and a group of water pores on leaf upper epidermis. (b) Cleared leaf, showing the venation pattern and three hydathodes, which are associated with vein-end junctions of the venation (arrowheads). (c) Scanning electron micrograph (SEM) of a young leaf, showing the laminar hydathode consisting of a giant trichome, numerous salt-glandular trichomes, and a group of water pores. (d) SEM micrograph showing a hydathode at a leaf tip of a young leaf. (e) SEM micrograph showing a mature laminar hydathode with a trichome, a remnant salt-glandular trichome, and a group of water pores. (f) SEMmicrograph of a hydathode, with chalk scales (arrowheads) dispersed on the outer surface of leaf after the guttate fluid evaporated. (g) SEM micrograph showing salt crystals (arrowhead) precipitated on outer surface of the water pores. (h) SEM micrograph showing fungal mycelia (arrowhead) growingon the outer surface of a hydathode. Some of them are even intruding into the inner parts of the hydathode through the water pore. Scale bars = 1 cm (a), 0.3mm (b), 1mm (c), 100 mm (d–g), and 10 mm (h) [Source: Chen and Chen 2005]. SG, salt-glandular trichome; T, trichome; VB, vascular bundle; and WP, water pore.

Phenomenon of Guttation and Its Machinery    13

of guttation, specialized multicellular epidermal structures are involved that are responsible for the generation of the force responsible for the initiation of guttation or exudation. In addition to guttation, the laminar hydathodes situated at the upper and lower parts of the leaves are involved in water loss from the leaves and its retention. One of the species of Myrothamnus genus, that is, Myrothamnus flabellifolius, has been marked with leaf teeth, which shows similar characteristics to that of hydathodes. Tracheary elements developing from the vein endings of each leaf tooth, subtend and extend into a cell cluster that is smaller in size as compared with the nearby mesophyll cells. The epithemstomata are larger in size than the stomata that are present on the leaf surface. Cytological studies showed the absorption of crystal violet (an indicator) by the stomata of leaves where no transpiration occurred, thus confirming the presence of water pores, whose number ranges from two to four for every hydathode. This makes it easily distinguishable in case of dehydrating leaves. In case of M. flabellifolius, laminar hydathodes are present. The short and wide tracheary elements join the outer edge of the abaxial leaf edge that lacks stomata and then reaches the crystal violet-penetrating regions. A fluorescent dye, calcofluor white, was used to trace the apoplastic water pathway. It has shown the existing role of both hydathode variants in case of foliar water uptake (that occurs in rehydration process) during sulforhodamine accumulation. This indicates retrieval of solutes from the apoplast found in the epithem, thus suggesting hydathodes to be a potential path for water loss in case of desiccated leaves (Drennan et al. 2009). Hydathode morphogenesis involves four different developmental stages: (a) initial, (b) cell division, (c) cell elongation and differentiation, and (d) maturation (Chen and Chen 2005, 2006, 2007). In the initial stage, a large trichome cell acts as the foundation for the development of hydathode. The primary epidermal initial cells responsible for the development of hydathodes are present around this trichome cell base. Then, the cell division begins, where anticlinal division is observed in the initial cells to form epidermal cells, followed by water pores and formation of subepidermal initial cells through anticlinal and periclinal divisions, which further differentiates into epithem, tracheid, and hydathode sheath layer. During elongation and differentiation, epithem evolves into lobe-shaped structures; the factors responsible for this development are still unknown. The cell mass consists of special arrangements of microtubules around pericytoplasm. Its cytoplasm contains digesting enzymes that act on cell walls, make them weak, and thus facilitate growth of the cell due to turgor pressure. Due to the development of lobe-shaped cells, the intercellular spaces of epithem markedly increase. In addition to this, the surface area of contact of the cells with the surrounding environment also increases because of its shape. Meanwhile, an increase in the number of water pores is observed on the epidermis, which occurs due to differentiation of tracheids. In the maturation stage, tracheids mature within the epithem, and the functionality of the water passage also develops between veinends and guttation. Basically, differentiation and maturation of epithem cells take place from under the water pore region to the vein-end region. Guttation becomes apparent at the vein-end region because of the presence of mature epithem cells. To examine epithem’s retrieval mechanism, the apoplastic water route tracer, that is, lanthanum nitrate, is used. Cytochemical experimentation revealed a probability of using coated-vesicle endocytosis in addition to proton pump by the epithem to absorb nutrients from the guttate. The active hydathodes that are present mainly in the epidermis are composed of all sorts of water-secreting or exudating organs. The mature hydathodes consist of specialized epidermal cells along with different unicellular and multicellular trichomes that have a direct link with the water-conducting system. As stated by Svetlikova et al. (2015), the active hydathodes exudate water

14   Guttation

following the same mechanism as that of root pressure. According to Haberlandt (1914), the name ‘active hydathodes’ was given because the energy source for water exudation comes from within the hydathodes along with the root pressure. The exudation from hydathodes is dependent on the pressure that persists in the water-conducting system. However, reports of stomata guttation in Gladiolus and of active water exudation in Vida faba L. could not be confirmed with experimental and anatomical investigations (Meidner 1977). 1.6.1.2  Ultrastructural and histological aspects A number of recent studies have been conducted on the morphology, anatomy, and ultrastructure of hydathodes of the following plant species:

• • • • • •

Physocarpus or bladder fruit (Lersten and Curtis 1982), Ferns (Sperry 1983), Asteraceae family plants (Lersten and Curtis 1985), Barley (Dieffenbach et al. 1980a), Wheat (Maeda and Maeda 1987), and Rice (Maeda and Maeda 1988).

Different categories of hydathodes have also been studied and reported in the literature, such as: • Laminar hydathodes: Pilea pumila (clearweed) and Urtica dioica (nettle) (Lersten and Curtis 1991), • Submerged hydathodes: Aquatic plants like Lobelia dortmanna and Sparganium emersum (Pedersen 1998) and F. formosana (Chen and Chen 2005; Haberlandt 1914; Lepschkin 1923). However, studies linked to grass lamina are limited. For example, in case of rice leaf, the ultrastructure of vessel elements is obscure. Thus, agricultural or horticulture plants are included in limited studies, taking into account the ultrastructure study of hydathodes. On the basis of the facts, the ultrastructure of laminar hydathodes and their surrounding cells in case of F. formosana have been stated (Chen and Chen 2005, 2006, 2007; Lersten and Curtis 1991). The research sets the foundation to understand the relation or link between water pores and vessel elements. Various techniques such as the clearing method, light microscopy, scanning electron microscopy, and transmission electron microscopy have been used to examine the morphology and ultrastructures of laminar hydathodes of F. formosana Maxim (Chen and Chen 2005; Lersten and Curtis 1991). Ultrastructural and cytological studies revealed the existence of numerous mitochondria, an extended endoplasmic reticulum system, and many small vesicles derived from Golgi bodies in epithem cells of a dense cytoplasm (Figure 1.5). As the epithem cells mature and age, the number of proliferate peroxisomes tends to increase. In addition to changes in these organelles, an abundance of plasmodesmata was observed on the contact surfaces of these cell walls between epithem cells. Moreover, variabilities in the structures of plasmalemmasome on the plasma membrane of the epithem cells were noted, which seem to arise from endocytosis through sequential plasmolysis–deplasmolysis cycle induced by recurrent transpiration and guttation during day and night, respectively. Thus, the hydathodes could be an ideal system for elaborate studies of endocytosis in plants (Chen and Chen 2005).

Phenomenon of Guttation and Its Machinery    15

Figure 1.5  Ultrastructures of laminar hydathodes in Ficus formosana Maxim. (a) Transmission electron micrograph (TEM) showing cross-section of a water pore on the leaf adaxial surface. The water pore is externally covered by two overlapping ridges of modified guard cells (arrowhead). Asterisk indicates the outside environment. (b) TEM showing paradermal section of the water pore with two modified guard cells. The aperture is permanently opened (O). (c) TEM of epithem cell, with conspicuous sinuous cell wall, vacuoles, peroxisomes, mitochondria, and abundant plasmodesmata between epithem cells. (d) Magnification of longitudinal section of plasmodesmata. (e) Magnification of cross-section of plasmodesmata. Arrowheads indicate vesicles. (f) TEM showing microtubules present near the periplasmic region of tortuous cell wall. Arrowheads indicate vesicles. (g) Magnification of an epithem cell, showing different organelles including a nucleus, peroxisomes, mitochondria, and chloroplasts. Arrowheads indicate vesicles. (h) Magnification of a partial epithem cell, showing three Golgi bodies, endoplasmic reticulum, and many vesicles (arrowheads). (i) TEM of sheath layer cells with a large central vacuole, and most of organelles and cytoplasm distributed far from near epithem side. Stars indicate the sheath layers. Scale bars = 2.5 mm (a–c, i), 0.5 mm (d–f), 1.25 mm (g), and 0.25 mm (h) [Source: Chen and Chen 2005]. C, chloroplast; CW, cell wall; ER, endoplasmic reticulum; G, Golgi body; IS, intercellular space; M, mitochondrion; Mt, microtubule; N, nucleus; O, water pore aperture; P, peroxisome; PD, plasmodesmata; S, starch; SG, salt-glandular trichome; and V, vacuole.

16   Guttation

Sinuous cell walls of epithem cells and their role The lobed structure of epithem cells enables the sinuous cell wall in increasing the surface area of contact with xylem sap. This leads to improvement in the rate of absorption of the epithem (Sattelmacher 2001). In a study on Chlorophytum comosum, an active reorganization of filaments in the leaf cells was observed due to the hyperosmotic stress (Komis et al. 2002). However, the induction of epithemal ontogenesis due to osmotic pressure and further filamental reorganization must be focused on (Telewski 2006). Endocytosis in epithem cells Following the transpiration process, the epithem’s cell membranes become unstable. This is because of the increased concentration of solutes in the xylem sap of the hydathodes. This results in high osmolarity in the cells. Such high salt concentration and osmotic stress can result in disturbed morphology of the plasmalemma, thus resulting in variability of plasmalemmasomes. Various other studies have stated invagination of the cells under stress condition, thus leading to formation of such variability (Chen and Chen 2005; Gordon-Kamm and Steponkus 1984; Oparka et al. 1990). During this time, the sinuous cell walls are enlarged so as to increase the surface area and help in the regulation of the ratio of membrane area/cell volume. A repeated cycle of guttation followed by transpiration leads to change in solute concentration, thus allowing another cycle of plasmolysis and deplasmolysis, which causes endocytosis (Oparka et al. 1990). A study by Nishizawa and Mori (1977, 1978) focused on endocytosis-induced membrane invagination by using the electronmicroscopic technique, which involved in-depth investigations of the entry mechanism of organic nitrogen into the root cells of rice. As a result of these studies, several invaginations of plasma membranes in the differentiating and elongating root-zone were observed. While classifying these invaginations into groups, they confirmed that endocytosis does occur in the root tissues of rice. Chen and Chen (2005) reiterated the findings of Nishizawa and Mori (1977, 1978) regarding the variability in plasmalemmasomes found in epithem cells and emphasized on hydathodes being an ideal system for dissecting the mechanism of endocytosis in plants. 1.6.2  Induction

of hydathodes by auxin

The mechanisms controlling the development of hydathodes and vascular differentiation in leaves were poorly understood, and therefore, Aloni (2001) suggested the ‘leaf venation hypothesis’ to explain how the vascular system in leaves and their hydathodes are induced and controlled. According to this hypothesis and its experimental analysis, hydathodes are induced through high auxin (indol3-acetic acid) concentrations (Aloni et al. 2003) (Figure 1.6). The auxin hormone, produced in young leaves, induces xylem differentiation and consequently also the hydathodes. Gradual changes in the locations of major auxin production sites during leaf primordium development control the initiation and developmental pattern of hydathodes. High auxin concentrations develop in the lobes in a basipetal pattern, first in the tip of a leaf and gradually downward along the leaf ’s margin to the base of the leaf. Early high auxin concentration at the elongating primordium tip induces the large apical hydathode. This auxin source also induces the water-conducting mid-vein of the leaf. Soon, the high primary auxin production in the upper lobe induces the differentiation of the hydathode at

Phenomenon of Guttation and Its Machinery    17

the upper lobe and its water-supporting large secondary bundle (Figure 1.6). Gradually, a new high auxin production site is created in a lower lobe, and this late auxin production site induces this lobe’s hydathode and its water-supporting secondary bundle (Aloni 2010). The auxin in differentiating hydathodes inhibits hydathode development below them, toward the leaf base (Aloni et al. 2003). It is significant that hydathode differentiation patterns can be induced experimentally by external auxin application (Aloni 2001), and the study of its regenerative biology deserves utmost attention.

Figure 1.6  Early stages of hydathode differentiation in leaf primordia of Arabidopsis thaliana (L.) Heynh., showing DR5::GUS gene expression in transformed plants, marking by the blue colour GUS staining the sites of high concentrations of the auxin hormone. (a) Centre of strong expression (black arrowhead) in a developing hydathode with decreasing expression (white arrowhead) towards the differentiating secondary bundle. (b) Gene expression (blue staining) at the margin (large arrowhead) in a more developed hydathode with four freely ending vessels (large arrow) differentiating toward the margin. Note some weak expression near the margin (small arrowhead) and GUS expression within the bundles (small arrowheads) where the auxin hormone is transported polarly [Source: Aloni et al. 2003].

1.6.3  Genetic

aspects of hydathodes

Undoubtedly, a gap exists in research at the genomic and molecular levels regarding the initiation and development of hydathodes, their distribution, and hydathodal index. In this context, Aloni et al. (2003) were the first to show that a local high auxin synthesis in a young leaf primordium induces hydathode formation in a leaf. However, few literature studies showed the need for the basic helix–loop–helix protein, named as MUTE, to initiate the production of various evolutionary structures that are linked to leaf stomata, that is, the hydathodes. Related studies were carried out on Gossypium hirsutum (Mexican cotton) and Arabidopsis thaliana (Thale cress) (Jaradat and Allen 1999; Pillitteri et al. 2008). The protein MUTE expresses itself at leaf and cotyledon tip, thus colocalizing with the auxin gradient. The protein was not regulated by auxin, and non-appearance of hydathode or water pores showed no effect on the auxin gradient. In case of the studied plant species,

18   Guttation

the protein is involved in the differentiation of hydathode pore as well as stomata (Peterson et al. 2010; Pillitteri et al. 2008). Further studies have stated that genetic evolution in hydathodes has resulted in mutation. Such mutations can be marked with defective polarity of the leaves or loss in the functionality of the YUC genes (auxin-producing gene) that leads to abnormality in leaf such as abnormal leaf margins along with less expanded leaves with few hydathodes. However, cell patches of epidermal cells resembling the hydathodes are present (Wang et al. 2011). Limited studies have researched hydathode development and its genetical changes. However, no clarity exists on how guttation occurs and what its functions are. It is hoped that in future, these gaps in knowledge and those described in earlier paragraphs will serve as a stimulant for further research. In summary, while closing this chapter, it may be mentioned that hydathodes, that is, mouths without lips, unlike stomata, remain permanently open and perhaps originated from the universal structure ‘stomata’. Guttation, occurring in a wide range of plants, remains one of the most powerful engines for chemical mobility in plants. Research on guttation is intrinsically interesting, but it also has theoretical and pragmatic implications discussed in detail in other chapters of this book. Although it is attracting the attention of a wider group of scholars, from botanical, ecophysiological, agricultural and pharmaceutical points of view, a high priority for holistic research strategy that recognises interdependency between the structure and function is to be accorded. Structural studies on the guttation organ, that is, hydathodes, have revealed that the structure of these organs is similar to that of stomata pores from which fluid exudation takes place during the guttation process. However, detailed studies on the structure of vascular systems and ultrastructure of hydathodes, especially in field crops, fruit and vegetable crops, and aromatic and medicinal plants, are required with a view to enhancing the efficiency of secretions for increased productivity of desired products for human use.

C h a p t er

2

Principles of Guttation and Its Quantification

2.1  Introduction Chapter 1 described the general features of guttation and the structural biology of hydathodes, that is, mouths of guttation, in the light of recent information. Now, in the next few pages, the principles governing guttation and its quantification will be explained in detail as because knowledge and understanding flowing from an event, physical or biological, that cannot be measured and expressed in quantities is meagre. The quantification of guttation is essential to efficiently understand its physiological functions, the ecological relevance, and the issues related to the phytopathological and agricultural implications and for the production of pharmaceuticals by guttation.

2.2  Natural guttation As stated in the previous chapter, guttation is a natural and spontaneous physiological phenomenon of sap exudation from the tips, margins, and adaxial and abaxial surfaces of leaves of a wide range of plant species. These exudations may occur either in intact plants in natural habitats in the field or polyhouses or may be induced in the laboratory or growth chambers or certain other enclosures. In this context, it is important to consider and describe the phenomenon of guttation, root exudation, and stump bleeding in an integrated manner, reflecting on root pressure as the driving force for these processes to occur.

20   Guttation

Guttation is usually visible when the rate of transpiration is reduced due to increased humidity levels or reduced light intensity that leads to closure of stomata, or both. When the rate of transpiration is low and absorption of water is high, the phenomenon of guttation is visible. In conditions of optimum soil nutrients and water supply, the rate of guttation can be high under high humidity levels and reduced light intensity. The predominant factors that have a direct and positive influence on the guttation process are the absence of wind and clouds and the presence of negligible humidity in the upper strata of the atmosphere to reduce the greenhouse effect; however, humidity needs to be high near the ground (Luo and Goudriaan 2000; Singh et al. 2009a). It is not always true that guttation will occur if the moisture content of the soil is high because other factors also affect guttation, such as wind, which usually has an adverse effect. Additionally, the guttation process is not limited to dark conditions and outdoors, as it can also occur in warm conditions, wet rooms, or industrial areas, where the humidity level is high. Even when guttation occurs it may not be visible on account of rapid evaporation from the sites of exudation or slow exudation or both. Furthermore, other conditions that initiate and enhance guttation include cool nights following warm days as during this period plant roots increase the absorption of nutrients as well as water— absorption occurs in the absence of any obstruction and the rate of transpiration also decreases. Exceptions to this case include lawn grass, maize, and rice plants, where guttation occurs under conditions of bright sunlight or during late hours just before the sunset (Singh et al. 2009a). The factors that lead to a decreased rate of guttation can be considered the same as factors that reduce root pressure, such as cold conditions, dry and aerated soil, and nutrient deficiency (Kramer and Boyer 1995). Guttation is frequent in areas that have early spring and late fall, as the nights are cooler than the days. Such places include irrigated agricultural lands and mountain valleys. In keeping with the importance of these soil and environmental conditions, Gaumann (1938) provided, rising soil temperature, as an explanation for the frequent occurrence of guttation during cool spring nights following warm days, in some plants. Several similar instances exist that explain the effect of soil and environmental conditions in regulating guttation. For example, during the spring season, the night temperature of the soil is usually high as compared with air which gets coupled with a high level of humidity. Thus, a reduced level of cuticular transpiration is observed in the night. The soil is warm, the plants absorb water from it, which further leads to a favorable water balance, thus leading to a formation of positive xylem pressure. This results in forcible exudation of guttation fluid from the hydathode water pores. In mountain valleys, the above-stated conditions prevail throughout the spring season as well as during the period showing vegetative growth of plants. Warming of soil may occur during the day due to high radiation followed by rapid air cooling during the night. This cycle of warming and cooling offers the optimum guttation condition (Frey-Wyssling 1941). In addition to this, the particular characteristics of highly humid nights and warm soil observed in the tropical regions are also favorable conditions for rapid guttation. Taking into account the mentions about the guttation process, there are some plants that naturally guttate through the release of water into the soil rather than into the atmosphere, which is referred to as ‘cloud stripping’, which is an important water source for creeks at few places. In these areas when the plants are cleared the creeks dry up impacting ecosystem balance immensely. The moisture exuded from Moso bamboo shoots is also classed as guttation, and during spring in South-east Queensland when the bamboo is shooting one can spot the pre-emergent shoots by the wet patches they create. This wetting process is considered to have agronomic significance as it softens the soil

Principles of Guttation and Its Quantification     21

ahead of rhizome tip, allowing the expanding rhizome to easily penetrate and emerge (author’s personal observations, 2008; while working as a Postdoctoral Research Fellow in the laboratory of Professor David Midmore at CQU, Rockhampton, Queensland, Australia).

2.3  Periodicity of guttation and bleeding Hofmeisteir (1862) was the first to report the occurrence of diurnal fluctuation in case of roots, and he also took cognizance of the fact that this cyclic activity occurs under constant conditions (Baranetzky 1877; Grossenbacher 1938). Such diurnal fluctuations can also occur due to periodic changes in the environment. Even under constant environmental conditions, uniform environment, studies show periodicity of activities in the cells (Bose 1923). The most important factor responsible for exudation is change in temperature, in addition to the external environment. This rhythmic phenomenon has been presented by Grossenbacher (1938, 1939) and Skoog et al. (1938). Cyclic fluctuations in bleeding intensity, magnitude of root pressure, and guttation rate exhibit practical complications while studying the effect of external factors on bleeding (Arisz et al. 1951). Plants displayed a regular 24-hour cycle of variations in the magnitude of root pressure even under a constant external environment (temperature and humidity). The peaks of pressures, as also reflected in the rates of exudation, were attained at periods usually close to noon and the low points in troughs near midnight. The application of auxin and beta indole acetic acid at different periods in the 24-hour cycle enhanced these rates of exudation from stem stumps (Skoog et al. 1938). Interestingly, while raising plants in experimental light continuously and capitating the stems at appropriate times, it was possible to invert the maxima and minima of fluctuations in pressure or exudation; but the 24hour cycle always persisted, presumably through the influence of active metabolic activities of the cells that could be interrelated with active uptake, transport, and accumulation of salt. However, the understanding of the mechanism behind periodic exudation and its association with root pressure is controversial. If a purely osmotic explanation for root pressure is accepted, then a diurnal cycle must result from a cyclic rate of secretion of solutes into the xylem followed by the osmotic influx of water. Decrease in root pressure might occur due to loss of solutes into the xylem with water, which might be due to the bleeding or reabsorption from trachea (Grossenbacher 1938). For proving the fact, there must be a combined action of auxin and periodic secretion as well as removal of solutes from xylem sap. However, experimentations on the exudation process, rate of total respiration, and composition of exudate did not reveal any relationship between auxin and the diurnal cycles. An indirect association exists between the auxin activity and exudation process, as it has already been confirmed that respirable substrate is a required substance for bleeding and salt accumulation.

2.4  Induced guttation Guttation, apart from the natural process, can be induced in intact land and aquatic plants or excised stems having leaves intact on them in the laboratory, growth cabinets, or coverings and enclosures by providing the required environment and instrumentation. If the roots of intact plants are placed in a dilute solution containing all the nutrient ions with good aeration at a favorable temperature,

22   Guttation

rapid guttation occurs and continues for hours in humid atmosphere (Raleigh 1946a). However, the guttation process is low when roots are exposed to a temperature lower than 5°C (Pedersen 1993, 1998). The following sections describe different ways for inducing guttation in plants. 2.4.1  Induction

of guttation in intact plant without application of pneumatic pressure

Under greenhouse conditions, guttation may be induced with abundant soil moisture in several species of plants by keeping the soil temperature in the range of 25–32°C and the relative humidity of the air nearly 100 percent. Raleigh (1946a) observed that tomato plants grown with complete nutrient solution guttated, whereas those in solutions lacking both nitrate and ammonium nitrogen did not guttate. Furthermore, tomato plants did not guttate if they were previously grown in complete solutions and thereafter transferred to solutions lacking nitrogen (nitrate or ammonium), phosphorus, potassium, calcium, and magnesium separately until deficiency symptoms appeared. Interestingly, supplying the lacking elements, such as nitrogen, phosphorus, and potassium, to each of the deficient solutions led to marked guttation on all plants except those supplied with calcium or magnesium. The author postulates that in this case, injury to the plants caused by deficiency of calcium and magnesium may have been responsible for the lack of guttation. Profuse guttation was induced in a shorter time with nitrate nitrogen than with ammonium nitrogen, which can be attributed to oxygen supply through the nitrate. Moreover, the absence of aeration and dilute nutrients prevent guttation; therefore, to induce guttation, these factors must be deployed. With the aeration of nutrient solution under high humidity, Eaton (1943) was able to induce exudation, resulting in guttation, from the stumps of Cedrus deodara and Thuja orientalis following decapitation after having been cultured under high salt conditions and then washed under tap water. 2.4.2  Induction of guttation in intact plant by applying pneumatic pressure Guttation has not only been studied in field plants in situ but also, as stated above, in plants growing in the greenhouse as well in a saturated atmosphere or under conditions of high atmospheric humidity. However, the drop disappears rapidly just after sun-up or after the sun strikes the leaves. Wei et al. (1999) and Tang and Boyer (2003) used a setup in which xylem pressure could be controlled by applying pneumatic pressures to the root of maize plants for the induction of guttation. Guttation droplets so produced can be seen along the margins of maize leaves (Figure 2.1). 2.4.3  Induction

of guttation in intact plant in a plastic enclosure

Pedersen (1993) carried out an experiment to induce guttation in case of Lobelia dortmanna (water lobelia) and Sparganium emersum (bur-reed), which are submerged aquatic plants. The experiment was performed in a plastic enclosure. Guttation was observed on the tip of the leaves through hydathodes. The exudated droplets were collected. Taking the experiment further, natural sediments were used for rooting the plants. Before the experiment, the level of water was reduced to below the shortest leaf ’s tip, which led to easy collection of the guttate. The tray with the plants was covered with a plastic film that helped in maintaining a humid atmosphere favorable to guttation. However,

Principles of Guttation and Its Quantification     23

Figure 2.1  The root of a four-week-old maize plant was placed in a root-pressure chamber so that the xylem pressure in the leaves could be changed to cause guttation by altering the pneumatic pressure in the root chamber [Source: Wei et al. 1999].

the experimental setup varied for plant species with different lengths of leaves. In this experiment, using water lobelia, the level of water was just below the shortest leaf tip, and the whole setup was covered with polyvinyl chloride (PVC) sheet with a 5-cm gas exchange space between the sheet and water level. This space started gathering water vapor, which led to the initiation of guttation from the hydathodes. Varied volumes of droplets, depending upon the plant activity, were collected with small glass capillaries (1 cm long) from leaves of chronologically different ages at 2- to 6-hourly intervals. However, this technique was modified for guttation measurements of S. emersum under field conditions. In this case, 1 mL of Eppendorf tubes were used to collect the guttate from leaf tips by making a slit in the tube. Later, the tube was sealed with water-resistant lanolin, which created a gas-tight chamber. To facilitate mounting, a large cylinder was placed around the plant and inserted into the sediment to pump out water using a submersible pump (operated with battery). Later, to restore the water level, the plastic cylinder was removed from the sediment once the Eppendorf tubes were in position. S. emersum showed a high level of guttation, and the guttate was collected after 1 hour of completion of the technique. 2.4.4  Induction

of guttation in intact plant inside bell jar

Herbaceous plants can also be included in induced guttation experimentation when favorable conditions are met, such as high absorption rate of water along with low rate of transpiration. The laboratory process to carry out this kind of experimentation involves high growth rate plant varieties of rice, wheat, tomato, or barley, which are placed inside a jar and are well watered. This condition leads to full saturation of the atmosphere within the jar, ceasing transpiration and causing guttation, which takes place for a short duration under such prevailing conditions.

24   Guttation

2.4.5  Induction of guttation in excised plant Klepper and Kaufmann (1966) induced guttation in the leaves of cut stems by fixing them in a pressure chamber; fluid was collected and osmotic potential determined thereof but no mention of quantitative measurement of guttation was found therein; perhaps they were not inclined to do it. The guttation from a branch of white pine (Pinus strobus L.), about 10 cm in length, was induced by applying a pressure of 200 kPa for 2 hours or 100 kPa for 4 hours (Figure 2.2). A high resistance to water flow through the xylem probably explains why guttation is never seen in pine under natural conditions. Exudation of water from detached pine needles is considered to occur due to processes distinct from those causing guttation. Figure 2.2  White pine shoot showing This artificially induced guttation probably is the first guttation with a pressure of 100 kPa applied observation of this phenomenon in pine. O’Leary (1966) to the cut stem [Source: Klepper and concluded that pressures of less than 100 kPa applied Kaufmann 1966]. in this manner produced normal guttation in many species. In further experiments with pepper leaves, a pressure of 10 to 20 kPa for 0.5 to 2 hours caused guttation resembling that of intact plants. However, a pressure of 30–40 kPa caused abnormal injection of the base of the leaf blades, without normal guttation. Pressures from 20 to 100 kPa applied to the base of the stem of pepper plants caused normal guttation from the leaves, whereas a pressure of 150 or 200 kPa resulted in rapid guttation from nodes and from cracks in the stem and petioles. In the absence of a pressure chamber, guttation can also be induced in excised plants by fixing them in a partially water-filled ordinary stove and then applying hand pressure, though these experiments were designed to study leaf rolling and unrolling phenomena in rice as an indication of pressurized root water transport to leaves through the shoot (Singh and Singh 1989; Singh et al. 2009a).

2.5  Measurement of guttation Unless stringent precautions are taken it is difficult to recover adequate volumes of guttation fluid and materials present therein for further studies without the accidental introduction of contaminants and partial loss of its volume. However, in view of its microbiological, phytopathological, ecological, physiological, biochemical, pharmaceutical, and agricultural implications (Chapters 7 and 8), particularly in such an advanced era of scientific revolutions, quantifying the guttate and determining its constituent elements are considered essential. In this area of research, many researchers succeeded in developing techniques for collecting the guttate and quantifying it (Komarnytsky et al. 2000; Pedersen 1993, 1994; Singh et al. 2008, 2009a; Wagner et al. 2004). Moreover, qualitative assessments of guttation were made through simple visual imaging, hand lense, microscopic imaging or by using electronic wetness-sensing detectors (Richards 2004). Quantitative assessment, on the other hand, was attempted either by collecting guttation fluid in micro-glass capillaries or in test tubes or Petri dishes as the fluid drips from leaves or through adsorption of exudates on adsorbent material such as blotting paper. These techniques are briefly described hereunder.

Principles of Guttation and Its Quantification     25

2.5.1  Qualitative

assessment

Wetting of leaves with guttation fluid has important phytopathological significance (Chapter 6). It is, therefore, necessary to study the shape and size of guttation droplets vis-a-vis dew deposition and evaporation, which can be done in different ways. 2.5.1.1  Image analysis of guttation droplets The shape of the droplets and their location on the convex versus concave sides of the plant coleoptiles or for that matter leaves as well, can be quantified using a model dominated only with surface tension parameters. Using this technique, Richards (2004) examined the droplets of guttation exudate of plants and recorded their images. 2.5.1.2  Measurement of shape of guttation droplets Often, in the morning on a sunny day, one can observe glare spots generally associated with the uncolored ‘rainbow’ caustics on account of sunlight reflected from the surface of dew or guttation drops. Adler et al. (2008) used this physical law for measuring the shape of guttation droplets seated on leaves. These dewdrops—or guttation drops—caused reflection rainbows due to places on the droplet (i.e. from an ‘inflection circle’) where the Gaussian curvature becomes zero (Lock et al. 2008). Though this technique is not quantitative in nature, it can be used for measuring the shape of guttation droplets and thus used for ecological studies. 2.5.1.3  Measurement of size of guttation droplets The size of guttation droplets, produced by leaf tips and margins, provides an approximate measure of guttation and is considered ecologically significant; for example, it impacts plant survival under semi-arid conditions. A study conducted by Hughes and Brimblecombe (1994) presented the average guttation droplet diameter to be 1.49 ± 0.16 mm as compared with 0.20 ± 0.02 mm for dew drops formed on the leaf of grass Holcus lanatus (Yorkshire fog). The results suggested that the average total guttation volume per blade of the grass in a single night is 1.0 ± 0.3 × 10 −7 dm3. This compares with dew, representing precipitation equal to 0.1 mm. Importantly, these authors suggested that in southern England, about 8 percent of the mean daily June-August net radiation energy would be required to evaporate the average dew- and guttation-derived leaf wetness, amounting to 0.25 ± 0.04 mm, which indicates the ecological significance of dew and guttation. 2.5.2  Quantitative

measurement

Quantitative measurements are essential for understanding the physiological functions, the ecological relevance with respect to soil–plant–animal–environment systems, and the phytopathological implications of guttation. Simple quantitative measurements can yield important information on concentration and total amounts of organic and inorganic solutes in guttation fluids. Quantitative measurement at field level on unit land area and unit time basis is, however, difficult at present, which suggests the need for attempting guttation modelling in the future. The following sections, however, briefly describe various laboratory and individual plant-based techniques for the quantitative measurement of guttation fluids.

26   Guttation

2.5.2.1  Measurement of guttation through mass collection of leaf drippings A number of workers have quantified guttation by collecting and measuring leaf drippings. Drippings can be collected in test tubes or adsorbed on blotting papers as they drip from the leaf tips. The collected materials can be used for different purposes, that is, for the study of the effects of ions on guttation intensity in plants (Raleigh 1946a), pathogenicity in crops (Smith and Olien 1978), or comparison of guttation with the amount of dewfall on crops (Luo and Goudriaan 2000). However, the true volume of the collected material is uncertain; therefore, determining a satisfactory index for the amount of guttation collected is difficult using this technique. Apparently, these methods lack precision because of incomplete collection of guttation fluid as only drops beyond a certain size will detach and fall, leading to fluid loss. In addition, the loss of water through evaporation during handling can change the concentration of dissolved solutes present therein. Further, in these cases, it is impossible to distinguish between fluids from leaf tips, edges, and surfaces, if one wishes to obtain such information, and contamination with dew is yet another confounding error. 2.5.2.2  Pedersen technique for measuring guttation Pedersen (1993), working at the University of Copenhagen in Denmark, developed an approach for guttate collection from the submerged aquatic plants, that is, L. dortmanna and S. emersum, which are kept in a highly humid atmosphere. The technique involved collection of water droplets from leaf tips by using pre-weighed micro-glass capillaries. Once the guttation fluid is collected, the increase in capillaries’ weight leads to quantification of the guttate volume (as stated in Section 2.4.3). The micro-glass capillary method can be implemented to examine water transport and transfer of hormones and nutrients in plants where no transpiration occurs, such as in the case of aquatic submerged plants. 2.5.2.3  Komarnytsky technique for measuring guttation Komarnytsky et al. (2000) developed a technique, while working in his US-based laboratory, that focused on collecting the guttate using a hand-held pipette or through vacuum suction into an aspirator bottle. This method has found application in evaluating the physiology of recombinant proteins secreted by the transgenic tobacco plant through the guttate (Nicotiana tabaccum L.), thus ensuring a nondestructive way for its continuous production. This approach can be adopted for further research and large-scale production of many essential pharmaceutical products that are meant to cure many ailments. 2.5.2.4  Wagner technique for guttation measurement This technique was developed by Wagner at the University of Kentucky (USA) and involves touching individual exudate droplets atop glandular secreting trichomes (GSTs) with a solvent-filled micro-capillary that readily dissolves exudate (Wagner et al. 2004). Before exudate removal, rapid rinsing of leaf surfaces with methylene chloride or acetonitrile is preferable, which neither hinders nor appears to leach components from within the leaf. As GSTs are surface protuberances, their exudates are more easily collected through mechanical means than those of cells or components of cells embedded in tissues within the plant. This method, although tedious, because of its ability to

Principles of Guttation and Its Quantification     27

make contact only around glands leaving other portions untouched, yields potentially pure exudate. This laboratory approach is quick and completely removes large quantities of exudate, allowing for accurate estimation of the exudate amount. However, this technique inherits the drawback of estimating non-trichome cuticular components of guttation fluids as well. Although this technique holds good promise for collecting chemicals present in GSTs, it cannot be used in a crop breeding program incorporating enhanced guttation trait under field conditions (Singh 2014a). 2.5.2.5  Singh technique for measurement of guttation Singh et al. (2008, 2009a) developed a guttation measurement method based on the quantitative perspective. The method is considered user-friendly, uncomplicated, accurate, noninvasive, and quick in generating results. It is also cost-effective and eco-friendly as it does not involve any toxic chemical substances. When compared with other quantitative measurement tools, the Singh technique saves considerable research time, as the instrumentation part is also simple and uncomplicated. The method uses blotting paper of varying sizes to soak the guttate from the exudation sites, such as the tip of the leaf or its margin or surface, before it trickles down. Following this, the soaked blotting paper is placed in air-tight glass vials and weighed. The guttate is evaporated by placing the opened vials in an oven for 24 hours at 80°C. Then the glass vials are sealed, cooled down using a desiccator, and weighed. The observed difference in the weight of the glass vials states the guttation amount. Making things a bit simple, the volume of guttation fluid that is determined gravimetrically and stated either for a single leaf, whole leaf area, or dry weight of leaf per unit time can also be stated volumetrically. This can be done by ignoring the content of the guttate (solutes) that can be considered as practically insignificant by weight. This technique can be implemented while selecting high guttation cultivars from a large germplasm pool, and it can be followed in transgenic breeding to obtain high-yielding varieties. This is because a positive correlation was found between sink strength of the panicle holding the grains and the guttation volume, especially in case of varieties of rice (Figure 2.3). Further research can be carried out on the exudated guttation fluid to determine its organic as well as inorganic contents by using suitable solvent elution methods. Before collecting the guttate, the blotting papers are washed with methyl or ethyl alcohol for sterilizing and freeing them from possible contaminants (Singh 2014a). The development of a technique for the quantification of guttation per unit land area and per unit time at the field level is presently a challenge, which should be addressed and evolved. However, for physiological, ecological, Figure 2.3  The relationship, for six rice cultivars, phytopathological, molecular biology, and between the rate of guttation during pre-heading stage relevant breeding activities, this technique and their panicle weights (the yield-sink potential) [Source: Singh et al. 2008]. holds promise.

28   Guttation

2.6  Guttation intensity The guttation amount is dependent on the guttation intensity and its duration, which in turn are regulated by the genetic makeup of the plant, along with various other environmental and abiotic factors (Singh 2016a, b). The guttation volume is also variable. The tropical rainforest trees grow in a warm and highly humid environment, thus resulting in guttation mimicking the effect of rain (Feild et al. 2003, 2005; Fisher et al. 1997). As stated above, the average of total guttate volume per blade of grass in a single night was 1.0 ± 0.3 × 10 −7 dm3. The value was similar to the amount of dewfall on the leaf surface of a short grass (Hughes and Brimblecombe 1994). Normally, guttation occurs at leaf tips as well as along the margins; however, in case of rice, it guttates profusely in situ during dusk, and through the tip, margin as well as adaxial and abaxial leaf surfaces (Singh et al. 2008, 2009a). Some of the tropical rainforests guttate at a high rate when the nights are highly humid and the water drips off the leaves (Feild et al. 2003, 2005). A single leaf of Colocasia antiquorum can guttate up to 100–250 mL in a single day (Dixon and Dixon 1931; Flood 1919; Stocking 1956a; Tazaki 1939). Some plants can contribute so much water to a channel in some areas through natural guttation, a condition called ‘cloud stripping’, that the creeks dry up when these plants are cleared. On the other hand, in the experiments conducted by Raleigh (1946a) on tomato, it was difficult to determine a satisfactory index for the amount of guttation as there was always likelihood of loss of much of the solution guttate during collection by placing a test tube under each drop. Yet, it was possible to collect 1 to 3 mL of solution from the plants at one time with a repeat of the collection every few hours. At times, guttation can be observed on the stems of plants, especially after leaf injuries or lenticels, fruits, flowers, and so on. The fungus named Pilobolus has a high guttation rate. Likewise, in case of Polyporus squamosus, water droplets are exudated from polypores. These fungi are parasitic and saprobic in nature, which grow on dead/living trunks of deciduous forest trees. They exhibit ‘dewatering’-like appearance, which in turn indicates intensive guttation relative to higher plants. This particular fungus offers a good example of guttation in the fungal group (Figure 1.2). The difference between bleeding and guttation in a variety of rice seedlings was presented by Fujii and Tanaka (1957). The intensity of guttation was high in case of rice varieties that matured late as compared with those that matured early. In case of early maturing varieties, the comparative guttation rates was 100, whereas it was 121 in case of intermediate maturing varieties, and 160 in case of late maturing varieties. Variations in the guttation process also occur due to the change in various parameters of culture solution, such as its composition and age. Thus, high guttation rates were observed in case of primary or young leaves of barley seedlings 6–7 days old that were cultivated using mineral nutrients solution (Dieffenbach et al. 1980a,b). The young leaves take up K+ ions from the medium, thus changing the concentration in the medium to below 1.5 mM. Additionally, the presence of K+ ions in the guttate is directly proportional to the concentration of K+ in the nutrient medium. The average guttation rate was found to be in the range of 1.4–2.4 mm3 h−1 per plant when K+ concentration remains within 10–20 mM; however, exuding plant varieties showcase 4.2–7.6 mm3 h−1 flow rate at 35–55 mM of K+ concentration. Pedersen (1993) carried out guttation studies on plant species like S. emersum and L. dortmanna, which are submerged plants with high guttation rates shown by their leaves. It was 10 times higher in case of the young leaves of S. emersum (2.1 µL leaf−1 h−1) compared with the youngest leaf of L. dortmanna (0.2 µL leaf−1 h−1). In S. emersum, the guttate fluid volumes were recorded to be 2.13

Principles of Guttation and Its Quantification     29

µL and 0.30 µL h−1 respectively in the first and second leaf, whereas the third leaf showed rare guttation. This showed the average guttation rate to be 0.006 µL h−1. The fourth leaf and all other leaves older to that showed no guttation. The variable guttation rate among leaves of different ages could probably be attributed to age-dependent changes in hydraulic conductance which indicates that the phenomenon is substantial and significant for the plant. A previous study focused on collecting guttate from genetically modified 2-month-old tobacco plants. The guttate’s volume was determined to be 1–2 mL/g of dry weight of the leaf per day, which consisted of 20 mg mL −1 (i.e. 40 mg/g leaf dw per day) of the total soluble protein (Komarnytsky et al. 2000). A similar study was also carried out with bean leaves and rice plant with a guttate volume of 6 mL/cm 2 and 25 mg/mL, respectively (Yarwood 1952). The rice leaf guttate consisted of copper-folin-positive materials (Ozaki and Tai 1962). Singh et al. (2009a) found a high guttation rate in case of rice that extended up to 271 µL/h per leaf tip. They observed that variation in the genotype determines significantly the rate of guttation which was 62–110 μL. Narendra Deva Rice Hybrid-2 (NDRH-2) cultivar showed the highest guttation volume as compared with the cultivar Mahsuri, which showed the lowest rate. The growth stage of the rice plant has a direct impact on the guttation rate. Observations recorded for guttation from leaf tips at tillering, heading, anthesis, milk, dough and maturity stages clearly brought out that guttation was maximum at anthesis (132 mL) followed by tillering (120 mL), heading and milk stages being almost equal (112 mL), while at dough stage it was minimal (82.7mL). At the maturity stage, no guttation was observed in case of rice plants. Conversely, during all the growth stages, the guttation progress was slower in the initial stages (11–20 µL per leaf tip), which later increased with time (83–132 µL). Thus, during most of the other stages, the guttation rate was moderate and usually varied between the two extreme values presented above. From a single vigorous 30–40 feet high Indian date palm (Phoenix sylvestris) tree, exudation or bleeding other than guttation under certain conditions has been found to yield as much as 5–6 L of sap in a single day and as much as 150 L of sap from the upper end of the trunk, although the average yield was about 25–75 L of sap in a season (Bose 1923). The highest bleeding, about 50 L per day, was found in Caryota urens. Losses of as much as 5–7 gallons of exudates were recorded for individual vines when new cuts were made every other day (Winkler et al. 1962). It is obvious from these descriptions that a wide range of variability exists in guttation rate and the chapter ends with the details of general principles of guttation, its induction, various techniques for its quantification and the variation in the intensity of natural and experimental exudation giving way for the next chapter on the mechanism of guttation.

C h a p t er

3

Mechanism of Guttation

3.1  Introduction As explained in Chapter 1, guttation is the process of exudation of liquid through permanently open pores called hydathodes located at the tips, edges, and surfaces of uninjured leaves in a wide range of plant species (Singh and Singh 2013; Singh 2013, 2014a,b, 2016a,b). This phenomenon is now known to play a significant role in the soil–plant–animal–environment systems (Chapter 7) and in the production of a number of recombinant proteins and pharmaceuticals, that is, drugs and vaccines, for animal and human use (Chapter 8). Despite these advances, the mechanism of this event is, however, not fully understood. What exactly triggers guttation when the environmental conditions favor low transpiration, particularly during night, is unknown. This chapter attempts to present the ‘cause and effect’ elements of events such as bleeding, oozing, exudation, and root pressure in an integrated manner to represent the whole guttation process.

3.2  Mode of guttation The mode of guttation constitutes a sequence of events supposedly originating in the roots, travelling through the shoots, and finally culminating in exudation as guttation from the hydathodes of leaves in a liquid form. For instance, transpiration from a vigorously growing tomato plant will cease under a bell jar following saturation of the atmosphere in the jar, but continued absorption of water through the roots and its ascent under pressure from below would result in a slow exudation of water from the tips and edges of the leaves. At night or briefly even before sunset, transpiration usually does not occur because the stomata of leaves close, but guttation appears as water drops. Guttation is mostly noticed in plants growing on well-irrigated land or in humid habitat. Water enters plant roots because their water potential is lower than that of the soil, and the continuance of this process leads to its accumulation in the plant, which creates hydrostatic pressure. This pressure forces some water

Mechanism of Guttation    31

to exude through the hydathodes as guttation drops. This pressure, which is essentially a ‘pushed up’ rather than a ‘pulled up’ phenomenon, provides the basis for the exudation force, giving rise to upward sap flow and finally its oozing out of leaves as guttation (Canny 1995, 2001; Singh and Singh 1989; Singh et al. 2009b). Hence, it is generally agreed that the guttation process occurs due to root pressure (Barrs 1966; Canny 1995, 2001; Dustmamatov et al. 2004; Klepper and Kaufmann 1966; Kundt 1998; Kundt and Gruber 2006; O’Leary 1966; Pedersen 1993; Zholkevich 1991). The condition mentioned above develops mostly during the night; hence, guttation often takes place before sunrise. The hydrostatic pressure in the xylem vessels, which depends on the resistance of the conduits, acts as a feedback in the ‘pumping activity’ of the root. However, it is unclear at the moment whether stem pressure or some other local pressures are also involved in guttation; this needs to be investigated in depth to unravel the mode of guttation or forces aiding it.

3.3  Mechanism of guttation From time to time, new hypotheses and theories, as it happens in science, have been put forward by various workers to explain the mechanism of upward movement of sap, a primary and necessary requirement for guttation to occur at least in vascular plants as the sap has to travel from the roots up to the tips and edges of leaves for its exudation as guttation droplets. These hypotheses, theories, and postulates constitute a part of the vital force theory or physical force theory or root pressure theory of the ascent of sap in plants. However, guttation from the coleoptile tips of cereals wherein vascular systems are not yet developed (Mcintyre 1994) and the fungi, particularly Polyporus sps. and Pilobolus sps., which guttate vigorously without or with under-developed vascular systems, appears to take place in either case through mechanisms other than those involved in the upward movement of sap (Tarakanova et al. 1985; Tarakanova and Zholkevich 1986) or inclusive thereof. The above-mentioned theories are discussed hereunder separately. 3.3.1  Ascent

of sap in plants: a key to exudation

3.3.1.1  Theories of ascent of sap Vital force theory of upward sap movement that leads to fluid exudation Vital force theories suggest that the ascent of sap, which results in fluid exudation, occurs due to the vital activity of plant tissues and living cells. Explaining the sap’s upward movement, Godlewski (1884) proposed a theory called the ‘relay pump theory’ and Bose (1923) propounded a theory called ‘propulsive pulsation theory’ (Shepherd 2012; Singh et al. 2009b). On presuming the results of vital activities, the root cells seem to be capable of water absorption against hydrostatic pressure under conditions of reserve food supply and active growth of roots. For example, vigorously growing vines in the spring exhibit excessive guttation, manifesting or expressing sap under pressure. Contextually, the theory of propulsive pulsation by Bose (1923), a physicist who later became a plant physiologist, popular as the father of Indian plant physiology (Shepherd 2012), is specifically interesting. The pulsatory activity of the inner cortical cells located in the endodermis was likened by him to that of ‘heartbeats’, and this activity naturally and automatically forces water into the xylem vessels, which continues to move upward (Shepherd 2012). Nevertheless, these theories of vital force were

32   Guttation

rejected, although erroneously (Wegner 2014), as living cells were considered inessential for the ascent of sap because when the living cells were destroyed with picric acid or HgCl 2 solution, a metabolic inhibitor, the ascent of sap still continued. However, the recent discovery of contractile proteins (Abutalybov and Zholkevich 1979; Abutalybov et al. 1980; Baluska and Volkmann 2008) and aquaporins (AQPs) (Kaldenhoff et al. 2008, 2014; Maurel et al. 2008; Steudle 2001), along with the effects of excision of roots and leaves, pneumatic application of pressure to the excised stem with leaves existing, and using metabolic inhibitors on the ascent of sap in rice (Singh et al. 2009b) have questioned the rejection, favoring a regeneration of the debate on the pulsation theory by Bose. This book’s author, like others, believes that the guttation process involves vital forces or activities for the development of root pressure (Kundt 1998; Kundt and Gruber 2006; Pickard 2003a,b; Singh 2016a,b; Wegner 2014, 2015a,b). Physical force theory of upward sap movement that results in fluid exudation This theory states that different physical principles like electro-osmosis, capillary action, imbibitional forces, atmospheric pressure, and cohesion-tension (Bohm 1893; Dixon and Joly 1895; Dixon 1914; Huber 1956; Levitt 1956; Milburn 1979; Nobel 2005; Plumb and Bridgman 1972; Steudle 2001; Tyree 2003) drive the ascent of sap, which results in guttation. However, whether the involvement of these physical forces, except cohesion-tension, leads to the upward movement of sap, particularly in tall trees, has not been experimentally confirmed. Lundegardh (1944), for example, could not observe a potential difference of more than 100 millivolts between the surface and inside of wheat roots. His calculations instead stated the requirement of a potential difference of about 150,000 millivolts for raising water to a height of 100 cm in tubes 0.63 µm in diameter, whereas the wheat root vessels are wider. It thus seems that electro-osmotic forces might be insufficient to maintain pressure differences of water in vascular plant membranes. Because earlier physiologists (Flood 1919; Priestley 1920; Tazaki 1939; White 1938) knew the phenomena of bleeding and guttation, they probably developed ideas that pressures developed in the xylem, due to a unidirectional inward flow of water, were responsible for fluid exudation. For the upward movement of sap, a theory about the polar movement of water based on permeable differences was also applied. However, none of the ideas succeeded in explaining the phenomenon the way it was necessary for guttation or bleeding in plants to occur. The cohesion-tension theory for the ascent of sap (Dixon and Joly 1895; Dixon 1914; Scholander et al. 1965; Steudle 2001; Tyree 2003) has been widely accepted, but not without contradiction, criticism, and controversy (Canny 1995; Kundt and Gruber 2006; Tanner and Beevers 1999, 2001). However, guttation generally occurs during the night, wherein transpiration, which creates a condition of cohesion-tension that generates a pulling force for sap to ascend, seemingly does not exist or exists at a low magnitude, which alone may not be enough to induce guttation (Kundt and Gruber 2006; Pedersen 1993, 1994, 1998; Tanner and Beevers 1999, 2001). Yet, its complementary contribution to guttation cannot be ruled out altogether (Tyree 2003). Thus, a combination of mechanisms probably leads to guttation. Root pressure theory of upward sap movement resulting in exudation Stephen Hales (1727) coined the term ‘root pressure’ and for the first time published an account of the phenomenon of heavy bleeding and bleeding pressures developed by a grapevine that was chopped

Mechanism of Guttation    33

off. This phenomenon, while explaining guttation, is considered an integration of water absorption, hydrostatic pressure, and upward translocation instead of separate processes. Contextually, a comprehensive account of the root pressure mechanism, its regulation, and its importance in plants was recently published by the author of this book (Singh 2016b), and those interested may refer to this study and those of Wegner (2014, 2015a,b) for the latest informative knowledge regarding this phenomenon. Guttation is also prioritized as the manifestation and expression of pressure in the roots and also called ‘exudation pressure’. Generally, the roots cause positive hydrostatic pressure through the absorption of ions from the soil and their collection in the xylem (Pedersen 1993, 1994; Stocking 1956b; Zholkevich 1991). The solutes that accumulate in the sap of the xylem lead to a negative osmotic potential, and the water potential in xylem decreases. The push for the absorption of water, which generates a positive hydrostatic pressure in the xylem, is caused by the lowering of xylem water potential (Figures 3.1 and 3.3). This pushes the water up and out of hydathodes as guttation, against the gravitational pull (Crafts and Broyer 1938; Dustmamatov et al. 2004; Pickard 2003a,b; Priestley 1920; Stocking 1956b; Taiz and Zeiger 2006; White 1938; Zholkevich 1991). The root pressure may lead to high rates of ion accumulation in plants, a consequence that cannot be avoided (Gaxiola et al. 2007; Morth et al. 2011; Palmgren 2001). The positive pressure within the xylem acts as an adaptive strategy developed at night that may help the gas bubbles dissolve, thus playing a role in reversing the deleterious effects of cavitation and embolism (Brodribb and Holbrook 2006; Holbrook et al. 2001; Singh et al. 2009b), keeping the water column in its place. Temperate climatic conditions are suitable for guttation, and the root pressure develops during warm nights, although water transport takes place during the day due to transpiration pull (Steudle 2001). During spring, a constant pressure is developed in a wide range of plant species, including herbaceous species and deciduous trees. Once leaves expand, water starts moving rapidly and the root pressure becomes undetectable although it has an invisible existence (Feild et al. 2005; Feild and Arens 2007). A fluctuation in root pressure always exists within a general range of 50–300 kPa depending on the species, growing conditions, environmental and edaphic factors, etc., which indicate seasonal and diurnal periodicity. 3.3.1.2  Magnitude of root pressure and exudation From time to time, efforts have been made by various workers to quantify directly or indirectly the root pressure developed in plants (Singh 2016b). The results of the quantification of root pressure varied depending on the techniques used, for example, the pressures measured with root pressure probe were higher by 0–0.5 MPa than those measured with the pressure chamber method. However, Wei et al. (1999) found no difference in the magnitude of root pressure measured using these two methods. As early as 1925, Sabinin reported that exudation ceased at an external osmotic pressure of 0.5–1.5 atm (50–150 kPa), whereas Tagawa (1934) found that an osmotic pressure of 1.9 atm (190 kPa) in the root medium was sufficient to prevent absorption through isolated bean roots, while 14.7 atm (1470 kPa) was required to prevent absorption through intact plants. Rosen (1941), in a similar experiment on onion, found that an external osmotic pressure of 1.8 (180 kPa) to 3.3 atm (330 kPa) prevented water absorption by the detopped root system, while that of 4.2 (420 kPa) to 5.7 atm (570 kPa) represented the limits above which intact roots of onion were unable to absorb water. On the other hand, it was suggested that the root pressure amounting to 64 kPa is sufficient to refill the embolized vessels of roots and stems just above the roots (Ewers et al. 1997). These results provide

34   Guttation

Figure 3.1  Morphology and anatomy of root systems and water and solute collection and transport. (a) Whole plant with root and shoot [Source: O’Toole and Chang 1978). (b) Root system [Source: http://www.greenmanspage. com/guides/logistics.html]. (c) Prolific root hairs [Source: https://www.pinterest.com/pin/506232814336189676/]. (d) Magnified view of root hairs [Source: http://www.daviddarling.info/encyclopedia/T/trichome.html]. (e) Radial flow of water and solutes in roots [Source: http://www.pleasanton.k12.ca.us/avhsweb/thiel/apbio/labs/ plant_transport.html].

evidence again that, in general, the maximum root pressure that most roots are capable of developing is between 2 (200 kPa) and 3 atm (300 kPa). Although root pressures amounting to 1 (100 kPa) to 2 atm (200 kPa) have frequently been observed (Knipfer et al. 2007; Steudle et al. 1987), higher values have also been occasionally recorded, for example, White (1938) reported pressures of 600–700 kPa in excised tomato roots. In fact, plants, depending on their nature of growth and habitat, require excess internal pressure, which can amount to 0.6 MPa in tomato plants, 1 MPa in grasses, or even up to 6 MPa in certain desert plants (Kundt and Gruber 2006; White et al. 1958). However, some of the high pressures observed within stems were undoubtedly a result of local stem pressures and not true root pressures (Kramer 1939). A pressure of 700 kPa, though capable of causing a flow

Mechanism of Guttation    35

of water in the xylem of tall trees, may not be sufficient to push the water in most of the tall trees in view of the existence of resistance to flow, cavitation, etc. However, work on excised roots may lead to errors when root pressures are quantified with respect to the intact plant. The shoot is a leaky plug that, by itself, affects the magnitude of the effective pressure. On the other hand, Davis (1961) recommended that high force exists in the living cells of the root, which causes the flow of sap. These magnitudes of root pressure will definitely not be problematic for horticultural as well as agricultural crops (Singh et al. 2009b; Singh and Singh 1989; Tanner and Beevers 1999, 2001), palm trees (Davis 1961), woody and vine-like lianas (Fisher et al. 1997), bamboos (Cao et al. 2012; Zachary 2009), or deciduous forest trees (Feild et al. 2005; Feild and Arens 2007) generating enough force on their own as an alternate option for and/or complementary as well as supplementary device to cohesion-tension mechanism as the natural situations arise for upward movement of sap in plants that go through rapid transpiration (Steudle 2001). The existence of positive pressures in the xylem is an evolutionary advantage that helps the gas bubbles dissolve, thus reversing the harmful effects of freeze- and drought-induced cavitation and embolism (Brodribb and Holbrook 2006; Holbrook and Zwieniecki 1999; Holbrook et al. 2001; Kaufmann et al. 2009; Tyree 2003a,b; Singh et al. 2009b; Zwieniecki and Holbrook 2009). 3.3.1.3  Pressure in the shoot and leaf causing bleeding and guttation The bleeding of sap from cuts, bore holes, and wounds in trees of some species do not act as attributes of root pressure but rather occur through local internal conditions within the stems or branches themselves. This phenomenon is well described by the flow of sap from cuts and bore holes in sugar maple (Acer saccharum). Sometimes, the amount of sap obtained from one single vigorous tree is 150 L. In summer, huge quantities of starch, sucrose, and hexose in the xylem are found to be associated with the bleeding phenomenon in sugar maple. During winter, before the flow of sap begins, hydrolysis occurs in the wood (Johnson 1945). Other examples of bleeding that results from local development of stem pressure are various kinds of palms. There has been a record of 10 L of sap in a day from Phoenix dactylifera and more than 1000 L of sap in a season from a single Indian date palm (Phoenix sylvestris) (Bose 1923). A single Palmyra tree (Borassus flabellifera) has the capacity of yielding up to 120,000 L of sap in its life time on account of accumulated carbohydrates in the stem. It is obvious that these exudations of sap can neither be dependent on root pressure nor is water being immediately supplied by the roots, but is due to withdrawal of sap directly from the trunk. This flow of sap and its mechanism are still not clear and understandable (Kramer 1949), however, there is no concrete reason behind such local pressures being not present causing guttation or exudation of fluid from the margins and tips of leaves that are not injured as bleeding in rice peduncle neck that influences quality of panicle-sink was noted (Quanzhi et al. 1999). 3.3.2  Mechanism

of root pressure

To familiarize the readers, the author of this book thinks it necessary to elaborate various components and chain of events involved in the development of root pressure, which is actually the driving force for guttation. As is known, roots not only absorb most of the water consumed by plants but also pump water into their shoots; the root pressure being the driving force for such pumping, which has been studied rather intensively. However, a series of highly contradictory hypotheses regarding the

36   Guttation

nature of root pressure have been advanced. Currently, mainly two mechanisms, that is, osmotic mechanism (Priestley 1920) and metabolic mechanism (Dustmamatov et al. 2004; Ginsburg and Ginzburg 1970, 1971; Oertli 1966, 1986; Singh 2016a,b; Wegner 2014, 2015a,b; Zholkevich 1991), or both in combination, have been advanced to explain the phenomenon of root pressure. They are discussed separately hereunder. 3.3.2.1  Osmometer model of root pressure According to this concept, the root behaves like an osmometer: xylem sap plays the role of a concentrated solution within the osmometer, whereas the endodermis together with all the root parenchyma cells plays the role of a semipermeable partition, which prevents an efflux of osmotically active solutes from xylem sap (House 1974; Kramer and Boyer 1995). Thus, as discussed earlier, only xylem sap sucks water and living cells do not promote water flow; on the contrary, they only resist it. It is known that guttation, as well as bleeding, occur due to root pressure in the xylem. Osmotically active solutes that are found in the lumen of dead xylem lead to water influx in root cells, which is dependent on water’s diffusion pressure that increases in xylem. Thus, exudation in case of cut stem takes place under a pressure which equates to difference in osmotic pressure inside the xylem vessels and that of the external medium through the diffusion of water. The root cells are multicellular as well as semipermeable in nature. The cells possess a turgor pressure, which is dependent on the internal osmotic pressure and external diffusion pressure of nearby cells; this creates a diffusion gradient till water conductivity exists (Arisz et al. 1951; Bai et al. 2007; Crafts and Broyer 1938; Eaton 1943; Lundegardh 1944, 1950; Priestley 1920; Sabinin 1925; Zhu et al. 2010). Sabinin (1925) presented the relation in the form of an equation: b = k (Ob − Om) Where b is equal to the rate of bleeding, k is the osmotic coefficient, and Ob and Om are the osmotic pressures of the xylem sap and the external medium, respectively (Arisz et al. 1951). It is assumed in this equation that solute concentration is the only factor influencing the diffusion pressure of water, both inside the xylem and in the external medium, that is, the roots must be growing in culture solution not in soil and no hydrostatic pressure exists in the xylem and no non-osmotic or electro-osmotic forces are acting within the root. Even if the process of bleeding is entirely an osmotic phenomenon, the continuation of the process depends on the movement of osmotically active materials into the dead xylem elements. This highlights two mechanisms, that is, secretion of salt into the xylem and osmotic influx of water. In the bleeding process, a certain amount of water binds to ions, hence a certain amount of movement of water is inherent in the process. In most ion channels, transport is in the single file mode, and indeed ions and water pass at a fixed stoichiometry (Figure 3.1), but this does not affect the osmotic process (Arisz et al. 1951; Lundegardh 1944, 1950). In addition to this, root metabolism shows a continual shift in the total concentration of such ions within the root cells. A starch–sugar equilibrium shift occurs, followed by the incorporation of nitrate into protein, with continuous influx and outflux of salt. All these factors lead to a continual shift in water balance in the living cells (Lundegardh 1950). However, the net water movement depends on differences in diffusion and osmotic pressures that function in the xylem cells and root, respectively. Thus, an osmotic mechanism of root pressure is dependent

Mechanism of Guttation    37

on salt transport into the xylem, and in guttating plants, salt must be loaded into the xylem at a rate that maintains the osmotic pressure of the xylem sap despite continuous dilution by water in case guttation is driven through osmosis. As stated above, if the bleeding obeys the osmotic laws, then the bleeding rate is a function of osmotic pressure formed in the external medium. Thus, these two factors seem to be indirectly proportional. For example, the bleeding stops when the osmotic pressure of the internal fluid is equal to that of the bleeding sap or osmotic pressure in the water absorption region. Many researchers have studied this phenomenon and stated that placing the roots in an osmotic solution of varying strengths showed a modified osmotic value of the exudate fluid (Arisz et al. 1951). Considering the exudate’s backward movement while placing the roots in a plasmolyzed solution, Heyl (1933) explained that the bleeding phenomenon is based on osmosis. van Overbeek (1942) stated that the exudate’s osmotic pressure observed in case of detopped tomatoes was low by 1 atm as compared with that of the mannitol solution, which halted the bleeding process. This might be due to the non-osmotic water oozing into xylem vessels. The inhibition effect was reversed with the addition of potassium cyanide (KCN). Eaton (1943) attempted to identify the ‘active component’ in the absorption of water. He explained the phenomenon of bleeding and root pressure based on variations found within the xylem vessel’s osmotic pressure and total osmotic and capillary forces present in the external medium. Eaton (1941) stated that cotton leaves exudated when the osmotic value of xylem vessels was high as compared with the external medium. According to his explanation, the cells absorb mannitol, and subsequently a higher concentration medium is needed to prevent bleeding in case mannitol remains unabsorbed. However, this explanation is wrong. As long as only osmotic forces are involved, the osmotic pressure of the cells separating the xylem and external solution is irrelevant and only mannitol movement into the xylem would affect the whole process. However, Arisz et al. (1951) did not conclude that mannitol is absorbed by the root cells. He rather showed that transferring the roots into mannitol solution lead to the secretion of salt into the xylem cells. Thus, a new equilibrium is reached, which prevents bleeding due to increased concentration of xylem sap. Sabinin’s equation, though it refers to steady-state situations, would, therefore, be valid only for instant modifications. Furthermore, modifications were observed in the composition and concentration of the external medium, which further affects the permeability of root cells to water (Arisz et al. 1951). This particular effect is found especially in high leaps or jumps in root habitat, i.e., when the roots are placed in a new environment a few times. The sudden change in osmotic pressure in the external medium has a significant effect on the bleeding rate. As per the findings of Arisz et al. (1951), these outcomes can be explained without accepting the fact of ‘active water secretion’. Several possibilities have been suggested by these researchers, which are as follows: (1) upward transportation of xylem sap to the injured region from the absorption region, and there is exchange of solutes and water (Eaton 1943); (2) if the water absorption region is larger than the salt absorption region, there will be influx of water, causing dilution, and (3) the secreted salt water may be transported into xylem along with the ions (Lundegardh 1950). Thus, phenomena such as root pressure, guttation, and bleeding are expressions of the active absorption of solutes (Klepper and Kaufmann 1966). Although root pressure and guttation presumably result from a high concentration of salts in the root xylem, the guttation fluid is dilute. Measurements of the osmotic potential of the guttation liquid and that of exudates at various plant heights in guttating plants indicate that salt is removed from the xylem

38   Guttation

from the upper part of plants, particularly the leaves. The osmotic potential of the guttation fluid is not influenced by the concentration of salt solutions forced through individual leaves by an applied pneumatic root pressure. This suggests that leaves play an important role in retrieving and removing salts from the xylem of guttating plants (Klepperand and Kaufmann 1966). In conclusion, such facts demonstrate the complicated nature of root pressure (Dustmamatov et al. 2004; Zholkevich 1991), and, briefly, the development of root pressure appears to follow or accompany active transport and accumulation of salts into the xylem of the conducting system of roots, where the osmotic value of roots rises above that of the external solution of the medium. Thus, the observations on guttation and bleeding may suggest a simple osmotic movement of water, with the cell activities favoring the antecedent or concomitant movement of solutes. However, this is not the complete explanation as a series of facts narrated above contradict the osmotic conception and simplified schemes of the osmometer mechanism of root pressure, equating the root pumping activity to the osmometer work. As narrated above, several findings reveal the complicated nature of this phenomenon, indicating that parenchyma cells actively participate in root pressure build-up. Yet, the relatively old conception of osmotic model is still wide spread. Obviously, the dictate, direction, and control of water flow by parenchyma cells needs further in-depth investigations for the elucidation of the mechanism of root pressure (Pickard 2003a,b; Singh 2016a,b; Wegner 2014, 2015a,b). 3.3.2.2  Metabolic model of root pressure A strong and productive group of Russian workers with Zholkevich as its head at the K. A. Timriyazev Institute of Plant Physiology in Moscow advanced a hypothesis popularly known as ‘metabolic concept’ of root pressure, apart from the osmometer concept discussed earlier, which, in fact, failed to justify entirely the development of root pressure through the uptake and upward movement of water and solutes (Dustmamatov et al. 2004; Dustamamatov and Zholkevich 2008; Zholkevich et al. 2007). This concept, in reality, indicates isotonic flow of water, or even the radial flow of water, against an osmotic pressure gradient that exists between the external medium and the guttation fluid or the exudate, which the root stump secretes; this supports the idea that the metabolic process is involved in the root pressure (Enns et al. 2000; Oertli 1966; Pickard 2003a,b; Schwenke and Wagner 1992; Zholkevich 1991). Contextually, Oertli (1966) was the first person to propose that the active water transport in plants occurs at the expense of the metabolic energy, defining the process characterized by the increased water potential, whose gain, he explained, must be dependent on the decline in free energy of some metabolic process. Israeli scientists Ginsburg and Ginzburg (1970, 1971) working with corn preparations subsequently stated that the flow of water consisted of two components, one osmotic and the other non-osmotic in nature, and the non-osmotic flow of water was inhibited by the metabolic inhibitor cyanide. The authors further studied and explained that no correlation existed between the flow of water and flow of solute, suggesting that the active transport of water contributes to root pressure. Data were accumulated in huge quantities by Russian workers who suggested the involvement of metabolic process triggered by virtue of G-proteins (also known as guanine nucleotide-binding proteins, a family of proteins that act as molecular switches inside cells and is involved in transmitting signals from a variety of stimuli outside a cell to its interior) during stimulatory action of neurotransmitters, adrenalin, and noradrenalin on water transport, root exudation, and consequently root pressure generation (Dustmamatov et al. 2004; Dustmamatov and Zholkevich 2008; Mozhaeva and Pil’shchikova 1971, 1972; Zholkevich 1991; Zholkevich et al. 2007).

Mechanism of Guttation    39

The Russian scientists, who worked intensively on the metabolic component of root pressure, consider ‘sleeves’, root segments after mechanical removal of the stele, especially appropriate to study the transport of water. They stated that the sleeve exudation only takes place at the expense of the metabolic component. The osmotic component is not present in the beginning. The osmotic component gradually begins to participate in the processes of transport as the exudate fills the hollow of the removed stele. The sleeve exudation rate of 5-day-old Zea mays seedlings with the roots proved to be twice as high as that of the whole root (Korolev and Zholkevich 1990; Zholkevich et al. 1989). The metabolic component that drove the Q10 value of sleeve exudation was close to 5, whereas that of the whole-root exudation rate was close to 3 only due to the functioning of the metabolic and osmotic components, simultaneously (the Q10 value of the exudation rate attributed to the osmotic component is close to unity). Although the sleeve exudation is double that of the whole root, its exudate osmotic pressure is three times lower and its cell hydraulic conductivity is almost constant as compared with the whole root. It contradicts the osmotic conception, according to which the sleeve exudation rate should be approximately three times lower than that of the whole root. The experiments with various inhibitors and stimulators enabled establishing a number of principally important facts. First, when the energy supply was cut off with 2,4-dinitrophenol (DNP) or when the contractile protein functioning was inhibited with cytochalasin B or colchicine, the exudation of sleeves was hindered considerably more than that of whole roots. The Q10 value of exudation by sleeves and the whole roots decreased to approximately 1. Second, hinderance to exudation under the action of the above-mentioned agents was observed against the background of increasing exudate osmotic pressure and cell hydraulic conductivity. Such disproportion was expressed much more clearly with the sleeve than with the whole root. Thus, exudation hindrance was observed under conditions when, according to the osmotic conception, the exudation should, on the contrary, increase. The increase in the osmotic pressure of exudate in these cases may be the consequence of a cell injury induced by the applied inhibitors. Such an injury decreases the capability of root parenchyma cells to retain solutes. The increased hydraulic conductivity may be due to a cell membrane injury induced by the applied inhibitors. Third, -indoleacetic acid, CaCl 2, adenine, and acetylcholine considerably stimulated both the sleeve and whole-root exudation and raised their Q10 value. However, the osmotic pressure of exudate decreased, and the cell hydraulic conductivity did not change (or even decrease) in these cases, which is clearly at variance with the osmotic conception of root pressure. Thus, experiments with sleeves clearly demonstrated the participation of the parenchyma cells in root pressure build-up and the real existence of the metabolic component of root pressure, the complicated nature of the metabolic component, and the close dependence of the metabolic component functioning on the energy supply, intracellular contractile system, cell membrane intactness, and probably mediator regulation as well. The metabolic component proved to be sensitive to metabolic and energy exchange regulators and to agents inducing structural disturbances in membranes (Ginsburg and Ginzburg 1970, 1971; Mozhaeva and Pil’shchikova 1972, 1978; Zholkevich et al. 1981; Zholkevich 1991). So, the metabolic component is reduced by KCN but stimulated by adenine; 2,4-dichlorophenoxiacetic acid in low concentrations enhances, but in higher concentrations reduces, the metabolic component, and so on. Experiments with the whole-root systems of 30- to 40-day-old Helianthus annus plants showed that the metabolic component is energy-dependent, as DNP strongly decreases it. Pipolphene, an anesthetic that destroys cell membrane structure due to calcium ion extrusion, abolishes the metabolic component. In ethylene diamine tetraacetic acid (EDTA) solutions, the metabolic component

40   Guttation

decreases. CaCl 2, an antagonist of pipolphene, which stabilizes the cell membrane structure, increases the metabolic component. Somewhat unexpected are the effects of d-tubocurarine (which induces muscle relaxation and immobility of animals) and its antagonist acetylcholine (a mediator of nervous excitement in animals): the former decreases, but the latter considerably increases the metabolic component (Zholkevich et al. 1981; Zholkevich 1991). The sensitivity of the metabolic component to acetylcholine and d-tubocurarine is in agreement with the experimental results, which established that camphor (a stimulator of heart contractions) essentially influences the metabolic component (Mozhaeva and Pil’shchikova 1978), and water transport in plants is sensitive to cytochalasin B and colchicine, the specific inhibitors of contractile proteins (Zholkevich et al. 1989). These facts enable direct participation of movable and sensory cell systems in water transport. The total root pressure and metabolic component are directly proportional to each other, whereas the osmotic pressure may be inversely proportional to them. Such correlations are the basis for the assumption that the metabolic component plays a leading role in root pressure build-up (Mozhaeva and Pil’shchikova 1978). Hence, it follows that living cells participate directly in root pumping activity. If the water transport mechanism is associated with metabolic component and with the participation of living cells, it seems that the water moves via these cells and not only via the apoplast. Experiments have been performed to demonstrate the water flow through symplastic and transvacuolar pathways of a decapitated root system (Boyer 1985; Ginsburg and Ginzburg 1970). It is important to be aware that parenchyma cells directly participate in the root pumping activity. At the same time, it must be remembered that this is associated with the nature of the metabolic component. It is necessary to remember that DNP, cytochalasin B, and colchicine considerably decelerate but do not stop exudation. However, the nature of exudation essentially changes, as its temperature coefficient (in the range 20–30°C) decreases under the effect of these inhibitors from 3 down to unity (Borisova et al. 1986). It is known that Q10 values provide us with integral information regarding the nature of the process under consideration. So, one may suppose that such a strong decrease in the Q10 value is due to the metabolic component elimination. Obviously, when the energy supply and intracellular contractile system are violated, exudation continues only at the cost of an osmotic constituent. This constituent is determined using relatively simple physical processes. When the metabolic and osmotic constituents function simultaneously, the Q10 value increases to 3. As this is an average value for the two constituents, one can assume that the Q10 value of the metabolic component is much higher than 3. Therefore, the metabolic component is itself of a rather complicated nature and probably connected with a chain of metabolic processes. On the horizon of root pressure research, Wegner in Germany proposed an ‘energetically driven uphill water co-transport’ hypothesis integrating osmotic and metabolic mechanisms of water transport in causing root pressure (Wegner 2014, 2015a,b). In this context, according to Wegner, AQPs are antagonistic to the uphill water transport because they tend to short-circuit the water potential gradient generated by water secretion, and under these conditions, AQPs mediate the backflow of water from the xylem to the parenchyma cells within the xylem, relying upon the free energy gradients generated by ions and sugars. This is evidenced from the fact that the energy supplied by photosynthate and reducing power from the shoot (ultimately adenosine triphosphate [ATP]) for such pumping activity did not exhaust for a number of days. Under such conditions, the ATP must have come from substrate reserves as well as from tissue degradation, exhibiting a remarkable degree of cell membrane integrity, critical in the maintenance of diffusion barrier existing between the xylem and the solution outside for such a long period. A co-transport of water and solutes could enable

Mechanism of Guttation    41

the drive of volume flow ‘energetically uphill’ against the free energy gradient of water energized by the solute gradient. Therefore, to keep the process going, the free energy released by the solute transported must exceed the energy required for water secretion (Wegner 2015a,b). Solutes released by xylem parenchyma cells to the sap are first retrieved and subsequently carried by using metabolic energy, which maintains the concentration gradient that triggers the water secretion (Figure 3.2a,b). The release of salts by co-transporters is considered an electroneutral process (Zeuthen and McAulay 2012), which would neither discourage nor interfere with K+ re-uptake by ion channels as more negative membrane potential is required than the Nernst potential of K+, which is maintained by proton pump activity (Figure 3.2a). Evidence for ‘simultaneous’ uptake and release of K+ from root tissue was obtained using refined radioactive tracer techniques (Britto and Kronzucker 2006). A rapid, apparently ‘futile cycling’ of ions is a common biological phenomenon at root membranes for ions like K+, Na+, and Cl−, which intensifies at enhanced levels of these ions. However, the futile cycling consumes some amount of metabolic energy, and its cost–benefit ratio for the plant must be investigated thoroughly and established. The water secretion may state a part of the explanation by the arranged thermodynamics of processes that cannot be reversed, containing combined ion and water transport by the cation–chloride co-transporter (CCC) type developed with intelligence for the quantitative expression of the hypothesis on root pressure (Wegner 2014, 2015a,b). Thus, the total volume flow across the plasma membrane of xylem parenchyma cells can be expressed as

Figure 3.2 (a)  Hypothetical interplay of membrane transporters in the plasma membrane of xylem parenchyma cells for water secretion. Coupling between water and ion transport occurs in a potassium chloride co-transporter (KCC) type that translocates K+ and Cl- together with a fixed number of water molecules. Note that this transport is electrically silent. The ions are at least partly recycled via a K+ inward-rectifying channel and a Cl- –2H+ symporter, respectively. These processes are energized by the activity of a H+-ATPase that maintains the H+ gradient and hyperpolarizes the membrane to values more negative than EK+. Aquaporins may to some extent short-circuit co-transport-driven water flow if their activity is not down-regulated. Note that all transporters have been demonstrated to coexist in the plasma membrane of root stelar cells. ΔVM=membrane potential of xylem parenchyma cell, EK+=Nernst potential for K+ [Source: Wegner 2014].

42   Guttation

Figure 3.2 (b)  Alternative model for water secretion that makes use of different water ion coupling ratios in outward- and inward-rectifying K+ channels. Arbitrarily, the K+ outward rectifier is thought to carry three water molecules together with one K+ ion, whereas the inward rectifier transports water and K+ on a 1:1 basis. Both rectifiers operate alternately, coordinated by membrane potential oscillations. In this way, futile K+ cycling is organized, which drives a net water flow from the cytosol into the apoplast. Note that this K+ cycling consumes metabolic energy when the membrane potential is hyperpolarized by proton pump activity. ΔVM=membrane potential of xylem parenchyma cell, EK+ = Nernst potential for K+ [Source: Wegner 2014].

the combination of two different components, that is, volume flow that the chemical potential of water forces and volume flow that the CCC-type transporters force (Teakle and Tyerman 2010). Significantly, the CCC-type transporters, which mediate water secretion in mammalian cells, have also been demonstrated in Arabidopsis and Oryza sativa. The proposed mechanism for the pressure of root could state an explanation of refilling of embolized vessels and contribute to long-distance transport of water in trees when the cohesion-tension mechanism of water ascent either fails or diminishes to a great extent. Thus, it is possible that few of the pathways for co-transport of water and ions (solutes) may coexist in the plant membrane for achieving the rate of water secretion that is needed under different conditions, as earlier stated by Zeuthen (2010) for epithelia also, although in reality it may be even more complex. Nevertheless, the current hypothesis requires rigorous testing and verification in plants of different habits, habitats, and statures growing in different ecological zones as plants differ significantly from animals in that they are taller, making water movement and its ascent difficult, and also they lose huge amounts of water through transpiration, which is not easily compensated for by water supplied through root pressure. Further, the cavitation caused by freeze–thaw and drought-stress and its subsequent embolism require vessels to be filled with xylem sap secreted by adjacent cells. The gas phase induced by cavitation in xylem vessels, it is believed, must be completely removed to regain functionality, and also some excessive pressure needs to appear on refilling for dissolving residual gas inclusions (Brodersen and McElrone 2013; Holbrook and Zwieniecki 1999; Nardini et al. 2011; Secchi and Zwieniecki 2011; Zwieniecki and Holbrook 2009). When the transpiration rate is low, tension occurs in the vessels adjacent to each other (Cao et al. 2012). The root pressure mechanism is considered in the recommendation of Wegner (2014, 2015a, b). However, for exudation to occur, in addition to the upward movement of sap, perhaps chemicomechanical stimuli sensing by hydathodes is also involved. To a certain degree, as mentioned earlier,

Mechanism of Guttation    43

the function of hydathodes resembles secretion, and therefore, it may also depend on metabolic energy supply, cell polarization, cell membrane intactness, and sensory systems such as actin and myosin and, probably, mediator regulation as well (Baluska et al. 2006; Baluska 2010; Staiger et al. 2000; Pickard 2003a,b; Zholkevich 1991). The issue of the participation of xylem parenchyma cells in root pumping activity that causes root pressure leading to guttation must be examined thoroughly in angiospermic and gymnospermic plants of different habits and habitats. 3.4  Integrated

view of sap movement and guttation

For understanding the mechanism of both the theory of osmometer and theory of metabolism of root pressure development, it is important to have an in-depth understanding of the transport of ions and/or neutral molecules in and out of cells. Fick (1855) has contextually stated in his first law that the movement of molecules through diffusion is always spontaneous, down a gradient of free energy or chemical potential, until equilibrium is reached. The movement of any substance, whether it is ions or molecules or solutes, occurs in two ways in plants, namely passive transport and active transport. ‘Passive transport’ occurs spontaneously downhill without the use of energy in the system. In this case, after obtaining equilibrium, no further net movements of solutes occur without applying a push or energy, and the movement of substances occurring against or up a gradient of chemical potential is called ‘active transport’, which is not spontaneous as it requires force or work to be done on the system by applying cellular energy driven by metabolic activities. Among the many ways of active transport, one is to combine transport with the hydrolysis of energy-rich molecule ATP. Water can be set to move across a semipermeable membrane against its water potential gradient when it is combined with the movement of solutes (Wegner 2014). Different membrane proteins can drag up to 260 molecules of water across the membrane with the transportation of each molecule of solutes such as sugars, amino acids, and other small molecules (Loo et al. 1996). This water transport mode occurring against the usual water potential gradient (i.e. toward a larger water potential) is accomplished because solute loses more free energy than it compensates as the gain of free energy by water. The net change in free energy remains negative under such conditions. Thus, the amount of water transported this way will generally be less when compared with the passive movement of water down its water potential gradient provided no physical barrier exists to the movement. As biological transport includes sap movement and guttation, it is important to know that they can be driven by major forces like gradients in concentration of solutes, hydrostatic pressure, and electrical fields. Proton transport mainly causes the membrane potential, and the excess voltage is provided by the electrogenic H+-ATPase of the plasma membrane (Palmgren 2001). The proteins transported across the biological membranes facilitate the passage of selected ions and other polar molecules, and they are provided energy through the electrochemical gradient of protons across these membranes. Transport protein refers to three main protein categories, namely channels, carriers, and pumps, which exhibit specificity for the solutes they transport by displaying a high degree of cell diversity. Channel proteins act as membrane pores specifically determined primarily by their biophysical properties, and these channel transmembrane proteins function as selective pores, letting molecules or ions passing through to diffuse across the membrane. As channels always conduct passive transport and as the specificity of transport depends on the pore size and electric charge more than on selective binding, channel transport is only limited to ions or water (Gaxiola et al. 2007; Morth et al. 2011; Pedersen et al. 2007). The channels are not always open; they contain structures known as gates,

44   Guttation

and the external signals like voltage changes, hormones, light, and post-translational modifications such as phosphorylation can open and close the pores. For example, voltage-gated channels open or close in response to changes in the membrane potential (Pedersen et al. 2007). On the other hand, carrier proteins bind to the molecule for transportation on one side of the membrane and release it on the other side. These proteins, unlike channels, do not contain pores extending entirely across the membrane. In a carrier-mediated transport, the substance for transportation is initially bound to a particular site on the carrier protein, and therefore, carrier proteins are specialize in functioning as transporters of specific ions or organic metabolites. A conformational change in the protein happens through binding, which exposes the substance to the solution on the other side of the membrane, and the process of transport is considered completed after the substance dissociates from the carrier’s binding site. Pumps perform primary active transport of solutes against their gradient of electrochemical potential by using energy directly, usually from ATP hydrolysis. The primary active water molecules are transported energetically uphill against the free energy of water by membrane proteins called pumps (Gaxiola et al. 2007; Singh 2016a,b; Wegner 2014, 2015a,b) although mostly all the pumps or carriers transport ions such as H+ or Ca 2+; however, other pumps that belong to the ATP-binding cassette family of transporters also contain large molecules. It must be noted that H+ ion is electrogenically pumped across the membranes of plants, fungi, and bacteria as well as tonoplasts and endomembranes in plants. The electrochemical potential gradient of H+ across the plasma membranes exists due to the cytosolic H+-ATPase and the vacuolar H+ pyrophosphatase (H+-PPase), while protons are pumped into the vacuole and the Golgi cisternae electrogenically, respectively. The most prominent pumps in plant plasma membranes are those of H+ or Ca 2+, which have an outward pumping direction. Hence, for the active uptake and transportation of mineral nutrients such as NO3-, SO42-, PO42-, amino acids, peptides, and sucrose (Bienert et al. 2011) and the export of Na+, which at high concentrations is toxic to plant cells, another mechanism is required. Further, the uphill transport of one solute related to or accompanied by the downhill transport of another could be another vital way through which solutes can be transported across a membrane against their electrochemical potential gradient. This type of carrier-mediated cotransport is referred to as secondary active transport, which is forced indirectly by pumps, and the active transport uses carrier-type proteins that derive energy directly through ATP hydrolysis or indirectly as symporters and antiporters (Morth et al. 2011; Singh 2016a,b; Wegner 2014, 2015a,b). The discovery and identification of the gene involved in active transport proved to be important, adding a lot to the elaborated form of molecular properties of transporter proteins (Jasinski et al. 2003). The existence of a family of transporter gene other than individual gene in the plant genome for all functions of transport is getting clearer with time. Varied transport characteristics, regulation mode, and expression of differential tissue are intact with a family of gene, which makes provision for plants with a remarkable degree of plasticity for acclimation to a wide range of environmental conditions (Arango et al. 2003). The plasma membrane H+-ATPase, like other enzymes, is regulated by a number of factors, which include substrate (ATP) concentration, pH, and temperature. In addition to these factors, some specific signals such as light, hormones, and pathogens can reversibly activate or deactivate H+-ATPase molecules. The protein enzymes such as kinases and phosphatases that add or remove phosphate groups to serine or threonine residues on C-terminal domain can also regulate the autoinhibitory C-terminal domain of H+-ATPase. The 14-3-3 proteins that are ubiquitous enzymemodulating proteins and whose function is to bind to the phosphorylated region are believed to

Mechanism of Guttation    45

displace the auto-inhibitory domain, leading to the activation of H+-ATPase. However, recent findings indicate that the plant plasma membrane H+-ATPase is activated through phosphorylation in the C-terminal auto-inhibitory domain with no requirement for regulatory 14-3-3 proteins (Piette et al. 2011). The osmotic pressure of the vacuole should be kept highly intact so that water enters properly through the central vacuole, and the tonoplast is responsible for the regulation of ion flow and metabolism between cytosol and vacuole; similarly, the plasma membrane is responsible for the uptake into the cell. This activity is performed using a new proton-pumping ATPase called vacuolar ATPase, also known as V-ATPase, which has structural and functional differences from the plasma membrane H+-ATPase (Isayenkov et al. 2010). The physiological role of the enzymatic proteins in water and solutions must be scrutinized and studied carefully to clearly elucidate, first, the energetically driven uphill transport of water from the growth medium and within the plant, followed by the continued osmotic withdrawal of water, ultimately building up hydrostatic pressure that results in the exudation of guttation fluid from leaves. 3.4.1  Water

forced upward-like mechanism of ascent of sap and guttation

Singh and Singh (1989) demonstrated that the water that moves upward in rice plant occurred primarily due to root pressure and not necessarily due to water potential gradients existing between the shoot and root of the plant. Of late, five different pieces of evidence exist of a mechanism which is analogous to a device that can force water upwards (Singh et al. 2009b). The authors drew a conclusion about the whole-plant level (Figure 3.3): roots have a high capacity for absorbing and pushing water up to the shoot, which depends on the physiological root activity (Eshel and Beeckman 2013; Passioura and Angus 2010). The physiological role of water transport mechanism when combined with Wegner’s ‘energetically driven uphill water co-transport’ (Wegner 2014, 2015a,b) that generates root pressure, as mentioned earlier, enables plants to overcome the problems of ascent of water, which is caused due to cavitation and embolism while freezing and/or intermittent dry and wet spells. The mechanisms constituting the strategy that is adaptive appear to facilitate ways of plant survival during unfavorable climatic conditions that lead to successful crop condition under stressful situations (McDowell et al. 2008; Singh et al. 2009b; Tyree 2003).

(a)

(b)

Figure 3.3  Photographs show the arrangement for investigating the effect of applied pneumatic pressure on water transport in intact rice plant. A cup with a conical shape about 5 cm deep was made around the intact stem at the height of 10 cm from the base just above a node. Some incisions were made in the stem by a sharp razor inside the cup filled with water. The entire cup assembly, with water in it, was spherically engulfed by hard rubber balloon-type of tubing by inserting it from the top of the plant and driving it down to bring the entire cup-assembly within it. Application of pneumatic pressure, in the stove connected by a rubber tube extending into the balloon [Fig. (b)], caused unrolling of leaves while in the absence of such pressure leaves did not unroll [Fig. (a)] [Source: Singh et al. 2009b].

46   Guttation

3.4.2  Compensating

pressure theory and guttation

Relatively recently, Canny (1995) contradicted the assumptions of the tensions in cohesion-tension theory measured in the xylem sap by using the pressure chamber. He stated the conditions under which plants did not get enough water, which was found to be at a pressure below −600 kPa, causing cavities rapidly. The average level of the threshold was about −200 kPa. That is the reason uncertain foundations in the light of Canny’s explanations of the cohesion theory were recapitulated despite their confirmation with the measurement of pressure chamber whose tension remained high in the xylem (Scholander et al. 1965). Canny said that no high tension and gradients of tension exist in the plant based on its height, and he further advanced the theory by saying that pressure of xylem operation is raised to a stable range by compensating tissue pressures (CTP), which press upon the tracheary elements. He also stated that the pressure of the tissue does not drive the transpiration stream, which is still driven by evaporation, protecting the stream from cavitation by showing evidence of pressure-positive roots, woods, and leaves (Canny 1990, 1995, 1997, 1998, 2001). He clearly stated that the water potential is not evaluated by the pressure chamber. The tension is due to compensating pressure applied to the xylem, keeping constant the upward flow of sap, aiding exudation as guttation, and not due to tension in the xylem. 3.4.3  Plant

hearts theory of ascent of sap and guttation

A renowned German astrophysicist Kundt (1998) and his colleague (Kundt and Gruber 2006) are of the opinion that favor the osmotic withdrawal of water by all plants from the soil through the root hair zone that lift it to heights up to about 140 m through the xylem. The endodermis jump caused by two layers of subcellular mechanical pumps called plant hearts, which are located in plasmodesmata in the endodermis and exodermis walls, are powered by ATPs, which push the water up in the plant. The continued supply of water accomplished by pumping of plant hearts helps upward transport through the whole plant and often out again in the form of guttation or exudation. These authors claimed that plants, small or tall trees, are dependent on these pumps or hearts for the intake of ground water, whose functioning may be subject to regulation by the involvement of contractile proteins (Staiger et al. 2000). These proteins create propulsive pulsation and oscillation (Bose 1923, 1927) aided by G-proteins that affect membrane permeability on account of energy supplied by ATP hydrolysis (Dustmamatov et al. 2004; Dustmamatov and Zholkevich 2008; Zholkevich 1991; Zholkevich et al. 2007). 3.4.4  Chemico-mechanosensory signal and

guttation

All living organisms naturally sense and respond to physical stimuli (Baluska et al. 2003; Baluska and Mancuso 2009; Ninkovic and Baluska 2010; Staiger et al. 2000). Collectively called as chemicomechanical stimuli, all the stimuli are related to each other closely. All these may be produced by pH gradient; hormones; mechanical loading by snow, ice, fruit, wind, and rainfall; turgor potential; membrane potential; temperature; light; touch; sound; etc., influencing cellular contractile proteins and gating of AQPs in the living cells. Both plants as well as animals are responsive to chemicomechanical signals, regardless of the taxonomic classification or habit of life, with prominent distinction between taxonomic groups related to the molecular components individually that exist in the microstructure of the internal cellular sensing network responded by an individual organism to

Mechanism of Guttation    47

each chemico-mechanical stimulus (Baluska et al. 2003; Telewski 2006). Hence, just like the stomata, the hydathodes have an important role to play to sense and drive changes in the environment when guttation commences (Hetherington and Woodward 2003; Pillitteri et al. 2008; Wang et al. 2011). Recently, a proposal of a plant-specific chemico-mechanosensory network was given, which remains within plant cells, having a similar presence in the animal system, as discussed earlier (Baluska 2015). It basically serves the purpose of unifying the hypothesis accounting for perceiving enormous chemico-mechanical signals (Hayashi et al. 2006; Telewski 2006). According to the assumptions, the leaf turgor changes and stresses mechanically on the cytoskeleton–plasma membrane–cell wall, also called as CPMCW, which acts as a chemico-mechanosensory network for plant cells. This concept is reliable as transpiration and guttation cause the plasmolysis and deplasmolysis of epithem cells of hydathodes, one after the other (Chen and Chen 2005, 2006, 2007); there is a chance that the hydathodal response, that is, the root pressure assists lead to guttation. These aspects are important and need attention to highlight the action mechanism of chemico-mechanosensory system for the commencement of guttation. 3.4.5  Light

signal and guttation

In the middle of the twentieth century, Engel and Friederichsen (1951, 1952, 1954) revealed how light and darkness influenced guttation, and Z. mays coleoptiles showed rhythms and periodicity in guttation under light stimulation. A rise in guttation was observed on exposure to light for a minimum of 2 hours after illumination, and a fall in guttation occurred in dark periods. This was seen regardless of the light and darkness process, that is, 12:12, 6:3, 3:3, or 2:2 hours of exposure. In 1:1 light–dark cycles nevertheless, this system was obscured by other growth processes. Their studies stated that the guttation reaction of oats to light and darkness was exactly opposite that of corn coleoptiles. These varied responses to light suggest the possibility of some additional components being involved in the system for the transformations of light stimulus into exudation of guttation. Furthermore, Szarek and Trebaez (1999), using ion channel and proton pump inhibitors, evaluated the membrane potential changes evoked by light to elucidate the nature and mechanism of the response and find a probable link of ions to guttation in gametophytes, particularly the ferns Asplenium trichomanes and Avena coleoptile, which exhibit guttation when illuminated (Mcintyre 1994). On the basis of the results, these studies find a possible role for Cl– and K+ fluxes in lightinduced guttation was suggested with its link to the need for energy for this phenomenon (Pedersen 1993, 1994) as well as oxidative gating of AQPs that affect permeability of water building osmotic pressure which drives guttation water out of hydathodes (Kim and Steudle 2009). The effect of light quality and laser light on the exact mechanism of the exudation of guttation still remains unknown. 3.4.6  Chemical communication between opposite plant poles and guttation The main physiological roles of xylem and phloem tissues in higher plants are to transport water, nutrients, and metabolites to different organs. However, the whole-plant events inclusive of stress responses and long-distance signaling are also affected by these tissues as xylem and phloem saps contain a number of sugars and proteins (Zimmermann and Ziegler 1997). Aki et al. (2008) recently used the available entire genomic information of rice plant to analyze the proteome of xylem and phloem saps derived from this crop and identified 118 different proteins and eight different peptides in

48   Guttation

xylem sap. Additionally, nucleobases and derivatives like cytokinin and caffeine are also translocated in the plant vascular system. One of the physiological functions of hydathodes is the retrieval of cytokinins from xylem sap in their epithem cells for preventing their loss during guttation. This is accomplished by the mediation of purine permeases (PUP), particularly AtPUP1 and AtPUP2, in Arabidopsis (Burkle et al. 2003). Thus, the presence of cytokinins due to PUP might help in the regulation of the phenomenon of guttation functioning partially or wholly in the system of signaling. Knowledge of long-distance signaling, xylem sap cytokinin concentrations, shoot auxin level, auxin transport, and auxin response seem imperative for the induction of guttation. Modern techniques of producing various types of mutants have immensely helped in understanding the mechanisms of physiological phenomena, including exudation, in plants. For example, mutants of garden pea (Pisum sativum) rms 1 to rms 4 are normal in auxin indole-3-aceticacid content as well as in its basipetal transport (Beveridge 2000). Nevertheless, three of the four mutants, rms 1, rms 3, and rms 4, have low concentrations of cytokinin attained from the xylem sap of roots. Some property of the shoot that is possibly a part of the feedback mechanism, which is induced by outgrowth of bud and its rudimentary state of absence or presence, causes reduction in cytokinin concentration of xylem sap. The auxin itself is unlikely to be a shoot-to-root feedback signal as its levels and transport were not connected with xylem sap cytokinin concentrations in various intact and grafted mutant and wildtype plants. Shoot and root stock of mutants rms 1 and rms 2 are regulated by the level or transport of graft-transmissible signals. Varied grafting studies and double-mutant analysis state that the rms 2 is in association with the regulation of the shoot-to-root feedback signal. Rms 1 seems to be in association with a second as yet unknown graft-transmissible signal that is hypothesized to move upward from the root to shoot. Nevertheless, in inhibiting branching following decapitation, exogenous auxin interacts with both of the signals that rms 1 and rms 2 regulate. The mechanism of action of rms 3 and rms 4 is presently less apparent, although the shoot largely appears to be the target for both of these signals. Along these lines, the study on the site of abscisic acid (ABA) biosynthesis is essential to link the understanding of the regulation of ABA metabolism to physiology and development of plants (Nambaru and Marion-Poll 2005). Especially, in contrast to stress-induced ABA accumulation, little is known about the role and function of ABA in plant growth and development under nonstressed conditions. In turgid tissues, the expression of AtNCEDs, AtABA2, and AAO3 genes is observed in vascular bundles. Koiwai et al. (2004) reported that protein is abundantly localized in phloem companion cells and xylem parenchyma cells of turgid plants. Therefore, vascular tissues are probably the main site of ABA biosynthesis in unstressed plants, and ABA and its precursors might be synthesized in vascular tissues and transported to target cells such as stomata and sites of root pressure and guttation. In addition to higher plants and fungi, ABA is synthesized in moss, fern, and algae. ABA is found in all divisions and classes of algae as well, including colorless species. In green algae Chlamydomonas reinbardtii, application of ABA enhances their resistance to the oxidative stress. Through its presence, ABA in vascular bundles, roots, and leaves might prove to be influential in gating of AQPs that results in increase in water permeability; therefore, its increase in transport causes water to exude as guttation. Exogenous application of ABA increases root hydraulic conductivity (Lpr) at both organ and cellular levels (Hose et al. 2000), which occurs because of up-regulation of AQP genes (Zhu et al. 2005). Thus, the Lpr enhancement could be a means for optimizing the water supply from the soil to the shoot. It also affects some of the other physiological and cellular adaptive processes, like osmotic adjustment and ion transport, and the induction of the production of metabolites or proteins that protect macromolecules from denaturation at lower

Mechanism of Guttation    49

potentials of water (Bray 2002; Chaves and Oliveira 2004). These ABA-regulated physiological processes have acted positively to facilitate plants for continuous growth and reproduction during periods of scarcity of soil water or for sustaining and recovering from dehydration, having its impact on crop yield (Thompson et al. 2007). Light might also play a similar role. Cytokinins (CK) and ABA originate in the roots of split-root samples of field-grown vines, and their concentrations and also the movement of chemical signals were found to be substantially altered during an irrigation cycle. Obviously, the water potential significantly affected the control of water balance of roots. Water redistribution may be helpful in surviving for longer periods by releasing chemical signals to support the activity of fine roots in soil. Further growth of roots may take place by augmentation and by water movement facilitation and by large distinction in ABA to cytokinin ration, which are famous for altering growth of root. A working hypothesis clearly states that the opposite plant poles, shoot tips and the root tips, from a morphogenetic perception have rapid communication with each other by producing and sending various specific signals as auxin produced in leaves acts as the main shoot signal (Aloni 2001, 2010; Aloni et al. 2003, 2005), whereas CK acts as the major root signal. Thus, the authors have demonstrated transpiration-dependent, on the one hand, and root pressure-dependent, on the other, transport of root-produced cytokinins in the xylem sap of transgenic Arabidopsis thaliana. In this context, it is of interest that Shabala et al. (2009) have shown stimulation of K+ uptake in barley roots by cytokinins produced in the root tip. Undoubtedly, the nature of guttation is complicated, and the interplay of phytohormones that the root and shoot generate in long-distance signaling, evidently seen by variations in xylem sap cytokinin concentrations, shoot auxin level, transport of auxin, and response of auxin, may be operative for induction of natural guttation. For example, chemical signals that originate from roots play a vital role in the root-to-shoot communication in the water movement from soil layers through roots and its distribution into the entire shoot to sustain physiological functions and plant growth. Further, hydathodes are reported to retrieve cytokinins from xylem sap in their epithem cells and aid in the prevention of their loss during guttation, which is accomplished by PUP, particularly AtPUP1 and AtPUP2, in Arabidopsis (Aki et al. 2008; Burkle et al. 2003). Thus, PUP that save the cytokinins get partially or wholly involved along with other hormones such as ABA in the system of signaling, which might play a vital role in commencing and controlling the guttation in a way that is yet to be understood. 3.4.7  Molecular

aspect of guttation: role of contractile proteins and AQPs

As mentioned earlier, Bose (1923, 1927) proposed an interesting theory called ‘propulsive pulsation theory’ for ascent of sap in plants. His theory is based on the assumption that the living cells around the xylem tissues undergo a sort of rhythmic contraction and expansion resembling heartbeats in animals. It is this rhythmic pulsation which is responsible for the upward movement of sap, he claimed. An instrument called ‘Cresograph’ was invented by Bose to locate these pulsations, whose electrodes were inserted deep into the plant stem, and the pulsations were graphically recorded. The oscillations imprinted on the graph very clearly indicated the pulsation activity of the cells. There is a great need to study these pulsation activities by using modern techniques, metabolic inhibitors, and pharmaceuticals to elucidate this theory as Kundt (1998) and Kundt and Gruber (2006) have recently advocated for the existence of plant hearts in plasmodesmata.

50   Guttation

However, recent investigations deploying respiratory inhibitors such as DNP and KCN have revealed the requirement of ATP synthesis as an essential step for the rapid transport of water in the xylem parenchyma and other microscopic structures such as tracheids and tracheae of living cells. These observations strongly lend support to the idea that the vital activity of living cells is involved for which the energy is required particularly for non-transpirational upward movement of sap, that is, root pressure. Water transport in the roots consists of two successive phases: water influx into cells (relaxation phase) and water extrusion in the xylem vessels (contraction phase) (Mozhaeva and Pil’shchikova 1972). Water extrusion decreases the cell’s water potential, and that lays the ground work for the uptake of a new water portion. Thus, water transport displays an impulsive rhythmicity. Additionally, apparent hydraulic conductance also reveals self-oscillations (Passioura and Tanner 1985). Rhythmic oscillations of water uptake and exudation are accompanied by alternating the contraction and relaxation of the root: maximum exudation coincides with a root contraction (Mozhaeva et al. 1979). Such a contraction is energy-dependent and depends on contractile proteins functioning. Actomyosin- and actin-like proteins were in fact detected in Cucurbita pepo and Helianthus annuus roots (Abutalybov et al. 1980; Abutalybov and Zholkevich 1981; Mozhaeva and Bulycheva 1971; Zholkevich et al. 1979). As the impulsive rhythmicity displays a self-oscillative nature of absorbing and pumping activity of a root system, the effect of various chemicals on water uptake and on exudation was investigated to reveal the nature of selfoscillations. The experiments were carried out with decapitated roots of 6- to 8-day-old Z. mays seedlings (Lazareva et al. 1986). Both exudation and water uptake revealed self-oscillations with periods of nearly 3 min, and the rhythms of exudation alternated with those of water uptake (i.e. water uptake and exudation are in antiphase). Under the influence of chemical agents, which reduce the metabolic component of root pressure, self-oscillations gradually cease, their amplitude decreases, and their period increases, whereby asynchronism is replaced by synchronism. The value of both, the water uptake and exudation rate, dropped from Q10 value 3 to 1. Such changes were observed under the action of DNP (which violates the energy supply) and of cytochalasin B, colchicine, p-chloromercuribenzoate (contractile proteins inhibitors), pipolphene, and ABA (which disturb the cell membrane structure and permeability). Only CaC12, which increases the metabolic component and stimulates àctomyosin-like protein, intensifies the self-oscillations, retaining their asynchronism. Thus, the data on root absorbing and pumping activity support the assumption concerning the leading role of the metabolic component in self-oscillations of water transport and water movement mainly through the involvement of contractile proteins of living cells of a decapitated root system. Zholkevich (1991) was, however, of the opinion that cell contraction or closing plasmodesmata should be connected with a pressure potential increase and therefore with a water potential increase. The water potential may increase to such a degree that it becomes positive when water extrusion begins. Thus, water moves along the water potential gradient but not against it. Owing to the participation of self-oscillations and cell polarization, local water potential gradients are created, and the water flow is directed and controlled. The question concerning endodermal jump in the water potential is still open to discussion (Borisova et al. 1986). With the passage of time, however, following scientific debate in the light of new findings, the vital force, that is, the propulsive pulsation theory originally advocated by physicist-turned-plant physiologist Bose (1923) could not survive experimental and theoretical analyses because of its so-called inability to fully explain and account for the long-distance transport of water in plants (Renner 1915). However, in the light of recent findings of Singh et al. (2009b), it would be of great interest to look into whether Bose’s pulsation-driven water transport and the recently proposed AQP-regulated water movement are

Mechanism of Guttation    51

one and the same thing or separate concepts preceding or succeeding one another, as these two physiological phenomena are affected similarly by a number of similar factors, that is, they are both greatly reduced in conditions of drought and enhanced with supply of water (Bose 1923; Cochard et al. 2007; Steudle 2000; van As 2007). The water finds its way from the xylem of root and stem, through the leaf to the stomata and hydathodes, which moves either through vessels, cell walls, and intercellular spaces apoplastically or makes way through cytosols crossing plasma membrane symplastically, passing from cell to cell through plasmodesmata for reaching to various tissues with living cells, contributing hugely to the overall leaf hydraulic conductance, even though all pathways stated are probably utilized to an extent. Contextually, the role that contractile proteins play in regulating water transport requires attention (Baluska and Volkmann 2008; Staiger et al. 2000; Zheng et al. 2009). The functions of contractile actomyosin- and actin-like proteins are probably related to self-oscillations of water transport involving rhythmic micro-oscillations of pressure potential of parenchyma cells that lead to their pulsations or alternate opening and closing of special channels in the symplasm (Abutalybov and Zholkevich 1979; Abutalybov et al. 1980; Dustmamatov et al. 2004; Mozhaeva and Bulycheva 1971; Mozhaeva and Pil’shchikova 1972). Bose (1923) stated that the ascent of water in plants is not possible without pulsation of parenchyma cells (Wegner 2014), which may include electrical pulses implicating these contractile proteins present therein. In case water transport is in fact something that depends on rhythmic cell contractions or reversible opening and closing of either plasmodesmata or other special channels, the polarization of the cell and of entire symplasm would require unidirectional flow of water, else the water is capable of moving in all directions. The role of AQPs present in the membranes is getting clearer in the transcellular flow of water and its regulation and balance of leaf water in normal conditions by modification of the cell membrane water permeability in response to different internal, external, and edaphic factors (Kaldenhoff et al. 2008, 2014; Kim and Steudle 2009; Maurel et al. 2008; Steudle 2001). The permeability of AQPs is regulative in response to intracellular pH, which rises in response to declined rates of respiration (Katsuhara et al. 2008). The conductance of AQPs present in the movement of water across roots gets modified due to rise in cytoplasmic pH. Not just this, other signals such as osmolarity and turgor also provide a mechanism for modification of their permeability to water in response to their fluctuation of local environment. Apart from how effective these signals are, high expression of genes for AQPs seen in the epidermis, endodermis, and xylem parenchyma could prove to be crucial for controlling the movement of water that affects root pressure and guttation in plants. The role of AQPs, hence, is seen as a multifaceted phenomenon (Azad et al. 2009; Heinen et al. 2009; Kaldenhoff et al. 2008, 2014; Katsuhara et al. 2008), and them being involved in the commencement of guttation as yet unknown creates a potential and formidable research area to stay updated about in future. Of significance, the activity of AQPs is also affected by a number of other factors such as water and salinity stresses, solute concentration, temperature, heavy metals, oxidative stress, and deprivation of nutrients to the roots (Mahdieh et al. 2008), which might impact root pressure and guttation simultaneously. 3.4.8  Energy

coupling in water and solute transfer during root pressure development resulting in guttation

It is common observation that if a stem is cut off or decapitated, fluid oozes out from the cut stump, and if roots are poisoned or deprived of ATP, oozing stops. However, no conclusive evidence of the

52   Guttation

active transport of water in such systems was available until the recent work of Wegner (2014) who elaborately explained the energy-dependent mechanism of such transport of water in plants (Wegner 2015a,b). In the absence of transpiration pull or when transpiration is low at night, or during the period of very high relative humidity, water is absorbed, as described earlier, by osmosis preceded by active absorption of ions at the cost of energy that the living cells supply (Gaxiola et al. 2007; Morth et al. 2011). The process of removal of water from the soil by the epidermal cells of the root and its subsequent movement into the xylem under sufficient pressure for producing ‘bleeding’ or ‘guttation’ is not entirely understood (Kundt 1998; Kundt and Gruber 2006). There is yet to be an agreement that root pressure, bleeding, and guttation include only osmotic forces and salt secretion. For exudation from cut stumps to take place, the root should actively respire, and though oxygen and sugar are necessary for this to take place (Lundegardh 1950), respiration does not entirely stop by adding vanadate, maybe because not all of the ATPases are inhibited (Pedersen and Sand-Jensen 1997; Sze 1984; Sze et al. 2002). It is also a possibility that only part of the guttation phenomenon depends directly on the conversion of energy in the roots. Though Wegner (2014; 2015a,b) has advanced our understanding significantly, more clear-cut experiments explaining how respiratory activity affect salts and water need to be devised. Such experiments are difficult but not impossible if water secretion takes place by a secondary active mechanism and is combined to the transport of ion. Temperature is another factor that supports guttation as water uptake declines when roots are subjected to low temperature (Pedersen 1993, 1994). Guttation is also adversely affected by anaerobic conditions or treatment of roots with respiratory inhibitors. For creating and maintaining the osmotic potential gradient at cell level, active pumping of the solute is needed for which ATP is needed. In most plant cells, the H+-ATPases are primary active transporters, and the proton-gradient is used by co-transporters and exchangers for active pumping, which leads to solute accumulation in root cells, causing energy-driven uphill transport of water in the xylem (Wegner 2014) (Figure 3.3). Thus, due to these two processes, there is a greater tendency for water to flow into the vascular bundle, thus building the hydrostatic pressure force that pushes the water upward. Undoubtedly, the mass movement of water from the root to the leaf is highly likely because of the evaporation–tension– cohesion mechanism in continuously transpiring plants and because chemical energy, that is, ATP is not required for this process. However, in the presence as well as absence of transpiration, ion transporters keep on pumping ions, which contributes to water uptake and its movement energetically uphill against the free energy gradient of water and transport happens unabated, as a consequence, which results in build-up of root pressure and exudation of guttation when opportunity arises (Singh 2016a,b; Wegner 2014, 2015a,b). As explained earlier, according to Wegner (2014), water flow conducted by AQPs and water secretion (irrespective of the mechanism) are antagonistic (Wegner 2014) as water secretion requires low hydraulic conductance of the membrane and down-regulation of AQP activity, whereas passive water flow is favored by up-regulation of AQP activity. It is clearly stated that the plant cells use the energy that respiration produces and drive absorption of water across plasmatic membrane, which is great evidence of the fact that respiratory inhibitors (DNP and azide) block absorption of water and respiratory promoters (sugar) enhance absorption of water. Concluding this, the metabolic absorption of water by plant cells happens in this manner, which results in root pressure development that culminates into guttation. Even though the guttation mechanism is not entirely understood, it is suggested that the process be modeled so as to take further steps in defining interdependent plant activities, as shown in Figure 3.4.

Abbreviations: ABA = abscisic acid, ADP = adenosine diphosphate, ATP = adenosine triphosphate, ER = endoplasmic reticulum, Pi = inorganic phosphate, PM = plasma membrane, V = vacuolar.

Figure 3.4  Singh model of guttation, proposed to account for the mechanism of guttation [Source: Singh 2016].

54   Guttation

3.4.9  Sum

of the mechanism at a glance

It is apparent that the entire process of guttation is ‘switched on’ either because of absolute reduction in or inhibition of transpiration due to high humidity, still air, favorable climatic and soil temperatures, abundant soil moisture, etc., following the perception of any one or combined chemico-mechanosensors such as pH gradients and hormones (ABA, auxin, or cytokinins) either singly or jointly, temperature, light, turgor potential, or membrane potential that influence and integrate the functioning of those contractile proteins and transporter proteins, that is, plasma membrane and vacuolar ATPases, which pave a path to energy-driven influx of solutes, metabolites, and water across membranes. Hormone indulgence, at this point of the phenomenon, influences the permeability of water as well as its energetic uphill transport, increasing positive hydrostatic pressure that is translated into root pressure, which is marked as critical. The pressure of the root developed in the xylem vessel and, as described initially, perhaps aided by forces located higher up in the shoot and leaves as well as intermingling xylem and phloem saps on the way, provide impetus to the upward flow of sap in the plant. Saps that reach the tracheary endings of the leaves remain available for sensing, chemically and/or environmentally, by hydathodes, appearing as permanently open pores that are least resistant to flow, which facilitate the flow of liquid out in the form of droplets or drippings or sometime streams, popularly known as guttation, under overall intrinsic and extrinsic master regulation of factors that may be genetic, internal, external, and edaphic conditions. 3.4.10  The

unknowns—a look at the future

From the above discussion, one might conclude that the way root pressure, guttation, and bleeding is observed, they appear as raising an argument for a simple osmotic movement of water forced by metabolic energy as a result of antecedent or concomitant solute/s movement. Nevertheless, this is not the entire scenario of the phenomenon, and simply explaining the process on the basis of osmotic relations does not sufficiently explain the need for the occurrence root pressure, bleeding, exudation, and guttation, which in fact only indicates the complex nature of causes and effects involved in these physiological events. Therefore, it is interesting to have an in-depth investigation of the structural biology of hydathodes inclusive of their initiation, differentiation, and root hairs and the regulation of pressure in the roots. Using functional magnetic resonance imaging and positron emission tomography methods to gauge the starting point of different signals that emerge from and reach out to these tissues to finally have an interaction for initiating guttation would be imperative, as imaging may show an activity milli- or micro- or even nano-seconds after it is activated, to begin (Holbrook et al. 2001; van As 2007). In the light of new frontiers in guttation research created by the work of Komarnytsky and his colleagues (Komarnytsky et al. 2000, 2004, 2006; Komarnytsky and Borisjuk 2012) using genetic tricks with tobacco, further work might enable a better understanding of the mechanism of guttation as to when and how the switch of guttation is turned ‘on’, left ‘on’, and switched ‘off ’, facilitating safer, quicker, and cheaper production of plant-based medicines for animal and human use. It is also evident that we have both an osmotic component and a metabolic component of root pressure and there is a delicate interplay between the two; it is a great challenge to find out how they are coordinated. Having discussed and presented a hypothetical model of the mechanism of guttation, its regulation by plant genetic makeup and other internal factors, external stimuli and edaphic factors will be detailed out in the next chapter.

C h a p t er

4

Regulation of Guttation

4.1  Introduction The phenomenon of guttation, like other physiological phenomena, is regulated by a number of internal and external factors. These factors mainly include the genetic makeup, growth and phenology of plants, hormonal and solute balance, temperature, humidity, light, and wind, which may conveniently be classified into the following heads and subheads.

4.2  Internal factors These factors originate within the plants themselves and play a significant role in influencing guttation from leaves. These factors are briefly categorized and described below.

4.2.1  Genetic factors 4.2.1.1  Species variability As stated earlier in Chapter 1, the phenomenon of guttation occurs in a wide range of plant species, which include herbaceous mesophytes, shrubs, and woody trees in angiosperms; gymnosperms; pteridophytes; algae; and fungi (Chen and Chen 2005; Lersten and Curtis 1991; Raleigh 1946a,b; Singh et al. 2009a; Sperry 1983; Stocking 1956a). Normally, significant guttation occurs in grass species including rice, wheat, barley, oats, and maize and other plant species such as tomato, balsam, Nasturtium, Colocasia, and Saxifraga and in some plants of Cucurbitaceae family as well; however, there is a high variability in guttate volume. Plants that exhibit guttation are seen to guttate through

56   Guttation

leaf tips, but in some plants, like the rice plant, guttation mainly occurs through the edges of the leaf, along with the surface, especially during the late hours of the day (Singh et al. 2008, 2009a). Few plant species exhibit profuse guttation under highly humid conditions, which appear as if water droplets are falling from the leaves (Feild et al. 2005). A single leaf of Colocasia antiquorum is capable of exuding up to 100–250 mL guttation water per day (Stocking 1956a). Guttation may also occur sometimes from the stems, generally through leaf scars or lenticels and flowers. The fungus Pilobolus is well known for its abundant guttation (Tarakanova et al. 1985; Tarakanova and Zholkevich 1986). Similarly, the fungus Polyporus squamosus also exudes droplets of water through its polypores profusely, which are similar to guttation in higher plants (Figure 1.2). Thus, these species of fungi, among others, present good examples of the occurrence of guttation. As for guttation variabilities, it is worth mentioning that the exudation from Moso bamboo shoots during spring, in Southeast Queensland of Australia, can be sighted by the wet patches they create, having considerable agronomic and economic significance. One of the important functions of guttation in rhizome sheaths is to moisten the soil ahead of the rhizome tip, allowing easy penetration and emergence by the expanding rhizomes, facilitating proper growth of shoots out of the soil. Engel and Friedericksen (1954) observed species differences in guttation of oats and corn in response to light and darkness, which were opposite to each other. Similarly, the morphology, anatomy, and relationship of extrafloral nectarines and hydathodes in two species of Impatiens (Balsaminaceae) have been studied by Elias and Gelband (1977). Lewis (1962) studied the variation in guttation fluids of three cereal species, namely, rye, barley, and wheat, and found a large variation in the growth of Claviceps purpurea when guttation fluids of these cereals were used separately in the growth media. These findings led Lewis’s laboratory to undertake further research for determining an array of substances and amounts thereof that would account for differential growth of C. purpurea, resulting from guttation fluids added to the growth media obtained from seedlings of cereals like Rosen rye, Genesee wheat, and Traill barley (Goatley and Lewis 1966). Importantly, with variable guttation rates among these cereals, barley accumulated inorganic elements the most, whereas wheat accumulated them the least, with the exception of iron, where rye was the highest accumulator and barley the lowest. Another important advancement was made by Stoller (1970), who used guttation fluid for the assessment of differential rates of amiben absorption, translocation, and metabolism in various crop species. It must be noted that amiben (3-amino-2,5-dichlorobenzoic acid) is widely used as herbicide for weed control in soybean plantation. The author determined the relative ability of several species to transport 14C-amiben absorbed by roots to the shoots. Further, in light of the existence of vast differences in translocation between two species, these plant species were utilized to investigate the metabolic basis for the differences observed. The variation in the accumulation of this substance was attributed to species differences in guttation. Further study was conducted to develop an understanding regarding the differential translocation of amiben. The study stated the difference in translocation of amiben among species by conducting an experiment with a tolerant variety of wheat species. The tolerant variety was involved in fixing 14C-amiben, in the form of nontransportable N-(carboxy-2,5-dichlorophenyl) glycosylamine (N-glucosyl amiben), in its roots. The ‘free’ amiben was translocated to the plant shoot. As compared with barnyard grass, wheat was found to be more efficient in root and shoot translocation of amiben to N-glucosyl amiben or amiben-X. Therefore, wheat translocated a higher concentration of amiben (50 mg L−1) for inhibiting elongation of the radicle in case of a 4-day-old plantlet as compared with barnyard grass (1 mg L−1).

Regulation of Guttation    57

Similarly, Japanese butterbur (Petasites japonicus var. giganteus) and Japanese knotweed (Polygonum cuspidatum) exuded large quantities of guttation fluid from their leaf edges (Mizuno et al. 2002). Growing Japanese butterbur on anultramafic soil showed presence of Ni (2.24 mg L −1) and Mn (0.32 mg L −1) in the guttate fluid. In case of Japanese knotweed, the amounts were 0.22 and 3.13 mg L−1 of Ni and Mn, respectively. This showed a higher Ni concentration in Japanese butterbur as compared with the other plant species, and the guttation occurred profusely through the leaf margins. However, it was just the opposite in case of Mn concentration, which was found to be six times higher in case of Japanese knotweed. The results showed a difference in the concentration of Ni and Mn in both the plant species. Also, concentrations of K, Mg, and Ca were found to be higher in case of Japanese butterbur guttate, which again was an indication of widespread variation in guttation among plants in the ultramafic rock regions. Recently, Singh et al. (2009a) observed profuse guttation in rice leaves, which exuded 271 µL h−1 tip−1, whereas under similar environmental conditions, no guttation was noticed in other 10 plant species, namely Bermuda grass (Cynodon dactylon), hyacinth bean (Dolichos lab-lab), okra (Hibiscus esculentus), papaya (Carica papaya), ridge gourd (Luffa acutangula), rose (Rosa sinensis), sorghum (Sorghum bicolor), snake gourd (Trichosanthes anguina), sponge gourd (Luffa cylindrica), and sugarcane (Saccharum officinarum). Apart from terrestrial plants, guttation also takes place in submerged aquatic plants (Pedersen 1993, 1994, 1998; Pedersen et al. 1997). The intensity of guttation, however, differs markedly between two species of submerged aquatic plants, with guttation rates being 10 times more (2.1 µL leaf−1 h−1) in Sparganium emersum than in Lobelia dortmanna (0.2 µL leaf−1 h−1) (Table 4.1). Species differences in guttation could be attributed to difference in vessel dimensions, number of vessels, flow velocity, pressure gradient, root pressure, hydraulic resistance, and hydraulic conductance, apart from variation in hormones, plant nutrients, and root metabolism. Further investigations on some of the above-mentioned aspects must be carried out to unravel the underlying causes of variation in guttation as why certain plants, for example, pines, fail to develop root pressures under apparently favorable conditions. Table 4.1  Estimates of flow velocities inside the xylem vessels, pressure gradients, root pressure, and hydraulic conductance in S. emersum and L. dortmanna. Median values and ranges in parentheses are shown apart from the mean guttation rate. Plant parameters h-1)

S.emersum

L. dortmanna

2.13 (0.43-3.66)

0.25 (0.02-0.67)

Vessel dimensions (r, µm)

6.3 (2.5-15.3)

3.2 (1.6-9.6)

Number of vessels (n)

55.5 (32-100)

11.0 (7-12)

23 (2-223)

84 (16-334)

3.4 (3.0-22.3)

159.0 (8.7-1 98.1)

1.1 (1.0-7.4)

7.5 (0.4-9.9)

2.7 × 10 –17 (9.8 × 10 -18 –7.4 × 10 -17)

1.4 × 10 -19 (1.1 × 10 -19 –2.5 × 10 –18)

Guttation rate (Q, µL

Maximum flow velocity (2V mean = Vmax, cm h-1 ) Pressure gradient (∆P, kPa m-1) Root pressure (P min, kPa) Hydraulic conductance (khr m4 Pa-1 s-1) Source: Pedersen (1993)

Median values and ranges in parentheses are shown apart from the mean guttation rate (Pedersen 1993).

58   Guttation

4.2.1.2  Genotypic variability Indeed, information is lacking on varietal differences in guttation among field crops. However, the rate of guttation differs widely, not only among species but among varieties thereof, too. For example, dwarf wheat cultivar ‘USU-Apogee’ was developed under a research project sponsored by National Aeronautic Space Agency (NASA) at the Utah State University, Logan (USA) and conducted by a group of crop physiologists for high yields in space. Interestingly, Bugbee and Koerner (2002) reported, among other characteristics, USU-Apogee having significantly high rates of guttation during dark periods, with its occurrence even during the light period when the stomata were partly closed by elevated CO2. Varietal and species variations in guttation were also found among Satsuma oranges and between Satsuma and grape fruit varieties, with the latter showing, only occasionally, traces of salt deposits due to guttation (Long et al. 1956). Curtis (1943) stated that the exploration of the possible influence of the guttated liquid upon the plant itself was the outgrowth of a breeding problem in which an attempt was made to produce a tipburn-resistant lettuce. Plant pathologists, physiologists, and horticulturists were all baffled from tipburn disease, which is a Ca-mediated physiological disease, ever since head lettuce became an important economic crop. Fujii and Tanaka (1957) examined the difference in the guttation and bleeding of seedlings of varieties of rice, which was positively correlated with the lateness of varieties, that is, the later the variety, the more was the rate of guttation. These authors investigated the differences in the physiological properties of rice varieties differing in their maturity time. The osmotic pressures of xylem sap collected from the roots of early-maturing varieties were lower than those of the latematuring ones. They also observed a similar tendency in bleeding as well as in guttation; studies on the respiration of roots of rice seedlings revealed the superiority of root activity of late-maturing varieties over that of early-maturing ones. These studies soon prompted Ogura (1958), who investigated the phenomena of bleeding and guttation of seedlings of upland rice varieties for comparison with lowland rice. The findings revealed that when both types of rice varieties were cultured together in upland and lowland conditions, bleeding in lowland rice was always superior to that in upland rice seedling till the third leaf stage, but the bleeding superiority of upland rice seedlings over that of lowland rice seedlings was recognized after the fourth leaf stage. The authors concluded that these variations were mainly attributable to the increase of root pressure in seedlings of upland condition and of water absorptive surface area of root in seedlings of lowland condition. It is worth noting that these growth stages of rice plants fall on the turning point of enhanced absorption of nutrients, which could be, among other factors, the cause of variation in guttation. Unfortunately, in most of the above studies, no efforts were made to measure guttation quantitatively. Of much interest, however, are the investigations of Singh et al. (2008), who recently developed an innovative method for quantitative measurement of guttation in experimental fields as well as in laboratories (refer to Section 2.5). The study found out a noteworthy variation in the genotype of NDRH-2 that showed guttation volume ranging between 62 and 110 μL within 30 min, with another cultivar Mahsuri showing the lowest values. Guttation rate showed a direct relationship with the panicle’s sink strength. The study outcome offers a prospect for gene mapping of those genes that are involved in controlling the desired traits, which can then be used for developing new and improved varieties with higher yield potential and increased guttation rate. In case of a genetically modified tobacco plant, recombinant protein production has been found that enhances phytosecretion efficiency (Borisjuk et al. 1999; Drake et al. 2009; Komarnytsky et al. 2000, 2004, 2006; Komarnytsky and Borisjuk 2012).

Regulation of Guttation    59

The reasons for varietal differences in guttation are not known but could be attributed to one or all of the differences in vessel dimensions, number of vessels, flow velocity, pressure gradient, root pressure, hydraulic resistance, hydraulic conductance, hormone physiology, and cell permeability (Pedersen 1993). As in the case of sap bleeding, varietal differences in guttation could also be attributed to variation in the starch, sucrose, and hexose concentrations in the stem as well—an aspect which has not been looked into at least in crops of economic importance. Still, several other important aspects remain to be studied, for example, what causes the conspicuous divergence of the bulk water flow to the youngest leaves as observed by Pedersen (1993). Larger vessels result in much lesser resistance to water transport because hydraulic conductivity is proportional to the fourth power of radius. This anatomical feature may be an important adaptation to root pressure, as the driving force of this pressure is considered potentially weaker compared with transpiration. Clearly, the hydraulic conductance must be high in these leaves, but it is not confirmed whether the regulation is distributed along the entire transport path or is confined to the hydathodes at the tips and edges of leaves. Furthermore, studies are required on the anatomical aspects of the vascular system of crop plants and varieties thereof to facilitate a breeding program for different ecological zones. 4.2.1.3  Phenological variability The phenology of plant growth has a significant effect on guttation. Accordingly, the stage of growth has a tremendous effect on the intensity of guttation in rice. Ogura (1958) stated that guttation rate was high in case of lowland paddy as compared with highland rice plantlets, with leaf development being at the third stage. However, it was just the opposite during the fourth stage of leaf development. Among the various stages of the leaf, guttation was found to be the highest during anthesis (132 µL), which was followed by the tillering stage (120 µL), heading and milk stages (112 µL), and, finally, the dough stage (82 µL). The maturity stage showed no guttation (Figure 4.1) (Singh et al. 2009a). During the initial leaf development, the guttation rate was slow with a magnitude of difference value between 11 and 20 µL per leaf tip within the stages. This later increased to a range of 83–132 µL. Whereas, the guttation intensity for all other stages remained moderate and was between the two extreme ranges stated above. The guttation rate is not only dependent on the phenological growth stages but, interestingly, also on the lamina surface—the ridged portion of the leaf lamina (Singh et al. 2009a) exudes more guttation fluid than the smooth portion of the same lamina Figure 4.1  The rate of guttation as affected by (Table 4.2). Similarly, adaxial surface of the leaf plant growth stages of hybrid rice (cv. NDRH-2) over lamina guttates more frequently than its abaxial a period of 30 min. The least significant difference (LSD 0.05) = 1.00 [Source: Singh et al. 2009a]. surface even if it is placed upside down manually.

60   Guttation

It may be speculated that these variations in guttation rates could be attributable to the variabilities in hydathodal distribution, chemico-mechanosensors perception, absorptive root surfaces, root pressure, and metabolic status of plants, which deserve in-depth studies in future. Table 4.2  Variations in guttation as revealed by different intact leaf portions of rice leaf at anthesis during 30 min (cv. NDRH-2). Leaf portion Leaf tip Leaf margin Ridged protion of leaf Smooth portion of leaf Adaxial surface Abaxial surface

Guttation fluid (µL) 98 151 86 44 135 81.5

Leaf manually turned upside down   Adaxial surface (facing earth)   Abaxial surface (facing sky)   LSD 0.05

132 82 2.4

Source: Singh et al. (2009a)

Guttation rates are also strongly dependent on leaf location, leaf length, and leaf age (Pedersen 1993). Guttation by barley leaves depended on the age and the nutrient composition of the culture solution (Dieffenbach et al. 1980a). As mentioned earlier, the primary leaves of 6- to 7-day-old seedlings grown on full mineral nutrients guttated the most. The newly growing leaves became saturated with K+ with less than 1.5 mM K+ in the medium, whereas K+ concentration of the guttated fluid still increased further with the increase in K+ concentration of the medium. As the concentration of K+ increased from 3 mM to 10–20 mM, the average values of guttation were found to be 1.4–2.4 mm3 h−1 per plant, but for exuding plants, the flow rates were 4.2–7.6 mm3 h−1 per plant, with K+ concentration of 35–55 mM. Pedersen (1993) conducted guttation studies on plant species like S. emersum and L. dortmanna, which are submerged plants with high guttation rates, shown by primary leaves. It was 10 times higher in case of the young leaves of S. emersum (2.1 µL leaf−1 h−1) as compared with those of L. dortmanna (0.2 µL leaf−1 h−1). In S. emersum, the youngest leaf guttate was 2.13 µL h−1, the second leaf guttate was 0.30 µL h−1, the third leaf showed limited guttation, and all other old leaves showed no guttation, thus showing an average guttation volume of 0.048 µL h−1. Such variation in the rates was due to the varying age of the leaves in hydraulic conductance, which makes this event significant for plants. This change can be spotted along the water pathway; however, modification in the conductivity of water in case of hydathode offers an apt mechanism of regulation. For example, increase in the age of the leaf leads to clogging of its hydathodes, which prevents further guttation. The pathway of water flow generally occurs where the resistance is the least, and thus, the younger leaves show profuse exudation of fluids. Two aquatic plants, namely L. dortmanna and Ranunculus fluitans, showed that there was blockage in the hydathodes due to secretion of gum-like substances (Mortlock 1952; von Minden 1899; Wilson 1947). Such diversion of the pathway of water from the hydathodes (because of the clogging) can result in availability of water in the actively growing regions of the plant. Hydathode blocking was also reported in old strawberry leaves (Takeda et al. 1991).

Regulation of Guttation    61

In this study, the primary reason of hydathode clogging was wax deposition. The study suggested that removing this wax from the old strawberry leaves resumes guttation from the hydathodes. All the above-stated findings suggest an age-dependent clogging of hydathodes, which cause uneven guttation among the leaves at various stages of growth. Further, Pedersen (1993) demonstrated a negative relationship between leaf length and the guttation rate, that is, the greater the length of leaves, the lesser the guttation rate. The variation in leaf length with age and position, providing thereby greater resistance to water transport, could be the answer for the skewed distribution of guttation rates. In case of S. emersum, the leaf length determines the guttation volume; however, this is different in case of L. dortmanna. With increase in the leaf length, an increase in hydraulic resistance occurs, which leads to reduced guttation rate in S. emersum (Pedersen 1998). In addition to the blockage caused by wax, with increase in leaf age, microbes accumulate around the hydathode pores, which results in further clogging. This has been confirmed through microscopic studies performed by Pedersen et al. (1997). Thus, it can be concluded that hydraulic performance in submerged aquatic plants cannot be considered inferior to that of terrestrial plants. 4.2.1.4  Hormonal variability Along with the concentrations of Na+ and K+, different plant hormones (abscisic acid [ABA], the cytokinins [CKs], zeatin, zeatin riboside, and the ethylene precursor 1-aminocyclopropane-1carboxylic acid) have been analyzed in xylem saps of leaves, a representative of guttation fluid in graft combinations of contrasting vigor, yielding important and useful information on the vigor and growth of grafted plants (Albacete et al. 2009). Depending upon the ratio of the combinations of these hormones, both the positive and negative effects on leaf biomass have been noted when applied exogenously (Borisjuk et al. 1999; Drake et al. 2009; Komarnytsky et al. 2000, 2004, 2006; Komarnytsky and Borisjuk 2012). Further, the exudate collected over a 3-hour period from decapitated hypocotyl stumps of cucumber seedlings treated with 100 µL L −1 ethylene for 16 hours showed increased peroxidase activity, but the amount of exudate released was less. These observations suggest that these saps contain a number of enzymes and natural plant hormones, including auxins, gibberellins, CKs, and ABA (Biles and Abeles 1991), and that their qualitative and quantitative variabilities may play a role in plugging vascular tissues, impacting plant secretions. The involvement of these hormones seems to be strengthened by the findings of Dieffenbach et al. (1980b), who reported that the addition of ABA at 10 −6 to 10 −4 M to the root medium increased the volume flow of guttation and exudation in barley seedlings due to increased water permeability. Conversely, benzyladenine (5 × 10 −8 to 10 −5 M) and kinetin (5 × 10 −6 M) progressively decreased the volume flow and export of K+ into guttation and exudation fluids. 4.2.1.5  Enzymatic variability Wilson (1923) stated the role of enzymes like catalase and peroxidase in the guttation process, taking into account maize (Zea mays) and oats (Avena sativa), and reductase enzyme in case of timothy (Phleum pratense). Shepherd and Wagner (2007) studied the biochemical properties of phylloplane, the antimicrobial protein that plays a significant role in the guttation process. Its release mechanisms into the guttate, through specific trichomes, have also been focused on by researchers.

62   Guttation

Guttate consists of various transport proteins, which have been observed in the case of exuded fluid collected from cultured Arabidopsis thaliana. This shows the presence of adenine and CK, which are translocated through a H+-coupled high-affinity purine transport system that consists of AtPUP1 and AtPUP2, which are similar in function to transporters found in Arabidopsis (Burkle et al. 2003). On examination, the promoter-reporter gene present in the epithem cells of hydathodes and stigma of siliquae showed the expression of AtPUP1. This shows that CK retrieval from xylem sap prevents fluid loss due to guttation. Slewinski et al. (2009) stated the importance of sucrose transporter SUT1 in case of sucrose-loading in the phloem that might interfere with the xylem, thus preventing guttation because of intertrafficking of these substances in case of maize leaves. In addition to this, mRNA localization in sieve tubes was found to be same as that of sucrose, thus highlighting its role in nutrient transport (Testone et al. 2009). Therefore, many qualitative and quantitative variations exist at the enzymatic level, and their role in guttation is still unknown.

4.3  External factors 4.3.1  Environmental

factors

A number of environmental factors affect the guttation process. These factors mainly include mechanical stimuli, temperature, humidity, and light, which have been detailed below. 4.3.1.1  Mechanical stimuli Recent studies have focused on knowing about the mechanical stimuli that is responsible for survival of living beings. There have been suggestions made for developing a plant-specific mechanosensory network related to that of the animal system network (Baluska 2015). A living cell can spot the variation between the mechanical forces exerted on it, such as the force exerted by snow, wind, rainfall, fruit, touch, and gravity. The hydration state is also one of these mechanical forces, which is referred to as the turgor pressure. As the above-stated study results suggest, all living cells respond to many such mechanical forces or signals, irrespective of their classification or habit, and the presence of certain molecular materials that form the microstructure of the internal cell network causes these responses (Baluska et al. 2003; Baluska 2010; Baluska and Mancuso 2009; Jaffe et al. 2002). This sensory network is the foundation for a unifying hypothesis, which may account for the perception of numerous mechanical signals affecting guttation (Monshausen and Haswell 2013) (Section 3.4.4). 4.3.1.2  Atmospheric temperature Temperature is one of the most important environmental factors influencing guttation. This phenomenon usually occurs when the environmental conditions lead to reduced transpiration, either on account of increased humidity or the closing of the stomata in reduced light or both, accompanied by abundant water and plant nutrients in the soil. Cool nights following warm days are also conducive to guttation (Gaumann 1938; Kramer and Boyer 1995). The daily periodicity in the pressures developed during warm days and cool nights was thought to be responsible for a pumping-like action, giving rise to variation in guttation, though just how this might work is unclear.

Regulation of Guttation    63

Although the external environment affects the extent and course of bleeding, Heyl (1933), for instance, believed that daily temperature fluctuations were the controlling factor for the autonomic nature of the rhythmic guttation (Grossenbacher 1938, 1939; Skoog et al. 1938). Relatively more recent work demonstrates that guttation being an energy-dependent process, temperature plays a dominant role in its regulation (Section 4.3.2.1). 4.3.1.3  Light Engel and Friederichsen (1951, 1952, 1954) have researched on the effect of light and its intensity on guttation. In an experiment, a light-induced wave was superimposed for 24 hours on the guttation cycle of maize. The periodicity of exposure to light was recorded while guttation was seen from the coleoptiles. There was an increased guttation rate with increased exposure to light, which was vice versa for low intensity. The various light/dark conditions used were 12:12, 6:3, 3:3, and 2:2. ‘Indigenous rhythms in guttation’ were not found in maize, as earlier spotted in the case of oats. Heimann (1952) showed a 24-hour periodicity in the bleeding rate of a 2-year-old Kalanchoe plant cut stem when grown under controlled temperature and 98–99 percent humidity. However, maximum exudation was observed post-24 hours of cut. High humidity and light-and-dark cycle conditions showed limited or no effect on exudation rate. However, if more than three leaf pairs were present on the plant, then light cycle showed an impact on the bleeding rate. However, previously, when the plants were grown in natural or artificial light-and-dark cycle, before cutting the top of the plant, these cycles showed fixing of time of the highest and lowest bleeding rates. Thus, timing of the cut or any minor change in temperature had no impact on the cycles. When the sunflower plants were grown on a normal day having 12-hour light:dark cycle, they showed a high level of bleeding at 12 noon and a minimum at 12 midnight. However, it was possible to induce a 12-hour cycle during the bleeding peak by letting the plants grow under artificial light at night and allowing the plants to grow in the dark during the day. This is referred to as ‘inverted day’, where maximum exudation occurs (Grossenbacher 1939). Furthermore, comparative experiments between bleeding and guttation showed that, under the same external conditions, the course of water loss by these two processes may be different (Heimann 1950, 1952). On one hand, bleeding is affected, under some conditions, by the time of cutting as described above, and, on the other hand, guttation is influenced by the light-and-dark cycle to which the leaves are subjected. The maximum rate of guttation was seen in case of dark cycle with minimal light, even when the level of humidity was high with constant temperature. However, this differed in case of sunflower plants, where bleeding rate was high during the day as compared with night, which could be due to its antiphasic nature with respect to bleeding and could be demonstrated by inserting one extended light (or dark) phase. Initiation of guttation occurred during night/dark, whereas it increased during the day, which was in line with the findings of Heimann (1950, 1952). Five-day-old dark-grown seedlings of barley were placed under light after 48 hours, with or without 1 μM diuron herbicide in the root medium (Riedell and Schmid 1987). Then, potassium was added uniformly to the root medium, and the plants were transferred to a site having high humidity. Guttation fluid was collected after 12 hours, and the potassium contents of roots, shoots, and guttation fluid were determined. Shoots of seedlings grown under light contained larger amounts of potassium but had smaller amounts of potassium in their guttation fluid than dark-grown seedlings. Interestingly, diuron reduced this light-stimulated increase in

64   Guttation

shoot potassium content and increased its amount in the guttation fluid. These results suggest that barley shoots do modify the ionic content of the xylem sap and that changes in the ionic contents of the xylem sap can be monitored by sampling guttation fluid. Pedersen (1993, 1994) studied the transport of water acropetally in case of submerged aquatic plants. He stated that tritiated water was channelized to the youngest leaves, which were in a state of active growth in case of S. emersum, that water transportation was dependent on exposure to light. The movement of water seemed to be energy dependent during dark hours, as the rate of transportation was low during this period. Like Pilobolus, gametophytes of the fern Asplenium trichomanes also display guttation under light regime. Changes in membrane potential brought about by light were measured in the presence of inhibitors of ion channel and proton pump to determine connectivity, if any, between membrane potential and guttation (Szarek and Trebacz 1999). Remarkably, the two anion channel inhibitors, that is, anthracene-9-carboxylic acid and niflumic acid, led to suppression of light-induced depolarization. However, K channel blockers, namely tetraethylammonium and Ba 2+, showed increased amplitude of light-induced membrane potential. In case of Ca channel inhibitors, such as La3+, Gd3+, diltiazem, nifedipine, and verapamil, no change in the membrane potential was observed. Likewise, H+ pump inhibitors, diethylstilbestrol, and vanadate, showed a minor effect, thus stating a possible involvement of chloride and potassium fluxes in case of guttation which is light dependent. The pale-colored oat seedlings show profuse guttation following their exposure to light for around 3–6 hours (Mcintyre 1994). This phenomenon of guttation occurs in all intensities of light that are available continuously; however, no guttation occurs in darkness. The study outcome showed that the length of oat coleoptiles reduced by around 30 percent due to continuous exposure to light. Thus, it can be confirmed from the above-stated fact that light-induced guttation in case of oat seedlings was caused by water exudation from short coleoptiles, which was indirectly linked with root pressure and the changes seen in it. The hypothesis stating the cause of phototropism, which was due to the impact of light on transpiration, was denied by the fact that Avena coleoptiles were found to be responsive to light, being an aquatic and submerged plant. Further research hinted the reason behind phototropism in Avena plant, which might be due to the light-induced guttation in the stomata (Mcintyre 1994). Evidence for this statement was obtained by measuring the amount of tritium that accumulates in the water jacket that surrounds the coleoptile when tritiated water is transported to the roots. Further investigation showed that the application of nail paint to coleoptiles resulted in immediate guttation from the stomata, which was then quantitatively measured using image analysis techniques. The analysis showed guttation occurring at the coleoptiles’ irradiated edge that was previously stimulated in its submerged condition by using blue light. Later on, a reduced rate of guttation was observed along with its uniform distribution when light intensity was reduced through complete water saturation with carbon dioxide. The coleoptiles showed small curvatures when they were kept under darkness and in a highly humid environment, which resulted in its presence towards the side from where guttation occurred through the water pores. Application of petroleum jelly on the leaf stomata under light exposure of coleoptiles showed stimulated response, stating a negative curvatures in the apical half of the coleoptile along with reduced positive curvature in the basal half. However, in parts where stomata did not exist, application of petroleum jelly showed no impact. Thus, it seems reasonable to hypothesize that light affects guttation by changing the membrane potential and permeability through oxidative gating of aquaporins (Cochard et al. 2007; Kim

Regulation of Guttation    65

and Steudle 2009; Mcintyre 1994). Furthermore, investigations into these aspects of guttation in agricultural and horticultural crops and their genotypes, with the focus on the energetics of uptake and transport of mineral nutrients, facilitated by membrane ATPases are warranted (Wegner 2014, 2015a,b). 4.3.1.4  Atmospheric humidity For the guttation rate to be high, the atmospheric humidity level must be high. Thus, when the soil and atmosphere are saturated with water, the guttation rate is high. For example, a high guttation rate is observed for a long period when roots of the plant are immersed in a salt solution containing mobile ions with continuous aeration at a constant optimum temperature. In addition to this, certain ions, like that of bromide and potassium, are indicators of guttation. Concentrations of these ions were found to be high under humid and dark conditions (Hoagland and Broyer 1936). Of no surprise, under conditions of high humidity, sometimes, deciduous rainforest trees guttate so vigorously that one might liken it to rainfall, but it is not so, nor is it real dew that condenses on them and falls as droplets (Feild and Arens 2007). 4.3.1.5  Wind Guttation water droplets exuded by the grass leaves are seen only when the air is saturated and wind velocity is absent. If the vapor source is the wet soil, wind always plays an adverse role. Guttation at nights is usually seen because of the movement of warm air from hotter to colder regions. Singh et al. (2009a) focused on studying the effect of certain environmental and edaphic factors like wind velocity and soil moisture stress on guttation in rice plants. The rice plants were grown in pots till the tillering stage. Following this, they were placed before a table fan 2 m apart so that differential wind velocities could be achieved by adjusting fan speeds at 0, 30, and 60 rpm. The experiment was conducted in a low light, just before the sunset. The exudate was collected for around 30 min, and a remarkable impact of wind velocity was observed on the guttation rate from leaf tips. At 0 rpm, the guttate volume was 100 µL, which further declined to 75 µL at 30 rpm. Only limited guttate was collected at 60 rpm (Figure 4.2). Thus, it states the importance of wind velocity as a regulatory factor in guttation, probably by shrinking the Figure 4.2  Effect of wind velocity on the rate of guttation size of droplets tremendously due to over a period of 30 min at tillering stage in hybrid rice (cv. NDRH-2). The least significant difference (LSD 0.05) = 3.91 rapid evaporation, making the guttation [Source: Singh et al. 2009a]. process almost invisible.

66   Guttation

4.3.2  Edaphic

factors

Apart from atmospheric and environmental factors described above, several soil factors also affect guttation. In the following subsections, the effects of each of these factors are described separately. 4.3.2.1  Soil and root temperature If the water taken up from the soil by the plants can no longer be evaporated through plant surfaces, it is excreted through the water stomata, that is, hydathodes. This process presupposes moist soil that is warmer than the air and high atmospheric humidity. Undoubtedly, therefore, soil and root temperatures play a dominant role in the regulation of guttation. Hoagland and Broyer (1942) observed a rapid guttation, which continued for a relatively long time in humid atmosphere at a favorable temperature, but little guttation occurring when roots were exposed to a low temperature (5°C). Although periodicity in bleeding appears to be an intrinsic occurrence of the maxima and minima, it depends on temperature (Skoog et al. 1938). Similarly, temperature around the roots of submerged aquatic plant S. emersum had a profound impact on the guttation rate (Pedersen 1993). It was found that on reducing the temperature from 15°C to 10°C, the guttation rate was also reduced by five folds. At 4°C, the guttation process completely seized. However, this process seemed to be reversible by increasing the temperature to 25°C. Different temperatures showed different Q10 values for guttation in S. emersum. The Q10 value was projected as 8.2 at 10°C and 1.4 at 25°C. The experiments confirmed that the driving force for guttation is restricted to the root and its metabolism (Fujii and Tanaka 1957). Realizing the significance and economic importance of guttation as a biological phenomenon, a research project sponsored by NASA of the United States, was undertaken to study this process by controlling relative humidity and root temperature on tipburn in lettuce. The symptoms of this disease developed later at the lower root temperature of 15°C than at the higher temperature of 23.5°C (Tibbitts 1986). Plants grown at root temperatures of 23.5°C had marginally higher calcium concentrations in the inner leaves than plants grown at root temperatures of 15°C. Although saturated humidity at night increased the growth rate, tipburn was reduced probably as a result of root pressures, as indicated by the intensity of guttation that developed during the dark period, promoting calcium transport to the young, expanding leaves. Nonetheless, the combination of low humidity during the day and saturated moisture conditions at night would act together to provide a large fluctuation in plant water potential, which would encourage calcium movement to the young leaves and delay tipburn. The small increase in calcium concentration observed with the higher root temperature was not great enough to have a significant effect in reducing tipburn injury. Thus, it appears that root temperature regulation between 15°C and 25°C will not be effective in preventing tipburn. Role of warm soil in determining the rate of guttation can be observed in case of plants that show high guttation rate during spring nights, which are cooler as compared with spring days (Gaumann 1938). During spring season, the soil temperature remains high as compared with the atmospheric temperature, mainly at night, which is accompanied with highly humid cool air. Such conditions favor cuticular transpiration at night, but its rate is reduced because of the optimum water balance by the plant, which results in the development of positive root pressure in xylem sap. Finally, guttation takes place through the water pores or hydathodes. In high valley regions, the above-stated conditions prevail in the spring as well as in the vegetative period under intense light radiation, which heats up the soil in the day and there is rapid cooling at night, which results in guttation (Frey-Wyssling

Regulation of Guttation    67

1941). This is why tropical forest areas favor rapid guttation due to the above-stated conditions, which are mostly prevalent in these areas. Hughes and Brimblecombe (1994) researched on the process of guttation and dew formation with relation to environmental significance. They found that guttation volume was linked with humidity in the air and temperature of the soil. In case of dew, the droplets’ diameter was directly associated with the amount of dew as well as the average of the total surface area of the droplet on the short grass. It was about 11 m 2 ground area. The average guttation was around 5 m 2. These results show that guttation and dewfall significantly added to the water status of the leaf. Guttation seems to be an energy-dependent process, and thus, it is affected by the temperature of both the soil and the roots. 4.3.2.2  Soil moisture As discussed earlier, the phenomenon of guttation is well observed in humid climate, where the moisture content of the soil is also high. All these conditions favor positive root pressure and low level of transpiration, thus high rate of guttation. However, plants that have their top portions removed do not always show guttation from the stumps. Such kind of phenomenon is observed when there is water deficit in the root. Such a condition does not always result in positive root pressure, as the roots require ample time to recover from this deficit situation. If water is added, oozing occurs. In an experiment conducted on Coleus, sunflower, and tomato plants, no guttation was observed in them when they were grown in sandy soil with 45 percent moisture available (Kramer 1941; Stocking 1945). This indicates that the development of positive root pressure required for guttation is hampered when plants are grown in sandy soil with moisture content of 45 percent or less. Adding polyethylene glycol (PEG) (400 MW) to the nutrient medium exhibits reduced osmotic potential in the xylem of the root system. This, in turn, creates an osmotic gradient for the water to enter into the roots and results in guttation at an osmotic potential of 4.8 bars (480 kPa) (Kaufmann and Eckard 1971). At any value lower than this, osmotic potential can lead to negative root pressure. Nearly 50 percent of the osmotic gradient resulted from increased ion concentration of K+, Na+, Ca 2+, and Mg2+ , along with PEG, in the root xylem. Along the lines of these investigations, Zaitseva et al. (1998), in an experiment with a single independent variable, examined the separate influence of capillary-sorptive (Pcs) and osmotic moisture pressure (Po) on the growth and guttation of barley, with the aim of examining the availability of soil moisture for plants, in a wide range of salinization (0.2–50 gL−1 NaCl and 0.2–66 gL−1 Na 2SO4) and moisture content (Pcs= −24 atm or −24.3 bars or −2.43 MPa). The study was carried out on samples of chernozem soil, moistened with the above two salt solutions to the values of field water capacity. It was shown that the relation between the relative growth and guttation of barley and the osmotic pressure was close to the linear one, that is, the equations derived were in agreement with modern concepts of water movement in the soil–plant system. Water stress inhibited guttation in rice, and it could only be noticed, if any, in the succeeding mornings (Singh et al. 2009a). These authors studied the effect of water stress on guttation in rice at the field level. For experimental studies, the water stress was developed through withholding of the irrigation supply to field crops that were at the anthesis stage. Guttation results were evaluated on a daily basis in the morning. In the evenings, no guttation was observed. Guttation volume was recorded during water stress periods by using the dye method (Knipling 1967) when the water potentials were −0.5, −1.0, −1.5, and −2.0 MPa at mid-day, which were recorded through experimentation in

68   Guttation

the succeeding mornings. The watered plants were considered as controls with no stress (−0.2 MPa). It was seen that water stress levies a negative effect on guttation (Figure 4.3). The guttation rate reduced drastically to about 3 µL at −2.0 MPa for 30 min, which later increased a bit to around 5 µL at −1.5 MPa of leaf water potentials that was recorded at mid-day on previous study days. However, the volume was high, that is, 19 µL, 56 µL, and 93 µL at leaf water potentials of −1.0, −0.5, and −0.2 MPa (watered), respectively. This stated a positive correlation between guttation rate and water availability or leaf ’s water potential. The natural mechanism for overcoming the water stress situation was also noted in case of the leaves in the morning at leaf water potential of −0.5 and −1.0 MPa, as recorded on previous days. Figure 4.3  Effect of water stress on the rate of These findings, together with those discussed guttation over a period of 20 min at anthesis stage earlier, clearly bring out the significant role in hybrid rice (cv. NDRH-2). The least significant of soil moisture in the regulation of guttation difference (LSD 0.05) = 1.7. Water stress was imposed by way of reduced root hydrostatic pressure by withholding the supply of irrigation water until leaf (Kramer and Boyer 1995), inhibited cell water water potentials fell to approximately (±0.08) −0.5, −1.0, −1.5, and −2.0 MPa during advancing stress periods at permeability (Heinen et al. 2009; Kaldenhoff mid-day preceding the mornings when the observations et al. 2014; Katsuhara et al. 2008; Maurel et on guttation were recorded [Source: Singh et al. 2009a]. al. 2008), and development of cavitation and embolism in plants (Brodribb and Holbrook 2006; Holbrook et al. 2001). In addition, soil moisture stress may also affect the sensitivity of hydathodes, impacting guttation. 4.3.2.3  Soil nutrients Plant nutrition also plays a role in guttation. The observations obtained under high humidity showed a high effect of exudation pressure in case of plants growing with high salt nutrient supply (Broyer 1951; Crafts and Broyer 1938). Hoagland and Broyer (1936) stated that the impact of carbohydrate reserves on absorption of salts also had a negative impact on root pressure. Researchers carried out experiments on barley plants and studied the root’s metabolic activities. They observed that a strong association exists between the amount of guttation fluid and the concentration of salt that was translocated to the shoots when roots were submerged in distilled water. In this case, the guttation process seized even if there was frequent aeration. In another set of experiment, the salt solution was diluted along with no aeration. This condition also exhibited a low guttation rate. In another condition, dilute salt solution, good aeration, and favorable temperature resulted in profuse guttation. Further study was conducted by Raleigh (1946a) on tomato plants that were grown in a complete nutrient solution. The plants showed guttation; however, plants grown in a nutrient medium devoid of N, P, K, Ca, and Mg showed no guttation. On addition of these lacking constituents to the medium,

Regulation of Guttation    69

all the plants, except those in the control solution, showed guttation. It can be hypothesized that plant injury due to Ca and Mg deficiency results in the absence of guttation. Thus, it confirms a relationship between the activity of the cell due to availability or non-availability of nutrients and rate of guttation. Raleigh (1946b) established exudation of glutamine in the guttate of rye grass, which was followed by the addition of ammonium chloride. The amount of guttation was related to the condition of grass before the addition of ammonium chloride. Conversely, plants deficient in nitrogen produce more glutamine as compared with dark green plants with the application of ammonium chloride. Baba (1957) conducted experiments on rice plants that contained silica in the exudate obtained from cut stems. The silica amount in the guttate was related to the amount of the elements added to the culture medium. The silica concentration ranged from 400 to 800 ppm in exudate sap and 100 to 300 ppm in the guttate. As green manure or mineral fertilizers contain more silica, the concentration of silica is also higher in case of plants that are grown in such medium. Additionally, injury to the root caused by addition of H 2S to the nutrient solution or addition of ammonium sulfate to soil leads to reduction in the guttation rate, along with reduced concentration of silica in the guttate fluid. Ivanoff (1961) studied the guttation amount in cantaloupe plants considering two different soil fertility levels. All study plants were set up in a greenhouse and rooted into sandy and loamy soil that had a naturally high content of and was deficient in N and P. Commercial fertilizer (formula 11-48-0) was added to a few plants at approximately 200 lbs per acre. Artificial conditions were created inside the greenhouse that favored guttation. Guttate production could be observed around the leaf margins on both fertilized and non-fertilized plants. After evaporation of the guttate, large deposits of minerals were found on the leaves of artificially fertilized plants as compared with other plants. Recently, Zwieniecki et al. (2004) demonstrated that phloem girdling led to a decrease in xylem ion concentrations, which, in turn, led to axial xylem hydraulic conductance. Phloem girdling showed no effect of such hydraulic conductance when similar measurements were done for branches perfused with KCl solution ( 140 mOsm kg−1). The study outcomes suggest a link between the hydraulic systems of phloem and xylem, which occurs due to change in the ion concentration of xylem cell sap. Subsequently, chemical materials move between phloem and xylem, which is believed to have a significant impact on exudation and root pressure (Section 3.4.6). It can, therefore, be stated that nutrition has a great impact on the guttation rate. 4.3.2.4  Soil aeration The presence of air in the soil system is essential for both soil flora and fauna, for their optimum respiration to occur. It has been described earlier that guttation is an energy-dependent phenomenon that requires oxygen for metabolic activities (Szarek and Trebacz 1999; Sze 1984; Sze et al. 2002). It was observed that when, as mentioned earlier, the roots were submerged in a dilute salt solution containing mobile ions and were well aerated while maintaining a favorable temperature, rapid guttation occurred (Broyer and Hoagland 1943). Water intake by vines in free water or in soils of fairly high water content may be slowed because of inadequate soil aeration, resulting in oxygen deficiency. The CO2 and N2 have a negative effect on guttation, whereas aeration improved guttation. This condition slows the respiration of the roots, thereby slowing growth; if the condition unfavorable to respiration persists, the roots disintegrate (Fujii and Tanaka 1957; Pedersen 1993, 1994). Experiments

70   Guttation

on barley showed that immersion of roots in distilled water shows limited guttation even on proper aeration. In case of a dilute salt solution without aeration, guttation was observed, but it was limited; however, in case of a dilute salt solution with aeration, profuse guttation was observed (Broyer 1951; Broyer and Hoagland 1943). It is, therefore, clear that aeration, though important (Szarek and Trebacz 1999; Sze 1984; Sze et al. 2002), functions only if other factors are not limiting. 4.3.2.5  Soil mycorrhizae Mycorrhiza is a symbiotic association of lichen and fungus. It has a root system containing root hairs that assist in absorbing nutrients and water from the soil. This phenomenon of absorption of contents is faster when the mycorrhizal fungi are associated with the roots (Smith et al. 2003; Smith and Read 2008). A strong correlation exists between the rate of guttation and mycorrhizae in the soil. However, its role in water absorption is not well studied (Lehto and Zwaizek 2011). The water uptake mechanism that results in increased uptake can be attributed to the extensive growth of the roots and increased surface area, that is, more than 3 m of the fungal hyphae, which can extend from every root. Enhanced functionality of aquaporin and, consequently, increase in hydraulic conductivity of roots in case of ectomycorrhizas were also studied under drought as well as watersufficient conditions (Barzaana et al. 2012). Nevertheless, in-depth study is required. The effect of mycorrhizae on water relations could be due to enhanced nutrient content, which, in turn, affects water uptake, positive root pressure favoring guttation, and guttation (Raleigh 1946a; Smith et al. 2003; Smith and Read 2008). In addition, the arbuscular mycorrhizal symbiotic plants bestowed with the ability of switching between apoplastic and cell-to-cell water transport pathways could acquire a higher flexibility in response to the guttation rate, but more work is required to elaborate its association with guttation. 4.3.2.6  Soil salinity and pollutant The salinity of the soil is known to increase the osmotic pressure of the soil solution, which interferes with water absorption through the roots. The inhibited water absorption affects the processes of both root pressure and guttation (Kramer and Boyer 1995). The guttate has been found to contain Zn, Cd, Ni, B, As, Se, and so on, along with few transporter proteins, which might be considered as a measure for detecting the content of toxic elements as well as enhancing the level of tolerance to such toxicity (Ghosh and Singh 2005; Meagher and Heaton 2005; Schmidt et al. 2009). Thus, this is one of the ways of excreting toxins through guttation, which depends on its intensity and magnitude. To conclude, the effect of crucial factors regulating the phenomenon of guttation has been studied in some detail. However, in view of increasing degrees of environmental pollution and other factors that affect climate change and lead to global warming, which are a threat to human civilization and existence, it would be rewarding to examine the impact of such factors on guttation, with a view to enhance quantum and quality of agricultural and forest production and products, in addition to improving environmental quality and ecological balance (Aloni 2001, 2010; Aloni et al. 2003, 2005; Feild et al. 2003; Feild and Arens 2007; Hughes and Brimlecombe 1994). From the above-stated evidence, it can be confirmed that guttation is primarily regulated by genetic modifications in the plants, along with edaphic and environmental factors to which the plants are exposed.

C h a p t er

5

Chemistry of Guttation

5.1  Introduction The liquids and deposits of guttation on hydathodes consist of organic as well as inorganic constituents that vary from time to time. In this context, stem exudation obtained through decapitation for chemical analysis can be equated, by and large, to guttation fluid. Similarly, xylem-sap constituents can be likened to guttation fluid constituents as well. The composition of these liquids is determined by the age, physiological activity, plant species and varieties, as well as the solute composition and concentration of the medium from which the plant absorbs. Most of the guttate and solute samples obtained from hydathodes were not collected under controlled conditions, and so, their composition test results, at best, indicate general trends. The composition of the guttated liquid seems to vary from pure water to a dilute solution of organic as well as inorganic solutes that constitute around 0.05–0.5 percent of the liquid. Curtis (1943) showed that guttate obtained from squash, cabbage, tomato, and cucumber are some of the best examples of the general order of magnitude of solute concentration and guttate’s composition. Nearly 50 percent of the solute was organic in nature, which ranged from 600 to 2500 mg L −1. The osmotic potential of guttated liquid obtained from leaves of cotton plants varied from −0.051 to −0.091 MPa for plants growing in the usual concentration of nutrient solution, whereas it was −0.013 MPa for plants grown in a dilute solution (Eaton 1943). Following heavy application of fertilizer to a lawn, the leaves of the grass were encrusted with a deposit of glutamine left by the evaporation of guttated liquid, and the leaves of some species of Saxifraga usually become coated with calcium salts (Curtis 1943). In this chapter are described the salient features of the chemistry of guttation fluids in some detail, which carry immense agricultural, pharmaceutical, nutriceutical, therapeutical, cosmeceutical, and commercial significance (Rybicki 2009).

72   Guttation

5.2  Organic constituents of guttation fluids Guttation fluids are known to carry several different organic materials such as sugars, amino acids, amides, purines, pyrimidines, reductants, various kinds of proteins (such as simple proteins, recombinant proteins, transporter proteins, and signal transduction proteins), enzymes (such as isoprenyl transferase, peroxidases, ATPases, and dehydrogenases), antibodies, ATP, mRNA, lipophilic materials, volatile oils, herbicides, insecticides, fungicides, alkaloids, and toxins (Fischer et al. 2004; Ma et al. 2005; Twyman et al. 2005) (Figure 5.1, Table 5.1). Goatley and Lewis (1966) determined the types and quantity of substances responsible for growth and differentiation of Claviceps purpurea raised in growth media containing guttation fluids obtained from seedlings of Rosen rye, Genesee wheat, and Traill barley, separately, by using different separation and analytical techniques such as chromatographic methods for amino acids and sugars, spot tests and spectrometric techniques for inorganic materials, and microbiological techniques for vitamins. The

Figure 5.1  Guttation fluid containing a number of organic and inorganic compounds including metabolites, enzymes, hormones, vitamins, salts, ions, nutrients, pathogens, etc., impacting plant behaviour [Source: Singh and Singh 2013].

Chemistry of Guttation    73

total sugar content was equal in rye and barley fluids but lower in wheat fluid, with glucose being the principal sugar component of the former two species and the concentration of galactose being the highest in wheat. Aspartic acid or asparagine was the amino acid mostly present in all the three fluids, with barley having the highest concentration and wheat having the lowest concentration of all amino acids among the three species. Barley contained the highest concentration of inorganic elements and wheat had the lowest of them, except for iron for which rye had the highest and barley the lowest. Barley had the highest amount of vitamins and other chemicals such as choline, p-aminobenzoic acid, thiamine, and uracil; rye had the highest amount of inositol and pyridoxine; and wheat had the lowest amount of choline and inositol. Proteins with signal transduction, putative transcription factors, and stress response factors, which include hormones and metabolic enzymes, have recently been seen in xylem and phloem saps that culminate into guttation fluid (Aki et al. 2008; Biles and Abeles 1991). These results suggest that nano-scale proteomics would be a potent tool for investigating and unravelling biological processes such as guttation and exudation in plants with ample intertrafficking of chemical substances from xylem to phloem and vice versa (Kundt and Gruber 2006). 5.2.1  Proteins

and enzymes

Several kinds of proteins and enzymes are found in the guttation fluid whose amount and composition depend upon the genetic makeup, health, and habitat of plant species. In this regard, the pioneering and highly appreciative works of Magwa and coworkers in South Africa are worth mentioning (Magwa et al. 1993; Magwa 1995). Some of these macromolecules are described in the following sub-sections. 5.2.1.1  Protein profile: new proteins Some proteins are naturally secreted into the plant guttation fluid. At the beginning of the past century, Wilson (1923) reported, for the first time, that the concentration of total solids obtained from maize (Zea mays) is as high as 1030 ppm and that from timothy (Phleum pratense) is 220 to 573 ppm. The water that was exuded from maize, oats, and timothy also had catalases, peroxidases, and materials that could cause reduction of methylene blue. However, reductases was present in timothy exudate, but not in maize exudate. More recently, different peroxidases have been partially characterized in the guttation f luids of strawberry (Fragaria ananassa), tomato (Lycopersicon esculentum), and cucumber (Cucumis sativus) (Biles and Abeles 1991). Phylloplane proteins The leaf surface, known as phylloplane, acts as an inter-kingdom crossroad in between plants and microorganisms, and antimicrobial biochemical secretion to aerial surfaces is considered one of the strategies of defense, with the help of which plants deter potential pathogens. The secondary metabolites present on leaf surfaces are documented properly, but antimicrobial phylloplane proteins that are biosynthesized (e.g. phylloplanins) by trichomes of a specific group have only recently been identified (Shepherd and Wagner 2007). These authors have physically described the structures

Table 5.1  Organic and inorganic constituents commonly found in guttation fluids of different plant species. A. Organic chemicals a. Plant-derived chemicals I. Carbohydrate A. Monosaccharides 1. Glucose 2. Fructose 3. Galactose, etc. B. Disaccharides 1. Sucrose C. Polysaccharides 1. Cellulose (cotton fiber) Goatley and Lewis (1966), Smart et al., (1998), Jaradat and Allen (1999), Kim and Triplett (2001), Slewinski et al. (2009)

II. Proteins 1. Simple proteins 2. Regulatory proteins 3. Signal transduction proteins 4. Transport proteins 5. Recombinant proteins 6. Antibodies 7. Defense proteins 8. Antimicrobial phylloplane proteins 9. Actomysine-like protein 10. Actine-like protein 11. Pathogenesis- related proteins 12. Recombinant immunoglobulins 13. Pharmceutical and technical proteins

III. Enzymes 1. Dehydrogenases 2. Peroxidases & isozymes 3. Glutaminyl cyclase 4. ATPases 5. gdhA 6. mRNA (Transcriptase)

Biles and Abeles (1991), Young et al. (1995), Ameziane et al. (2000), Gay and Tuzon (2000), Mizuno et al. (2002), Burkle (2003), Nolte et al. (2004), Pilot et al. (2004), Magwa et al. (1993), Mihucz et al. (2005), Kerstetter et al. (1998), Ghosh and Singh Borisjuk et al. (1999), (2005), Meagher and Komarnytsky et al. (2000, Heaton (2005), 2004, 2006), Gaume et Sutton et al. (2007), al. (2003), Grunwald et al. Tapero et al. (2007), (2003), Fischer et al. (2004), Schmidt et al. (2009), Shepherd and Wagner (2007), Testone et al. (2009), Aki et al. (2008), Slewinski et Harada et al. (2010) al. (2009), Drake et al. (2009), Rybicki (2009), Pascal et al. (2009), Hehle et al. (2011)

IV. Amino acids & Amides 1. Glutamate 2. Aspartate 3. Glutamine 4. Aspargine

V. Lipoids VI. Secondary 1. Fatty acids Compounds 2. Resins 1. Sesquiterpene Sparrow et al. 2. Antidepressant 3. Monoterpenes (2007) 4. Sclareol 5. Volatile oils Curtis (1944 a) 6. Pesticides (diuron, Ameziane et al. griseofulvin, (2000), Pilot et al amiben, peramine, (2004) durelle, etc.) Stokes (1954), Riedell and Schmid (1987), Harris (1999), Giddings et al. (2000), Kim et al. (2003), Shawki et al. (2006), Koulman et al. (2007), Sparrow et al. (2007), Valente and Bologna (2011)

VII. Hormones 1. Auxins 2. Gibberellins 3. Cytokinins 4. Abscisic acid 5. Ethylene Pedersen (1998), Dodd et al. (2004), Aloni et al. (2005), Fletcher and Mader (2007), Thompson et al. (2007)

VIII. Vitamins 1. Water- soluble vitamins B & C 2. Lipid- soluble vitamins A, E & K Goatley and Lewis (1966), AsensiFabado and MunnéBosch (2010)

IX. Alkaloids 1. Cyanogenic glucosides such as dhurrin 2. Diglucoside such as dhurrin-6glucoside Selmar et al. (1996), Tattersall et al. (2001), Koulman et al. (2007)

b. Microbial chemicals: toxins and mycotoxins produced by bacteria, fungi and viruses. Young et al. (1995), Gay and Tuzon (2000), Scott et al. (2004), Gareis and Gareis (2007) B. Inorganic chemicals such as salts, ions and nutrients: Na+, K+, PO43+, NH4+, NO3 –, SO42–, CO32–, HCO3 –, Cl –, B, Zn2+, Mg2+, Mn2+, As3+, Al3+, Si4+, etc., found in guttation fluids, leaf leachates and rain water washings. Wilson (1923), Curtis (1943, 1944a, b), Ivanoff (1963), Necmi (2005).

Source: Singh and Singh (2013)

Chemistry of Guttation    75

for various biochemistry on the phylloplane and have also provided a brief discussion about surface defenses against animals that are based on protein. Having reviewed the present literature that is related to antimicrobial phylloplane proteins and mechanisms of their release to the phylloplane, and delivering them into fluid of guttation from hydathodes, it is hoped that further research will pave a way for the advancement in our understanding of the phylloplane and phylloplane proteins for useful bio- and nano-technological interventions. These aspects, therefore, need to be studied in depth, both extensively and intensively, for food and horticultural crops and varieties thereof, particularly in South Asian, African, and Latin American countries, including Caribbean countries. It is advisable to first initiate these studies, to investigate the production and secretion of such and other proteins at cultivar levels of various guttating agricultural crops, with a view to addressing issues related to food and nutrition security. Transporter proteins Recently, many proteins have been detected in the guttation fluid that are transferrable in nature, and the expression profile measurement of Pi, K+, and NO3− transporters along the longitudinal axis of the barley leaf showed that some of the transporters are expressed properly at the hydathode, but no specific variation was observed for most of the transporters along the leaf phyllotaxy. The transporters facilitate longitudinal ion gradients in the mechanism; their development takes place in leaves, and their physiological functions have been recently discussed by Nagai et al. (2013). Of course, this area of research needs in-depth studies to provide new insights into guttation protein chemistry. Cytokinins and derivatives transporter Nucleobases and their derivatives like CKs and caffeine are placed differently in the vascular system of plants. Burkle et al. (2003) found that adenine and CKs are moved by one common H+coupled high-affinity purine transport system. In this case, AtPUP1 and AtPUP2, present in the hydathodes’ epithem and siliqua’s stigma, generate energy-dependent high-affinity adenine uptake in yeast, whereas AtPUP3 remained undetectable. Similarly, the results of PUP-mediated uptake of adenine was inhibited by CKs, which indicated that CKs are transport substrates, and measurements that use the method of tracer demonstrated that AtPUP1 could mediate uptake of radiolabeled trans-zeatin (t-Z). Furthermore, adenine strongly inhibited CK uptake, and 6-chloropurine poorly inhibited isopentenyl adenine (iP) uptake. Numerous physiological CKs, inclusive of trans- and cis-zeatin, were highlighted to be efficiently competitive for AtPUP2-mediated adenine uptake, suggesting that AtPUP2 is capable of mediating CK transport. Furthermore, AtPUP1 mediates the transfer of caffeine and ribosylated purine derivatives in yeast. The AtPUP2 promoter drives ß-glucuronidase reporter activity of genes in the phloem of Arabidopsis leaves, which indicates a role in long-distance transfer of adenine and CKs. Nevertheless, AtPUP3 promoter activity was seen only in pollen. Obviously, these PUPs that are closely related to each other are mentioned differently in Arabidopsis, and two of the PUPs have properties similar to that of the adenine and CK transport system. These results, no doubt, inspire and encourage further work on transporters of other hormones as well.

76   Guttation

Sucrose transporter Most plants have sucrose exported from leaf source to sink tissues that import carbon for their growth and metabolism to survive. Apoplastic phloem-loading species highly need SUTs for sucrose transportation into the phloem (Slewinski et al. 2009). Genetic and biochemical evidence indicates that in a lot of dicot plants, SUT1-type proteins lay their functions in loading the sucrose into the phloem. Nevertheless, it is unclear whether SUT1 plays a role in phloem loading in monocot plants, as the rice (Oryza sativa) and sugarcane (Saccharum officinarum) SUT1 orthologs do not lay their functions in phloem loading of sucrose. Along these lines, SUT1 gene was cloned from maize (Z. mays) and was portrayed to have expression and biochemical activity consistent with a hypothesized role of phloem loading. Further, Slewinski et al. (2009) determined the biological function of SUT1 in maize, and specifically for this purpose, a sut1 mutant was isolated and characterized. It was interesting that sut1 mutant plants hyper-accumulated carbohydrates in leaves that were mature portraying chlorosis of leaf with premature senescence. Additionally, sut1 mutants had significantly reduced stature, modified and accumulated biomass and its partitioning, delayed flowering, and stunted development of tassel. Treatments like cold-girdling of leaves of wild-type (WT) for blocking transport of phloem phenotypically simulated the action of sut1 mutants, which supported maize SUT1 in exporting sucrose. Further application of 14C-sucrose to abraded sut1 mutant and WT leaves showed that sucrose export had diminished in sut1 mutants in comparison with WT. These collective data signify that SUT1 is critical not only for phloemloading efficiency of sucrose but also for loading sucrose into xylem saps. Hence, guttation fluid may also serve other physiological processes. Expectantly, transporter proteins for other sugars may be found in guttation fluid as well. Furthermore, class 1 KNOTTED1-like transcription factors (KNOX) are popular regulators of plant development. Nevertheless, information on class 2 KNOX is limited (Testone et al. 2009). These authors attempted the peach KNOPE3 gene cloning, belonging to a family of few members of class 2 and having its location at 66 cM in the Prunus spp. G1 linkage group. Transpiration stream-fed leaflets cause up-regulation of KNOPE3 at 3 hours after absorption of fructose, glucose, and sucrose and at 12 hours after absorption of sorbitol. Hence, both the expressions associated with phloem and gene modulation that had sugar in it suggested that KNOPE3 might be playing a role in translocation of sugar, and guttation fluid proves to be good experimental material for this purpose while agro-relevant organs like drupe were in their developing stages. Toxic element transporters As stated earlier, few of the transporter proteins for toxic elements such as Zn, Cd, Ni, B, As, and Se were recently detected in plant exudates. These proteins have been implicated in enhancing the tolerance of plants by restricting these toxic elements from getting absorbed, that is, exclude or exude (Ghosh and Singh 2005; Meagher and Heaton 2005; Schmidt et al. 2009). Sutton et al. (2007) recently identified Bot1, a BOR1 ortholog, as the gene for tolerance of superior boron toxicity in plant exudates of the Algerian barley landrace Sahara 3771. High-resolution mapping technique was used to locate Bot1 at the tolerance locus, which provided a high capacity for tolerance in yeast. Bot1 transcript levels identified in barley tissues were indeed found to be capable of both limiting the net entry of boron into the root, on the one hand, and its enhanced disposal from leaves through hydathode guttation,

Chemistry of Guttation    77

on the other. Hence, enhancing tolerance for elemental toxic effects by restricting absorption and exudation and by disposal through production of special transporter proteins is significant in crop production on marginal lands and wastelands. For direct practical utility, for example, the nickel (Ni) hyper-accumulator Alyssum murale, in the form of commercial crop for phytoremediation and phytomining, has been developed from Ni-enriched soils (Tappero et al. 2007). Mihucz et al. (2005) stated arsenic speciation of the xylem sap of cucumber plants (Cucumis sativus L.) and came up with a number of arsenic compounds in the sap of plants, ultimately finding a path towards the guttation fluid, which serves the purpose of detoxification. Thus, guttation is a process of excreting harmful elements like Ni and As in excessive amounts and thus promoting plant growth. Nutrient transporters The plasma-membrane H+-ATPases present a topography of the membrane and general action mechanism with other P-type ATPases but have a different property of regulation (Palmgren 2001). Workers in Denmark, Belgium, and Germany have advanced, remarkably, the study in this field which includes the identification of the complete H+-ATPase gene family in Arabidopsis (Duby and Boutry 2009; Bobik et al. 2010). The techniques of reverse genetics used for the analysis of H+-ATPase function revealed a better understanding of post-translational regulation by 14-33 proteins of the pump’s activity (Duby et al. 2009; Yang et al. 2015). Further, novel insights into H+ mechanism of transport (Buch-Pedersen and Palmgren 2003; Niittyla et al. 2007), and progressive structural biology and enzymology of H+-ATPases that are related to ions and uptake of solutes, a prerequisite for root pressure development that causes guttation, were also obtained (Duby and Boutry 2009; Duby et al. 2009; Pedersen et al. 2007). In summary, plasma-membrane H+-ATPases constitute a family of proton pumps operated through ATP hydrolysis and are seen in the plasma-membrane of plants and fungi, playing a significant role in nutrient transfer into the cell. The plasma-membrane H+-ATPases extrude positive charges (H+) and are therefore electrogenic enzymes; thus, they create a membrane potential gradient (negative on the inside). The electrochemical gradient established in such a way across plasma membrane provides a driving force to solutes for making their way into the cell. Cations, anions, and neutral solutes can get into the cell through various carrier proteins, as described earlier, by which transport is energized by the concomitant proton uptake (Sections 3.3.2.2 and 3.4.8). Thus, most of the hundreds of membrane-bound transport proteins that have been recognized in plants are energized indirectly through the plasma-membrane H+-ATPases. Hence, they have been traditionally assumed to be general end points of all pathways of signaling that affect polarization and transport of membrane (Merlot et al. 2007). Signal transduction proteins In fact, the involvement of G-proteins in different pathways in different organisms is extensive (Baluska and Volkmann 2008; Bolle 2004; Sopory et al. 2012; Trusov and Botella 2016), and the purpose here is not to give a detailed account of these aspects. GRAS proteins (G = Gibberellic Acid Insensitive [GAI]; RA = Repressor of GAI [RGA]; S = Scarecrow [SCR]) are a recently discovered family of plant-specific proteins named after GAI, RGA, and SCR; the first three of its members

78   Guttation

have been isolated. Although the Arabidopsis genome focuses on encoding at least 33 GRAS protein family members, few GRAS proteins have been characterized so far. Yet, it is getting clearer with time that GRAS proteins play vital roles in diversified processes such as signal transduction and maintaining and developing meristem. However, the pertinent question as to how G-proteins are involved in signal transduction pathways in plants remains unanswered. In this context, of particular interest is the fact that evidence is accumulating regarding the presence and role of G-proteins in root pressure development for guttation in plants (Dustmamatov et al. 2004; Dustmamatov and Zholkevich 2008; Ma et al. 1990, 1991; Mozhaeva and Pil’shchikova 1972; Zholkevich 1991; Zholkevich et al. 2007). Recombinant proteins, antibodies, and antigens The first recombinant human serum albumin was expressed in tobacco and potato (Sijmons et al. 1990), and simultaneously, demonstration of the fact that the plants were able to express recombinant antibodies was of great interest (Hiatt et al. 1989). As of now, more than a quarter of the available drugs are derived from plants or have plant extracts in them (Raskin et al. 2002). Surprisingly, this area of scientific studies progressed so rapidly that about 100 therapeutic and diagnostic recombinant proteins and vaccines were produced from various plants, including tobacco, cereals, legumes, fruit and vegetable crops, fodder crops, edible foliage crops like lettuce and spinach, oilseeds, and aquatic or unicellular plant species grown in bioreactors (Ma et al. 2003; Twyman et al. 2003). The reasons for this momentum are that plants have benefits over traditional systems of production based on microbial or mammalian cells, particularly in economic terms, scale of production, safety, and practicality (Borisjuk et al. 1999; Drake et al. 2009; Fischer et al. 2004; Komarnytsky et al. 2000, 2004, 2006; Komarnytsky and Borisjuk 2012; Ma et al. 2003; Twyman et al. 2005). Currently, some highly reputed companies have taken a step forward in investigating and exploiting the potential of recombinant vaccines, antibodies, and all other therapeutic entities derived from plants (Section 8.2). Pathogenesis-related proteins It is a general observation that when guttation occurs, the plant surface is wetted, facilitating epiphytic living motile bacteria to move and eventually enter the interior of plants through the hydathodes. It is of interest to know whether plants develop a protection mechanism against motile bacteria near the hydathodes, as xylem and phloem saps are inclusive of various proteins, and such a protection mechanism could use the well-known pathogenesis-related (PR) proteins. Indeed, an analysis of the guttation fluid has demonstrated a clustering of approximately 200 proteins, primarily with isoelectric points (pIs) in the acidic pH range (Grunwald et al. 2003). In these studies proteins, identified, belonged mostly to the family of PR proteins suggesting a role in plant protection against invaders. The protein profile of the guttation fluid was, however, remarkably modified by treating plants with methyl jasmonic acid, which suggests that the protein composition of the guttation fluid is controlled by both internal and external stimuli. Of late, Aki et al. (2008) conducted a shotgun analysis of the proteome of phloem and xylem saps attained from rice crop. These authors recognized 118 varieties of proteins and eight varieties of peptides in the xylem sap, which were highly likely to be found in the guttation fluid moving up through the shoot, and

Chemistry of Guttation    79

107 other varieties of proteins and five varieties of peptides in the phloem sap. It is evident that signal transduction proteins, putative transcription factors, and stress response factors as well as metabolic enzymes have been identified in these saps and exudates. These studies are inclusive of both basic and practical essence, highlighting the indulgence of hydathodal secretion of defense proteins against pathogens (Singh 2014b). Enzymes Peroxidases and isozymes Among various classes of enzymes, peroxidases perform oxidative catalysis that cross-links and polymerizes certain organic compounds by hydrogen peroxide and other organic peroxides. Biles and Abeles (1991) studied xylem-sap proteins gathered by means of pressure extrusion from twigs of apple (Malus domestica Borkh), peach (Prunus persica Batsch), and pear (Pyrus communis L.). This sap consisted of a number of acidic peroxidases and various other proteins. Stem exudates and guttation fluid were the two sources of xylem sap used in this study. Interestingly, peroxidases similar to each other were also recognized in the exudates of stem and guttation fluids of strawberry (Fragaria x ananassa Duch.), tomato (Lycopersicum esculentum L.), and cucumber (Cucumis sativus L.). Determining isoelectric focusing activity of gels portrayed that two peroxidases (pI 9 and pI 4.6) were found in stem exudates in the first 30 min post-excision, and the subsequent samples of stem exudate had only the pI 4.6 isozyme. The pI 4.6 peroxidase isozyme was also seen in both the root tissue and guttation fluid. These observations indicate that roots are responsible for production and secretion of the pI 4.6 peroxidase into the xylem sap. For further investigation, Cucumber seedlings, as mentioned earlier, were treated with 100 µL ethylene for 16 hours, and the exudate from decapitated hypocotyl stumps was gathered over a 3-hour period. Treatment with ethylene increased the peroxidase activity of stem exudates and inhibited the amount of exudate that was released. These observations indicate that xylem-sap peroxidase plays a role in plugging damaged vascular tissue. Studied otherwise, isoperoxidases (EC 1.11.1.7) in the guttation fluid of Helianthus annuus showed up as two bands, G1 (slower moving) and G2 (slightly faster), following disc gel electrophoresis and again as two bands, SG1 (M 39000) and SG2 (M 36000), on sodium dodecylsulfate (SDS) gels (Magwa et al. 1993). These were electrophoretically indifferentiable from the corresponding isoperoxidases, R1 and R2 (on non-SDS gels) and SR1 and SR2 (on SDS gels), found in bleed extracted from just above the root (root bleed). Additionally, these isozymes showed that their representation by SR1 and SR2 were also seen in root apoplast washes. Guttation fluids were also found to accumulate cationic peroxidase attained from xylem vessels induced during incompatible interactions with Xanthomonas oryzae pv. oryzae in rice (Young et al. 1995). A cationic peroxidase, PO-C1 (molecular mass 42 kD, pI 8.6), was made pure and organic, and amino acid sequences from its chemically cleaved fragments were determined, which exhibited a high percentage of identity with deduced sequences of peroxidases from rice, barley, and wheat. The anti-PO-C1, an antibody having reacted only with a protein of the same mobility as POC1 in extracellular and guttation fluids, was gathered 24 hours after infection from plants that had previously undergone throughly incompatible responses. For better insight into the mode of infection, polyclonal antibodies were pulled up to an 11-amino acid oligopeptide (PO-C1) that was obtained from a domain where the sequence of the cationic peroxidase was divergent from similar peroxidases. In the responses that were compatible, the antibodies did not find PO-C1 until 48

80   Guttation

hours after infection. By using immunoelectron microscopy, it was shown that PO-C1 accumulates within the apoplast of mesophyll cells and within the cell walls and vessel lumen of xylem elements of plants undergoing interactions that were incompatible. The agriculturally and ecologically important studies conducted by Kerstetter et al. (1998) demonstrate that peroxidases deposits exists in guttation f luid gathered from Bermuda grass hybrids 419 and Tifway 2 Cynodon dactylon L. × Cynodon transvaalensis Davy, which are seasonally warm C4 grasses, and Kentucky bluegrass (Poa pratensis L.), which is a seasonally cool C3 grass. Peroxidase activity in guttational fluids gathered from grasses early in the morning was in the range of 80–120 µg L −1. The pI values were in the range of 4.3–8.3 with 14 isozymes that were detected using guaiacol and hydrogen peroxide as substrates. Peroxidase extraction from soil supported the growth of Bermuda grass, and it was found to be the most abundant in the top 5 cm layer (activity was in the 6.8–16 purpurogallin units/g range). In the presence of fulvic and humic acids, these peroxidases were stable and active; on evaluating, it was found that peroxidase activity did not decline in winter when the grass seems to be dormant, which indicates that peroxidases released into the soil remain active for a considerable time. Moreover, peroxidases found in guttation fluids and plant exudates have also been implicated in plant disease resistance (Gay and Tuzun 2000) (Section 7.3.7). Presumably, not only the earlier mentioned proteins and enzymes but a number of other vital proteins also exist in the guttation fluid that need to be explored to elaborate the role of guttation in ecological distribution and plant productivity. Other enzymes Apart from peroxidases, other enzymes such as catalase, reductase, isopentyl transferase, and ATPases have also been detected in guttation fluids of various plant species such as oats, maize, and timothy. The amount of these enzymes depend on plant species, their phenological growth stage, growing conditions, nutrition, etc. (Wilson 1923). For example, Klein (1913) reported that nitrites were absent in the guttation fluid of Boehmeria utilis and Fuchsia sp. leaves when it was evaluated right after its excretion, but they appeared after 6 to 8 hours and further developed into ammonia. This loss of nitrates and subsequent appearance of nitrites and ammonia was ascribed to the action of molds and bacteria. It might appear that few other possibilities exist in such a reduction. Enzymes that reduce nitrates are seen in various plants; exuding water comes in contact with living cells of the roots, stems, and leaves. It may effectively reduce nitrates into nitrites and ammonia. The existence and role of various H+-ATPases has, also, been briefly pointed out earlier (Sections 3.3.2.2 and 3.4.8). 5.2.2  Nucleobases

and RNAs

As mentioned earlier, nucleobases such as purines and pyrimidines, ribonucleic acids, and derivatives like CKs and caffeine are translocated in the plant vascular system. Parts of these organic molecules ultimately find their way into guttation fluids (Aki et al. 2007; Biles and Abeles 1991; Burkle et al. 2003). Studies such as this have undoubtedly advanced our knowledge about the role of these compounds; yet, much remains to be understood about their role in guttation and vice versa (Singh and Singh 2013).

Chemistry of Guttation    81

5.2.3  Amino

acids and amides

The nature and composition of exudates from rye grass are of specifically different types which involve principally the glutamine synthesis. Greenhill and Chibnall (1934) stated certain conditions in which perennial rye grass fertilized with ammonium sulfate produced white colored exudate on the upper leaf blades, which contained glutamine at high concentration. This was the first experimentation of its kind. This kind of exudate was observed in case of pastures when ammonium salts were absorbed by roots mainly during spring season. Curtis (1944a) showed the formation of a high quantity of chemical deposits on the leaf blades of lawn grass in the guttate, especially glutamine. Nearly 20 percent of these insoluble deposits seemed to be organic materials. Some of these deposits were also formed on some fruits that were exposed to light (Ivanoff 1941). Activation of Arabidopsis thaliana-tagged mutant, glutamine dumper1 (gdu1), was recognized to collect salt crystals at the hydathodes (Pilot et al. 2004). Chemical analysis showed that, in contrast to the amino acid mixture generally existing in guttation droplets, the crystals primarily consisted of glutamine (Gln). Gdu1 was cloned and seen to have encoded a novel 17 kD protein consisting of a single putative transmembrane span. Gdu1 was clearly observed in the vascular tissues and in hydathodes. Gln levels rose, particularly, in the xylem sap and leaf apoplasm, whereas the content of few amino acids increased in the leaves and phloem sap. Selective secretion of Gln from the leaves may, nevertheless, lay the explanations through an enhanced release of these amino acid from cells. Thus, the authors were able to induce overexpression of Gdu1 in transgenic plants. These kinds of studies help in highlighting the secretory mechanisms for amino acids in plants. Further, a locus gsr1 (glutamine sensing regulatory), inferred for regulating gene expression as a response to exogenous glutamine, has also been identified in mutant A. thaliana (Meyer et al. 2006). The gsr1 locus may participate in regulating responses to endogenous and exogenous glutamine in a way that focuses on a particular organ. Such organs can, coordinately, help in regulating the activities of genes, enzymes and metabolic pathways that determine cellular nitrogen, carbon, and bio-energetic (redox) status of leaf tissues that play a decisive role in enhancing the yield. Such studies must be extended to investigate the status, control, and regulation of other macro- and micro-nutrients used as fertilizers in crops and various agricultural and horticultural activities. 5.2.4  Carbohydrates A number of monosaccharides, such as glucose, fructose, and galactose, and disaccharides, such as sucrose and sugar alcohols, have been found in guttation fluids (Goatley and Lewis 1966; Slewinski et al. 2009; Zimmermann and Ziegler 1975), and other sugars may be expected to be present in exudation fluids depending upon plant species and environmental and soil factors. However, the specific role of these sugars found in guttation fluid and other exudates in the regulation of plant growth, development, and survival is a matter of speculation, which needs specific studies for clarification. 5.2.5  Lipids,

lipoides, alkaloids, glucosides, and toxins

A number of lipid compounds, that is, fatty acids, sesquiterpenes, anti-depressants, monoterpenes, sclareol, and volatile oils, are found in the guttation fluid, and some of the compounds are not clearly understood (Sparrow et al. 2007). As for mycotoxins, Gareis and Gareis (2007) studied 8 of 11

82   Guttation

ochratoxigenic isolates of Penicillium nordicum and Penicillium verrucosum, whose guttation droplets, analyzed by high-performance liquid chromatography revealed the presence of ochratoxin A (OTA) and B (OTB) in high concentrations. The toxins’ concentrations were seen to range from 92.7 to 8667.0 ng for OTA and 159.7 to 2943.3 ng for OTB mL−1 in the guttation fluids. Nevertheless, considerably lower levels of toxins were seen in corresponding samples of the underlying mycelia (9.0–819.3 ng OTA and 4.5–409.7 ng OTB g−1) and agar that was free of fungus (15.3–417.0 ng OTA g−1 and 12.7–151.3 ng OTB g−1). This demonstrates that high amount of mycotoxins could be excreted from toxigenic Penicillium isolates into guttation droplets, which could be taken into account for the intensity variation of the disease, suggesting the practical utility of these findings for plant protection (Singh 2014b). Furthermore, a novel cyanogenic diglucoside was isolated from guttation droplets of young seedlings of Sorghum bicolor and identified as dhurrin-6-glucoside (Selmar et al. 1996). Compared with dhurrin, which is the major cyanogenic glucoside in sorghum leaves, dhurrin-6-glucoside occurs only in low concentrations in mature plants but in significant amounts in young seedlings, supporting the hypothesis that these compounds represent metabolites of cyanogenic monoglucosides, which translocate within the plant. The absorption, translocation, distribution, exudation, and guttation of 14C-glyphosate has also been studied through solution treatment of foliage, and it is found to be present in the organs of water hyacinth (Eichhornia crassipes) plant. Further, asymptomatic fungi (Neotyphodium spp. endophytes), growing in the intercellular spaces of the grasses which live in association with these fungi, produce alkaloids that protect the grass, functioning as a defense strategy against grazing by mammals and insects. One of these alkaloids is peramine, which appears to be synthesized continuously by the endophyte, but does not progressively accumulate (Koulman et al. 2007). However, the mechanism of its removal by further metabolism or any other process has not been reported. Extending these investigations further, the cut leaf fluid and guttation fluid of various grass–endophyte associations (Lolium perenne with Neotyphodium lolii, Festuca arundinacea with Neotyphodium coenophialum, and Elymus sp. with Epichloe sp.) were analyzed, and although peraminein was detected in the cut leaf fluid of all grass–endophyte associations, it was not detected in the guttation fluid of any of the associations studied. These authors also detected lolines and ergot peptide alkaloids in some associations and claimed this to be the first report showing the mobilization of fungal alkaloids into plant fluids by the host-plant in grass–endophyte associations. 5.2.6  Pesticide

residues

In modern agriculture, a number of pesticides are being used to control the infestation of insect pests and weeds. Studies have been conducted (Stokes 1954; Stoller 1970) on guttation droplets collected from leaf tips of plants and analyzed for the presence of the radiolabeled pesticides used. The tests clearly indicated the presence of pesticides in the exudates. Further, the effects of adjuvants on the elution rate were also investigated and related to the known biological profile of the fungicide, when used in combination with adjuvants. This method having practical utility, using experimental xylemmobile fungicide as a model molecule, may be developed as a means for investigating formulation behavior in vivo. However, not all xylem-mobile fungicides elute significantly. It is clear from the above discussion that guttation fluid and plant exudates contain a number of organic molecules and, expectantly, many more macromolecules still remain to be identified, offering vast scope for future investigations in the organic chemistry of plant secretions (Section 7.3.5).

Chemistry of Guttation    83

5.3  Inorganic constituents of guttation fluids: cations, anions,

and salts Guttation fluids contain several elemental ions, mineral constituents, and salts having potassium (K), calcium (Ca), magnesium (Mg), manganese (Mn), boron (B), selenium (Se), nickel (Ni), silicon (Si), aluminum (Al), chloride (Cl), ammonium (NH4), sulfate (SO4), phosphate (PO4), carbonate (CO3), bicarbonate (HCO3), nitrate (NO3), etc., or their combinations in different proportions. The composition and concentration of these inorganics depend upon the plant species and growing atmospheric and edaphic conditions. Guttation is, undoubtedly, associated with salt absorption and salt movement into the xylem before being exuded out of uninjured leaves as fluid. Therefore, the liquid of guttation is not pure water but a dilute solution of organic and inorganic salts (Curtis 1943, 1944a,b; DiTomaso 1998; Duncan et al. 1993; Ivanoff 1963; Klein 1913). In most cases, the guttation fluid contains nutrient salts generally seen in the plant sap, sugars, other organic substances, enzymes, etc., with some notable exceptions, nevertheless. De Saussure (1804) evaluated these accumulated chemicals on leaves. He stated that the guttate contained deliquescent salts that seemed to have similar properties as that of calcium muriate in combination with magnesia. These salts were readily precipitated by silver nitrate, potassium oxalate, and the alkaline carbonates, but not by Baryta water (barium hydroxide solution used as a reagent), which showed no modification on application of fire. Saline and earthy metals made up least one-third of the weight of salts. They were wrapped in a white substance, which was insoluble in alcohol and was also hydrophobic in nature. Duchartre (1859) showed the presence of KCl, Ca(CO3)2, and mucilaginous substances in the guttate of Colocasia antiquorum. Unger (1861) conducted chemical analysis of the minerals that were exudated in huge amounts from the leaves of Saxifraga crustata. At times, the leaves were found to be coated with scaly incrustations in which dilute acids were shown to dissolve 4.14 percent of Ca(CO3)2 and 0.82 percent of magnesium carbonate. They showed a direct link with the leaf ’s sap ducts. As guttation fluids contain a number of different cations, anions, and salts, their hydrogen ion concentration (pH) is likely to differ with the kind of plant, its phenological growth period, and the substratum where the growth takes place. For insights into these aspects, maize, oats, and timothy were made to grow under conditions that were sterile, on the similar types of substratum, and in addition to that, timothy was also made to grow on five types of substrata. The exudate water was gathered from all the plants everyday; its hydrogen ion concentration ranged from 5 to 8.2 (Klein 1913). Nobbe and Siegert (1864) studied the formation of white incrustations on the leaves as well as stems of buckwheat and barley, when they were grown in the nutrient medium. The study results showed KCl, and low level of phosphates and lime with nitrates. Marloth (1887) experimented on salt deposits exudated from Tamarix leaves and found that it contained 51 percent Ca(CO3)2, 12 percent MgSO4, 4.7 percent NaCl, 17.2 percent sodium nitrate, and 3.8 percent sodium carbonate. Klein (1913) reported that nitrates are present in exudates of Z. mays, Bochmeria utilis, and Fuchsia spp. and absent in the freshly excreted water; but within 6 or 8 hours, nitrites appear. He opined that these nitrites resulted from the action of bacteria or mold. Wilson (1923) evaluated the exudate from maize seedlings and timothy plants and observed many organic and inorganic materials in them. The total amount of solids in maize seedlings was 1030 ppm; from timothy, it was 573 ppm in one collection and 220 ppm in another, and about half of these solids were organic matter. Nitrates, nitrites, sugars, peroxidases, catalases, and reductases in the exudates were found by him. On the other hand, he hypothesized that perhaps reductases was present in sufficient amounts in

84   Guttation

exudates to convert nitrates to nitrites and ammonia without involving bacteria or molds. Dixon (1914) stated that the exudate from Colocasia antiquorum had freezing point equal to that of distilled water, with conductivity lower than that of tap water. The findings showed that exudation, which appears osmotically inactive, must be secreted through metabolic activity. Pavilinova (1926) found evidence of Ca (17 percent) in guttate obtained from maize grown in water cultures with concentrations proportional to that of the culture solution. Shardakov (1928) also provided evidence of Ca, K, Cl, and PO42− in the exudate. Arens (1934) stated that when distilled water was sprayed on the leaves, these minerals diffused out through the leaf cuticles. Ivanoff (1941) conducted dry chemical analysis of Citrus leaves, and the soluble portion showed presence of Cl−, SO42−, and Ca, along with insoluble portions containing carbonates of Ca and Mg, Si and Fe, and Al 2O3. Curtis (1943) focused on vegetable plants and analyzed the guttation fluids obtained from them. The total solids, approximately 50 percent of which were organic, in summer squash, tomatoes, and cabbage were 2500, 600, and 600 ppm, respectively. Many inorganic ions have been observed in the guttate, such as ammonium, phosphate, K, Mg, Cl−, and SO42−, but Ca and nitrate were found to be the more abundant. Guttates containing 80 percent inorganic matter and remaining percent organic matter showed Ca concentration between 100 and 750 mg L−1, while nitrate was at 250 mg L−1. The pH ranged between 7.3 and 6.1, and the inorganic materials included 31.3 percent Ca, 14.6 percent insoluble Si, 5.2 percent Fe and Al 2O3, 1.3 percent Mg, and 6 percent water. If Ca and Mg were present as carbonates, then 23.9 percent of CO2 might be present along with the remaining constituents, which have been identified as phosphates. Some soluble materials like K salts might have been washed away by rain and dew prior to sample collection. As Ca was found abundantly, it can be stated that the loss of this vital element from leaves might have an impact on abscission layer of fruit pedicel, causing the fruit to drop. Vigorov (1954) found 7–9 day-old wheat seedlings having 1.8 mg mL −1 of dry substance in the guttation drops, which consisted of 5–10 mg mL −1 ammonium nitrogen and 40–45 mg mL −1 phosphorous compounds. The author observed that the guttation intensity is completely dependent on illumination, soil humidity, nitrogen content, and other nutrient elements. The ammonium salts that get introduced into the soil increase the excretion of nitrogen compounds through guttation. As for the presence of organic substances in the fluids, the abundant growth of microorganisms, including fungi, in guttation drops is also proof (Krasil’nikov 1961). In this context, guttation fluid and bleeding sap of members of associations of Odontites with barley or Stellaria media were analyzed for phosphate, nitrate, calcium, potassium, amides, and amino acids (Govier et al. 1968), and the results suggest that the shoots of the hemiparasite and its host can absorb, selectively, the solutes supplied from their roots, with mesophyll cells and glandular hairs of the leaf being particularly active in this respect. Relatively recent studies conducted on trees demonstrated that salt cedar and Tamarix contained 41,000 ppm solids in their guttation sap (DiTomaso 1998; Duncan et al. 1993). Tamarix collects salt in special glands in its leaves and then excretes it onto the leaf surface. These salts gather in the surface layer of the soil when plants drop their leaves. Brotherson and Feild (1987) concluded that Tamarix contained NaCl under its canopy as an allelochemical agent. Along the banks of regulated rivers that do not experience annual flooding and scouring anymore, surface soils become more saline, over time, due to excretions of the leaves (Busch and Smith 1993). Plants also excrete various volatile substances with the aid of special glands or through guttation. The following subsections briefly describe some of the elements generally found toxic to plants, which are found in leaf leachates and exudation saps.

Chemistry of Guttation    85

5.3.1  Silica Silica concentrations in stump exudation sap range from 400 ppm to 800 ppm and that in guttation sap range from 100 ppm to 300 ppm, and the amount of guttation sap increased with increase in silica concentration in the culture solution to some level (Baba 1957). The concentration of silica in both guttation sap and exudation was more in the plants grown with stable manure consisting of more silica than in plants grown with mineral fertilizer or green manure. Heavy addition of both H 2S to the solution and ammonium sulfate to the soil caused root injury, leading to retardation in exudation and guttation and also decline in the amount of silica in these saps. These results indicate that silica concentration in the exudation and guttation saps is closely linked with the concentration of available silica in culture media or soil solution. Moreover, the properties of both exudation and guttation fluids and the amount of silica in these saps can be used to determine the activity of the root. 5.3.2  Boron Rajaratnam (1972) studied the distribution of boron following application of the element to the palm. Results indicate that boron moves in one direction from the roots to the leaflet, and from the leaflets, some boron can be lost through guttation. The practical implications of the results, with regard to boron application methods and leaf analysis as a diagnostic tool, lie in the fact that the boron guttated is likely to be significant in the re-cycling of boron through the soil–root system. However, both limiting and toxic soil concentrations of the essential micronutrient boron present major limitations to crop production worldwide. As mentioned earlier, in an important study of agricultural significance, the recent identification by Sutton et al. (2007) of Bot1, a BOR1 ortholog, as the gene responsible for the superior boron-toxicity tolerance of the Algerian barley landrace Sahara 3771 (Sahara) and the identification of this gene at the tolerance locus through high-resolution mapping is a landmark in genetic engineering of crops. Importantly, tolerance to boron toxicity was positively associated with Bot1 gene copies, and the tolerant genotype produced substantially more Bot1 transcript that encodes a Bot1 protein with a higher capacity to provide tolerance in yeast. 5.3.3  Nickel,

cobalt, manganese, zinc, and magnesium

An interesting and important applied research was performed in Sparks’s laboratory in Delaware, United States, which succeed in developing Ni-hyper-accumulator A. murale as a commercial crop for phytoremediation and phytomining Ni from metal-enriched soils (Tappero et al. 2007). They studied the guttation fluid for co-tolerance, metal accumulation, and localization in A. murale that are exposed to metal co-contaminants. A. murale hyper-accumulated Ni and Co (>1000 µg g−1 dry weight) from mixed-metal systems. Zinc was not hyper-accumulated. Elevated Co or Zn concentrations did not modify accumulation or localization of Ni. Microscopic images presented uniform distribution of Ni in leaves and localization of Co across leaf tips and margins. On further evaluation, the images revealed that leaf epidermal tissue was rich in Ni but lacked Co, that Co was localized in the apoplasm of leaf ground tissue, and that Co was sequestered on surfaces of the leaf across the tips and margins. Mineral precipitate rich in cobalt, formed on leaves of Cotreated A. murale, and processes specialized with a biochemical related to Ni (hyper) tolerance in A. murale, however, do not confer (hyper) tolerance to Co. A. murale relies on a different metal storage

86   Guttation

mechanism for Co (exocellular sequestration) than for Ni (vacuolar sequestration). Petasites japonicus var. giganteus (Japanese butterbur) and Polygonum cuspidatum (Japanese knotweed) exude guttation fluid in large quantities from the edge of the leaf (Mizuno et al. 2002). The concentrations of Ni and Mn in the guttation fluid from P. japonicus var. giganteus that were grown on ultramafic soil were 2.24 and 0.32 mg L−1, respectively, on an average, whereas those in the guttation fluid from P. cuspidatum were 0.22 and 3.13 mg L −1, respectively. The Ni concentration in the leaves of P. japonicus var. giganteus was about five times higher than that of P. cuspidatum, especially at the leaf edges, and the Mn concentration in the leaves of P. cuspidatum was about six times higher than that of P. japonicus var. giganteus. Thus, the concentrations of Ni and Mn in both the guttation fluid and leaves of P. japonicus var. giganteus were opposite to those of P. cuspidatum. The concentrations of K, Mg, and Ca in the guttation fluid from P. japonicus var. giganteus were higher than those in the guttation fluid from P. cuspidatum in the ultramafic rock area. Mizuno et al. (2003) further studied the distribution of Ni and Zn in the leaves of Thlaspi japonicum, a Ni-hyper-accumulator, which was grown on ultramafic soil and found a high concentration of Ni and Zn in its shoot. The concentration of Ni was the highest (3424 mg kg−1) in the lower epidermis, which contained many stomata, followed by the edge of the leaf, upper epidermis, which contained few stomata, and mesophyll. Contrastingly, the Zn concentration was the highest (615 mg kg−1) in the upper epidermis followed by the lower epidermis and was the lowest at the edge of the leaf. Using a microscope, the dimethylglyoxime-stained Ni compound was seen as rod-shaped crystals, primarily around the stomata and the projections of the edge of the leaf. Additionally, a considerable amount of Ni was excreted through the guttation fluid (0.67–1.33 mg L−1), whereas the Zn concentration in the guttation fluid was low (0.01–0.10 mg L −1). In leaves that appeared mature, the Ni content did not fluctuate during the growth period, while the Zn content decreased once around the summer solstice, reaching the highest level in mid-summer, and declined thereafter. In leaves that appeared young, however, Ni and Zn contents increased to a level that was same as that in mature leaves in mid-summer and declined after early September. The data recommend that Ni is translocated with the transpiration stream and concentrated around the stomata, and that the excess amount of Ni is excreted through the guttation fluid in T. japonicum, a Ni-hyper-accumulator. This mechanism, however, may not be operational for Zn accumulation in this plant, and Zn present in mature leaves may be imparted to young leaves on demand to grow rapidly. McNear et al. (2005) used the fluorescence computed microtomography method, which was appropriate to determine the compartmentalization and concentration of metals of intact leaf, stem, and root samples, and observed that Ni was deposited in stem and leaf dermal tissues and combined with Mn in distinct regions in association with Ca-rich trichomes on the surface of the leaf of the Ni-hyper-accumulator A. murale ‘Kotodesh’. Ni was also observed on the tips of the leaves, which was a possible result of release of excess Ni with guttation fluids. These results are constant with a movement model where Ni is separated from the soil through the finer roots, carried to the leaves through the stem xylem, and imparted separately throughout the leaf through the veins to dermal tissues, trichome bases, and, in some cases, the tip of the leaf. 5.3.4  Calcium The guttation deposits contain in general CaCO3 and apparently several other common salts too. The salts from peppers were hygroscopic and contained little or no CaCO3 (Ivanoff 1944). Bugbee

Chemistry of Guttation    87

and Koerner (2002), who developed the dwarf wheat cultivar USU-Apogee for high yields in the outer space of the Earth, observed that significant amounts of calcium (Ca) can be inter-moved through guttation. The segregating lines with the smallest leaves had the least chlorosis. Analysis of the tissue through inductively coupled plasma emission spectrophotometry showed adequate calcium in the top 30 percent of small leaves (0.4 percent Ca), but inadequate amounts (0.05 percent Ca) in large leaves with USU-Apogee with smaller flag leaves (11–20 cm long, depending on temperature) than those of YecoraRojo and Veery-10 (20–30 cm long), inheriting calcium concentrations in their leaves, accordingly. Calcium deficiencies, such as tipburn in lettuce (Lactuca sativa) and blossom endrot in tomatoes (Lycopersicum esculentum), caused by guttation are common in environmentally controlled production of crop because Ca has low mobility in phloem sap and is, thus, insufficiently inter-moved to meristems that grow rapidly. Foliar Ca applications and increased root-zone Ca do not affect meristems because they do not find their placement in the meristematic leaf tissue (Marschner 1995). 5.3.5  Arsenic As mentioned earlier, Mihucz et al. (2005) studied arsenic speciation of the xylem sap of cucumber (Cucumis sativus L.) plants, and different compounds like arsenite, arsenate, and dimethylarsinic acid of this element were seen in the sap of the plants. These arsenic compounds are likely to be present in the guttation fluid and other exudates, which may play the role of detoxifiers and may also recycle through the soil–root system. 5.3.6  Aluminum Bertrand et al. (1995) examined how low pH and 2 mM Al affect the growth of sugar maple (Acer saccharum Marsh.) seedlings over a 13-week period. At week 9, the total leaf area of Al-treated seedlings decreased by 27 percent; nevertheless, by week 13, leaf area was the same for seedlings of all treatments. None of the other growth parameters evaluated were negatively affected by the treatments at either week 9 or week 13. The evaluation of the concentration of ABA in the xylem sap, which indicates the tree stress in the field, showed that it was not impacted by any of the treatments, although its concentration was the highest in times of high evaporative demand in June and August. The authors concluded that the period of exposure to Al is crucial when assessing the threshold for Al toxicity, as plants are capable of acclimating to an Al concentration through compartmentalization, which was considered toxic previously. All the elements and their salts found in guttation fluids have not been mentioned here for brevity, and those interested in knowing about these are advised to refer to the literature on these topics described earlier in this chapter.

5.4  Hormones Apart from organic and inorganic constituents described above, guttation fluids and root and stem stump exudates are also known to contain natural plant hormones such as auxins, gibberellins (GAs), CKs (benzyladenine, kinetin, and zeatin [Z]), ABA, and ethylene (Fletcher and Mader

88   Guttation

2007; Thompson et al. 2000, 2007). In addition to these hormones, brassinosteroids, jasmonates, salicylic acid, etc., may also be present in the guttation fluid and exudation saps but, to the best of my knowledge, have not been reported in the literature related to sap exudation and guttation. Vaadia’s laboratory in Israel (Itai and Vaadia 1965; Itai et al. 1968) found root exudates to contain CK-like activities in sunflower, and Skene (1967) reported the occurrence of CKs and GAs in the plant exudates of grapevines. The physiological significance of these hormones and allied compounds in root exudates is now well known, which may regulate, among other functions, bud break and growth of aerial parts of the vine and other plant species as well (Paleg 1965). Both terrestrial and aquatic plants have a transport system that is efficient for acropetal water, inorganic nutrients, and hormones for satisfying their needs for active growth, and this system has been influenced by the stage of development of the leaf hydathodes (Pedersen et al. 1997). Plant growth regulators indole-butyric acid, 6-benzylaminopurine, and kinetin are known to increase rhizosecretion of Guy’s 13 (Drake et al. 2009). The effect of growth regulators was, however, variable as the root dry weight of hydroponic plants increased because of alpha naphthalene acetic acid and indolebutyric acid, while the CKs benzyl aminopurine and kinetin leveled up rhizosecretion without having any effects on the mass of the root. It would be interesting to investigate the impacts of GAs and auxins on guttation, considering how important they are for distinguishing and developing stomata and hydathodes (Aloni 2001, 2010; Aloni et al. 2003, 2005; Peterson et al. 2010; Pillitteri et al. 2008; Wang et al. 2011). Although limited literature exists on these hormones in context to guttation and sap exudation, the following sub-sections intend to cover the hormonal aspects of these exudations in brief. 5.4.1  Auxins As described earlier, bleeding occurs periodically, which appears to be automatic in origin. A crisply increased temperature determines when exactly the maxima and minima of this periodicity occurred, but the application of auxin indole-3-acetic acid (IAA) to the cut stumps of sunflower pumped up bleeding throughout the cycle of 24 hours during the initial few days of bleeding. Later, the effect of auxin was more pronounced during the maximum bleeding period (Skoog et al. 1938). It should further follow that auxin acts in conjunction with the timely secretion and removal of solutes from the xylem stream. To understand the genetic regulation of bud outgrowth in Pisum sativum L. (garden pea), Beveridge (2000) used four ramosus mutants with increased basal and aerial nodes’ branching. Studies were conducted on long-distance signaling, concentrations of xylem-sap CK, shoot auxin level, auxin movement, and response of auxin. A fresh branching control model was under construction, assuming two graft-transmissible signals that were additions to auxin and CK. It was seen that mutants rms1 to rms4 were not deficient in IAA or in the basipetal movement of this hormone. On the other hand, three of the four mutants, rms1, rms3, and rms4, had low CK concentrations in the sap of root xylem. The author emphasized that this xylem-sap CK concentration reduction was caused by a shoot property and may play the role of a feedback mechanism induced by an aspect of bud outgrowth. The shoot-to-root feedback signal is not likely to be auxin itself, as levels of auxin and movement were not in correlation with xylem-sap CK concentrations in varied intact and grafted mutant and plants that were wild in nature (Dodd et al. 2004; Morris et al. 2001). Mutants rms1 and rms2 were acting in shoot and rootstock and regulated the level or movement of graft-transmissible

Chemistry of Guttation    89

signals. Different grafting studies done by these authors and double-mutant analyses have associated rms2 with the regulation of the shoot-to-root feedback signal. The mutant rms1 was in association with a second graft-transmissible signal, which was not known and was postulated to move in the root-to-shoot direction. Exogenous auxin application interacted with both of the signals that rms1 and rms2 regulate, inhibiting the branching after decapitation. The action of rms3 and rms4 was less apparent at this stage, though both were observed to act largely in the shoot. Obviously, scant information is available in this area of research, which, undoubtedly, deserves more concerted efforts to unravel its basic and applied implications in plant physiology and horticulture, including aromatic and medicinal plants, using guttation and exudation fluids as analytical tools. 5.4.2  Abscisic

acid and ethylene

ABA has been traced in vascular bundles, roots, and leaves, which might influence, among other things, gating of aquaporins, resulting in increased permeability of water; hence, its increased transport forces the water to exude as guttation. It is, therefore, pertinent to have some greater insights into ABA functioning in plants. ABA may reduce growth by limiting assimilation (Blum 2005; Thompson et al. 2007), but it may also improve growth during soil-water deficit by increasing the status and turgor of the cell or may influentially help in growth and development by directly involving signaling pathways and crosstalk with other hormones. Exogenous application of ABA and the study of ABA-deficient mutants have indicated that ABA can have both positive and negative impacts on growth, depending on tissue, applied concentration, and its interaction with the environment (Barbour and Farquhar 2000). It has also been reported that for maintaining growth during water deficit, ABA antagonizes the growth inhibitory impacts of ethylene in tomato shoots (Sharp et al. 2000), Arabidopsis shoots (LeNoble et al. 2004), maize roots (Spollen et al. 2000), and also while filling grain in rice (Yang et al. 2007). Nevertheless, this topic is still controversial as the maize leaf expansion regulation through water deficit, during the night, was not regulated by either ABA or ethylene (Voisin et al. 2006). Exogenously applied ABA is also popular for increasing the been reported root hydraulic conductivity (Lpr), both at the organ and cellular levels (Hose et al. 2000), and this is at least partly explained by an up-regulation of aquaporin genes (Zhu et al. 2005). Enhancing Lpr is a way of optimizing the water delivery right from the soil to the shoot. ABA also impacts a wide range of other cellular adaptive processes, like osmotic adjustment and transport of ion, inducing production of metabolites or proteins that act as protecting macromolecules from denaturation at lower water activities (Bray 2002; Chaves and Oliveira 2004). These processes that regulate ABA have evolved to permit plants to continue their growth and reproduce during periods of low water availability or for sustaining and recovering from dehydration, but they may also have an effect on crop yield in the context of their implications for agronomic utility (Thompson et al. 2007). The process of how transgenic genotypes with elevated ABA content are produced and analyzed provides an approach to investigate the function of ABA in whole plants that has advantages over either the use of periodic or short-term exogenous application of ABA, or for the study of ABA-deficient mutants where ethylene impacts (Sharp 2002) and wilting have major negative effects on how they grow. Chemical signals, modified under and originating from roots may hence have a major role to play in the rootto-shoot communication of most plants. Further, water movement from layers of soil may be helpful for saving chemical signals as a response to support the activity of fine roots in soil or soil solutions

90   Guttation

of diminished water activity. Hence, guttation may be utilized as a non-destructive way to study the flow of water and mineral ions from the roots and be compared with parallel measurements of root exudation (Dieffenbach et al. 1980b). 5.4.3  Cytokinins Historically, the identification and importance of CKs and bioactive forms of CK, mainly of Z, zeatin riboside (ZR), and iP, for shoot development were discovered more than five decades ago (Amasino 2005; Kieber and Schaller 2010). Extensive investigations on root exudate CKs were carried out in Vaadia’s laboratory way back in the second half of the 1960s, signifying their role in root physiology (Itai and Vaadia 1965; Itai et al. 1968), and the legacy of these traditions in Israel is still being maintained by the brilliant works of Aloni and coworkers (Aloni 2001, 2010; Aloni et al. 2003, 2005). Though plant endogenous CK synthesis was suspected (Holland 1997), the recognition of genes that encode ATP/ADP isopentenyl transferase (IPT) is now established properly in Arabidopsis (Miyawaki et al. 2006). The strongest IPT expression was seen in the root cap columella (IPT5), and there was systemic expression in the phloem all over the plant (IPT3) (Aloni et al. 2005). Nevertheless, the sites of production and availability of bioactive-free CK have not yet been recognized unequivocally. According to concepts that are well known, CKs have their production in roots (Letham 1994) and in young shoot organs too (Muller and Leyser 2011; Taiz and Zeiger 2002). In transgenic tobacco plants, modified with ipt genes from Agrobacterium tumefaciens, it was seen that de-repression of tetracycline-inducible ipt genes led to 100-fold higher total CK concentrations in leaves than in tobacco plants that are wild in nature. The fact that the bulk of the CK goes through synthesis in the rootcap, and is exported through the xylem and collects at sites of highest transpiration, where cuticles are not yet seen or do not shield against loss of water, is supported by the apparent absence of free CK in the buds of plants protected from wind and the typical upward declining gradients of free and conjugated CKs (Aloni et al. 2005; Dodd et al. 2004). The latter group of authors have amply demonstrated transpiration-dependent upward movement of root-produced CKs in xylem sap of transgenic A. thaliana. The fundamental and applied studies of workers, mentioned earlier, on chemical signals from roots have concentrated on Auxin, ABA and CKs. A better, stable, isotope dilution protocol, which enables analysis of ABA and CK from the same tissue sample, was developed. Analysis of CKs has focused on Z, ZR, zeatin glucoside, and iP. Roots are relatively not accessible, specifically in situations of field, and hence, for enabling an access that is easier to go to the roots of vines grown on fields, split-root vines were planted in a trench that was refilled with sandy soil (Stoll 2000; Stoll et al. 2000). This generated a homogenous soil substrate and did not stop the root growth while still permitting access to roots under conditions of the field. Analyses of root samples of vines grown on field indicated that CKs and ABA may have their origins in the roots, and their concentrations can be substantially modified during a cycle of irrigation. Findings of alternate conditions of soil water showed that ABA in the roots on the ‘dry’ side was significantly higher when compared with the ‘wet’ side. Due to reduction in CKs of vines on the ‘dry’ side, the ratio between ABA and CK was substantially altered during an irrigation cycle. Factors like xylem-sap ZR concentration and shoot auxin levels as evidenced in rms5 mutant pea plants may manage water relations, therefore guttation in plants. Further, rise in root-derived hormonal status in leaves and exudates taken from them correlated with delayed leaf senescence and, therefore, may result in increased photosynthetic

Chemistry of Guttation    91

potential (PSII efficiency), which leads to increase in yield. Thus, ABA appears to increase guttation, whereas CKs decrease it. As mentioned earlier, Morris et al. (2001) and Dodd et al. (2004) conducted excellent studies on xylem-sap ZR concentration and shoot auxin levels in rms5 mutant plants of pea in comparison with rms1 and WT. Mutants rms1 and rms5 act closely at the biochemical or cellular level for controlling branching. Branching was inhibited in reciprocal epicotyl grafts between rms5 or rms1 and WT plants, but it was not inhibited in reciprocal grafts between rms5 and rmsl seedlings. The weakly transgressive or slightly additive phenotype of the rmsl–rms5 double mutant provides further evidence for this interaction. Like rms1, rms5 rootstocks have less xylem-sap CK concentrations, and rms5 shoots are not deficient in IAA or 4-chloroindole-3-acetic acid. The genes rms1 and rms5 interact similarly with other rms genes. Results of reciprocal grafting studies with rmsl, rms2, and rms5, in association with with the fact that root xylem-sap CK concentrations become less in rms1 and rms5 and elevated in rms2 plants, indicate that rms1 and rms5 may play a controlling role in a different pathway than rms2 controls. As mentioned, these studies are indicative of rms1 and rms5 regulating a novel graft-transmissible signal involved in the management of branching and other growth responses. Dodd et al. (2004), continuing their work on pea (P. sativum L.), experimented with rms2 and rms4 and found that branching mutants had higher and lower xylem-cytokinin (X-CK) concentrations, respectively, relative to WT plants. As sap-flow rate increased, X-CK decreased in WT and rms2 but did not get altered in rms4. When grown at 5.0 mM N, X-CKs of rms2 and rms4 were 36 percent higher and six-fold lower, respectively, than WT at sap-flow rates that were equal to whole-plant transpiration. Photoperiod CK delivery rates (the product of transpiration and X-CK) got reduced more than six-fold in rms4. Because the low N treatment reduced transpiration of all genotypes, photoperiod CK delivery rates also got reduced in all genotypes. The leaf had similar growth response to N deprivation in all genotypes, despite the differences in both absolute and relative X-CKs, and findings indicate that shoot N status is more vital in the regulation of leaf expansion than xylem-supplied CKs. Hence, induced decline in X-CK and rate of transpiration of rms2 under N deprivation indicate that modifications in xylem-supplied CKs may alter the use of water. Under such conditions, the guttation process may also be impacted, and a pertinent question that comes up is whether photoperiod CK has any link with the initiation of guttation. 5.4.4  Gibberellins Paleg (1965) highlighted the physiological significance of GAs in plants, which was followed by a number of subsequent papers on this topic (Daviere and Achard 2013; Yamaguchi 2008). However, the role of GAs in guttation and vice versa is not known. High concentrations of GA7 in apical and lateral buds and low concentrations of GAs (1, 3, and 9) in xylem sap combined with xylem-sap CKs may be regulatory for water relations; therefore, guttation occurs. Fletcher and Mader (2007) used high-performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry and analyzed many plant hormone groups in small samples for physiology study of abnormal vertical growth (AVG) in Macadamia integrifolia (cv. HAES344). CKs, GAs, ABA, and auxins were detected in xylem sap and lateral and apical buds. The method of extraction separated compounds with high sensitivity in positive (CKs) and negative (ABA, auxins, and GAs) modes of QToF-MS/MS. CK profiles were different in xylem sap and apical and lateral buds, regardless of the symptoms of

92   Guttation

AVG. Trans-zeatin riboside played a dominant role in the sap of normal and AVG trees (4 and 6 pmol g−1 FW, respectively). Isopentenyl adenine (similar to 30 pmol g−1 FW) was found to be the most abundant CK in apical buds, and t-Z (22–24 pmol g−1 FW) and iP (24–30 pmol g−1 FW) were seen to be the most abundant in lateral bunds. In apical buds, levels of t-Z in AVG trees were higher (13.88 vs. 6.6 pmol g−1 FW, p < 0.05) and lower in sap (0.16 vs. 0.51 pmol mL −1, p < 0.005) as compared to trees that were normal. In lateral buds, ABA was 1.9 times higher (p < 0.001) than AVG. IAA was below quantification level, whereas indole-3-butyric acid was present continuously. GA7 played a dominant role of GA in apical and lateral buds of all trees (100–150 pmol g−1 FW). GAs 3, 4, and 9 were present continuously at low concentrations (