Osmoprotectant-Mediated Abiotic Stress Tolerance in Plants: Recent Advances and Future Perspectives [1st ed. 2019] 978-3-030-27422-1, 978-3-030-27423-8

Table of contents :
Front Matter ....Pages i-xi
Osmoprotectant-Related Genes in Plants Under Abiotic Stress: Expression Dynamics, In Silico Genome Mapping, and Biotechnology (Éderson Akio Kido, José Ribamar Costa Ferreira-Neto, Manassés Daniel da Silva, Vanessa Emanuelle Pereira Santos, Jorge Luís Bandeira da Silva Filho, Ana Maria Benko-Iseppon)....Pages 1-40
Proline Metabolism and Its Functions in Development and Stress Tolerance (Maurizio Trovato, Giuseppe Forlani, Santiago Signorelli, Dietmar Funck)....Pages 41-72
Regulation of Proline Accumulation and Its Molecular and Physiological Functions in Stress Defence (Giuseppe Forlani, Maurizio Trovato, Dietmar Funck, Santiago Signorelli)....Pages 73-97
Exogenous Proline-Mediated Abiotic Stress Tolerance in Plants: Possible Mechanisms (Mohamed Zouari, Ameni Ben Hassena, Lina Trabelsi, Bechir Ben Rouina, Raphaël Decou, Pascal Labrousse)....Pages 99-121
Biosynthesis and Degradation of Glycine Betaine and Its Potential to Control Plant Growth and Development (Elisa M. Valenzuela-Soto, Ciria G. Figueroa-Soto)....Pages 123-140
Exogenous Glycinebetaine-Mediated Modulation of Abiotic Stress Tolerance in Plants: Possible Mechanisms (Tianpeng Zhang, Xinghong Yang)....Pages 141-152
Roles of Endogenous Glycinebetaine in Plant Abiotic Stress Responses (Pirjo S. A. Mäkelä, Kari Jokinen, Kristiina Himanen)....Pages 153-173
Biosynthesis and Degradation of Trehalose and Its Potential to Control Plant Growth, Development, and (A)biotic Stress Tolerance (Le Cong Huyen Bao Tran Phan, Patrick Van Dijck)....Pages 175-199
Proline, Glycinebetaine, and Trehalose Uptake and Inter-Organ Transport in Plants Under Stress (Suriyan Cha-um, Vandna Rai, Teruhiro Takabe)....Pages 201-223
Transgenic Plants Overexpressing Trehalose Biosynthetic Genes and Abiotic Stress Tolerance in Plants (Zsófia Bánfalvi)....Pages 225-239
The Role of Proline, Glycinebetaine, and Trehalose in Stress-Responsive Gene Expression (Merve Kahraman, Gulcin Sevim, Melike Bor)....Pages 241-256
Seed Osmolyte Priming and Abiotic Stress Tolerance (Danny Ginzburg, Joshua D. Klein)....Pages 257-267
Relationship Between Polyamines and Osmoprotectants in the Response to Salinity of the Legume–Rhizobia Symbiosis (Miguel López-Gómez, Javier Hidalgo-Castellanos, Agustín J. Marín-Peña, J. Antonio Herrera-Cervera)....Pages 269-285
Engineering Polyamine Metabolic Pathways for Abiotic Stress Tolerance in Plants (Susana de Sousa Araújo, André Luis Wendt dos Santos, Ana Sofia Duque)....Pages 287-318
Fructan Metabolism in Plant Growth and Development and Stress Tolerance (Alejandro del Pozo, Ana María Méndez-Espinoza, Alejandra Yáñez)....Pages 319-334
Back Matter ....Pages 335-342

Citation preview

Mohammad Anwar Hossain  Vinay Kumar · David J. Burritt  Masayuki Fujita · Pirjo S. A. Mäkelä Editors

OsmoprotectantMediated Abiotic Stress Tolerance in Plants Recent Advances and Future Perspectives

Osmoprotectant-Mediated Abiotic Stress Tolerance in Plants

Mohammad Anwar Hossain Vinay Kumar  •  David J. Burritt Masayuki Fujita  •  Pirjo S. A. Mäkelä Editors

Osmoprotectant-Mediated Abiotic Stress Tolerance in Plants Recent Advances and Future Perspectives

Editors Mohammad Anwar Hossain Department of Genetics and Plant Breeding Bangladesh Agricultural University Mymensingh, Bangladesh

Vinay Kumar Department of Biotechnology Modern College Pune, Maharashtra, India

David J. Burritt Department of Botany University of Otago Dunedin, Otago, New Zealand

Masayuki Fujita Laboratory of Plant Stress Responses Kagawa University Kagawa, Kagawa, Japan

Pirjo S. A. Mäkelä Department of Agricultural Sciences University of Helsinki Helsinki, Finland

ISBN 978-3-030-27422-1    ISBN 978-3-030-27423-8 (eBook) https://doi.org/10.1007/978-3-030-27423-8 © Springer Nature Switzerland AG 2019 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, express or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Switzerland AG The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland

Preface

In nature, plants are constantly challenged by various abiotic and biotic stresses that can restrict their growth, development, and yields. In the course of their evolution, plants have evolved a variety of sophisticated and efficient mechanisms to sense, respond to, and adapt to changes in the surrounding environment. A common defensive mechanism activated by plants in response to abiotic stress is the production and accumulation of compatible solutes (also called osmolytes). These include amino acids (mainly proline), amines (such as glycinebetaine and polyamines), and sugars (such as trehalose and sugar alcohols), all of which are readily soluble in water and nontoxic at high concentrations. The metabolic pathways involved in the biosynthesis and catabolism of compatible solutes and the mechanisms that regulate their cellular concentrations and compartmentalization are well characterized in many important plant species. Numerous studies have provided evidence that enhanced accumulation of compatible solutes in plants correlates with increased resistance to abiotic stresses. New insights into the mechanisms associated with osmolyte accumulation in transgenic plants and the responses of plants to exogenous application of osmolyte will further enhance our understanding of the mechanisms by which compatible solutes help to protect plants from damage due to abiotic stress and the potential roles compatible solutes could play in improving plant growth and development under optimal conditions. Although there has been significant progress made in understanding the multiple roles of compatible solute in abiotic stress tolerance, many aspects associated with compatible solute-mediated abiotic stress responses and stress tolerance still require more research. As well as providing basic up-to-date information on the biosynthesis, compartmentalization, and transport of compatible solute in plants, this book will also give insights into the direct or indirect involvement of these key compatible solutes in many important metabolic processes and physiological functions, including their antioxidant and signaling functions, and roles in modulating plant growth, development, and abiotic stress tolerance. In this book, Osmoprotectant-Mediated Abiotic Stress Tolerance in Plants: Recent Advances and Future Perspectives, we present a collection of 15 chapters written by leading experts engaged with compatible solute-induced abiotic stress v

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Preface

tolerance in plants. The main objective of this volume is to promote the important roles of these compatible solutes in plant biology, by providing an integrated and comprehensive mix of basic and advanced information for students, scholars, and scientists interested in, or already engaged in, research involving osmoprotectant. Finally, this book will be a valuable resource for future environmental stress-related research and can be considered as a textbook for graduate students and as a reference book for frontline researchers working on the relationships between osmoprotectant and abiotic stress responses and tolerance in plants. Mymensingh, Bangladesh  Mohammad Anwar Hossain Pune, India  Vinay Kumar Dunedin, New Zealand  David J. Burritt Kagawa, Japan  Masayuki Fujita Helsinki, Finland  Pirjo S. A. Mäkelä

Contents

Osmoprotectant-Related Genes in Plants Under Abiotic Stress: Expression Dynamics, In Silico Genome Mapping, and Biotechnology��������������������������������������������������������������������������    1 Éderson Akio Kido, José Ribamar Costa Ferreira-Neto, Manassés Daniel da Silva, Vanessa Emanuelle Pereira Santos, Jorge Luís Bandeira da Silva Filho, and Ana Maria Benko-Iseppon Proline Metabolism and Its Functions in Development and Stress Tolerance����������������������������������������������������������������������������������������   41 Maurizio Trovato, Giuseppe Forlani, Santiago Signorelli, and Dietmar Funck Regulation of Proline Accumulation and Its Molecular and Physiological Functions in Stress Defence����������������������������������������������   73 Giuseppe Forlani, Maurizio Trovato, Dietmar Funck, and Santiago Signorelli Exogenous Proline-Mediated Abiotic Stress Tolerance in Plants: Possible Mechanisms����������������������������������������������������������������������   99 Mohamed Zouari, Ameni Ben Hassena, Lina Trabelsi, Bechir Ben Rouina, Raphaël Decou, and Pascal Labrousse Biosynthesis and Degradation of Glycine Betaine and Its Potential to Control Plant Growth and Development����������������������  123 Elisa M. Valenzuela-Soto and Ciria G. Figueroa-Soto Exogenous Glycinebetaine-Mediated Modulation of Abiotic Stress Tolerance in Plants: Possible Mechanisms������������������������  141 Tianpeng Zhang and Xinghong Yang Roles of Endogenous Glycinebetaine in Plant Abiotic Stress Responses����������������������������������������������������������������������������������������������  153 Pirjo S. A. Mäkelä, Kari Jokinen, and Kristiina Himanen

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Contents

Biosynthesis and Degradation of Trehalose and Its Potential to Control Plant Growth, Development, and (A)biotic Stress Tolerance������������������������������������������������������������������������������������������������  175 Le Cong Huyen Bao Tran Phan and Patrick Van Dijck Proline, Glycinebetaine, and Trehalose Uptake and Inter-Organ Transport in Plants Under Stress��������������������������������������  201 Suriyan Cha-um, Vandna Rai, and Teruhiro Takabe Transgenic Plants Overexpressing Trehalose Biosynthetic Genes and Abiotic Stress Tolerance in Plants������������������������������������������������  225 Zsófia Bánfalvi The Role of Proline, Glycinebetaine, and Trehalose in Stress-Responsive Gene Expression ����������������������������������������������������������  241 Merve Kahraman, Gulcin Sevim, and Melike Bor Seed Osmolyte Priming and Abiotic Stress Tolerance����������������������������������  257 Danny Ginzburg and Joshua D. Klein Relationship Between Polyamines and Osmoprotectants in the Response to Salinity of the Legume-Rhizobia Symbiosis������������������  269 Miguel López-Gómez, Javier Hidalgo-Castellanos, Agustín J. Marín-Peña, and J. Antonio Herrera-Cervera Engineering Polyamine Metabolic Pathways for Abiotic Stress Tolerance in Plants������������������������������������������������������������  287 Susana de Sousa Araújo, André Luis Wendt dos Santos, and Ana Sofia Duque Fructan Metabolism in Plant Growth and Development and Stress Tolerance����������������������������������������������������������������������������������������  319 Alejandro del Pozo, Ana María Méndez-Espinoza, and Alejandra Yáñez Index������������������������������������������������������������������������������������������������������������������  335

Editors Biography

Mohammad  Anwar  Hossain is a Professor in the Department of Genetics and Plant Breeding, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh. He received his B.Sc. in Agriculture and M.S. in Genetics and Plant Breeding from Bangladesh Agricultural University, Bangladesh. He also received an M.S. in Agriculture from Kagawa University, Japan, in 2008, and a Ph.D. in Abiotic Stress Physiology and Molecular Biology from Ehime University, Japan, in 2011, through Monbukagakusho Scholarship. As a JSPS Postdoctoral Researcher, he has worked on isolating low-phosphorus stresstolerant genes from rice at the University of Tokyo, Japan, during the period of 2015–2017. His current research interests include the isolation and characterization of abiotic stress-­responsive genes and proteins, physiological and molecular mechanisms of abiotic stress response and tolerance with special reference to oxidative stress, antioxidants and methylglyoxal metabolism and signaling, generation of stress-­tolerant and nutrient-efficient plants through breeding and biotechnology, and cross-stress tolerance in plants. He has over 50 peer-reviewed publications and has edited 9 books, including this one, published by CRC press, Springer, and Elsevier.

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Vinay  Kumar is an Associate Professor in the Department of Biotechnology, Modern College of Arts, Science and Commerce, Ganeshkhind, Pune, India, and a Visiting Faculty at the Department of Environmental Sciences, Savitribai Phule Pune University, Pune, India. He obtained his Ph.D. in Biotechnology from Savitribai Phule Pune University (formerly University of Pune) in 2009. For his Ph.D., he worked on metabolic engineering of rice for improved salinity tolerance. He has published 40 peer-reviewed research/review articles and edited 4 books, including this one, published by Springer and Wiley. He is a Recipient of Young Scientist Award of Science and Engineering Board, Government of India. His current research interests include elucidating molecular mechanisms underlying salinity stress responses and tolerance in plants. David  J.  Burritt is an Associate Professor in the Department of Botany, University of Otago, Dunedin, New Zealand. He received his B.Sc. and M.Sc. (hons) in Botany and his Ph.D. in Plant Biotechnology from the University of Canterbury, Christchurch, New Zealand. His research interests include oxidative stress and redox biology, plant-based foods and bioactive molecules, plant breeding and biotechnology, cryopreservation of germplasm, and the stress biology of plants, animals, and algae. He has over 100 peerreviewed publications and has edited 4 books for Springer and 3 for Elsevier. Masayuki Fujita is a Professor in the Department of Plant Science, Faculty of Agriculture, Kagawa University, Kagawa, Japan. He received his B.Sc. in Chemistry from Shizuoka University, Shizuoka, and his M.Agr. and Ph.D. in Plant Biochemistry from Nagoya University, Nagoya, Japan. His research interests include physiological, biochemical, and molecular biological responses based on secondary metabolism in plants under biotic (pathogenic fungal infection) and abiotic (salinity, drought, extreme temperatures, and heavy metals) stresses, phytoalexin, cytochrome P-450, glutathione S-transferase, phytochelatin, and redox reaction and antioxidants. He has over 150 peer-reviewed publications and has edited 10 books including this one.

Editors Biography

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Pirjo S. A. Mäkelä is a Professor in the Department of Agricultural Sciences, University of Helsinki, Finland. She received her M.Sc. and Ph.D. in Crop Science from the University of Helsinki, Finland. Her research interests include physiological, biochemical, and agronomical responses of plants to abiotic stresses, such as water deficit and salinity, as well as ways to minimize the effects of abiotic stresses on yield formation and quality of yield. She is also interested in active learning in higher education. She has over 70 peer-reviewed publications and has edited 3 books including this one.

Osmoprotectant-Related Genes in Plants Under Abiotic Stress: Expression Dynamics, In Silico Genome Mapping, and Biotechnology Éderson Akio Kido, José Ribamar Costa Ferreira-Neto, Manassés Daniel da Silva, Vanessa Emanuelle Pereira Santos, Jorge Luís Bandeira da Silva Filho, and Ana Maria Benko-Iseppon

1  Introduction In nowadays, agriculture faces multiple challenges, including the adoption of sustainable methods to provide food for a growing urban population, in addition to the increase of bioenergy needs. Moreover, plants are subject to a variety of stresses, leading to damages that can negatively influence vegetative and reproductive development and compromise their yields, causing economic losses. In the last decades, the modernization of methods and tools enforced in agriculture has developed simultaneously with civilization. Scientific advances applied in traditional plant breeding have increased genetic gains of cultivated plants improving their yields and their resistance/tolerance to the environmental stresses around the world. Environmental stresses are classified into biotic stresses, those caused by organisms such as bacteria, fungi, viruses, nematodes, insects, or higher eukaryotes (e.g., weed and herbivores), or abiotic stresses, caused by nonliving organisms, including physical or chemicals stressors, such as high or low temperatures, drought or floods, and salinity, among others. These stressors can act alone or often combined, such as droughts and high temperatures. Plants under stress need to adapt in order to survive. They respond to the environment by modifying the expression of their genes to best suit the stressful situation and minimize the damages. The dynamics of the genes global expression determine the plant response to the stress-derived stimulus. Briefly, the stress stimulus is recognized by the receptors in plant cell membranes, and a generated signal is transmitted and amplified in a cascade that culminates in the activation of specific genes. Those gene expressions will constitute the plant response to the stress. During the signaling process, enzymes and receptors are activated or deactivated through phosphorylation and dephosphorylation by É. A. Kido (*) · J. R. C. Ferreira-Neto · M. D. da Silva · V. E. P. Santos J. L. B. da Silva Filho · A. M. Benko-Iseppon Department of Genetics, Federal University of Pernambuco, Recife, PE, Brazil © Springer Nature Switzerland AG 2019 M. A. Hossain et al. (eds.), Osmoprotectant-Mediated Abiotic Stress Tolerance in Plants, https://doi.org/10.1007/978-3-030-27423-8_1

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p­ rotein kinases and phosphatases (Ardito et al. 2017). Finally, activated transcription factors (TFs) inside the nucleus will recognize specific cis-regulatory elements in the promoters of the genes that will be expressed, helping them to modulate their expression. Besides the TFs, gene expressions might be influenced by CoRegs (co-­ regulator proteins; Burdo et al. 2014). These proteins, unlike TFs, do not interact directly with the DNAs but interfere with gene regulation by protein-protein interactions, even interacting with TFs (Chevalier et  al. 2009). They also restrict or release DNA access, behaving as histone modifiers (Fang et al. 2014) or chromatin remodelers (Han et al. 2016). Therefore, gene expression resulting from complex interactions acts on metabolic pathways or other processes such as RNA interference (RNAi, Saurabh et al. 2014), to respond to the triggering plant stress stimulus. Over time, plant evolving under unfavorable growth conditions presented molecular, biochemical, and physiological adjustments. Some of these alterations were induced by new alleles associated with isoforms neofunctionalizations, increasing plant variability. Such context resulted in a range of combined strategies, which plants access to minimize damages caused by environmental stresses. In general, plants under abiotic stresses rely on genes from three broad categories (Hossain et al. 2016): (a) Genes transcribing regulatory proteins, such as kinases and TFs, which are widely reported in plant responses. (b) Genes related to water channel proteins and ion transporters. (c) Genes linked to the protection of essential membranes and proteins such as chaperones, heat shock proteins, and osmoprotective osmolytes. This chapter regards genes related to osmoprotectants reported in plant transcriptomic studies, their regulation under abiotic stress, their genomes mapping, potential pathways, and, finally, their experiences as transgenes in order to improve plant breeding.

2  Osmoprotectant Definition, Classification, and Roles Some inorganic ions in ideal concentrations contribute to the biochemical functions, but in high amounts, they disrupt protein functions. Diversely, osmoprotectants are small, electrically neutral, and highly soluble organic compounds with low toxicity. They can accumulate high amounts in the cells, balancing the intracellular with the external environment when in an unfavorable osmotic condition. Due to their high solubility and little interference in the cellular metabolic pathways, they are also known as compatible solutes. The DEOP database (Dragon Explorer of Osmoprotection Pathways; Bougouffa et al. 2014) is an online resource on osmoprotectants and its associated pathways (http://www.cbrc.kaust.edu.sa/deop/). According to the DEOP web index, osmoprotectants are classified into three distinct classes: (i) those containing quaternary ammonium compounds (QACs) and deriva-

Osmoprotectant-Related Genes in Plants Under Abiotic Stress: Expression Dynamics…

a Polyamines

b Aminoacids

c Carbohydrate

1. Putrescine 2. Spermidine 3. Spermine

1. Proline

1. Threalose 2. Fructan

d Betaines

e Sugar alcohol

1. Glycine betaine

1. Inositol (and its phosphorylated derivatives)

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Fig. 1  Main classes of plant osmoprotectants and some representatives compounds

tives, e.g., polyamines and betaines (such as glycine betaine); (ii) those containing amino acids and derivatives, e.g., proline and ectoine; and (iii) those containing sugars and derivatives, e.g., oligosaccharides (sucrose, trehalose, raffinose, stachyose, verbascose), fructan [fructose polymers; oligosaccharides or polysaccharides (>10 units)], and sugar alcohols (polyols: glycerol, inositol, arabitol, maltitol, sorbitol, mannitol, and D-ononitol). Further, based on the DEOP data, which involved scientific manuscripts published until 2014, covering more than 1160 organisms (including microorganisms, plants, and animals), a total of 135 osmoprotectant compounds were identified (Bougouffa et al. 2014). The major classes of plant osmoprotectants with representatives described in this chapter, whose expressions of their related genes have been reported, are shown in Fig. 1. Essentially, plants face two situations when under an abiotic stress, such as salt stress (Singh et al. 2015): (a) an osmotic stress due to the higher Na+ concentration in the rhizosphere, which decreases plant water potential, and (b) a nutritional imbalance caused by ionic stress, in which the higher concentration of Na+ and Cl− limits the availability and assimilation of essential nutrients. Thus, in plants under hypertonic conditions resulting from high NaCl, a flow of water occurs from the inside to the outside of the cell. This situation increases the concentration of the cellular constituents. A high concentration of ions can disrupt proteins, shifting the balance to their unfolded forms. In this case, protective osmolytes accumulated on the surfaces of proteins help to stabilize their structures, tensioning them back to their native structure. Therefore, these osmolytes are recognized as osmoprotectants, due to this protective role against osmotic and saline stresses. In plants under abiotic stress causing denaturation of macromolecular (proteins and membranes), osmoprotectants such as proline can improve protein stability by binding to hydrogen bonds without affecting the other functions (Slama et al. 2015). Further, trehalose, another osmoprotectant, can stabilize macromolecules, such as the bilayer structure of membranes, by binding to hydrogen bonds in the polar

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groups of membranes and proteins, preserving their integrities (Pereira et al. 2004). However, this characteristic varies depending on the osmoprotectant considered. Additionally, some osmoprotectants present chaperone-like activities in order to keep both protein structures and functions. Those compatible osmolytes are also named chemical or molecular chaperones (Slama et al. 2015). Besides, in plants exposed to salinity and drought, osmoprotectants can accumulate in the cells, helping to maintain cellular turgor and driving the gradient for water uptake to sustain cell volume by osmotic adjustment. In this regulation, the cell tends to compartmentalize ions in the vacuoles; at the same time, it begins to synthesize and accumulate osmoprotectants in the cytoplasm, such as proline, to maintain the osmotic balance between these compartments (Gagneul et al. 2007). Nevertheless, when a cell undergoes osmotic stress, its redox potential is disturbed, and generated an excessive induction and accumulation of reactive oxygen species (ROS). ROS are by-products of the oxygen metabolism linked to electron transport (Bae et al. 2011). These reactive species [superoxide (O2−), hydrogen peroxide (H2O2), and hydroxyl radicals (OH)] are important in cell signaling once in adequate amounts but in excessive volume cause peroxidation of lipid, oxidation of proteins, and damage of nucleic acids. Further, they can inactivate antioxidative enzymes and even culminate in cells and plant deaths. Therefore, ROS regulation is crucial to avoid cytotoxicity and oxidative damage. Some of the compatible solutes can protect plants from oxidative damages by directly scavenging for ROS or protecting the enzymes from the antioxidative process (Slama et al. 2015). Other osmoprotectants functions in plant responses to abiotic stresses, especially concerning the drought and salinity tolerance, are found in the review reported by Singh et al. (2015).

3  The Basic of Plant Transcriptomic Studies A transcriptomic study is an excellent option to look in the potential of osmoprotectants, notably their related genes, in a plant responding to an abiotic stimulus. In general, these studies allow the preview of the gene expression profile of an organism, organ, tissue, or cell, after applying a given stimulus. These global patterns are usually contrasted by comparing the stressed or treated profile with those corresponding to the negative control without the stimulus. Several methodologies (e.g., Northern blot, EST, SAGE and derivatives, microarray, RNA-Seq) can be used to generate libraries expressing these profiles (Kido et al. 2016). Some aspects will be briefly commented below. Transcriptomic libraries when properly generated and sequenced, using deep-­ sequencing (NGS, next-generation sequencing) technologies, provide millions of reads. After data quality inspections and the removal of adapters and low-quality bases, these reads allow (a) tag annotation, as those tags (26 pb) generated by the

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SuperSAGE technique (Matsumura et  al. 2012),  or (b) transcriptome assembly (RNA-Seq data), using de novo strategy or based on a reference genome, generating the final assembled transcripts or unigenes (Wang et al. 2009). In both cases, tags or transcripts/unigenes must be adequately annotated, considering similarity levels with previously annotated sequences, using BLAST alignments. In this context, molecular targets involving osmoprotectant-related genes could be identified and selected considering: (a) The tag or unigene annotation. (b) The tag or unigene regulation (induced or repressed), considering their frequencies in two circumstantial libraries (stress treatment versus control). (c) The expression modulated by the tag or unigene; the fold change (FC) value representing the ratio of the normalized frequencies, usually tpm (tags per million), or FPKM (fragments per kilobase of transcript per million mapped reads) considering two comparing libraries. Furthermore, after appropriated statistical analysis (p-values) comparing the normalized frequencies of the tag or unigene based on the contrasted libraries, attention should be given to those identified as differentially expressed gene (DEGs). In this process, the p-values are corrected in order to minimize the type I error (Li et al. 2012), using the FDR (false discovery rate) method or similar. That correction diminishes false-positive episodes of differential expressions since the probability of false positives increases due to the high number of tests performed. Another advantage of a genomic-scale approach is the fact that many plant genomes and transcriptomes data, including those from crops under experimentally controlled stress, are deposited in public databases. The GenBank at NCBI (the National Center for Biotechnology Information; https://www.ncbi.nlm.nih.gov/), the DNA Data Bank of Japan (DDBJ; https://www.ddbj.nig.ac.jp/index-e.html), or the European Nucleotide Archive (ENA; https://www.ebi.ac.uk/ena) are constantly receiving biomolecules sequences from current projects. Therefore, these substantial databases provide bioinformatic tools, online analysis, and downloads of datasets allowing reliable annotations of DEGs, tags, or unigenes, in addition to other analysis. Diversely, although transcriptomic studies address the global expression of genes, the identification of those related to osmoprotective osmolytes is not a simple task. In transcriptomic studies, despite the detection of many genes comprising several functional metabolic categories, only a few tags or unigenes have already been more detailed. In general, the most observed are those genes related to DEGs showing relevant modulation based on the in silico analysis. Thus, if osmoprotectants are not the primary focus, then few osmoprotectant-related genes are unveiled. Essentially tags or unigenes that are strongly expressed after an applied stimulus are the mostly noted. Usually, the same set of genes is reported to be associated with plant abiotic stress profile. A compilation of some known osmoprotectants and their potential related pathways are shown in Table 1.

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Table 1  Some pathways associated with osmoprotectant biosynthesis in plants Pathway Choline biosynthesis Ectoine biosynthesis Fructan biosynthesis Glycine betaine biosynthesis L-glutamate biosynthesis L-proline biosynthesis Mannitol biosynthesis Myo-inositol biosynthesis Putrescine biosynthesis Sorbitol biosynthesis Spermine biosynthesis Sucrose biosynthesis Trehalose biosynthesis

Related compound/class Amine and polyamine/choline Amine and polyamine/ectoine Sugar/fructan Amine and polyamine/glycine betaine Amino acid/L-glutamate Amino acid/L-proline Polyol/sugar alcohol Myo-inositols Amine and polyamine/putrescine Polyol/sugar alcohol Amines and polyamines/spermine Sugar/sucrose Sugar/trehalose

4  Osmoprotectant-Related Genes and Associated Pathways Despite the importance of osmoprotectants in plants and the scientific advances over the years, a database compiling most of the generated information was not available until 2014, when Bougouffa et al. (2014) performing intensive data mining (more than 900,000 scientific articles) compiled 141 osmoprotectant compounds from 1160 organisms (microorganisms, plants, and animals). The authors connected osmoprotectant with potential pathways (biosynthesis or degradation) affecting these osmolytes (834), including reactions (1883), genes (3529), and proteins (4899). Concerning those compounds, only 34 remained not correlated with the identified pathways or reactions. This unique initiative resulted in the DEOP website (http://www.cbrc.kaust.edu.sa/deop/index.php), which is a database dedicated exclusively to osmoprotectants and their possible associated pathways. Based on the site’s background information, the focus of the authors was to study the potential of microorganisms accumulating osmoprotectants to become cell factories. Another concern was the potential transference of such functional capability into other organisms through synthetic biology. Besides those already mentioned features, the available information provides perspectives covering microorganisms-­ plant interactions, with both organisms acting together against adverse conditions in the rhizosphere and soil environment. Such information can greatly assist studies of functional, comparative, and evolutionary genomics aspects involving osmoprotective genes. The searches performed on DEOP relational tables scrutinize pathways derived from the KEGG (Kyoto Encyclopedia of Genes and Genomes; https://www.genome. jp/kegg/) and MetaCyc databases (Metabolic Pathway Database, https://metacyc. org/). The MetaCyc database is a cured bank containing more than 2570 pathways of almost 3000 organisms from the various domains of life (Caspi et al. 2018). A compound can be associated with pathways representing osmoprotectant

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7

Fig. 2  Venn diagram based on numbers of entries related to osmoprotectant-associated pathways [biosynthesis (final or intermediate product)/ degradation] prospected from DEOP database (http://www.cbrc. kaust.edu.sa/deop/index. php)

b­ iosynthesis (related to the final or intermediate product; also, reversible or not), osmoprotectant degradation, and other osmoregulation. Furthermore, since it is possible to download data from the DEOP site, the entries related to the biosynthesis pathways of osmoprotective compounds as final products (December 2018) totalized 120, while those addressing intermediate products were 205, and those covering degradation were 140 (Fig. 2). Some of the 120 identified entries are listed in Table 1, and the described pathways typically associate sugars and its derivatives (e.g., sugar alcohol), amines and polyamines, and amino acids, highlighting these compounds as osmoprotectants. According to the DEOP relational tables associated with the applied research, plant species presenting data associated with the pathways presented in Table 1 are listed in Table 2. The identified pathways and plants comprised mostly the biosynthesis of proline (14 plant species), sucrose (9), trehalose (8), and putrescine (8). In turn, plants concentrating studies focusing on those pathways (Table 1) comprised Nicotiana tabacum (9), Arabidopsis thaliana (7), Oryza sativa (7), and Triticum aestivum (5) (Table 2). Moreover, the set of reactions predicted in the pathways listed in Table 1, as well as the others available in the DEOP database, allow the identification of genes and enzymes associated with a specific osmoprotectant compound; that association is supported by the MetaCyc database, a comprehensive source of diagrams showing the enzymes involved in such reactions. Therefore, genes encoding the related enzymes identified above are good candidates to be noted in a transcriptomic study, as well as their expression after a signalized stress. Once some gene candidates are identified as DEGs, its expression still needs to be validated by a second method. Usually, the RT-qPCR (real-time reverse transcription-polymerase chain reaction) technique is performed; after all, it is considered a reference method in such cases (Provenzano and Mocellin 2007). After the validation process is done, the reliable candidates become promising to be applied as functional molecular markers to

8

É. A. Kido et al.

Table 2  Plant species presenting osmoprotectants data and associated pathwaysa based on the DEOP database (http://www.cbrc.kaust.edu.sa/deop/index.php) Plant species Arabidopsis thaliana Avena sativa Brassica napus Glycine max Helianthus tuberosus Hordeum vulgare Lycopersicon esculentum Malus x domestica Nicotiana tabacum Oryza sativa Phaseolus vulgaris Pisum sativum Populus sp. Solanum tuberosum Spinacia oleracea Triticum aestivum Vigna aconitifolia Zea mays Subtotal

Pathwaysa 1 2 3 4

5 x

x x

7 x

8

x x x x

x

x

6 x

x x

x x x x

9 x x

10

11 x

12 x

x x

x x

x x x

x x x x

x x

x x x

1

1

4

2

1

x x x x x 14

x x

x

2

1

8

2

1

x 9

13 Subtotal x 7 1 1 x 3 1 4 1 2 x 9 x 7 x 2 1 1 x 2 3 x 5 1 x 3 8 54

Biosynthesis pathwaysa: (1) choline; (2) ectoine; (3) fructan; (4) glycine betaine; (5) L-glutamate; (6) L-proline; (7) mannitol; (8) myo-inositol; (9) putrescine; (10) sorbitol; (11) spermine; (12) sucrose; (13) trehalose

assist selection steps in plant breeding programs or to be evaluated in transgenic assays, helping breeders to develop new cultivars or varieties.

5  Expression of Osmoprotectant-Related Genes Plants under abiotic stresses presented osmoprotectant-related genes modulating their expressions after the stress stimulus. Regarding salinity stress, at least 15 scientific articles covering 2015 until the beginning of 2019 presented osmoprotectant-­ related genes analyzed in 12 plant species. The investigated plant species comprised classic model plants (e.g., A. thaliana, M. truncatula, N. tabacum), important cultivated worldwide crops (e.g., G. max, O. sativa, S. bicolor), and other lesser-known plants (e.g., Bacopa monnieri, Chenopodium album) (Table 3). The experimental assays described in those articles were quite diverse, covering plants at different growth stages that were submitted to the NaCl salt, which molarities comprised from 75 to 400 mM, and the time of exposure ranging from less than 1 hour to days or even weeks. Some of the studies also looked at the influence of

Osmoprotectant Proline Trehalose

Putrescine/polyamine Proline Proline Glycine betaine Proline Raffinose Glycine betaine Proline Putrescine/polyamine

NaCl 200 mM (0.5, 1, 3, 6 h) NaCl 250 mM (12, 18, 24 h) NaCl 200 mM (3 weeks) NaCl 200 mM (3 weeks) NaCl 200 and 300 mM (60 days)

NaCl 400 mM (2, 4, 6 h) NaCl 100 and 200 mM (48 h) NaCl 100 and 200 mM (48 h) NaCl 75 mM (0, 14 days)

Oryza sativa Raphanus sativus Raphanus sativus Sorghum bicolor

NaCl 300 mM (0, 24, 48 h) Putrescine/polyamine NaCl 100 and 250 mM (1 h, 24 h, 15 days) Glycine betaine NaCl 150 and 300 mM (30 days) Glycerol, hydroquinone NaCl 50 mM (3 weeks) Proline NaCl 200 mM (2, 4, 6, 12, 24, 45 h) Myo-inositol NaCl 250 mM (2, 4, 6, 9, 12 h) Proline NaCl 250 mM (2, 4, 6, 9, 12 h) Proline NaCl 200 mM (0.5, 1, 3, 6 h) Putrescine/polyamine NaCl 200 mM (0.5, 1, 3, 6 h) Putrescine/polyamine NaCl 200 mM (0.5, 1, 3, 6 h) Polyamine

Stress treatment NaCl 300 mM (6 h) NaCl 300 mM (0, 6, 12 h)

Malus hupehensis Medicago truncatula Nicotiana rustica Nicotiana rustica Nicotiana tabacum/Arabidopsis

Glycine max Ipomea batatas Lilium regale Lilium regale Malus hupehensis Malus hupehensis Malus hupehensis

Plant species Arabidopsis Arabidopsis/ Gossypium hirsutum Chenopodium album/tobacco Chenopodium quinoa Glycine max

RS5 BADH P5CS OAT

P5CS MIPS1 P5CS2 P5CS1, P5CS3 ADC2, ODC1 ADC1 SAMDC1-5, SPDS2-6 SPDS1, SPDS4 P5CS2 P5CS, PDH BADH P5CS1

ADC1, ODC2 BADH GSTU4

Target genes P5CS1, P5CS2 TPS11

Table 3  Published scientific reports presenting regulation of osmoprotectant-related genes in plants under salt stress

UR DR UR UR

(continued)

Gong et al. (2018) Li et al. (2017) Rajaeian et al. (2017) Rajaeian et al. (2017) Ibragimova et al. (2015) Jung et al. (2017) Sun et al. (2016) Sun et al. (2016) de Freitas et al. (2019)

Ren et al. (2018) Zhai et al. (2015) Wei et al. (2016) Wei et al. (2016) Gong et al. (2018) Gong et al. (2018) Gong et al. (2018)

UR UR DR UR DR UR DR UR UR UR UR UR

Wang et al. (2017) Jiang et al. (2016) Kissoudis et al. (2015)

UR UR UR

Reg. Reference UR Alavilli et al. (2016) UR Wang et al. (2016a)

Osmoprotectant-Related Genes in Plants Under Abiotic Stress: Expression Dynamics… 9

Stress treatment NaCl 75 mM (0, 14 days) NaCl 75 mM (0, 14 days) NaCl 75 mM + proline,30 mM (0.5–14 days) NaCl 75 mM + proline,30 mM (0.5–14 days) NaCl 75 mM + proline,30 mM (0.5–14 days) NaCl 300 mM (6 h)

Target genes P5CS2 ProDH OAT P5CS2 P5CS1, ProDH P5CS1, P5CS2

Osmoprotectant Proline Proline Putrescine/polyamine Proline Proline Proline

UR

UR

DR

Reg. DR UR UR

Alavilli et al. (2016)

de Freitas et al. (2019)

de Freitas et al. (2019)

Reference de Freitas et al. (2019) de Freitas et al. (2019) de Freitas et al. (2019)

Reg. (gene regulation), ADC (arginine decarboxylase), BADH (betaine aldehyde dehydrogenase), GSTU (glycerol and hydroquinone), MIPS (myo-inositol-­1phosphate synthase), OAT (ornithine-δ-aminotransferase), ODC (ornithine decarboxylase), P5CS (Δ1-pyrroline-5-carboxylate synthase), PDH/ProDH (proline dehydrogenase), RS (raffinose synthase), SAMDC (S-adenosylmethionine decarboxylase), SPDS (spermidine synthase), TPS (trehalose 6-phosphate synthase)

Arabidopsis

Sorghum bicolor

Sorghum bicolor

Plant species Sorghum bicolor Sorghum bicolor Sorghum bicolor

Table 3 (continued)

10 É. A. Kido et al.

Osmoprotectant-Related Genes in Plants Under Abiotic Stress: Expression Dynamics…

11

other factors besides NaCl, such as PEG, phytohormones, proline, and ethanolamine, among others, independently or combined with the salt. In that set of reports, the highlighted osmoprotectant compounds comprised the amino acid proline, the glycine betaine, the polyamines (putrescine and spermidine), the sugars (trehalose, oligosaccharides), and sugar derivatives (e.g., myo-inositol). Also, a total of 12 different osmoprotectant-related genes were investigated about their regulation after salt-stress exposure. Concerning drought stress, covering the period 2015–2019, at least 14 scientific articles also reported osmoprotectant-related genes modulating their expression after dehydration stress treatment. The experimental assays described in that researches showed different stress application methods, involving natural drought conditions (Yang et  al. 2015c), drought simulated by root dehydration (0–72  h; Singh et al. 2015), suppression of irrigation (Rickes et al. 2019; Dastogeer et al. 2018), withholding water assay (Chen et al. 2016), addition of polyethylene glycol (30% PEG 6000 solution; Yadav et  al. 2018), and even dehydrated fruits (grape ­berries) (Conde et  al. 2018) (Table  4). These studies embraced 15 plant species, including the reference plants and crops already mentioned, and other lesser-known plants, such as Stipa purpurea (Yang et al. 2015c) and Ziziphus nummularia (Yadav et al. 2018]. Also, this set of reports encompassed 21 genes related to osmoprotectants, such as the amino acid proline, the glycine betaine, the polyamines (putrescine and spermidine), the sugars (sucrose, raffinose, and trehalose), and polyols (sorbitol and mannitol). Some of the investigated genes are presented in Table 4, taking into account their expression after drought or salt stress.

5.1  Amino Acid Proline In respect to proline as an osmoprotectant, and considering plants responding to salt stress, eight scientific manuscripts presented expression results of only two genes. One of them encoded P5CS (Δ1-pyrroline-5-carboxylate synthase) and the other PDH (proline dehydrogenase). Only the first gene takes part in the proline biosynthesis pathway. The P5CS (EC 2.7.2.11/1.2.1.41) reduces glutamic acid to γ-glutamic semialdehyde (GSA), and GSA is converted spontaneously by P5CR (Δ1-pyrroline-5-carboxylate reductase) into Δ1-pyrroline-5-carboxylate (P5C). Finally, P5C is converted to proline by P5CR (Szabados and Savoure 2010). The other gene codifies the enzyme PDH (EC 1.5.5.2) that catalyzes proline degradation after plant dehydration. In general, except for some P5CS isoforms and depending on the analyzed tissue, the upregulation of transcripts of both genes are observed in roots after the salt application (Table 3). Still, considering proline, now taking into account plants responding to drought stress, the same genes were investigated (Table  4). About nine articles presented P5CS expression showing upregulation after drought stress, and only one research (Dastogeer et al. 2018) investigated the PDH expression, also noting the upregulation of the transcript. Interestingly, Dastogeer et al. (2018) pointed fungal ­endophytes

Spinacia oleracea

P5CS1, P5CS3 P5CS2 ADC1, ADC2, ODC1

Proline Proline Putrescine/ polyamine Putrescine/ polyamine Proline

GolS2 SOT1 P5CS SIP1 S6PDH BADH, CMO, SAMDC

ADC, CPA, ODC

RFO Polyol Proline Raffinose Polyol Glycine betaine

Putrescine/ polyamine

PDH1

P5CR, P5CS

Drought (6 days withhold water) Drought (irrigation suppressed; 0, 4, 7, 9 days) Irrigation suppressed (0, 4, 7, 9 days) Irrigation suppressed (0, 4, 7, 9 days) Irrigation suppressed (0, 4, 7, 9 days) Withholding water (0, 5, 8 days) + re-watering (after 1, 4 days). NaHS application in S. oleracea seedlings Withholding water (0, 5, 8 days) + re-watering (after 1, 4 days). NaHS application in S. oleracea seedlings

Medicago truncatula Nicotiana benthamiana Oryza sativa Prunus persica Prunus persica Prunus persica Prunus persica Spinacia oleracea

P5CS

Proline

ODC2

GolS

Raffinose

Proline

Withholding water (9 days)

Malus hupehensis

Target genes P5CS

Osmoprotectant Proline

Withholding water (8 days)

NaCl 200 mM (0, 1, 3, 6, 12, 24 h)

Lilium regale Lilium regale Malus hupehensis

Jatropha curcas

Glycine max

Stress treatment PEG 6000 [osmotic potential: −0,50 Mpa] (10 days) PEG 6000 [osmotic potential: −0,50 Mpa] (10 days) PEG 6000 10% (2 days); osmotic potential (−0,50 Mpa) Withholding water (7 days) Withholding water (7 days) NaCl 200 mM (0, 1, 3, 6, 12, 24 h)

Plant species Glycine max

Table 4  Published scientific reports presenting osmoprotectant-related gene regulation in plants under water-deficit stress

UR

UR DR UR UR UR UR

UR

UR

DR

UR ns UR

UR

UR

Reg. UR

Chen et al. (2016)

Jung et al. (2017) Rickes et al. (2019) Rickes et al. (2019) Rickes et al. (2019) Rickes et al. (2019) Chen et al. (2016)

Dastogeer et al. (2018)

Antoniou et al. (2018)

Gong et al. (2018)

Wei et al. (2016) Wei et al. (2016) Gong et al. (2018)

Reference Vaishnav and Choudhary (2018) Vaishnav and Choudhary (2018) Yang et al. (2015b)

12 É. A. Kido et al.

P5CS BADH ADC, ODC DHS, SAMDC, SPDS PLT1, SDH MTD GolS1 GolS

Proline Glycine betaine Putrescine/ polyamine Polyamine Polyol Polyol/ mannitol Raffinose Raffinose Proline Proline

PEG 6000 30% (6, 12, 24, 48, 72 h)

PEG 6000 [with osmotic potential of −0,50 Mpa] (10 days) P5CS

P5CS

FBPase, SPS1, TPS

Sucrose

Withholding water (0, 5, 8 days) + re-watering (after 1, 4 days). NaHS application in S. oleracea seedlings Naturals drought conditions Naturals drought conditions Withholding water (>11 days); exogenous polyamine application Withholding water (>11 days); exogenous polyamine application Fruit dehydrated (5, 11 days) Fruit dehydrated (5, 11 days) Fruit dehydrated (5, 11 days) PEG 6000 30% (6, 12, 24, 48, 72 h)

UR

UR

Vaishnav and Choudhary (2018)

Yadav et al. (2018)

Conde et al. (2018) Conde et al. (2018) Conde et al. (2018) Yadav et al. (2018)

Ebeed et al. (2017)

UR UR n.s. UR UR

Yang et al. (2015c) Yang et al. (2015c) Ebeed et al. (2017)

Chen et al. (2016)

UR UR UR

UR

Reg. (gene regulation), BADH (betaine aldehyde dehydrogenase), CMO (choline monooxygenase), DHS (deoxyhypusine synthase), SPDS (spermidine synthase), SAMDC (S-adenosylmethionine decarboxylase, PLT (polyol transporter), SDH (sorbitol dehydrogenase), SOT (sorbitol transporter), S6PDH (sorbitol6-phosphate dehydrogenase), MTD (mannitol dehydrogenase), P5CR (Δ1-pyrroline-5-carboxylate reductase), P5CS (Δ1-pyrroline-5-carboxylate synthase), PDH (proline dehydrogenase), ADC (arginine decarboxylase), CPA (N-carbamoylputrescine amidohydrolase), ODC (ornithine decarboxylase), GolS (galactinol synthase/myo-inositol 3-alpha-D-galactosyltransferase), SMO (sterol C-4 methyl oxidase), SIP (raffinose synthase), SPS (sucrose-phosphate synthase), FBPase (fructose-1,6-bisphosphatase), TPS (trehalose 6-phosphate synthase), RFO (raffinose)

Vitis vinífera Vitis vinífera Vitis vinífera Ziziphus nummularia Ziziphus nummularia Glycine max

Triticum aestivum

Stipa purpurea Stipa purpurea Triticum aestivum

Spinacia oleracea

Osmoprotectant-Related Genes in Plants Under Abiotic Stress: Expression Dynamics… 13

14

É. A. Kido et al.

and inoculated virus conferring drought tolerance to Nicotiana benthamiana plants through osmolyte modulation and expression of host drought-­responsive genes.

5.2  Glycine Betaine Regarding glycine betaine (GB) and plants responding to the salt stress, the single gene investigated in the researched period encoded BADH (betaine aldehyde dehydrogenase; EC 1.2.1.8), which presented upregulation in most of the manuscripts (Table 3), and only one BADH downregulated (Sun et al. 2016). Besides this DR regulation, the analyzed radish transcriptome (Raphanus sativus L.) in response to salt stress (0, 100, and 200 mM NaCl for 48 h) presented 29 induced DEGs associated with osmoprotectants (threshold of |log2Ratio|  ≥  1 with FDR  ≤  0.001 and p-value ≤ 0.05), including 9 P5CS candidates and 5 (induced) of 7 trehalose-related ones (Sun et al. 2016). Concerning the drought stress, besides the BADH gene, another induced gene evaluated in GB biosynthesis pathway was CMO (choline monooxygenase; Chen et  al. 2016]. In higher plants, choline is converted by CMO (EC 1.14.15.7) into betaine aldehyde, which is then catalyzed by BADH into GB (Chen et  al. 2016; Takabe et al. 2006).

5.3  Polyamines Concerning the osmoprotectant polyamines (PAs), which have some functions similar to plant growth regulators, five investigated genes covered this issue in plants responding to salt stress: ADC (arginine decarboxylase), ODC (ornithine decarboxylase), OAT (ornithine-δ-aminotransferase), SAMDC (S-adenosylmethionine decarboxylase), and SPDS (spermidine synthase) (Table 3). The ADC, ODC, and OAT genes are directly involved with the putrescine biosynthesis pathway, while SAMDC and SPDS are involved with spermidine pathway. The osmoprotectant putrescine (Put) can be synthesized directly from ornithine by ODC (EC 4.1.1.17) or indirectly, through a series of intermediates following arginine decarboxylation by ADC (EC 4.1.1.19). Most of the ADC and ODC transcripts are induced in responses to salt stresses (Table  3). Upregulation is also observed in OAT transcript, target only analyzed by de Freitas et al. (2019) in their study of S. bicolor after the stress of 75 mM NaCl, 14 days after the salt application. In respect to drought stress, several manuscripts (Chen et  al. 2016; Ebeed et  al. 2017; Gong et al. 2018) relate the upregulation of ADC and ODC transcripts. The osmoprotectant spermidine (Spd) is synthesized from Put by successive additions of aminopropyl groups catalyzed by SPDS (EC 2.5.1.16). In the other hand, the aminopropyl is provided by decarboxylated S-adenosylmethionine, a metabolite synthesized by SAMDC (EC 4.1.1.50). The SAMDC enzyme is also

Osmoprotectant-Related Genes in Plants Under Abiotic Stress: Expression Dynamics…

15

implicated in cysteine and methionine metabolism, as well as the arginine and proline metabolism (https://www.genome.jp/dbget-bin/www_bget?ec:4.1.1.50). Based on the genome-wide study reported by Gong et al. (2018) in apple (Malus hupehensis Rehd.), the MhSAMDC1 and MhSPDS1 genes were induced not only by salt but also by other treatments (alkaline, abscisic acid, cold, and dehydration), suggesting that these genes have relevant roles in plant stress responses. About drought stress, also SAMDC and SPDS presented upregulation after stress application (Chen et al. 2016; Ebeed et al. 2017). However, concerning Chen et al. (2016), the enhancement of plant drought tolerance with effects on polyamines and soluble sugar contents also derived from NaHS application before the stress exposure. The CPA (N-carbamoylputrescine amidohydrolase) and DHS (deoxyhypusine synthase) genes were also investigated concerning responses to drought stress. Both genes are induced in plants under such stress (Chen et al. 2016; Ebeed et al. 2017). The CPA (EC 3.5.1.53) is implicated in Put generation from N-carbamoylputrescine (https://www.genome.jp/dbget-bin/www_bget?ec:3.5.1.53), while DHS (EC 2.5.1.46) participates in Spe degradation using it as a substrate. Wang et al. (2003) report that suppression of DHS delays premature leaf senescence induced by drought stress in A. thaliana among other pleiotropic effects.

5.4  Carbohydrates Furthermore, in plants responding to salt stress, osmoprotectant carbohydrates (sugars) are the oligosaccharide raffinose and the complex sugar trehalose. Both were represented by the RS (raffinose synthase) and TPS (trehalose-6-phosphate synthase) genes, respectively. These genes participate directly in the raffinose and trehalose biosynthesis pathways, and both are induced after the applied salt stress (Jung et al. 2017; Wang et al. 2016a). In relation to plants responding to drought stress, the genes investigated, TPS and SIP (also a raffinose synthase; Rickes et al. 2019), presented upregulation after the applied stress (Chen et al. 2016). The RS enzyme (EC 2.4.1.82), as predicted in the galactose metabolism (https:// www.genome.jp/kegg-bin/show_pathway?ath00052+AT1G55740), converts galactinol into raffinose. In turn, the TPS enzyme (EC 2.4.1.15) participates in the trehalose biosynthesis in plants, generating T6P (trehalose-6-phosphate) from glucose-6-phosphate and UDP-glucose, with the subsequent dephosphorylation of T6P to trehalose by TPP (trehalose-6-phosphate phosphatase) (Cabid and Leloir 1958). Besides RS (SIP) gene, the GolS (galactinol synthase/myo-inositol 3-alpha-D-­ galactosyltransferase) is another induced gene observed during dehydration events and associated with the galactose metabolism. In the biosynthesis of raffinose family oligosaccharides (RFOs), the enzyme galactinol synthase (EC 2.4.1.123) catalyzes the first step converting UDP-galactose and myo-inositol to galactinol, and this will be further converted to raffinose by the RS (SIP) enzyme. Overexpression of AtGolS2 in transformed Arabidopsis plants showing reduced leaves transpiration

16

É. A. Kido et al.

presented increased endogenous galactinol and raffinose (Taji et  al. 2002). The overexpression of GolS transcripts was observed in several manuscripts (Table 9.4). Additionally, another two genes associated to sugar biosynthesis were induced in plants responding to drought, but in this case, plant tolerance was improved by NaHS that was applied to Spinacia oleracea seedlings as pretreatment (Chen et al. 2016). The investigated genes were SPS (sucrose-phosphate synthase) and FBPase (fructose-1,6-bisphosphatase). According to the KEGG database, in the starch and the sucrose metabolism, the substrate UDP-glucose is converted by SPS (EC 2.4.1.14) into sucrose-6-P which is converted by SPP1 (sucrose-phosphatase 1) to sucrose. In turn, FBPase enzyme (EC 3.1.3.11) converts the substrate D-fructose-1,6-­ bisphosphate into D-fructose 6-P, which is a compound involved with many pathways, including galactose, and also starch and sucrose metabolisms. Based on the results, the NaHS pretreatment improved plant tolerance, modulating the expression levels of genes associated with sugar biosynthesis, and also polyamines, as mentioned before. Sugars, such as sucrose and trehalose, replace water molecules on the surfaces of proteins allowing them to preserve their conformations and, therefore, to restore their functions after rehydration (Hoekstra et al. 2001).

5.5  Sugar Alcohols When talking about the osmoprotectant sugar alcohol myo-inositol, the represented gene is MIPS (myo-inositol-1-phosphate synthase), in plants responding to salt stress. The induced gene, after the salt-stress exposition, participates directly in the myo-inositol biosynthesis pathway (Zhai et  al. 2015). The MIPS enzyme (EC: 5.5.1.4) catalyzes the conversion of D-glucose-6-phosphate to 1 L-myo-inositol-1phosphate. The conversion is rate limiting in the biosynthesis of all inositol-­ containing compounds. Myo-inositol plays an essential role as a structural basis for generating second messengers useful in signal transduction (Gillaspy 2011). Also, inositol serves as a crucial component of the structural lipid phosphatidylinositol (PI) and its various phosphates, the phosphatidylinositol phosphate (PIP) lipids. Considering plants responding to drought stress, MIPS gene was not investigated in the set of researched articles. About polyols (polyhydric alcohol), including sugar alcohols such as sorbitol and mannitol, genes associated with these pathways were not presented in the set of manuscripts covering plants responding to salt stress. Nevertheless, Conde et  al. (2018) reported a study covering dehydrated grape berries. In that study, most of the genes were associated with sorbitol, and they encoded SDH (sorbitol dehydrogenase, EC 1.1.99.21), S6PDH (sorbitol-6-phosphate dehydrogenase; EC 1.1.1.140), and two polyol transporters, SOT (sorbitol transporter) and PLT (polyol transporter). Based on gene expression results, all of them presented upregulation after the applied stress (Table 4). Another investigated gene encoded MTD (mannitol dehydrogenase, EC 1.1.1.255) and it was also induced (Conde et al. 2018). The MTD enzyme converts D-mannitol into D-mannose, a compound implicated in several

Osmoprotectant-Related Genes in Plants Under Abiotic Stress: Expression Dynamics…

17

pathways, including galactose, fructose, and mannose metabolisms (https://www. genome.jp/dbget-bin/www_bget?cpd:C00159). In the polyol pathway, the unused glucose is reduced by aldose reductase to sorbitol, which is subsequently oxidized to fructose by SDH. After that, fructose can be phosphorylated by fructokinase and subsequently metabolized via dihydroxyacetone phosphate or glyceraldehyde to D-glyceraldehyde 3-phosphate, which is a substrate in the glycolysis process. In turn, S6PDH acting on D-sorbitol 6-­phosphate generates D-fructose 6-phosphate (fructose and mannose metabolism), a compound implicated in many KEGG pathways (https://www.genome.jp/dbget-bin/get_ linkdb?-t+pathway+cpd:C05345). The interrelation between sorbitol and sucrose supply due to its gene expression is observed in transgenic apple altered with S6PDH cDNA (Kanamaru et al. 2004).

6  I n Silico Genome Mapping of Osmoprotectant-Related Genes The osmoprotectants and their genes related to proline (P5CS1; P5CR1), trehalose (TPS1; TPPB, trehalose-phosphatase), glycine betaine (BADH1; CMO), cysteine (SAT1, serine acetyltransferase; OASTL1, O-acetyl serine (thiol) lyase), and myoinositol (MIPS1) were in silico mapped on genomes of six plant species, including model plants (P. patens, A. thaliana), monocots (S. bicolor and O. sativa), and dicots (G. max, P. vulgaris). The mapping allowed a comparative analysis among them. The investigated genes were chosen based on a previous study with transcripts identified using 26 bp SuperSAGE unitags expressed in soybean roots after air dehydration in time intervals ranging from 0 up to 150 min (Kido et al. 2013). Apart from P5CS1 which is absent in moss P. patens (bryophyte), the most basal species analyzed, the remaining genes were identified in virtual chromosomes of all six plant species (Table 5). The analysis brought up that 33 loci were identified in P. patens (Table  5) on 21 of 27 chromosomes total (Fig. 3A), while A. thaliana, which is a compact angiosperm genome with five chromosomes, due to the loss of DNA by unequal homologous recombination (Devos et al. 2002), mapped 43 loci (Table 5 and Fig. 3B). This information points to the relevance of osmoprotectants in cellular homeostasis maintenance through plant evolution. Mosses and flowering plants evolution diverge in more than 400 million years (MYA, Nishiyama et al. 2003). In turn, considering the two legumes (Fabaceae family), soybean (Glyxine max) presented 112 loci (Table 5, Fig. 3D), while the common bean (Phaseolus vulgaris) presented 58 loci (Table 5. Figure 3C). It is worth mention that G. max (2n = 40) has almost the double of chromosomes of P. vulgaris (2n  =  22), as a result of two genome duplications events, at approximately 59 and 13 million years ago (Schmutz et al. 2010). Therefore, soybean is a highly duplicated genome with nearly 75% of the genes present in multiple copies (Schmutz et al. 2010).

18

É. A. Kido et al.

Table 5  Loci numbers of genes associated with osmoprotectants∗ biosynthesis in six plant genome species Genome Physcomitrella patens Arabidopsis thaliana Glycine max Phaseolus vulgaris Sorghum bicolor Oryza sativa

P5CS1 P5CR1 BADH1 CMO TPS1 TPPB INPS1 OASTL SAT1 Loci 0 1 11 1 4 7 2 3 4 33 2

1

13

1

10

1

3

8

4

43

7 4

2 1

48 13

1 3

20 12

2 9

4 2

18 8

10 6

112 58

2 2

1 1

11 16

1 1

7 11

13 13

2 2

11 12

1 6

49 64

∗Osmoprotectants [gene(s)]: proline∗ [P5CS1 (delta(1)-pyrroline-5-carboxylate synthetase), P5CR1 (delta(1)-pyrroline-5-carboxylate reductase)], Glycine betaine∗ [BADH1 (betaine aldehyde dehydrogenase), CMO (choline monooxygenase)], Myo-inositol∗ [INPS1 (myo-inositol 1-phosphate synthase)], Trehalose∗ [TPS1 (trehalose-6-phosphate synthase), TPPB (trehalose-­ phosphatase)], Cysteine∗ [SAT (serine acetyltransferase), OASTL (O-acetyl-serine(thiol)lyase)]

The other two analyzed genomes, representing grasses (Poaceae family), presented 49 loci (Sorghum bicolor; subfamily Panicoidae; Table 5 and Fig. 3E) and 64 loci (O. sativa; subfamily Oryzoidae; Table 5 and Fig. 3F). Some synteny and collinearity comparing the two genomes showed 1 block involving the chromosomes 1 of sorghum and 10 of rice (same gene order for P5CS1, SAT1, and TPS1) and other 2 blocks (chromosomes 8 of sorghum and 12 of rice and chromosomes 9 of sorghum and 5 of rice). Also, another difference is observed involving the gene SAT1 presenting only one copy in sorghum, while it shows six copies in rice (Table 5). Concerning P5CR1 and CMO genes, most of the species had only one locus, with P5CR1 duplicated in soybean, and CMO with three loci in P. vulgaris (Table 5). The consequence of these two extra copies needs further investigation. On the other hand, BADH1, TPS1, and OASTL mapped at multiple loci (Table  5), probably reflecting events of duplications, which is one of the sources of new gene generation. Once a duplicate segment is subjected to lower selection pressure in subsequent mutations, it may lead to new functions (Sankoff 2001). Based on this assumption, polyploid species, such as soybean and modern sugarcane (Garcia et al. 2006), which is highly polyploid and aneuploid, as a result of interspecific crosses within the Saccharum complex, are valuable sources of genes/alleles with potential to increase plant fitness in response to biotic and abiotic stresses.

7  Osmoprotectant-Related Genes as Transgenes Regarding the scientific manuscripts addressing osmoprotectant-related genes, they acquired biotechnological relevance for the agriculture area, making them attractive targets to be manipulated also taking into account their participation in plant stress responses (Tables 3 and 4). The impact of this relevance is revealed by data mining

Osmoprotectant-Related Genes in Plants Under Abiotic Stress: Expression Dynamics…

20

TPS1

1 BA

ST

PBB TPPP S1 T IP M S1 MIP PB 4 TP B TPP

21

L

1 DH BAAT1 S S1 TP

P5C R1 MIP S1

1 TPS 1 BADH TPPB BADH1 TPS1

DH1

BA

SAT1

MIPS1 BADH1 SAT1 BADH1

5

19BADH1

BADH1 BADH1

CMO TPS1

6

OASTL

BAD

1

TPS

TL AS 17O ADH1 B

TP

S1

TP S1 M BAIPS1 DH 12

4 OASTL

7

S1

1

1 1

B P A S 5 DH O ATCS1 1 AS 1 TL

P5CS

2

3

BADH1

SA T1 P MIP5CS OA S1 1T PP BA ST B L DH 1

1

TL

R1

OAS

P5C

12 BADH1

SAT 1 T B PS1 OAADH1SAT1 ST BA L DH

P5

BA BA DH1 DH 1

TPPB

13

14

CMO

15

L ST

1

CS

BA TPDH S 1 P5 SA 1 M CST1 BA IPS11 DH 1

2

1

OA

19

--

S1 1 TPAT S

TL OAS R1 P5CSTL OA

1 DHH1 BAAD STL 1 B A S O 5C P

O

CS

20

d

T1L SS TP OASTL OA OASTL TPS1

11 S1

TP

CM

OASTL BADH1

9

0

TPS1

1 PB TP5CR1 P PB TP

TPS1

1 DH BA PPBB T P 1 TP AT S

10

CMO TPS1

11

c

L ST L OAAST 1 O ADH B 1 SAT

1

DH

BA

8

P5

BADH

1

3

1 CR P5 STL OA

1 IPS 1 S1 M DH 17 TPPPB BA T BADTPS1 C 1 S H1 B MO TP ADH H1 ASTL 1 BAD O BADH TPS1 SAT11

P5 CS 1 BA BADDH1 TPSH1 1

4 H1 BAD 1 TPS 1 BADH TPS1

16

3

TPPB

15

TPS1

1

1 CST1 P5 SA H1 1 D S BA TP

11

12

OAS TL TPS1

7

OASTL

1

S1

BADH1

TP TP PB PB

MIP

OASTL

B TPDPH1 BA

11 BA

STL

B 1 TPPPB TPS TPPS1 T DH1 BAPB TP PB TP TPPB TPPB

8

T1 T SA AS O

DH

2 OA

9

10

12

CMO

BADH1

f

OASTL OASTL

5

6

10

OASTL TPPB BADH1

10

T TPPPB PB

1

L

2 1 DH 1 BAADH B PPB 1 T ADH B PB TP S1 TP

MIP

S1

SAT1 SAT1 MIPS1

BADH1 TPS1 TPS1 TPS1 9 TPPB

TPPB

3

BA

DH

1

L

4

BADH1

6

H1

5

CM O OA OASSTL OASTTL L

1 DH

L

1 DH B BA TPP

5

H1

OAST

ST

S1 P5C SAT1 TPS1 TPS1 OASTL OASTL TPPB

6

OA

8

1 H D A B B PP T

4

BAD

PB 1 AD TPADH B B PPB T

B B AD OAAD H1 STH1 L

S1 H1 TPPPB BAD H1 T BAD

BA

B BAADH1 T DH1 O PS1 P AS P 5C TL TP5CRS1 OA S1 1 ST L

7

L

PB

BA

3

OAST

TP

TPS1 PB DH1 TP

7

9

PB TPSTLL A ST L LO A T ST L O AS S1 OAAST O TPCS1 5 O S1 1P TP AT S

e

H1 BAADDH1 B DH1 BA

S1

TP

SAT1

O OAAST ST L L

PB

TP

13 BA T DH BA PS 1 DH 1 1

A

O

1

L ST A O 1 H D 8 S1 A B 1 TP DHH1 BAAD 1 1 B AT H S AD H1 B AD B

L ST

6

TPS 1 S B AT1 P5ADH BAD CS 1 B H1 AD 1 T H1 BAPS1 7 O B A D B A S H1 TP ADDHTL S1 H1 1 B M BAAD IP D H S1 H 1 1

DH

BA

14

4

1

CS

TPS1 BADH1 MIPS1BADH1

BADH1 TPPB BADH TPS1 1 BADH

1 BADH H1 BADDH1 BA DH1 BA

8 P5

5

SAT1

B BAADH1 DH 1 S 1 T AT1 OAPS1 T P P5 S CS TL S1 P 1 OA5C STR1 L

SAT1 P5CS1

SAT1 OASTL TPS1 OASTL

BADH1 CMO TPS1 BADH TPS1 1 TPPB BADH BADH1 SAT1 1 BAD H1

TP S1 BA D MIP H1 S1

TPPB TPPB

H1

P5

SA T1

O OAAST ST L L CS 1

TP

16

9

TP TPSS1 1

5

1

DH

OA

3

ST H1 L

18

B TPADH1 S1

2

1 SAT

T1 SA

1 TPS

BA DH 1 B BAADH1 DH 1

1

b

H1 T1 SA

23

24 BAD

22 OBAAD

27

26

25

a

19

Fig. 3  In silico mapping of loci covering osmoprotectant-related genes in six plant genomes (A, Physcomitrella patens; B, Arabidopsis thaliana; C, Phaseolus vulgaris; D, Glycine max; E, Sorghum bicolor; F, Oryza sativa). Syntenic relationships showed by color lines: red [proline: P5CS1 (Δ1-pyrroline-5-carboxylate synthetase); P5CR1 (Δ1-pyrroline-5-carboxylate reductase)]; purple [glycine betaine: BADH1 (betaine aldehyde dehydrogenase); CMO (choline monooxygenase)]; orange (myo-inositol: MIPS1 (myo-inositol 1-phosphate synthase)); green [trehalose: TPS1 (trehalose-6-phosphate synthase); TPPB (trehalose phosphatase)]; blue [cysteine: SAT (serine acetyltransferase); OASTL (O-acetyl-serine(thiol)lyase)]

20

É. A. Kido et al.

in the NCBI/PubMed (https://www.ncbi.nlm.nih.gov/pubmed/), with free access to the MEDLINE, a database presenting citations and manuscript summaries in life science. Searching by keywords (“osmoprotectant name AND plant stress AND transgenic”), and considering the osmoprotectants presented in Fig. 1 (proline, glycine betaine, polyamines, sugars, and sugar alcohols), looking for scientific ­manuscripts published in the last decade, excluding reviews and genes not directly involved in osmoprotectant biosynthesis pathways, the results were very inspiring (Fig. 4).

7.1  Amino Acid Proline In light of amino acids as osmoprotectants and taking into consideration transgenic (or syngenic) plants, proline is the most studied amino acid. Several scientific manuscripts attest its participation in plant stress responses. Curiously, when associated with transgenic plants, the text mining results showed that most of the reports do not address genes from the proline biosynthesis pathway (data not showed), but other genes. Some of these genes, whose biotechnological potentials were investigated, encode the protease inhibitor chymotrypsin (Tiwari et al. 2015), transcription factors (NAC, Liu et al. 2017b), and even structural proteins, such as dehydrin, DHN-5 (Saibi et al. 2015). In those reports, proline was analyzed and associated as a positive reference of osmoprotectant acting under stressful conditions. Thus, the increase in proline content after plant post-transformation followed by stress application was a positive sign of the transgene impact. Besides those cases, the applied data mining identified five manuscripts covering genes encoding key enzymes of the proline biosynthesis in plants, basically P5CS and P5CR (Fig. 4 and Table 6). Transgenic plants of Panicum virgatum, G. max, S. bicolor, and A. thaliana, superexpressing P5CS or P5CR after stress stimulus, have been reported in abiotic stress responses to salt, heat, and drought (Table 6). In transgenic plants of P. virgatum, the physiological impacts of the P5CS gene are associated with higher ROS scavenging levels (Guan et al. 2018). However, regarding P5CR, its physiological impact still needs to be clarified.

7.2  Glycine Betaine As for betaines, the referential osmoprotectant is glycine betaine (GB). Furthermore, genes codifying the enzymes choline oxidase (CO, EC 1.1.3.17) and betaine aldehyde dehydrogenase (BADH, EC 1.2.1.8) were the most studied in transgenic plants reports (Fig. 4 and Table 7). Nevertheless, based on the glycine, serine, and threonine metabolisms, the osmoprotectant betaine is synthesized involving two conversions: (1) choline into betaine aldehyde and (2) betaine aldehyde into betaine. In higher plants, CMO (choline

Osmoprotectant-Related Genes in Plants Under Abiotic Stress: Expression Dynamics…

Spermine

Spermidine

Putrescine NM: 11

NM: 12

NM: 13

TS: Arginine decarboxylase

TS: S-adenosylmethionine

TS: S-adenosylmethionine

decar-boxylase TP: Arabidopsis thaliana and Sola-num lycopersicum TM: Agrobacterium-mediated

TP: Arabidopsis thaliana TM: Agrobacterium-mediated AS: Drought and dehydration

AS: Drought and salt

BS: F. oxysporum, Al. solani, B.

BS: F. oxysporum, A.solani,

cinerea, F. oxysporum, and P. syringae

decar-boxylase

TP: Arabidopsis thaliana and

Sola-num lycopersicum TM: Agrobacterium-mediated

AS: Drought and salt BS: F. oxysporum, A.solani

B. cinerea, F. oxysporum, P. syringae, and M. sexta

Threalose

Proline Prol NM: 5

Fructan

NM: 6 TS: Trehalose-6-phosphate

TS: P5CS and P5CR

NM: 3 TS: Fructan

synthase

TP: Arabidopsis thaliana, Panicum virgatum, Glycine max, and Sorghum bi-color TM: Agrobacterium-mediated AS: Salt, heat, and drought BS: Not avaiable

TP: Arabidopsis thaliana and Oriza sativa

6-fructosyltransferase TP: Triticosecale wittmack and Ni-cotiana tabacum

TM: Agrobacterium-mediated AS: Salt, drought, and chilling

TM: Agrobacterium-mediated AS: Cold, salt, and drought

BS: Botrytis cinérea and P. syringae

BS: Not avaiable

Glycine betaine

Inositol and phosphorylated derivatives

NM: 22

NM: 22

TS: L-myo-inositol-1-phosphate

TS: Choline oxidase and

synthase TP: Arabidopsis thaliana and Nico-tiana tabacum TM: Agrobacterium-mediated

betaine aldehyde dehydrogenase

TP: Arabidopsis thaliana and Sola-num lycopersicum

AS: Drought and salt

TM: Agrobacterium-mediated AS: Drought and salt

BS: Not avaiable

BS: Not avaiable

Polyamine

Carbohydrate

21

Aminoacid

Sugar alcohol

Betaine

Fig. 4  Data mining results covering the most representative osmoprotectants in plants by compound class (colored boxes), based on manuscripts total number (NM), target genes (TS), transgenic method (TM), transformed plant species (TP), and the analyzed stress: abiotic (AS) or biotic (BS)

22

É. A. Kido et al.

Table 6  Plants genetically modified (GM) with transgenes associated to the osmoprotectant proline Stress Reg. Tol. treatment UR > Heat and Drought UR < Heat

Transgene donor species Arabidopsis thaliana|P5CR

GM plant Glycine max

Arabidopsis thaliana|P5CS

Arabidopsis thaliana Arabidopsis UR thaliana Panicum UR virgatuma Sorghum bicolora UR

Phaseolus vulgaris|P5CS1 & P5CS2 Puccinellia chinampoensis|P5CS Vigna aconitifolia|P5CS

>

Salt

>

Salt

>

Salt

Reference De Ronde et al. (2004) Lv et al. (2011) Chen et al. (2013) Guan et al. (2018) Reddy et al. (2015)

Reg. (transgene regulation), UR (upregulated), DR (downregulated), Tol. [increasing (>) or decreasing ( Drought Spilanthes oleracea|BADH

Ipomoea batatas

UR

>

–

>

UR

>

Arthrobacter globiformis|CO Solanum lycopersicum Aphanothece Nicotiana tabacum halophytica|2-MGMT Aphanothece Nicotiana tabacum halophytica|GSMT Arthrobacter globiformis|CO Oryza sativaa

UR

>

–

>

Bacterial|CO

UR

>

UR

>

UR

>

UR

>

–

>

Populus alba × Prosopis glandulosaa Methanohalophilus Arabidopsis portucalensis|GSMT thalianaa Methanohalophilus Arabidopsis portucalensis|SDMT thalianaa Arthrobacter globiformis|CO Solanum lycopersicum Arthrobacter globiformis| CO Solanum lycopersicum

Reference Cheng et al. (2013) Salt, oxidative Fan et al. and cold (2012) Drought and Goel et al. Salt (2011) Drought He et al. (2011) Drought He et al. (2011) Drought Kathuria et al. (2009) Drought, cold, Ke et al. and salt (2016) Drought and salt Drought and salt Heat

Lai et al. (2014) Lai et al. (2014) Li et al. (2011b) Li et al. Low-­ phosphate salt (2011b) stress (continued)

Osmoprotectant-Related Genes in Plants Under Abiotic Stress: Expression Dynamics…

23

Table 7 (continued) Transgene donor species Oryza sativa|CMO

GM plant Nicotiana tabacum

Arabidopsis thaliana|BADH10A8 Arabidopsis thaliana|BADH10A9 Aphanothece halophytica|2-MGMT Aphanothece halophytica|GSMT Oryza sativa|BADH

Arabidopsis thaliana Arabidopsis thaliana Gossypium hirsutuma Gossypium hirsutuma Oryza sativa (MT)

Bacterial|CO

Brassica chinensis

Suaeda liaotungensis|BADH

Stress Reg. Tol. treatment UR > Salt UR




C




–

>

UR

>

Solanum lycopersicum Suaeda corniculata|BADH Arabidopsis thaliana Arthrobacter globiformis|CO Solanum lycopersicum Salicornia europaea|CMO Nicotiana tabacuma

UR

>

–

>

Bambusa vulgaris|CMO

C

>

UR

>

UR

>

Arthrobacter globiformis|CO Solanum tuberosum UR

>

Sesuvium portulacastrum|ALDH10 Bacterial|CO

Bambusa vulgaris (MT) Arabidopsis thaliana water lilya

Reference Luo et al. (2012) Salt Missihoun et al. (2014) Salt Missihoun et al. (2014) Salt Song et al. (2018) Salt Song et al. (2018) Drought, cold, Tang et al. and salt (2014) Salt and heat Wang et al. (2010) Salt Wang et al. (2013) Drought and Wang et al. salt (2016) Salt Wei et al. (2017) Salt Wu et al. (2010) Salt Yamada et al. (2015) Drought and Yang et al. osmotic (2015a) Cold Yu et al. (2018) Drought Cheng et al. (2013)

GM (genetically modified plant), Reg. (transgene regulation), UR (upregulated), DR (downregulated), C (constant), Tol. [increasing (>) or decreasing (

Drought

UR

>

UR

C

Drought (FRE) Salt

C

C$

Drought

UR UR

> >

Cold (FRE) Drought

UR

>

C

>

Osmotic, drought, cold Drought

C

>

Drought

Citrus sinensis

DR

>

Salt

Arabidopsis thaliana Hordeum vulgare

UR

C$

Drought

UR

>

Drought

UR C DR

> >


Heavy metal Cold Salt and drought Heat

UR

>

Cold

C

>

Drought

UR

>

Heat

Alcázar et al. (2010) Sagor et al. (2013)

UR

>

Salt

Alet et al. (2011)b

C

>

Drought

DR

>

Salt

Espasandin et al. (2014) Wang and Liu (2016)

Malus domestica|SPDS Panorpa communis Medicago sativa|SAMS1 Nicotiana tabacum Nicotiana tabacum|SPDS Nicotiana tabacum (MP) Saccharomyces Solanum cerevisiae|SAMDC lycopersicum Yeast|SAMDC Solanum lycopersicum Arabidopsis Arabidopsis thaliana|ADC2 thalianaa Arabidopsis Arabidopsis thaliana|SPMS thalianaa Avena sativa|ADC Arabidopsis thalianaa Avena sativa|ADC Leptotes tenuis Citrus sinensis|PAO

Stress Reg. Tol. treatment UR > Drought

Citrus sinensis

Alet et al. (2011)b Peremarti et al. (2009) Guo et al. (2014) Molina-Rueda and Kirby (2015) Wang et al. (2011) Alcázar et al. (2010) Espasandin et al. (2014) Wang and Liu (2016) Peremarti et al. (2009) Montilla-Bascón et al. (2017) Wen et al. (2010) Guo et al. (2014) Choubey and Rajam (2018) Cheng et al. (2009) Goyal et al. (2016)

(continued)

26

É. A. Kido et al.

Table 8 (continued) Transgene donor species Datura stramonium|SAMDC Human|SAMDC Leymus chinensis|SAMDC Medicago sativa|SAMS1 Saccharomyces cerevisiae|SAMDC Saccharomyces cerevisiae|SAMDC Yeast|SAMDC

GM plant Arabidopsis thaliana Lycopersicon esculentuma Arabidopsis thaliana Nicotiana tabacum Solanum lycopersicum Gossypium barbadenseb Solanum lycopersicum

Stress Reg. Tol. treatment UR C$ Drought UR

>

UR

>

Salt, drought, cold Cold and salt

C UR

> >

Cold Heat

UR

>

Drought

UR

>

Cold

Reference Peremarti et al. (2009) Hazarika and Rajam (2011) Liu et al. (2017b) Guo et al. (2014) Cheng et al. (2009) Momtaz et al. (2010) Goyal et al. (2016)

Reg. (transgene regulation), UR (upregulated), C (constant), C$ (faster plant recovery), Tol. [increasing (>) or decreasing ( Drought UR




UR

>

UR

>

UR

>

UR

>

UR

>

Reference Joo et al. (2014) Cold Wang et al. (2016a) Drought Han et al. (2016) Salt, PEG, and Li et al. cold (2011a) Drought, cold, He et al. and salt (2015) Cold Diedhiou et al. (2012) Cold Diedhiou et al. (2012) Cold and salt Bie et al. (2012)

GM (genetically modified), Reg. (transgene regulation), UR (upregulated), Tol. [increasing (>) or decreasing ( >

Arabidopsis thaliana|IP6K

Lycopersicon esculentuma

–

>

Brassica napus|PLC2

Canolaa

–

>

Brassica napus|PLC2 Cicer arietinum|IMPase

Canolaa Arabidopsis thalianaa

UR UR

> >

Cicer arietinum|MIPS

Arabidopsis thalianaa

–

>

Glycine max|OEP

Arabidopsis thalianaa

–

>

Glycine max|SAL1 Glycine max|IMT

Arabidopsis thalianaa Arabidopsis thalianaa

DR –

> >

Human|5-ptases

DR

>

Ipomoea batatas|MIPS

Lycopersicon esculentuma Ipomoea batatasa

UR

>

Medicago falcata|MIPS

Nicotiana tabacuma

UR

>

Mesembryanthemum crystallinum|IMT Oryza sativa|MIOX

Nicotiana tabacuma

UR

>

Oryza sativaa

–

>

Oryza sativa|IMPase Oryza sativa|MIPS Populus euphratica|MIPS Porteresia coarctata|MIPS

Nicotiana tabacuma Oryza sativaa Populus euphraticaa Nicotiana tabacuma

UR UR UR –

> > > >

Porteresia coarctata|MIPS Spartina alterniflora|MIPS Thellungiella halophila|IPK

Nicotiana tabacuma Arabidopsis thalianaa Brassica napusa

UR – –

> > >

Zea mays|PIS Zea mays|PIS

Zea maysa Nicotiana tabacuma

UR UR

> >

a

Stress treatment Drought Cold, salt, heat, pyrene Drought, cold, and oxidative Cold Drought Salt, PEG or paraquat, cold Salt and osmotic Drought and salt Salt Salt and osmotic Light Drought, salt Cold, salt, drought Salt PEG and mannitol Cold Salt Salt and Cu Salt

Reference Alcázar et al. (2010) Lisko et al. (2013) Zhang et al. (2009)

Nokhrina et al. (2014) Georges et al. (2009) Saxena et al. (2013)

Kaur et al. (2013) Ahn et al. (2018) Ku et al. (2013) Ahn et al. (2011) Alimohammadi et al. (2015) Zhai et al. (2015) Tan et al. (2013 Patra et al. (2010) Duan et al. (2012) Zhang et al. (2017) Kusuda et al. (2015) Zhang et al. (2018) Chatterjee et al. (2010) Patra et al. (2010) Joshi et al. (2013) Zhu et al. (2009)

Salt Salt Salt, osmotic, and oxidative Drought Liu et al. (2013) Drought Zhai et al. (2012)

Reg. (transgene regulation), UR (upregulated), DR (downregulated), Tol. [increasing (>) or decreasing (9) also the reverse reaction can be detected, because P5C is highly labile at elevated pH (Rena and Splittstoesser 1975) (unpublished data by G. Forlani). Unfortunately, proline-dependent formation of NAD(P) H by soluble plant extracts at high pH is often erroneously interpreted as ProDH activity (see below). Most plant species contain a single P5CR gene, and in Arabidopsis, a P5CR:GFP fusion protein was detected exclusively in the cytosol (Funck et al. 2012). After cell fractionation, the major part of the P5CR activity was detected in the soluble protein fraction in many plant species, whereas some authors also reported P5CR activity in chloroplast-enriched fractions (Murahama et  al. 2001; Noguchi et al. 1966; Rayapati et al. 1989). It remains to be clarified if P5CR, which lacks a conserved chloroplast transit peptide in all analyzed genomes, can be imported into plastids by an unconventional mechanism or can be partially attached to plastids during isolation. P5C is formed nonenzymatically by cyclization of glutamate-5-semialdehyde (GSA), which is an equilibrium reaction in aqueous solution. Two plant enzymes are known to produce GSA: ornithine-δ-aminotransferase (OAT) and P5C synthetase (P5CS). The first plant OAT gene was isolated by trans-complementation of an Escherichia coli strain unable to synthesize P5C with a cDNA clone from Vigna aconitifolia (Delauney et al. 1993). OAT is localized in the mitochondria and uses pyridoxal phosphate as cofactor in the transfer of the δ-amino group of ornithine to α-ketoglutarate, yielding GSA and glutamate (Funck et  al. 2008; Roosens et  al. 1998; Stránská et al. 2008). For mammalian OAT, it has been shown that it also catalyzes the reverse reaction in certain tissues, although the chemical equilibrium is far to the side of GSA and glutamate (Strecker 1965). The second enzyme, P5CS, reduces glutamate to GSA in a two-step reaction consuming ATP and NADPH. All land plants and animals have bifunctional P5CS enzymes, whereas prokaryotes and some unicellular green algae and fungi have separate γ-glutamyl kinase and glutamyl-γ-phosphate reductase enzymes (Fichman et  al. 2015; Hu et  al. 1992; Zhang et  al. 1995). A previous report describing the occurrence of prokaryote-like γ-glutamyl kinase and glutamyl-γ-phosphate reductase genes in tomato (Solanum lycopersicum) was most likely an artifact, because no such genes are present in genomic sequences of tomato (Fujita et al. 1998). In the initial reaction, the γ-glutamyl kinase domain of P5CS uses ATP to phosphorylate the γ-carboxy group of glutamate, and in the second reaction, glutamyl-γ-phosphate

Proline Metabolism and Its Functions in Development and Stress Tolerance

45

is reduced to GSA under consumption of NADPH. The γ-glutamyl kinase activity is inhibited by millimolar concentrations of proline and mutations identified in ­bacterial enzymes were used to engineer feedback-insensitive variants of P5CS in plants (Hu et al. 1992; Zhang et al. 1995). The first plant P5CS gene was isolated by complementation of a proline synthesis-deficient E. coli strain (Hu et  al. 1992). Most plant species have at least two P5CS isoforms, of which one can be regarded as a housekeeping gene, while others are induced by stress to enable proline accumulation (Kim and Nam 2013; Signorelli and Monza 2017; Székely et  al. 2008; Turchetto-Zolet et  al. 2009; Wang et  al. 2014). Initial characterization of the Arabidopsis P5CS proteins by GFP fusion indicated that both isoforms are cytosolic in non-stressed plants but may be imported into plastids upon osmotic stress (Székely et al. 2008), whereas our own data indicate exclusive cytosolic localization (Funck et al. 2019). Similarly, GFP fusions of two out of three P5CS isoforms from Medicago truncatula co-localized with the small subunit of ribulose bisphosphate carboxylase/oxygenase in root hairs, but like in the Arabidopsis P5CS sequences, no typical chloroplast transit peptides are present in the protein sequences (Kim and Nam 2013). By cell fractionation and Western blot, corn (Zea mays) P5CS2 was detected exclusively in the cytosol and not in the organelle fraction (Wang et  al. 2014). Because glutamate, ATP, and NADPH can be used as substrates by many different enzymes (e.g., glutamine synthetase or P5C dehydrogenase, see below), a specific assay of P5CS activity in crude plant or organelle extracts has not been reported so far, and thus the subcellular localization awaits biochemical confirmation.

2.2  Proline Degradation Enzymes For the degradation of excess proline that is not used for protein synthesis or as compatible solute, also a single enzyme is known in plants. Proline dehydrogenase (ProDH), previously also referred to as proline oxidase, is an FAD-containing enzyme at the inner mitochondrial membrane, which oxidizes proline back to P5C while transferring the obtained electrons to the mitochondrial electron transport chain and thus fueling respiratory ATP production (Elthon and Stewart 1981; Huang and Cavalieri 1979; Schertl et al. 2014). Structural studies of Put1, the ProDH of baker’s yeast (Saccharomyces cerevisiae), strongly indicate that the electrons are transferred via the tightly bound FAD cofactor to ubiquinone (Moxley et al. 2017; Wanduragala et al. 2010). A ProDH gene from Arabidopsis has been independently identified by homology searches and by screening for genes that respond rapidly to changes in the water status (Kiyosue et al. 1996; Peng et al. 1996; Verbruggen et al. 1996). Many plant genomes contain a single ProDH gene, while an early genome duplication in the Brassicaceae led to two isoforms in Arabidopsis with further multiplications occurring in Brassica species (Faes et al. 2015; Funck et al. 2010; Mani et al. 2002). ProDH activity has been exclusively detected in mitochondria unless the reverse reaction of P5CR at high pH was erroneously assigned to ProDH

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(see above; (Huang and Cavalieri 1979; Schertl et al. 2014). C-terminal GFP fusion proteins of both Arabidopsis ProDH isoforms were targeted to the mitochondria in stably transformed plants, whereas a transient transformation assay provided evidence for chloroplast targeting of ProDH2 (Funck et al. 2010; Van Aken et al. 2009). P5C produced by ProDH has three potential fates: It can be converted to proline by P5CR, or it can be linearized to GSA and be converted to ornithine by OAT or to glutamate by P5C dehydrogenase (P5CDH, recently suggested to be renamed as glutamate semialdehyde dehydrogenase, GSALDH, to better reflect the actual substrate and the evolutionary relationship to the aldehyde dehydrogenase family (Tanner 2019)). The latter is the last metabolic enzyme that is discussed here in detail. P5CDH activity was first characterized in corn mitochondria, and 20 years later the first P5CDH gene from Arabidopsis was identified by functional cloning (Deuschle et al. 2001; Elthon and Stewart 1981). In most annotated plant genomes, P5CDH is a single-copy gene except for polyploid species (Ayliffe et  al. 2005; Deuschle et al. 2001; Korasick et al. 2019). Biochemical analyses of isolated corn mitochondria provided evidence for two P5CDH isoforms with distinct pH optima (Elthon and Stewart 1982), and for the single-copy P5CDH gene in the Zea mays genome, several predicted splicing variants are annotated (NCBI gene ID: 100193220). P5CDH is a soluble enzyme in the mitochondrial matrix and prefers NAD+ over NADP+ as electron acceptor during the oxidation of GSA to glutamate (Forlani et al. 1997). In many bacteria, ProDH and P5CDH activities are combined in a single enzyme that allows direct substrate channeling, and recently it was reported that also the two plant enzymes might be physically linked through interaction with the inhibitory protein DROUGHT AND FREEZING RESPONSIVE GENE 1 (DFR1; (Ren et al. 2018). Especially in fragrant rice but also in some other plant species, proline and P5C were also identified as potential precursors for the production of 2-acetyl-1-­ pyrroline, the main constituent of the typical flavor (Wakte et al. 2017; Yoshihashi et al. 2002). However, a recent metabolomic and genomic study in rice proposed putrescine-derived 4-aminobutanal as immediate precursor for 2-acetyl-1-pyrroline and challenged the direct involvement of proline or P5C (Daygon et al. 2017).

3  Proline Transport and Metabolic Pathways Proline is most likely synthesized exclusively in the cytosol but is needed for protein biosynthesis also in mitochondria and plastids. In leaves of osmotically stressed potato (Solanum tuberosum) plants, the highest concentrations of proline were reported for chloroplasts (Büssis and Heineke 1998). Additionally, a substantial part of protein degradation occurs in the vacuole, and proline degradation takes place in mitochondria. After release from stress, the high concentration of proline rapidly decreases, primarily by ProDH- and P5CDH-dependent degradation (Deuschle et al. 2004; Nanjo et al. 1999b). Therefore, efficient transport proteins for proline must exist in most intracellular membranes, but their molecular identity is only beginning to be revealed.

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3.1  Intracellular Proline Transport Isolated mitochondria from monocot seedlings can use proline and P5C/GSA as substrates for respiratory O2 consumption, but in Arabidopsis mitochondria proline-­ dependent respiration was only detected when the expression of ProDH1 was stimulated by proline treatment prior to the isolation of mitochondria (Boggess et  al. 1978; Cabassa-Hourton et al. 2016; Elthon and Stewart 1982). No carriers for proline or P5C/GSA in mitochondria have been molecularly identified so far. However, biochemical analyses provided evidence that the import of proline into mitochondria is dependent on a proton gradient and at least two transporters, a proline uniporter and a proline/glutamate antiporter, are present in the inner mitochondrial membrane (Di Martino et al. 2006; Elthon et al. 1984). Recently, several members of the mitochondrial carrier family (MCF) were shown to mediate glutamate transport, but no evidence for proline transport activity has been obtained yet (Monne et al. 2018; Porcelli et al. 2018). Even less data is available on amino acid transport across the chloroplast membranes, where so far only a malate/glutamate antiporter (DiT2 in the inner envelope) and a transporter for neutral amino acids in the outer envelope (OEP16) have been characterized (Pohlmeyer et  al. 1997; Renné et  al. 2003). In the vacuolar membrane of yeast, the family of AMINO ACID VACUOLAR TRANSPORTERS (AVT) has been characterized, and recently it was shown that Arabidopsis homologues AVT3A and AVT3C complement the defects of avt3/avt4 double mutant yeast cells (Fujiki et al. 2017). These proteins are localized in the vacuolar membrane in Arabidopsis and functional studies suggested that they mediate the ATP-dependent export of several amino acids, including proline, from the vacuole. This suggestion is in agreement with evidence from potato showing that proline concentration in the cytosol can be 260-fold greater than in the vacuole, indicating the presence of an active transport system (Büssis and Heineke 1998).

3.2  Pathways for Proline Biosynthesis and Degradation The lack of information about the activity and specificity of transport proteins in intracellular membranes makes it difficult to draw definite conclusions about the metabolic pathways of proline biosynthesis and degradation that occur in  vivo. Biosynthesis of proline from glutamate by the sequential action of P5CS and P5CR appears to be the predominant pathway, especially for stress-induced proline accumulation. Accordingly, both P5CS and P5CR are essential genes in Arabidopsis, and double mutations in P5CS1 and P5CS2 are gametophytic lethal, as no fertile p5cs1/p5cs2 mutant pollen is formed, while homozygous p5cr mutant embryos were observed but aborted at a very early developmental stage (Funck et al. 2010; Mattioli et  al. 2012). These observations demonstrate that no other pathway can produce sufficient amounts of proline for successful sexual reproduction. An alternative pathway of proline biosynthesis from ornithine has been assumed based on radiotracer studies, co-expression analyses, and analogy to mammals (da Rocha

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et al. 2012; Mestichelli et al. 1979; Roosens et al. 1998). To actually bypass P5CS activity, this pathway depends on export of GSA/P5C produced from ornithine by OAT in mitochondria, which was detectable in isolated corn mitochondria but is difficult to assess in vivo due to the high reactivity and inherent instability of GSA/ P5C at neutral pH (Elthon and Stewart 1982; Mezl and Knox 1976). When mitochondria were incubated with proline, the production of glutamate was two orders of magnitude higher than GSA/P5C production, and also in Arabidopsis plants treated with external proline, GSA/P5C content stayed below the detection limit of 50  nmol/g fresh weight unless p5cdh mutants were used (Boggess et  al. 1978; Deuschle et  al. 2004). Much higher GSA/P5C contents were reported in a study using plants overexpressing ProDH and, together with unchanged GSA/P5C to proline ratios, were interpreted as evidence for a proline-GSA/P5C cycle between the cytosol and mitochondria (Miller et al. 2009). However, Miller et al. (2009) did not provide evidence that the employed color reaction is specific for GSA/P5C in crude plant extracts and the production of glutamate by both OAT and P5CDH makes it difficult to exclude that ornithine only stimulates the P5CS-dependent pathway of proline biosynthesis. Similarly, there is at present no evidence that proline degradation might yield ornithine instead of glutamate in plants, as it has been proposed for certain mammalian tissues (Ginguay et al. 2017).

3.3  Intercellular Proline Transport In contrast to organellar proline transporters, which remain elusive so far, numerous proline transporters were identified that are localized in the plasma membrane. Several members of the amino acid/auxin permease (AAAP) family mediate amino acid-proton symport (Dinkeloo et al. 2018). Among these, members of the amino acid permease (AAP) and lysine histidine transporter (LHT) subfamilies transport proline along with a rather broad range of both neutral and charged amino acids (Fischer et al. 1995; Hirner et al. 2006). Members of the proline transporter (ProT) subfamily have rather narrow substrate specificity and transport proline, glycine betaine, or γ-aminobutyric acid (GABA) (Lehmann et al. 2011). A recent analysis of Arabidopsis aap1 mutants suggested that AAP1 could contribute to the uptake of proline from the growth substrate (Perchlik et al. 2014; Wang et al. 2017). However, little is known about the concentrations of free proline in natural soils and its relevance for plant nutrition or communication, indicating that the major function of AAPs and ProTs is the redistribution of proline within the plant. Amino acids are transported in both the xylem and the phloem, and several members of the AAP family were shown to contribute to phloem loading in source leaves or retrieval of amino acids along the transport route (Tegeder and Hammes 2018). Apoplastic phloem loading requires that amino acids are released by source cells into the intercellular space and also loading of the dead xylem vessels in roots requires export of amino acids and other solutes by the surrounding living cells. In 2012, the SILIQUES ARE RED 1 (SIAR1/UMAMIT18) protein was identified in Arabidopsis as the first transporter that can mediate bidirectional amino acid transport, depending on the

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electrochemical gradient across the membrane (Ladwig et al. 2012). SIAR1 is part of a protein family with 44 members in Arabidopsis, of which several members have been characterized in the meantime as broad specificity amino acid exporters and which were therefore named “usually multiple amino acids move in and out transporters” (UMAMITs) (Besnard et al. 2016, 2018; Müller et al. 2015). Also, plasmodesmata are a possible route for amino acid transport between connected cells, but to our knowledge, no specific transport mechanisms have been described so far.

4  P  roline Biosynthesis and Degradation: Spatial and Temporal Regulation The diverse functions of proline metabolism, which must sustain the variable requirements of protein synthesis, while playing multiple additional physiological functions and responding to environmental and biotic stimuli, are reflected in, and derived from, an elaborate network of gene and enzyme activity regulation (Fig. 2). Most of the known regulatory mechanisms for proline metabolism operate at the transcriptional level and appear to distinguish between stress and normal physiological conditions. Key to this distinction is the capability to detect and respond to different inputs via several signaling pathways that result in expression or activation of specific transcription factors (TFs). Accordingly, the promoters of all genes coding for proline metabolic enzymes that were characterized so far are especially rich in confirmed or predicted TF recognition elements (Fichman et al. 2015; Zarattini and Forlani 2017). Further regulatory mechanisms were found to act epigenetically or posttranscriptionally on gene expression, or allosterically on enzyme activities. Far less is known about posttranslational modifications or regulated degradation of proline metabolic enzymes and transporters. Most of the knowledge about regulatory mechanisms of proline metabolism has been obtained by analysis of Arabidopsis wild-type plants or mutants. As indicated above, duplications and functional diversification of proline metabolic genes occurred several times independently in different plant taxa. Therefore, it is at present unknown how much of the knowledge obtained in Arabidopsis can be directly transferred to different species (Mattioli et al. 2018; Signorelli and Monza 2017; Turchetto-Zolet et al. 2009). We will focus on the knowledge gained for Arabidopsis and indicate it specifically, when data from other plants are described as well.

4.1  R  egulation of Genes Coding for Proline Biosynthesis Enzymes As discussed above, the short pathway converting glutamate into GSA and P5C into proline is the most important and probably unique route of proline synthesis in higher plants. Compelling evidence indicates that P5CS, the first enzyme of glutamate-­derived proline synthesis, is under most conditions the rate-limiting

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Fig. 2  The regulatory network controlling proline metabolism. Enzymes are given in blue letters and metabolic fluxes as solid black arrows. The uncertain transport of P5C/GSA across the mitochondrial membrane is indicated by a dashed arrow. Green and red lines indicate induction and repression, respectively. Lines ending at an enzyme name indicate regulation of gene expression, while lines ending at the metabolic flux indicate posttranslational regulation. For reasons of simplicity, low water potential and high ionic strength are depicted as a single regulatory unit, although they probably use partly independent signaling cascades. ABA abscisic acid, αKG α-ketoglutarate, BR brassinosteroids, DFR1 drought and freezing regulated gene 1, GDH glutamate dehydrogenase, Glu glutamate, GSA glutamate-5-semialdehyde, OAT ornithine-δ-aminotransferase, OCD ornithine-cyclodeaminase, Orn ornithine, P5C pyrroline-5-carboxylate, P5CDH, P5C dehydrogenase, P5CR P5C reductase, P5CS P5C synthetase, Pi Phosphate, Pro proline, ProDH proline dehydrogenase, TCA tricarboxylic acid cycle, ΨW water potential

enzyme of proline synthesis in higher plants. This evidence derives from the strict correlation between P5CS expression and proline accumulation (Hu et  al. 1992; Peng et al. 1996; Savouré et al. 1995; Strizhov et al. 1997; Yoshiba et al. 1999) and from the effects of P5CS overexpression (Kavi Kishor et al. 1995; Per et al. 2017) and antisense inhibition (Nanjo et al. 1999b) or knockout mutations (Mattioli et al. 2008; Székely et al. 2008). Accordingly, the overall rate of proline biosynthesis is predominantly determined by the temporal and spatial regulation of P5CS gene expression. The two Arabidopsis P5CS genes are located on chromosome 2 and 3 and share the same genomic structure with 20 exons sharing a nucleotide identity ranging

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from 80% to 94%. A higher degree of difference is found in the promoter regions, the 5′ and 3′ untranslated sequences and introns, including variations in putative splicing sites, which might give rise to four different P5CS1 and two different P5CS2 transcripts included in the current annotation (ARAPORT11) of the Arabidopsis genome (https://www.Arabidopsis.org). The four different /P5CS1/ transcripts are derived from two splice variants and two alternative transcription initiation sites, while in P5CS2 a single-splice variant skipping exon 3 is annotated. Skipping of exon 3 of P5CS1 produces nonfunctional transcripts and has been experimentally confirmed as potential mechanism underlying differential drought tolerance of several natural accessions from different regions (Kesari et al. 2012). The expression of P5CS1 and P5CS2 in Arabidopsis has been analyzed by northern blot, in situ hybridization, and analysis of transgenic plants carrying promoter-­ GUS and promoter-gene-GFP fusion constructs (Abrahám et al. 2003; Fabro et al. 2004; Mattioli et al. 2018; Mattioli et al. 2009; Strizhov et al. 1997; Yoshiba et al. 1999). There are some minor discrepancies between the results, most likely attributable to different cultivation conditions and analysis techniques. The prevailing picture is that both isoforms have partially overlapping expression patterns, whereby P5CS1 is expressed more strongly in aboveground tissues and differentiated cells, whereas P5CS2 expression levels are highest in regions of active cell division. In flowers, both P5CS isoforms are almost exclusively expressed in developing microspores and pollen (Mattioli et al. 2018). P5CS1 and, to a lesser extent, P5CS2 transcription is rapidly induced by drought and salt stress, with light and abscisic acid (ABA) as key inducing signals (Abrahám et al. 2003; Feng et al. 2016; Strizhov et al. 1997). An independent pathway seems to mediate induction of P5CS1 expression in response to phosphate starvation (Aleksza et al. 2017). Proline-, brassinolide-, and phospholipase-dependent signaling were identified as negative regulators of P5CS expression and probably contribute to the rapid downregulation after relief from stress (Abrahám et al. 2003; Thiery et al. 2004). Bioinformatic analyses of the promoters of P5CS1 and P5CS2 showed that the promoter of P5CS1 is enriched in putative binding sites for TFs related to abiotic stress, such as ABA response elements, AP2/EREBP, ERF2, DREB/CBF, and MYB binding sites (Fichman et  al. 2015). The promoter of P5CS2, on the contrary, is enriched in putative regulatory elements for TFs related to biotic stresses such as HD-HOX, AP2/EREBP, MYB, WRKY, and bZIP (Fichman et al. 2015). Additionally, the promoter of P5CS2 contains putative binding sites for TFs related to flowering time, such as SQUAMOSA PROMOTER BINDING-LIKE (SPL) and bHLH factors, and related to pollen development and function such as WRKY2 and WRKY34 (Mattioli et al. 2018). Most studies on stress-induced or developmental accumulation of proline report good correlation between P5CS transcript levels and proline content, indicating that proline biosynthesis is predominantly regulated at the level of transcription. Early studies describing proline accumulation in tomato and grapevine in the absence of increased P5CS transcript levels can now be explained by the presence of second P5CS isoforms in these species that were unknown at the time (Fujita et al. 1998; Stines et al. 1999). More direct evidence for post-transcriptional regulation of P5CS expression was obtained by computational identification of matching micro-RNAs

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in potato and chickpea (Shui et  al. 2013; Yang et  al. 2013). Expression levels of some micro-RNAs during stress were negatively correlated with P5CS transcript levels, but direct proof for their involvement in P5CS regulation is still missing. Epigenetic regulation caused by modulations of the methylation pattern of specific genes may also contribute to P5CS regulation. Changes in DNA methylation induced by environmental stresses or by developmental stimuli are known to modulate both plant stress tolerance and developmental processes, respectively (Bastow et al. 2004; Chinnusamy and Zhu 2009; Karan et al. 2012; Richards 2006). In rice, differential methylation of a P5CS gene has been proposed as a mechanism for trans-generational stress memory (Zhang et al. 2013). As mentioned above, another level of regulation is added by allosteric inhibition of the γ-glutamyl kinase activity of plant P5CS proteins by proline (Hu et al. 1992; Zhang et al. 1995). It is so far unknown if and how feedback inhibition of P5CS may be overcome in tissues or conditions where proline accumulation is desired. Immunoblot analyses of Arabidopsis P5CS1 protein levels in different protein phosphatase 2C mutants indicated the presence of posttranslational modifications or mechanisms to regulate protein stability (Bhaskara et al. 2015). The concept of P5CS catalyzing the rate-limiting step in proline biosynthesis is contested by several studies reporting higher proline content, especially under stress conditions, upon overexpression of P5CR (De Ronde et al. 2004; Ma et al. 2008; Szoke et al. 1992). The most detailed analysis of P5CR expression has again been performed in Arabidopsis. In particular, a P5CR promoter-GUS fusion construct showed ubiquitous expression with the highest expression levels in areas of active cell division, in guard cells, and in reproductive tissues, especially pollen and developing seeds (Hua et al. 1997). Similarly, a P5CR promoter-gene-GFP fusion construct was expressed ubiquitously in leaves and roots, with highest expression in the root tip (Funck et al. 2010). The 5’-UTR of P5CR was found to mediate posttranscriptional regulation by stabilizing P5CR transcripts under heat and drought stress while at the same time inhibiting translation, resulting in unchanged protein levels despite strongly increased transcript levels, thus raising the question how P5CR keeps up with increased P5CS-mediated GSA/P5C production during stress (Hua et al. 2001). The biochemical properties of P5CR might solve this apparent conflict, as the activity of purified P5CR was stimulated by high ion concentrations when NADPH was available as electron donor (Forlani et al. 2015; Giberti et al. 2014). Phosphoproteomics studies have revealed two directly adjacent phosphorylation sites at T237 and S238 of Arabidopsis P5CR, but information about the possible function of P5CR phosphorylation is not available (Schulze et al. 2015). As discussed in Sect. 3.2, it is at present unclear whether OAT-mediated production of GSA constitutes an alternative route for proline biosynthesis or whether it stimulates proline synthesis merely by increasing the level of glutamate. The spatial distribution of OAT expression has not been analyzed in Arabidopsis, while in pea the highest activity was detected in cotyledons, followed by true leaves, roots, and seeds (Taylor and Stewart 1981). In pine seedlings, OAT transcript levels were highest in the radicle and peaked transiently after germination (Canas et al. 2008). In young Arabidopsis and rice seedlings as well as in radish cotyledons and cashew

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leaves, OAT activity or gene expression was induced in response to salt or drought stress (da Rocha et al. 2012; Liu et al. 2018; Roosens et al. 1998; You et al. 2012). Rice OAT expression was additionally induced by heat, ABA, brassinolide, and auxin treatment (You et  al. 2012). Arabidopsis oat knockout mutants developed normally and had unchanged proline content but were unable to utilize arginine as nitrogen source for growth (Funck et al. 2008). In contrast, deletion of OAT in rice caused fertility defects and lower proline content together with general symptoms of nitrogen deficiency (Liu et al. 2018). In summary, the available data supports an essential role of OAT in recycling of nitrogen from arginine degradation, but does not demonstrate or exclude the existence of an alternative route for proline biosynthesis.

4.2  R  egulation of Genes Coding for Proline Catabolic Enzymes Since the transporters that mediate the uptake of proline into mitochondria have not been molecularly identified, we know virtually nothing about the regulation of this transport. Once cytosolic proline is imported into mitochondria, it can either be used for mitochondrial protein synthesis or it can be oxidized to glutamate by the sequential action of ProDH and P5CDH (see Sect. 2.2). Copy numbers of ProDH genes have not been thoroughly analyzed in available genomes except in Brassicaceae, where an early family-specific genome duplication produced two copies that were further multiplied in the genus Brassica (Faes et al. 2015). In Arabidopsis, the best-­ characterized species, it was shown that both genes, ProDH1 and ProDH2, encode functional proteins with nonredundant but partially overlapping functions (Funck et al. 2010). P5CDH is encoded by a single-copy gene in Arabidopsis and in cereals, while no systematic searches in other plant genomes were reported (Ayliffe et al. 2005; Deuschle et al. 2001). As for proline biosynthesis, most studies on temporal and spatial regulation of proline catabolism were performed in Arabidopsis and we will therefore focus on this species, being aware that this knowledge might not be readily transferred to other plants with different gene copy numbers. ProDH1, the more extensively characterized proline catabolic gene, is after a weak and transient induction repressed by dehydration but is rapidly and strongly induced by rehydration (Kiyosue et al. 1996; Peng et al. 1996; Verbruggen et al. 1996). In addition, ProDH1 expression is induced by proline and hypoosmolarity and during HR-mediated pathogen defense but repressed by hyperosmolarity (Cecchini et al. 2011; Kiyosue et al. 1996; Monteoliva et al. 2014; Verbruggen et al. 1996; Yoshiba et al. 1999). More interesting in respect to plant development is the pattern of ProDH1 expression under non-stressed conditions. Weak constitutive expression of ProDH1 was observed in most organs of Arabidopsis, while in root tips and in flowers, particularly in pollen grains, stigmata, carpels, and developing seeds, the promoter activity was higher (Nakashima et al. 1998). Analysis of orthol-

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ogous ProDH1 genes in Brassica species revealed a very similar expression pattern (Faes et  al. 2015). These findings are particularly interesting because they imply that the molecular mechanisms that reduce proline degradation and support accumulation under stress may be quite different from those active in proline accumulation during reproductive development. Detailed analysis of the Arabidopsis ProDH1 promoter revealed an ACTCAT motif responsible for proline and hypoosmolarity-mediated induction of ProDH1 (Nakashima et al. 1998; Satoh et al. 2002). The ACTCAT motif is a typical binding site for basic leucine zipper (bZIP) TFs of the S1-group, and among these AtbZIP53 and AtbZIP1 were shown to physically interact with the promoter of ProDH1 and to mediate induction of gene expression in response to proline, hypoosmolarity, and low sugar or energy levels (Dietrich et al. 2011; Satoh et al. 2002; Weltmeier et al. 2006). The activity of the ProDH1 promoter was shown to be additionally controlled by the interaction between ARR18 and bZIP63, the former being a type-B response regulator that functions as a positive osmotic stress response regulator in Arabidopsis seeds, the latter a negative regulator of seed germination upon osmotic stress (Veerabagu et al. 2014). Furthermore, ROS- and redox-mediated signaling was reported to regulate ProDH1 expression, but the precise mechanisms remain to be determined (Shinde et al. 2016). Immunoblot analyses of ProDH1 protein levels in leaf extracts or isolated mitochondria yielded multiple bands, indicating that ProDH1 may be subject to posttranslational modifications or alternative processing during mitochondrial import (Bhaskara et al. 2015; Cabassa-Hourton et al. 2016; Schertl et al. 2014). The pattern and regulation of ProDH2 expression appear largely different from ProDH1: ProDH2 promoter activity was mainly detected in vascular tissue and in the abscission zone of sepals, petals, and stamina (Funck et al. 2010). In contrast to ProDH1, transcript levels of ProDH2 were induced during senescence and by salt stress, whereas the repression by high sugar concentrations and the induction by proline and during pathogen defense were similar for both isoforms (Cecchini et al. 2011; Funck et al. 2010). Similar to P5CS1, expression of ProDH2 was induced by phosphate starvation (Aleksza et al. 2017). Averaged over the entire seedlings or tissues, the expression level of ProDH2 was much lower compared to ProDH1, and accordingly, deletion of ProDH2 had no influence on the capacity of isolated mitochondria for proline-dependent respiration, whereas for mitochondria isolated from prodh1 mutants, proline-dependent respiration was undetectable (Cabassa-Hourton et al. 2016; Funck et al. 2010). The strong and specific expression of ProDH2 in the vascular system and its strong downregulation in the presence of sucrose are ­consistent with the report of Hanson et al. (2008) that identified ProDH2, along with ASPARAGINE SYNTHETASE1 (ASN1) as two of the early targets of bZIP11, a transcription factor induced by SUCROSE NON-FERMENTING1 RELATED KINASE1 (SnRK1) in response to energy deprivation (O'Hara et al. 2013; Weiste et al. 2017). SnRK1 and bZIP11 also provide a direct link between proline metabolism and trehalose signaling and metabolism, which is discussed in more detail in Chap. 8 of this book.

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Arabidopsis mutants for either ProDH1 or ProDH2 (Funck et al. 2010; Nanjo et  al. 2003), as well as transgenic plants with antisense-mediated repression of ProDH1 and ProDH2 (Cecchini et al. 2011; Mani et al. 2002), have been generated. While no phenotypic or developmental aberrations were observed under normal conditions, these mutants exhibited enhanced proline accumulation under stress conditions, but stress tolerance and pathogen defense were weakened (Cecchini et al. 2011; Sharma et al. 2011). An unexpected, and as yet unexplained, observation is the hypersensitivity of prodh1 mutants to exogenous proline under non-stressed conditions (Funck et al. 2010; Nanjo et al. 2003). Toxicity of proline supply was also observed in non-stressed wild-type plants but was proposed to be linked to ProDH activity, with either excess P5C production or excess electron load on the mitochondrial electron transport chain as harmful effects (Hellmann et  al. 2000; Miller et al. 2009). A crucial role in preventing proline toxicity was attributed to P5CDH, either by P5C/GSA detoxification or by withdrawing P5C/GSA from the proposed P5C-­ proline cycle (Deuschle et  al. 2004; Deuschle et  al. 2001; Miller et  al. 2009). P5CDH expression was observed constitutively in Arabidopsis leaves, where it increased with leaf age, and to a lesser extent in roots (Deuschle et al. 2004). In reproductive organs, P5CDH was strongly expressed in pollen, developing embryos, and aborted seeds. External proline supply stimulated P5CDH expression, although with slower kinetics compared to ProDH1, whereas no prominent changes in transcript levels were observed in response to salt stress or pathogen infection (Cecchini et al. 2011; Deuschle et al. 2001; Monteoliva et al. 2014). In contrast to Arabidopsis P5CDH, the P5CDH gene from flax (Linum usitatissimum) was strongly induced by pathogen attack, but only when the plant encountered a virulent rust strain that did not elicit a hypersensitive response (Ayliffe et al. 2002; Mitchell et al. 2006). No upstream elements of P5CDH regulation have been identified so far, but transcript levels might be regulated posttranscriptionally by double-strand RNA formation with transcripts from the overlapping SIMILAR TO RCD ONE 5 (SRO5) (Borsani et al. 2005). Recently, a posttranslational mechanism for the regulation of ProDH and P5CDH activity was identified: Binding of DFR1 to both ProDH and P5CDH was found to inhibit their enzymatic activity and could thus explain how proline accumulation and high levels of ProDH expression can occur simultaneously (Ren et  al. 2018). Strong expression of DFR1 was observed in inflorescences and in response to salt, drought, and cold stress. In summary, the knowledge about the regulation of genes and enzymes involved in proline metabolism supports important contributions to stress tolerance and pathogen defense but also to sexual reproduction and other developmental processes both in the absence and presence of stress. Regulation of proline metabolism was found to occur at multiple levels, and therefore we need to be careful when inferring physiological functions from mere correlations between proline content, gene expression, and phenotypic observations.

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5  D  evelopmental Processes Influenced by Proline Metabolism 5.1  Proline and Plant Development The idea that proline may be an active player in plant development, besides being one of the 21 amino acids used for protein synthesis, began to be accepted only at the end of the last century, when different groups detected, under non-stressed conditions, large amounts of proline in the reproductive organs of some plant species (Chiang and Dandekar 1995; Fujita et al. 1998; Mutters et al. 1989; Schwacke et al. 1999; Venekamp and Koot 1988; Walton et  al. 1991). Similarly, upregulation of proline biosynthesis genes was reported in flowers, fruits, and seeds of plants not subjected to evident biotic or abiotic stress (Armengaud et  al. 2004; Fujita et  al. 1998; Schmidt et al. 2007; Schwacke et al. 1999; Vansuyt et al. 1979). Overall, these data indicated that proline levels could locally increase even in the absence of stress. In the vegetative Arabidopsis rosette before floral transition, for example, Chiang and Dandekar (1995) found a percentage of proline, relative to the total amino acidic pool, ranging from 1% to 3% in striking contrast to up to 26% in reproductive tissues after the floral transition (Chiang and Dandekar 1995). A similar result was reported by Schwacke et al. (1999) who measured a proline content in tomato flowers 60 times higher than in any other organ analyzed. The striking difference in proline concentrations between vegetative and floral tissues suggested that proline might play a special role in plant reproduction while raising the problem of the origin of the accumulated proline. As described in Sect. 3, the distribution of proline in plants is subjected to a complex regulation, involving long-distance transport between tissues through vascular vessels (Girousse et  al. 1996), active transport from cell to cell and between different cell compartments (Lehmann et al. 2011; Rentsch et al. 1996; Schmidt et al. 2007; Schwacke et al. 1999), direct synthesis within target tissues (Chiang and Dandekar 1995; Mattioli et  al. 2018), selective catabolism (Kiyosue et al. 1996; Nanjo et al. 1999b), and the rates of protein synthesis and degradation (Hildebrandt 2018). The complexity of these regulations, by itself, was suggestive of some special importance of proline in plant development, particularly in the reproductive phase. Indeed, although proline is relatively common in plant proteins, because of the frequent occurrence of long stretches of proline and/or hydroxyproline residues in a number of cell wall proteins, such as extensins, arabinogalactan-proteins, and hybrid proline-rich (Hyp/Pro-rich) proteins (Kavi Kishor et al. 2015), it seemed unlikely that, under non-stressed conditions, such large amounts of proline would be accumulated only for the requirements of protein synthesis. However, differently from stress-induced proline accumulation, a phenomenon generally considered beneficial to plant cells, proline accumulation in the absence of stress drew little attention and was mostly attributed to some type of prior or undetected stress. Chiang and Dandekar (1995), for example, hypothesized that the high content of proline found in anthers and pollen grains of Arabidopsis could

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function as a compatible osmolyte to protect pollen grains from the water stress caused by the natural process of dehydration during pollen maturation. A significant step toward the understanding of the role of proline in plant development came from the study of the hairy root syndrome induced by infection with the soil bacterium Rhizobium rhizogenes, formerly known as Agrobacterium rhizogenes (Trovato et al. 2018). The capability of R. rhizogenes to reprogram plant development and induce de novo root synthesis on differentiated tissues has been long studied as a paradigm of plant development control and relies on the integration of a transfer DNA (T-DNA) into the plant genome. It turned out that rolD, one of the four “root locus” (rol) genes in the T-DNA responsible for hairy root induction, codes for an ornithine cyclodeaminase (OCD), which converts ornithine into proline and ammonium (Trovato et al. 2001). This finding, along with the above-cited proline accumulation in floral organs of plants grown in optimal conditions, disclosed a novel role for proline in plant development, and we now know that proline is critically involved in a number of developmental processes, such as root elongation, floral transition, pollen fertility, and embryo development.

5.2  Germination Seed germination, a developmental process of enormous physiological and economic relevance, has been sometimes reported to be positively correlated with proline accumulation, particularly under stress conditions, although the observations are rather scarce and a clear-cut demonstration of the involvement of proline in germination is still lacking. Because of the beneficial role that proline accumulation, or more probably proline metabolism, exerts on plant cells under stressful conditions, it may be difficult to distinguish a generic improvement of stress tolerance from a specific effect on seed germination. Notwithstanding this, a limited number of authors have reported that the accumulation of proline and/or the upregulation of proline biosynthesis genes can improve seed germination rates. Roosens et al. (2002) reported that overexpression of Arabidopsis OAT increased proline biosynthesis and germination rates in transgenic tobacco (Nicotiana tabacum) plants under osmotic stress conditions. Similarly, transgenic tobacco plants overexpressing a feedback-insensitive variant of Vigna aconitifolia P5CS accumulated high levels of proline and exhibited higher germination rates under stress (Zonglie et al. 2000). A few reports also described positive effects of proline pretreatment on germination rates (Hua-long et al. 2014; Kubala et al. 2015; Posmyk and Janas 2007). However, this procedure, known as “osmopriming,” can also function with different compatible solutes and might be dependent on the provision of a carbon and nitrogen source rather than a specific effect of proline. The more convincing and exhaustive report claiming a positive role of proline metabolism on Arabidopsis germination comes from a study published by Hare et al. (2003) who observed that proline biosynthesis and the oxidative pentose phosphate pathway (OPPP) were induced in parallel during Arabidopsis seed germination.

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Antisense inhibition of P5CS1 (which most likely silences both P5CS1 and P5CS2 expression due to the high sequence similarity) delayed seed germination, whereas external proline supply inhibited germination and this inhibition was relieved by addition of artificial electron acceptors. Hare et al. (2003) proposed that proline biosynthesis served to lower the NADPH/NADP+ ratio, which is known to stimulate the OPPP in many organisms and may be needed to provide sufficient ribose for nucleotide synthesis in the geminating seed (Shetty and Wahlqvist 2004).

5.3  Root Growth In addition to being an essential component of protein biosynthesis in any growing tissue, proline also seems to play a role as a modulator of cell division, especially in the root elongation zone (Biancucci et al. 2015; Wang et al. 2014). This novel role ascribed to proline is not completely surprising as the elongation of the hairy roots induced by transformation by R. rhizogenes was originally ascribed to the action of rolD, later recognized as a proline producing OCD (Trovato et al. 2001; White et al. 1985). A specific requirement for proline metabolism was also reported in the elongation of Arabidopsis and corn primary roots at low water potential (Verslues and Skarp 1999; Sharma, 2011). Sharma et al. (2011) proposed that proline synthesized and accumulated in leaves was transferred to the root, where it was degraded to provide energy and building blocks for sustained root growth. In non-­stressed Arabidopsis seedlings, exogenous proline supplementation, at micromolar concentration, was shown to induce root elongation and branching (Mattioli et al. 2009). Contrarily, exogenous supply of proline at millimolar concentrations inhibited root growth with symptoms resembling programmed cell death (Hellmann et al. 2000). Arabidopsis mutants with strongly reduced capacity to synthesize proline (p5cs1/p5cs1;P5CS2/p5cs2) displayed reduced root growth by reduction of the area of active cell division in the root meristem (Mattioli et al. 2009). In both Arabidopsis and corn p5cs mutants, reduced root growth was correlated with decreased expression levels of cyclins and other cell cycle-related genes, suggesting a link between proline or proline biosynthesis and cell cycle regulation (Mattioli et al. 2009; Wang et al. 2014).

5.4  Flowering After the first demonstration of the importance of the rol genes in hairy root induction (White et al. 1985), rolD/OCD from R. rhizogenes has been overexpressed in tobacco, tomato, and Arabidopsis (Bettini et al. 2003; Falasca et al. 2010; Mauro et al. 1996). The ectopic expression of rolD, driven by its own promoter, was subjected to a complex developmental regulation and eventually led to early flowering and formation of increased numbers of flowers (Trovato et  al. 1997). Transgenic tobacco plants

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expressing rolD under the control of its own promoter reached anthesis 60 to 75 days before untransformed plants, produced abundant and long-lasting inflorescences, and exhibited an overall altered morphology with height reduction and bract-like leaves (Mauro et  al. 1996). In addition, in  vitro flower formation on tissue explants was stimulated in rolD transgenic plants, presumably by RolD-­mediated conversion of ornithine to proline (Mauro et al. 1996; Trovato et al. 2001). Switchgrass (Panicum virgatum) plants overexpressing a heterospecific P5CS gene flowered earlier than control plants and produced more tillers after mowing (Guan et al. 2018). In transgenic Arabidopsis plants overexpressing an additional copy of P5CS1 driven by the strong CaMV 35S promoter, the time until flowering induction was shortened and axillary coflorescences proliferated, especially in short-day conditions (Mattioli et al. 2008). The overexpression of the transgenic P5CS1 copy was only transient though, and soon after the floral transition, a downregulation of both P5CS1 and P5CS2 took place, likely because of gene silencing (Mattioli et al. 2008). Unfortunately, most of the numerous studies on P5CS overexpression in other plant species focused on drought and salt stress tolerance and did not systematically analyze flowering. Consistent with a role of proline synthesis in flowering induction, upregulation of both proline biosynthesis (P5CS, P5CR) and transport (ProT) genes has been reported, under normo-osmotic conditions, in reproductive organs, such as flowers, inflorescences, and anthers (Savouré et al. 1997; Schwacke et al. 1999; Verbruggen et al. 1993). Intriguingly, also the expression of the proline catabolic genes (ProDH, P5CDH) was reported to increase in reproductive tissues in the absence of stress (Deuschle et al. 2001; Verbruggen et al. 1996), in contrast with the steep downregulation of these genes observed under stress conditions (Kiyosue et al. 1996; Peng et al. 1996). It is as yet unknown, whether upregulation of proline catabolic genes by high proline concentrations under non-stressed conditions causes rapid metabolic cycling or whether posttranscriptional mechanisms like the interaction with DFR1 limit the rate of proline degradation (Ren et al. 2018). The antisense expression of P5CS1 (likely affecting both P5CS1 and P5CS2, see above) has been shown to inhibit Arabidopsis bolting (Nanjo et  al. 1999a). Ambiguous observations have been reported for insertional mutants: in two labs, growth and flowering of p5cs1 single mutants were not different from wild-type plants, whereas in a third lab, a delay in the onset of bolting was observed (Funck et al. 2012; Mattioli et al. 2008; Székely et al. 2008). For p5cs2 single mutants and near-double mutants (p5cs1/p5cs1, P5CS2/p5cs2), a generally slower development was observed (Funck et al. 2012; Mattioli et al. 2012). Similarly, silencing of P5CS2 expression in Lotus japonicus produced several lines with defects in flower and seed formation (S. Signorelli, unpublished observations). Altogether, these data indicate that both P5CS1 and P5CS2 can modulate flowering and suggest that proline plays a role in floral transition, bolting, and coflorescence emergence. In Arabidopsis, and probably all flowering plants, multiple signaling pathways respond to a range of environmental (photoperiod, cold, heat) and endogenous (metabolites, gibberellin, age) stimuli and converge to induce the conversion of ­vegetative shoot meristems into floral meristems (Khan et al. 2014; Srikanth and Schmid 2011). CONSTANS, one of the master regulators of the photoperiodic

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pathway of flowering induction, has been identified as an inducer of P5CS2 in Arabidopsis (Samach et al. 2000). FLOWERING LOCUS C (FLC) was identified as inducer of P5CS1 along with ELONGATED HYPOCOTYL 5 (HY5), which was found to be a crucial factor in the light-dependent induction of P5CS1 by stress, indicating that proline may indeed contribute to the light-dependent regulation of flowering (Abrahám et al. 2003; Chen et al. 2018; Feng et al. 2016; Hayashi et al. 2000). Recently, a number of plant species, belonging to different taxonomic groups, have been reported to flower rapidly after exposure to a wide range of different stressors (Wada and Takeno 2010). Since the responses to many types of stress involve proline accumulation, it is tempting to speculate that stress-induced flowering and proline-induced flowering in non-stressed plants may rely on a common mechanism. The distribution of proline under normal physiological conditions, however, seems partly different from that found under stress conditions: In Arabidopsis, a locally and temporally confined increase of proline in the shoot apical meristem at floral transition has been reported, whereas, under stress conditions, proline is accumulated at high levels in all the tissues of the plant (Mattioli et al. 2008). Overall, the body of accumulated evidence points to proline as a modulator of floral transition, although its mechanism of action, the genes involved in this process, and the interaction with other regulatory pathways still need to be revealed in detail.

5.5  Pollen Fertility Among floral organs, the highest proline contents have been observed in the pollen of many plant species including Arabidopsis, tomato, dandelion (Taraxacum officinale), willow (Salix sp.), and petunia (Petunia hybrida) (Auclair and Jamieson 1948; Chiang and Dandekar 1995; Hong-qi et al. 1982; Schwacke et al. 1999). In grass pollen, proline was the most abundant amino acid, accounting for up to 1.65% of pollen dry weight (Bathurst 1954). Proline was the most abundant amino acid in anthers of devil’s trumpet (Datura metel) and the only one found to increase during pollen development (Sangwan 1978). In addition to this correlative evidence, recently proline has been shown to be essential for pollen development and fertility by two research groups, who independently reported that in Arabidopsis p5cs1/ p5cs2 double-mutant pollen was misshaped and infertile (Funck et al. 2012; Mattioli et al. 2012). The morphological abnormalities were accompanied by lack of storage compounds and nuclei and appeared late in pollen development, starting from stage 11 of anther development. The requirement for proline biosynthesis was specific for pollen, because only the pollen failed to transmit both p5cs mutant alleles simultaneously, whereas p5cs1/p5cs2 double mutant egg cells showed almost no compromised fertility. Importantly, exogenous L-proline, supplemented in planta to developing anthers of p5cs1/p5cs1 P5CS2/p5cs2 near-double mutant plants, allowed the formation of fully developed and fertile p5cs1/p5cs2 double mutant pollen (Mattioli et al. 2012). Quite surprisingly, Arabidopsis plants carrying mutations in P5CR, the gene coding for the second and final step of proline biosynthesis, are embryo lethal, but

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not male sterile, presumably because P5CR is an exceptionally long-lived protein (Funck et al. 2012). High expression of the specific proline transporter ProT1 in the pollen of tomato and Arabidopsis raised the question, whether proline is imported during pollen development or is synthesized cell autonomously (Grallath et al. 2005; Schwacke et al. 1999). By targeting P5CS expression to different tissues in Arabidopsis anthers, Mattioli et al. (2018) demonstrated that only proline synthesized within developing pollen grains can fully restore fertility of p5cs1/p5cs2 double mutant pollen. Consistently, both P5CS1 and P5CS2 genes exhibit a strong and specific expression in microspores and pollen grains but are essentially unexpressed in surrounding sporophytic tissues of the anther, as shown by β-glucuronidase (GUS) analysis, and inferred by bioinformatic analysis of P5CS1 and P5CS2 promoters (Mattioli et al. 2018).

5.6  Embryo Development The analysis of p5cs2 knockout mutants in Arabidopsis has disclosed an essential role of proline in plant embryogenesis. Three research groups have independently isolated and characterized two p5cs2 T-DNA insertion mutants (Funck et al. 2012; Mattioli et  al. 2009; Székely et  al. 2008). Quite surprisingly, despite the high sequence similarity shared by the two paralogous genes, and although the same pattern of expression was detected for both P5CS1 and P5CS2 transcripts by in situ hybridisation of sections of shoot apical meristems and embryos (Mattioli et  al. 2009; Székely et al. 2008), p5cs2, but not p5cs1 mutants, are embryo lethal suggesting a specific role of P5CS2 or posttranscriptional repression of P5CS1 activity during embryogenesis. P5CS2-GFP fusion proteins were uniformly distributed in the cytosol of Arabidopsis embryos, whereas P5CS1-GFP formed cytoplasmic speckles, possibly indicating that P5CS1 is inactivated by aggregation (Székely et al. 2008). Spraying flowers with proline, induction of P5CS1 expression by salt stress, or in vitro cultivation of immature seeds allowed rescuing homozygous p5cs2 mutants, which were retarded in development but produced viable seeds under favorable conditions (Funck et al. 2012; Mattioli et al. 2009; Székely et al. 2008). The reason why homozygous p5cs2 embryos die only in the siliques of heterozygous, but not of homozygous, mutants is not yet fully understood. Potentially, the slowly developing homozygous mutants are aborted when the faster-growing wildtype and heterozygous embryos in neighboring seeds reach maturity. In addition, microscopic analysis of the malformed p5cs2 embryos revealed various aberrations typically associated with a defective cell cycle, such as anomalous orientations of cellular division planes, indicating that low proline levels may similarly inhibit cell cycle progression in embryos as in the root meristem (Mattioli et al. 2009). In corn pro1 mutants, in which the independently evolved P5CS2 of corn is inactivated, storage compounds in the seed endosperm of homozygous mutant seeds were strongly reduced, but formation of viable embryos still occurred (Wang et al. 2017). However, without exogenous proline supply, pro1 homozygous mutants were seedling lethal and successful propagation has not been reported.

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In addition to P5CS2, also P5CR, the enzyme involved in the second and final step of proline synthesis, is essential for embryogenesis as shown by Funck et al. (2012), who characterized two p5cr mutants, found in the Salk collection and annotated as embryo lethal in the SeedGenes database (SeedGenes Project. http://seedgenes.org). Intriguingly, any attempt to rescue homozygous p5cr embryos by proline supplementation was ineffective, differently from p5cs2 mutants. Expression of a P5CR-GFP fusion protein under control of the endogenous P5CR promoter, which is active in developing embryos, reverted the embryo-lethal phenotype, while CaMV-35S-driven overexpression of P5CR-GFP in vegetative tissues was ineffective (Funck et al. 2012). These results indicate that, similarly to the situation in the pollen, long-distance transport of proline cannot fully substitute for local biosynthesis in tissues that critically depend on proline.

5.7  Role of Proline Metabolism in Development Under Stress The accumulation of proline both during certain developmental processes and in response to stress is frequently regarded as two different phenomena which, accordingly, have been treated as separate chapters in this book. However, the large increase in proline content observed in reproductive tissues of most plant species is similar to that observed after many different types of stress, thus posing the question whether the function of proline may be similar in both cases. This seemingly simple question is particularly difficult to tackle because there are some hypotheses but no generally accepted idea of what the function of proline may be, neither under stress conditions nor during development. In addition, also the concept of stress is not as simple as it may seem. What is stress? According to Lichtenthaler (1998), any “unfavorable condition or substance that affects or blocks a plant’s metabolism, growth, or development is regarded as stress.” It may well be that many “normal” physiological conditions, such as seed and pollen maturation or high-­intensity light, are more demanding for a plant than mild environmental stress, such as a transient period of drought or a moderate reduction of the average temperature. According to Chiang and Dandekar (1995), stronger proline accumulation was observed in tissues with low water content, such as embryos and pollen grains, which successfully entered a developmentally induced process of desiccation without loss of cellular and tissue viability. The most probable benefit of proline accumulation in these tissues is based on the kosmotropic properties of proline by which it helps to protect enzymes and membranes of plant cells with low water content (see Chap. 3). In contrast, the increase in proline concentration following osmotic stress may not be sufficient to protect cells because, as discussed in the following chapter, the amount of proline accumulated is typically not sufficient to counterbalance the decrease in the cellular osmotic potential. Another possibility is that proline in (some) reproductive tissues may be a precautionary measure in case of future adverse conditions in order to protect important plant organs and improve the fitness of the species. Consistent with this hypothesis is the observation that p5cs1 mutants have no aberrant

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phenotype under normal conditions but exhibit hypersensitivity to salt and hyperosmolarity stress (Sharma et  al. 2011; Székely et  al. 2008). On the other hand, as described in detail in the following chapter, regardless of proline accumulation being a response to stress or part of a developmental program, it remains still unclear if its beneficial effects are mediated by accumulation per se or by increased metabolic turnover. As indicated above and discussed in more detail in the next chapter, biosynthesis and degradation of proline have the capacity to change the redox state of the cytosol and the mitochondria, respectively, and may additionally modulate the levels of reactive oxygen species. Since far less details are known about the regulation of proline metabolism during normal development, it is at present difficult to predict downstream effects, although it is tempting to speculate that the accumulation of proline under stress and its accumulation during development are two sides of the same coin. Further studies on proline-dependent signal transduction and actual flux rates of proline metabolism and of the exchange of proline between different tissues or cell types will be needed to fully understand how proline exerts its stress protective and developmental functions.

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Wanduragala S, Sanyal N, Liang X, Becker DF (2010) Purification and characterization of Put1p from Saccharomyces cerevisiae. Arch Biochem Biophys 498:136–142. https://doi. org/10.1016/j.abb.2010.04.020 Wang G et al (2014) Proline responding1 plays a critical role in regulating general protein synthesis and the cell cycle in Maize. Plant Cell 26:2582–2600. https://doi.org/10.1105/tpc.114.125559 Wang T, Chen Y, Zhang M, Chen J, Liu J, Han H, Hua X (2017) Arabidopsis AMINO ACID PERMEASE1 contributes to salt stress-induced proline uptake from exogenous sources. Front Plant Sci 8:2182. https://doi.org/10.3389/fpls.2017.02182 Watanabe S, Tanimoto Y, Yamauchi S, Tozawa Y, Sawayama S, Watanabe Y (2014) Identification and characterization of trans-3-hydroxy-l-proline dehydratase and Delta(1)-pyrroline-2-­ carboxylate reductase involved in trans-3-hydroxy-l-proline metabolism of bacteria. FEBS Open Bio 4:240–250. https://doi.org/10.1016/j.fob.2014.02.010 Weiste C et  al (2017) The Arabidopsis bZIP11 transcription factor links low-energy signalling to auxin-mediated control of primary root growth. PLoS Genet 13:e1006607. https://doi. org/10.1371/journal.pgen.1006607 Weltmeier F et al (2006) Combinatorial control of Arabidopsis proline dehydrogenase transcription by specific heterodimerisation of bZIP transcription factors. EMBO J  25:3133–3143. https://doi.org/10.1038/sj.emboj.7601206 White FF, Taylor BH, Huffman GA, Nester EW (1985) Molecular and genetic analysis of the transferred DNA regions of the root-inducing plasmid of Agrobacterium rhizogenes. J  Bacteriol 164:33–44 Yang J, Zhang N, Ma C, Qu Y, Si H, Wang D (2013) Prediction and verification of microRNAs related to proline accumulation under drought stress in potato. Comput Biol Chem 46:48–54. https://doi.org/10.1016/j.compbiolchem.2013.04.006 Yoshiba Y, Nanjo T, Miura S, Yamaguchi-Shinozaki K, Shinozaki K (1999) Stress-responsive and developmental regulation of Delta(1)-pyrroline-5-carboxylate synthetase 1 (P5CS1) gene expression in Arabidopsis thaliana. Biochem Biophys Res Commun 261:766–772 Yoshihashi T, Huong NT, Inatomi H (2002) Precursors of 2-acetyl-1-pyrroline, a potent flavor compound of an aromatic rice variety. J Agric Food Chem 50:2001–2004 You J, Hu H, Xiong L (2012) An ornithine δ-aminotransferase gene OsOAT confers drought and oxidative stress tolerance in rice. Plant Sci 197:59–69. https://doi.org/10.1016/j. plantsci.2012.09.002 Zarattini M, Forlani G (2017) Toward unveiling the mechanisms for transcriptional regulation of proline biosynthesis in the plant cell response to biotic and abiotic stress conditions. Front Plant Sci 8. https://doi.org/10.3389/fpls.2017.00927 Zhang CS, Lu Q, Verma DP (1995) Removal of feedback inhibition of delta 1-pyrroline-5-­ carboxylate synthetase, a bifunctional enzyme catalyzing the first two steps of proline biosynthesis in plants. J Biol Chem 270:20491–20496 Zhang CY, Wang NN, Zhang YH, Feng QZ, Yang CW, Liu B (2013) DNA methylation involved in proline accumulation in response to osmotic stress in rice (Oryza sativa). Genet Mol Res 12:1269–1277. https://doi.org/10.4238/2013.April.17.5 Zimorski V, Ku C, Martin WF, Gould SB (2014) Endosymbiotic theory for organelle origins. Curr Opin Microbiol 22:38–48. https://doi.org/10.1016/j.mib.2014.09.008 Zonglie H, Karuna L, Zhongming Z, Verma DPS (2000) Removal of feedback inhibition of Δ1-Pyrroline-5-Carboxylate Synthetase results in increased proline accumulation and protection of plants from osmotic stress. Plant Physiol 122:1129–1136. https://doi.org/10.1104/ pp.122.4.1129

Regulation of Proline Accumulation and Its Molecular and Physiological Functions in Stress Defence Giuseppe Forlani, Maurizio Trovato, Dietmar Funck, and Santiago Signorelli

1  Introduction Among the building blocks needed for protein synthesis, proline is the only cyclic amino acid, i.e. a secondary amine in which two carbons are bonded to the amine nitrogen forming a five-membered heterocyclic ring. Because of this, proline plays a unique role in determining protein structure by influencing backbone folding and stability (Ge and Pan 2009). Proline is synthesized in a two-step reduction and cyclization of glutamate (Fichman et al. 2015), although other possible routes have been hypothesized to occur in some plant species (da Rocha et al. 2012). Preliminary evidence supporting an at least partial localization of the anabolic pathway in the chloroplast (Szekely et al. 2008) has not been further supported; thus proline production is regarded as a cytosolic process. As a consequence, free proline is present in the cytoplasm, where homeostatic levels depend on the balance between the rates of its production and utilization for protein synthesis, but also on its translocation to the mitochondrion (Di Martino et al. 2006), where proline is oxidized back to glutamate via an equally short catabolic pathway. The biosynthetic route is controlled mainly through feedback regulation of the enzyme catalysing the first step, namely,

G. Forlani (*) Department of Life Science and Biotechnology, University of Ferrara, Ferrara, Italy e-mail: [email protected] M. Trovato Department of Biology and Biotechnology, Sapienza University of Rome, Rome, Italy D. Funck Department of Biology, Division of Plant Physiology and Biochemistry, University of Konstanz, Konstanz, Germany S. Signorelli (*) Department of Plant Biology, Universidad de la República, Montevideo, Uruguay e-mail: [email protected] © Springer Nature Switzerland AG 2019 M. A. Hossain et al. (eds.), Osmoprotectant-Mediated Abiotic Stress Tolerance in Plants, https://doi.org/10.1007/978-3-030-27423-8_3

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δ1-pyrroline-5-carboxylate (P5C) synthetase (P5CS) (Hu et  al. 1992; Hong et  al. 2000). The turnover of amino acids into proteins and vice versa seems rapid, since similar proportions of 14C were found in unstressed cells following 14C labelling (Dong et al. 2018). Besides the involvement in protein synthesis, proline is believed to play a main role in the plant response to several (a)biotic stresses. Starting from the 1960s of the last century, hundreds of papers have reported rapid accumulation of high free proline levels in cells subjected to a variety of stress conditions (Delauney and Verma 1993; Hayat et al. 2012; Verbruggen and Hermans 2008) (Table 1). In response to stress, protein synthesis is reduced, favouring amino acid accumulation (Dong et al. 2018). Notwithstanding this, in most cases the rise of free proline content largely exceeds those of the other proteinogenic amino acids. Accordingly, genetically modified plants with increased intracellular proline content showed higher stress tolerance (Per et al. 2017). The specific increase in proline content may derive from either a reduction in its mitochondrial oxidation (Nanjo et al. 1999), an increase of its biosynthesis (Zhang et al. 1995) or import from other tissues (Lehmann et al. 2010; Wang et al. 2017) via phloem sap. The latter seems to contribute to developmentally related variations in proline homeostasis, as in reproductive organs (Biancucci et al. 2015), whereas increased biosynthesis appears the main mechanism occurring after the exposure to abiotic stress, such as drought, salinity or freezing (Yoshiba et al. 1995, 1997). Because P5CS is feedback inhibited by millimolar concentrations of proline, and homeostatic proline level results from the combination of synthesis, catabolism and transport rates, the accumulation of increased levels of proline in the cytosol requires fine regulation of its metabolism under stress. Consistently, an impressive number of putative cis-regulatory elements recognized by different classes of transcription factors were found in the promoter region of the involved genes (Fichman et al. 2015; Zarattini and Forlani 2017).

2  R  egulation of Proline Accumulation Under Stress Conditions A variety of diverse abiotic and biotic stress conditions were found to induce the accumulation of proline in plants (Szabados and Savouré 2010). Proline accumulation was suggested to be due to an increase of the de novo biosynthesis rather than lower catabolism or greater protein degradation (Chiang and Dandekar 1995; Szabados and Savouré 2010; Hildebrandt 2018). Under stress, proline is mainly biosynthesized from glutamate by two enzymatic steps consuming NADPH preferably and catalysed by P5CS and P5C reductase (P5CR) (Forlani et al. 2015c; Giberti et al. 2014). Most plants have two P5CS isoforms, one housekeeping and the other inducible, the latter being mainly responsible for proline accumulation under stress (Signorelli and Monza 2017). Proline accumulation is more significant in photosynthetic tissues (e.g. leaves) but also takes place in roots (Sharma et al. 2011; Signorelli et al. 2013a; Verslues and Sharp 1999). At the cellular level, proline accumulation is

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Table 1  Stress-induced accumulation of intracellular proline levels in response to stress conditions in some selected plant species

Species Nicotiana tabacum Nicotiana tabacum Solanum lycopersicon Capsicum annuum Arabidopsis thaliana Arabidopsis thaliana Arabidopsis thaliana Oryza sativa Oryza sativa Trifolium pratense Lotus japonicus

Zea mays

Lotus corniculatus

Pro content (fold increase) 48 246 445 40

Stressor NaCl (171 mM) NaCl (342 mM) NaCl (428 mM) 30% relative humidity PEG 20% PEG 25% PEG 30% PEG 25%

Tissue Cultured cells

Cultured cells

38 244 506 200

NaCl (120 mM)

Rosette leaves

8–10

Wilted leaves Cultured cells

NaCl (150 mM)

14-day-old seedlings Low ΨW (−1.2 MPa) Seedlings

160

NaCl (100 mM) NaCl (200 mM)

Seedlings Seedlings

2–10 2–3

Drought (42% hydric index reduction) Drought (45% relative water content reduction) Drought (33% relative water content reduction) Growth in medium at −1.6 MPa

Wilted leaves

139

Leaves

18

Roots

35

Apical millimeter of corn root tips Drought (50% hydric Wilted leaves index reduction)

16

Reference Binzel et al. (1987)

Sano and Kawashima (1982) Handa et al. (1983)

del Socorro Santos-­ Díaz and Ochoa-­ Alejo (1994) Chiang and Dandekar (1995) Signorelli et al. (2016)) Shinde et al. (2016) Lv et al. (2015) Bertazzini et al. (2018) Signorelli et al. (2013b) Signorelli et al. (2013c)

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While several Solanaceae showed hundredfold increases in free proline content, much lower concentrations were found in other plants

suggested to be greatest in the chloroplast, followed by the cytoplasm (Büssis and Heineke 1998). P5CR was shown to be induced by saline stress (Verbruggen et al. 1993); however, this was challenged by Yoshiba and co-workers (1995), who found no response of P5CR to salt stress or dehydration and attributed proline accumulation to increased P5CS1 expression. Therefore, P5CR is not believed to play a critical role in proline accumulation under osmotic stress. However, the biochemical properties

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of P5CR suggest that under some conditions also P5CR activity might become limiting (Giberti et al. 2014). Although proline dehydrogenase (ProDH, also known as proline oxidase or POX) is not as determinant as P5CS1 concerning proline accumulation under stress, the oscillation of gene expression in the light/dark cycles and downregulation under dehydration, saline, and osmotic stress contributes to enhanced proline accumulation (Hayashi et al. 2000; Verslues and Sharma 2010). Other factors, such as light or reactive oxygen species (ROS), also need to be present for proline accumulation to take place. Early studies demonstrated that proline content oscillated according to light/dark cycles (Hayashi et al. 2000), and even stress-induced proline accumulation was shown to be dependent on light (Sanada et al. 1995; Díaz et al. 2005; Aleksza et al. 2017). The light dependence of proline accumulation can be attributed, at least in part, to the need of reducing power for its biosynthesis, which is generated during photosynthesis and might be transferred into the cytosol by different redox shuttles. However, later reports showed that the expression of P5CS1 is also affected by light and is mediated by Elongated Hypocotyl 5 (HY5) (Feng et al. 2016; Lee et al. 2007). HY5 is a basic leucine zipper (bZIP) transcription factor, controlling a plethora of processes, such as development, abiotic stress, ROS, and hormone signalling, in a light-dependent manner (Gangappa and Botto 2016; Signorelli et al. 2018). Thus, the light dependence of proline accumulation is not just due to the requirement of NADPH but also to signalling reasons. This finding of the role of HY5 in mediating P5CS1 expression also points out how important the coordination of proline metabolism with other processes is. Similarly, the expression of the main enzymes controlling proline accumulation, P5CS1 and ProDH, was suggested to be dependent on the Respiratory Burst Oxidase Homologue (Rboh, NADPH oxidase in animals) activity (Ben Rejeb et al. 2015). In short, the authors showed that the hydrogen peroxide (H2O2) produced as a consequence of Rboh and SOD activity is necessary for proline accumulation to occur. The simple pharmacological scavenging of H2O2 was enough to attenuate proline accumulation under stress conditions (Ben Rejeb et al. 2015). This finding demonstrated that proline accumulation is also regulated, at least in part, by ROS signalling. ROS seem to be produced under most stress conditions. Thus, in a physiological context, while light can be a determinant factor for proline accumulation to take place or not, the role of ROS is probably more complex, and variable concentrations of different ROS species may influence proline biosynthesis differentially. The production of the phytohormone abscisic acid (ABA) is induced under stress conditions in plants, and ABA acts as a signalling molecule to mediate the adaptation of the plant to the new environment. In rice, ABA is known to mediate proline accumulation (Sripinyowanich et al. 2013); however, in arabidopsis (Arabidopsis thaliana), proline accumulation can be either ABA-dependent or ABA-independent depending on the stress condition (Savouré et al. 1997; Zarattini and Forlani 2017). Recently, a wild variety of barley was shown to accumulate higher proline levels under drought compared to the cultivated one. The authors showed that the difference between the ancestral and the cultivated variety was one nucleotide in the

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sequence of the P5CS1 promoter, which modified an ABF (ABA-responsive elements (ABRE) binding factors) binding site, making the cultivated barley to be unresponsive to ABA (Muzammil et al. 2018). Likewise, in arabidopsis, the 5’ UTR region of AtP5CS1 contains ABF binding sites. Other cis-responsive elements (CRE), such as AP2/EREBP, ERF2, DREB/CBF, and MYB binding sites are also found in the promoter of P5CS1 genes of different species (for detailed reviews, see Fichman et al. 2015; Zarattini and Forlani 2017). This wide variety of CRE in the promoter of the main gene responsible for proline synthesis explains why proline accumulation is a conserved response observed in a broad range of conditions in plants.

3  Osmoprotective Role of Proline Despite the plethora of papers describing a stress-induced increase of free proline concentration, and the evidence of a statistically significant relationship between this increase and increased stress tolerance, the mechanisms by which high levels of this amino acid may benefit the cell are far from being fully understood and conclusively proven. The early hypothesis of a central osmotic role for proline, i.e. that high levels of this compound may avoid water withdrawal to the apoplast by lowering the cellular water potential (ΨW), seems inconsistent with the relatively low absolute concentration reached in several cases and has been recently questioned (Bhaskara et al. 2015; Forlani et al. 2018; Kavi Kishor and Sreenivasulu 2014; Ben Rejeb et al. 2014; Sharma et al. 2011; Signorelli 2016). Notwithstanding this, many authors still consider a function of proline as a “compatible osmolyte” as an established fact.

3.1  Proline as Compatible Osmolyte A famous quote says that if you repeat a lie often enough, people will believe it, and you will even come to believe it yourself. In the scientific literature, we could say that if a sound hypothesis is repeated in many papers, with citation to other previous reports, at a certain point, scientists will start to believe that it has been proven somewhere in the past, and do not verify whether it fits with their experimental system or whether their results are consistent with it. This is probably true for the “osmotic role” of proline accumulation in response to hyperosmotic stress conditions. Because of early reports describing in some plant species a striking increase of its intracellular level under drought or salt stress (Table 1), from a given moment onwards, any statistically significant increase in proline concentration has been interpreted as a genuine contribution to osmotic compensation. Is this interpretation sound? Let us make some calculations. Because in a differentiated plant tissue the measurement of both the apoplastic water volume and the external water potential

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(ΨW) is not easy (Lohaus et al. 2001), the use of a cell suspension culture may facilitate the estimates. Cells growing in the widely used Murashige and Skoog (MS) medium with 30 g L−1 sucrose are in osmotic equilibrium with it, at an external ΨW of about −0.52 MPa (Fig. 1a). Since cellular concentration (ΨS) usually corresponds to an osmotic pressure (Π) ranging from −0.7 to −1.2  MPa (Handa et  al. 1983; Ikeda et al. 1999), this allows cell turgidity, with a turgor pressure (ΨP) of about 0.1 to 0.7  MPa. The addition of 25% polyethylene glycol (PEG-6000) to the culture medium, a condition that has been found permissive in many cases (i.e. plant cells can adapt to these conditions [e.g. del Socorro Santos-Díaz and Ochoa-Alejo 1994]), lowers the external ΨW to −2.0 MPa (Fig. 1b). To avoid water withdrawal, the cell has to increase its internal osmolyte concentrations to obtain a Π decrease of 0.8 to

Fig. 1  Osmotic compensation in rapidly dividing cells. Suspension-cultured cells growing in MS medium are in equilibrium with an environmental water potential of about −0.52 MPa (panel a). The addition of an osmoticum, for instance, 25% PEG 6000, causes a dramatic decrease of the external water potential (ΨW) (b). To adapt, the intracellular concentration (ΨS) should increase consistently, so as to avoid loss of turgor pressure (ΨP) and the consequent occurrence of plasmolysis (c). Because only about 20–30% of the overall cell fresh weight is attributable to the cytoplasm (d), to obtain osmotic compensation solely by proline accumulation, an increase of cytosolic free proline concentration as high as 80–130 μmol (g fw)−1 would be required

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1.3  MPa, corresponding (at 25  °C) to an increased osmolarity of about 320–520 mmol·L−1 (Fig. 1c). In most studies, proline content is measured on a fresh or dry weight (fw or dw) basis following cell extraction with sulfosalicylic acid and expressed as μmol (g fw or dw)−1 (e.g. Aleksza et al. 2017). If, as most likely, proline accumulation occurs mainly in the cytosol, the evaluation of its osmotic value would require quantitation of the cytoplasmic volume. Concerning this, we should consider that in actively proliferating cultured cells, the vacuole accounts for only 5–15% of total volume but that nucleus, mitochondria, and plastids are a significant fraction of the cell. Also, considering that the apoplastic water may represent 10–40% of water content (Binzel et al. 1987; Speer and Kaiser 1991), to a good approximation, we can consider the cytoplasmic water as 20–25% of the overall fresh weight (Fig. 1d). This would imply that if proline would be the only osmolyte produced and accumulated, to achieve osmotic compensation, its content should increase by at least 80–130  μmol (g fw)−1. In fact, in most cases, the reported increase does not exceed 5–10 μmol (g fw)−1 (e.g. Poustini et al. 2007), and often it is less than 1–2 μmol (g fw)−1 (e.g. Bertazzini et al. 2018). Consistently, in some excellent and pioneer works in which most of the main components of the cellular sap have been quantified at the same time, the contribution of proline to the required increase of osmolarity was estimated as not exceeding 3–15%, despite the fact that its concentration had shown a hundredfold increase over controls (Binzel et  al. 1987; Handa et al. 1983; del Socorro Santos-Díaz and Ochoa-Alejo 1994). Only in the apical millimeter of corn roots growing at a water potential of −1.6 MPa, proline accumulation reaching about 120 μmol (g fw)−1 accounted for almost half of the osmotic adjustment (Voetberg and Sharp 1991). In wheat plants, the contribution made by proline was estimated to be equivalent to 0.07 MPa, whereas that made by K+ and Na+ was 0.21 and 0.45 MPa, respectively (Poustini et al. 2007). Of course, any significant input to the attainment of a suitably high cellular Ψs concurs to ameliorate the osmotic unbalance. However, because of the implicated values, the significance of a proline accumulation lower than 5  μmol (g fw)−1 (approximately equivalent to 0.06 MPa Ψs in the cytosol) should be regarded – in our opinion – as marginal for an effective osmotic compensation.

3.2  Proline as Stabilizer for Enzymes and Membranes Given that proline seems unlikely to fully compensate the osmotic unbalance produced under stress, the doubtless beneficial effect of higher proline levels in the cell facing hyperosmotic conditions should also rely on other possible mechanisms. Several hypotheses have been proposed. One of the most commonly accepted is a kosmotropic (= anti-chaotropic) activity of proline. In solution, proteins, and in general all macromolecules, are surrounded by highly ordered water molecules that lower entropy, allowing the attainment of proper three-dimensional folding and, in case of an enzyme, the achievement of the catalytically active conformation (Fig.  2a). Osmotic stress-induced water withdrawal from the cell or

Fig. 2  Proposed mechanism for protein protection by proline. In solution, enzymes are surrounded by water molecules that interact with the hydrophilic regions of proteins, mainly through the formation of hydrogen bonds, allowing the attainment of proper three-dimensional folding and the achievement of the catalytically active conformation (panel a). Increased concentration of ions, and/or chaotropic substances, reorders the water molecules around them and reduces the interactions with the protein, resulting in protein unfolding and activity loss (panel b). Proline is believed to exert an anti-chaotropic effect and stabilize proteins by helping to maintain a proper water solvation shell around them (panel c). This effect may rely, for instance, on the hydrophobic interaction of the pyrrolidine ring with hydrophobic surfaces, thereby increasing hydrophilic areas, or on stabilization of the water molecules that interact with the protein through hydrogen bonds. The hydrogen bonds are represented by dashed lines. Thicker hydrogen bonds represent stronger interactions

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h­ yperosmolarity-­driven ion influxes cause a progressive increase of chaotropic substances in the cytosol, leading to protein unfolding and activity loss (Fig.  2b). Proline and other very soluble and well-hydrated molecules with little tendency to aggregate, such as the sugar α,α-trehalose, the quaternary ammonium compound glycine betaine, and 3-dimethyl-sulfonopropionate, at high concentrations exert a kosmotropic effect and stabilize the structure of macromolecules in solution by helping to maintain a proper hydration shell around them (Fig. 2c). It was postulated that proline forms aggregates by stepwise stacking and hydrophobic interaction of the pyrrolidine ring with hydrophobic surface residues of proteins, thereby increasing their hydrophilic area (Schobert and Tschesche 1978). An increased water-binding capacity of the proline-protein solution has therefore been invoked to explain alleviation of the noxious effects of water stress effects on protein activity and stability (Arakawa and Timasheff 1983, 1985). Moreover, proline has also been reported to favour protein renaturation and avoid protein aggregation by similarly trapping the folding intermediates in a supramolecular assembly (Samuel et  al. 2000). A great unknown, also in this case, is whether local concentrations high enough to ensure these beneficial effects may be reached in vivo inside the cell. On the other hand, when the phase behaviour of 1-palmitoyl-2-oleoyl-phosphatidylethanolamine was examined in aqueous dispersions containing a range of sodium salts, the lipid phase properties exhibited a graded response analogous to that of the Hofmeister series causing protein salting out (Sanderson et  al. 1991). Therefore, it is likely that at low water potential kosmotropic agents may stabilize membranes as well. Consistently, proline, betaine and trehalose were found to increase the area/molecule of three membrane phospholipids, therefore acting as stabilizer agents. In particular, data suggested that, due to its amphipathic nature, proline may interact with phosphatidylcholines through intercalation between phospholipid head groups (Rudolph et al. 1986).

4  Proline as a Potential ROS Scavenger Abiotic and biotic stresses affect ROS homeostasis, usually resulting in an overproduction of specific ROS species. To avoid oxidative damage, plants respond by enhancing enzymatic and non-enzymatic antioxidant systems. As proline accumulates under stressful conditions, some authors hypothesized that proline protects against ROS. In good agreement with this idea, proline was shown to protect against the most potent ROS, the hydroxyl radical (•OH; Smirnoff and Cumbes 1989). Later, proline was shown to enhance photochemical activity in thylakoid membranes (Alia and Mohanty 1991). Moreover, the same group demonstrated that proline attenuates malondialdehyde (MDA) formation in cotyledons of Brassica juncea under saline stress (Alia and Mohanty 1993), zinc stress (Alia and Saradhi 1995), and UV stress (Saradhi et al. 1995). Based on the lower oxidative damage observed in plants treated with proline, the authors proposed that proline plays an essential role in non-enzymatic detoxification of free radicals, although no evidence of direct

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reaction between proline and oxidant molecules, or their potential products, were obtained (Alia and Saradhi 1995). Proline was also shown to reduce MDA formation in thylakoids during exposure to intense light and was suggested to either react with singlet oxygen (1O2) or reduce its formation (Alia and Mohanty 1997). In 2001, the same group presented compelling evidence suggesting that proline could attenuate the singlet oxygen-mediated 2,2,6,6-tetramethylpiperidin (TEMP) oxidation, claiming they demonstrated that proline is a very effective singlet oxygen quencher (Alia and Matysik 2001). This work finished stamping the antioxidant label on proline. In an elegant work, Hamilton and Heckathorn (2001) demonstrated that the primary cause of mitochondrial electron transport disruption by saline stress is oxidative damage in complex I and Na+ toxicity in complex II. The authors showed that proline was unable to protect complex I, whereas non-enzymatic antioxidants, such as glutathione, tocopherol, and ascorbic acid, and enzymatic antioxidants, such as superoxide dismutase (SOD), ascorbate peroxidase (APX), and catalase (CAT), did it. As SOD showed the highest protection of the complex I, the authors concluded that superoxide (O2•−) was causing most of the oxidative damage of complex I. Altogether, these results challenge the idea that proline is an antioxidant and suggest that proline does not protect against superoxide. In addition, proline was shown to induce the activity of CAT, peroxidase, and polyphenol oxidase (Öztürk and Demir 2002), suggesting that some of the observations of proline amelioration of oxidative damage could be related to an enhancement of the enzymatic antioxidant machinery. The connection between proline accumulation and the oxidative damage/antioxidant response under different stress conditions was evaluated in many different plant species; however, a clear link between proline accumulation and oxidative stress was difficult to establish, as the results were opposite in many cases. This controversy might be explained by the fact that while proline accumulation is suggested to protect against the oxidative damage, vast evidence was also provided showing that excess proline can be a pro-oxidant, as it induces ROS production via mitochondrial electron leakage (Deuschle et al. 2004; Lv et al. 2011; Miller et al. 2009a). The latter is well established in animals (Donald et  al. 2001; Elia et  al. 2017; Liu et al. 2009; Polyak et al. 1997). In turn, proline-induced ROS generation can induce the antioxidant response, acting as priming agent, adding an extra layer of complexity. For instance, the exogenous treatment with proline induced the activity of antioxidant enzymes in cultured tobacco BY2 cells (Hoque et  al. 2007; Hossain and Fujita 2010; Islam et al. 2009), but could not directly protect against superoxide or H2O2 (Hoque et al. 2007). Concerning endogenous proline accumulation, two arabidopsis non-proline-accumulating mutant lines, p5cs1–2 and p5cs1–4, were shown to have higher H2O2 and lipid peroxidation upon saline stress, whereas the enzymatic antioxidant activities showed no uniform results (Szekely et al. 2008). The lack of direct evidence about the putative antioxidant properties of proline was in part attributed to the difficulty to work with ROS, due to their high reactivity. Although proline was suggested to quench singlet oxygen (Alia and Matysik 2001),

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the mechanism had not been described. In order to determine whether this quenching was chemical or physical, some of us investigated these putative mechanisms of quenching using direct real-time measurement of singlet oxygen by its luminescence at 1270 nm. Surprisingly, proline was not able to quench singlet oxygen, neither chemically nor physically (Signorelli et al. 2013b). In an attempt to reproduce the observations from Alia and Matysik (2001), we found out that the EPR signal of singlet-O2-dependent TEMP oxidation becomes less stable in the presence of proline, explaining why the authors apparently detected lower TEMP oxidation activity, which led to the misinterpretation that proline was scavenging singlet oxygen and avoiding formation of oxidised TEMP. Similarly, the reaction mechanisms of proline with hydroxyl radicals were investigated to assess whether hydroxyproline is a potential product of this interaction. Using computational chemistry, we found out that proline would rapidly react with a hydroxyl radical through hydrogen abstraction (H-abstraction). When the H-abstraction occurs on a C atom, it is more likely to yield P5C as a non-radical product than hydroxyproline (Signorelli et al. 2014). As P5C is converted back to proline by P5CR, we suggested a Pro-Pro cycle in which proline could scavenge two hydroxyl radicals and being regenerated with the consumption of one NADPH (Signorelli et al. 2014). One of the essential characteristics of antioxidants, such as glutathione and ascorbate, is that enzymes can recycle them. By the Pro-Pro cycle, proline was for the first time able to share this typical feature of antioxidants. When the H-abstraction on the nitrogen atom was evaluated, we found that this reaction was energetically preferred by hydroxyl radicals and would result in the decarboxylation of proline (Signorelli et  al. 2015). The potential non-radical product was shown to be δ1-pyrroline, a ǖFE;-aminobutyric acid (GABA) precursor. This provided a link between proline and GABA involving non-enzymatic reactions under stress conditions (Signorelli et  al. 2015). It is worth mentioning that, due to the non-enzymatic nature of the reaction, this link would never explain the concomitant accumulation of proline and GABA, which is most likely due to similar upstream regulators of their biosynthesis. As the second most reactive ROS, singlet oxygen, was not able to react with proline, we wondered whether proline was able to react with more stable ROS and reactive nitrogen species (RNS). In both in  vivo and in vitro experimental conditions, we observed that proline was not able to scavenge superoxide, nitric oxide (•NO), nitrogen dioxide (•NO2), and peroxynitrite (ONOO−; Signorelli et al. 2016). This challenged the idea that proline is accumulated under stress to act as an antioxidant. There is at present no evidence to suggest that proline is more likely to react with hydroxyl radicals than most other organic molecules. Further research is needed to understand how significant the contribution of proline to hydroxyl radical scavenging is in physiological conditions. With the current evidence, we believe that the observed beneficial effects of proline on oxidative damage are more likely to be due to its direct stabilization effect on membranes and proteins (Sect. 3.2) and/or the capacity to activate the antioxidant defence (further in Sect. 6) than to a direct role as an antioxidant.

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5  Effect of Proline Accumulation on Redox Balance Proline synthesis from glutamate requires a double equivalent amount of reducing power. When needed to accumulate to high concentrations, the increased synthetic rate is therefore capable of lowering NAD(P)H availability inside the cytosol. Conversely, the mitochondrial catabolism of proline provides reducing equivalents in the form of an FADH2 moiety bound to the ProDH enzyme (that is believed to transfer electrons directly to the respiratory chain) and an NADH molecule formed during the subsequent P5C oxidation (Fig.  3). As a consequence, a reciprocal dynamic relationship has been hypothesized between proline metabolism and the redox status of the cell, i.e. the possibility that any variation in proline homeostasis may result in a corresponding change in NAD(P)H/NAD(P)+ ratio and (more recently) that also the opposite may be true. Moreover, as the two pathways occur spatially separated in the cytosol and in mitochondria, the interconversion of

Fig. 3  The potential P5C-proline cycle. Proline synthesis in plants proceeds in the cytosol through a two-step pathway, while proline is oxidized back to glutamate in the mitochondrion. It has been hypothesized that the intermediate in both routes, P5C, may be transported through the inner mitochondrial membrane. If so, a cycle may be established in which the interconversion of P5C and proline may transfer reducing equivalents from the cytosol to the mitochondrion without the expense of cytosolic ATP, directly fuelling the respiratory chain by the activity of ProDH. Arginine catabolism might also contribute to this cycle by generating P5C through transamination between ornithine and α-ketoglutarate. GDH glutamate dehydrogenase, OAT ornithine-δ-aminotransferase, P5C δ1-pyrroline-5-carboxylate, P5CDH P5C dehydrogenase, P5CS P5C synthase, P5CR P5C reductase, ProDH proline dehydrogenase, RC respiratory chain, TCA tricarboxylic acids

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g­ lutamate and proline could be used as a mean to transfer reducing equivalents between these cell compartments.

5.1  Proline-Glutamate Interconversions in Redox Balance The rapid activation of the oxidative pentose phosphate pathway that has been reported in response to drought, salt, and temperature stress conditions can significantly increase the cytosolic NADPH/NADP+ ratio (Esposito 2016). Moreover, as several NAD kinases were found to be calmodulin-dependent, Ca2+ fluxes early occurring following the exposure to many types of stress may alter the NADP(H)/ NAD(H) ratio (Li et al. 2018). Because of the functional properties of the enzymes involved in proline biosynthesis, these metabolic stress responses are expected to impact proline homeostasis greatly. P5CR, which can use in vitro both NADH and NADPH as the electron donor, showing higher maximal catalytic rate but a lower affinity with NADH, has been found to be highly sensitive to changes in the ratio of phosphorylated versus non-phosphorylated pyridine dinucleotides. The NADH-­ dependent activity of the plant P5CR is very sensitive to NADP+, being already inhibited at physiological NADP+ concentrations (Giberti et al. 2014). Moreover, high proline and salt levels were found to inhibit the enzyme when NADH was the co-factor, whereas the NADPH-dependent reaction was unaffected or even stimulated (Forlani et al. 2015b; Giberti et al. 2014; Ruszkowski et al. 2015). Concerning P5CS, which is believed to catalyse the rate-limiting step in the short anabolic pathway (Fichman et al. 2015), much less is known. However, it seems to use preferentially, if not exclusively, NADPH rather than NADH (Fichman et al. 2015; Zhang et al. 1995). Moreover, preliminary data showed an even higher sensitivity of P5CS to NADP+, with 50% inhibition at a NADPH to NADP+ ratio of 1.5 (Forlani 2017). As a consequence, increased NADPH/NADP+ and NADP(H)/ NAD(H) ratios are expected to increase the biosynthetic rate and the homeostatic level of proline inside the plant cell, without the need and before any transcriptional activation of the corresponding genes. Consistently, the Rboh inhibitor diphenyleneiodonium was found to induce high levels of proline accumulation (Shinde et al. 2016), although Rboh is necessary for the transcriptional induction of proline accumulation (Ben Rejeb et al. 2015). An unexpected effect of the impairment of very-­ long-­chain fatty acid synthesis on proline homeostasis was shown to be mediated by effects on redox status rather than signalling functions of lipid metabolism enzymes or intermediates (Shinde et al. 2016). A recent report showed the binding of MYB-­ type transcription factors Phosphate Starvation Response1 and PHR1-Like1 to P5CS1 regulatory sequences in wild-type arabidopsis plants subjected to phosphate starvation (Aleksza et al. 2017). The consequent gradual increase in proline content could also be reasonably related to a reduction in the NADP(H)/NAD(H) ratio, although this aspect was not investigated in detail. All these results showed that the cellular redox status influences proline metabolism. Nevertheless, the opposite also holds true, as increased rates of proline

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s­ ynthesis (or oxidation) may influence in turn the NAD(P)H/NAD(P)+ ratio. The interconnection between high levels of proline synthesis during stress and regulation of the adenylate redox status was early hypothesized to maintain NAD(P)+/ NAD(P)H ratios at values compatible with metabolism under normal conditions and to reduce stress-induced cellular acidification (e.g. Hare and Cress 1997). This hypothesis has found more detailed substantiation in several recent studies. Although not definitely proven, proline production dissipating excess reducing equivalents was proposed to act as a compensatory strategy to sustain photosynthesis and prevent photoinhibition under excess light in arabidopsis mutants lacking a chloroplast NADP-dependent malate dehydrogenase (Hebbelmann et  al. 2012). Trapping reducing power through enhanced proline biosynthesis has been proposed to limit the generation of ROS and the consequent cell damage (Ben Rejeb et  al. 2014). Tissue-specific differences in proline metabolism and function in maintaining a favourable NADP+/NADPH ratio, where proline synthesis in photosynthetic tissues regenerates NADP+, while its catabolism in meristematic and expanding cells is needed to sustain growth by increased availability of energy and reducing power, were found to take place during drought adaptation in arabidopsis (Sharma et al. 2011).

5.2  The P5C-Proline Cycle More recently, a puzzling result has been reported in an increasing number of studies: the activation under stress of genes in both the proline anabolic and the catabolic pathways. Under these conditions, only transfer of redox equivalents to mitochondria but no proline accumulation would be achieved with the expense of one cytosolic ATP per cycle, since proline is oxidized as soon as it is synthesized, and does not accumulate in the cell (or accumulates much less than expected based on the enhancement of its biosynthetic rate). In PEG-treated arabidopsis seedlings, P5CS1 was induced about 20-fold (Sharma and Verslues 2010), but a five-fold increase was evident also for P5C dehydrogenase (P5CDH, recently proposed to be renamed as glutamate semialdehyde dehydrogenase; Korasick et  al. 2019), the enzyme catalysing the second and last step in the mitochondrial oxidation of proline (Forlani et al. 1997; Forlani et al. 2015a), and for ornithine aminotransferase (OAT), the enzyme deaminating ornithine to yield P5C (da Rocha et al. 2012) (Fig. 3). In some instances, microarray and RT-PCR data showed the concurrent transcriptional activation of ProDH and P5CR. During cold acclimation in arabidopsis, the steady-­ state mRNA levels for P5CSs markedly increased after 12 h of exposure to 4 °C but then declined to basal levels after 96 h of cold treatment, while transcript level of ProDH1 continuously increased; P5CDH mRNA level was unaffected and that for P5CR increased slightly throughout (Kaplan et al. 2007). If the product of proline oxidation in the mitochondrion, P5C, may trespass the membrane and enter the cytosol, this would cause an apparently futile cycle between glutamate and proline, feeding electrons from cytosolic NADPH to the respiratory chain (Fig.  3).

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Overexpression of ProDH in tobacco and arabidopsis or impairment of P5C oxidation in the arabidopsis p5cdh mutant was reported not to change the cellular proline to P5C ratio under both normoosmotic and stress conditions, leading the authors to suggest that excess P5C produced in the mitochondrion may be reduced to proline in the cytosol (Miller et  al. 2009b). This so-called P5C-proline cycle involving ProDH and P5CR, conclusively demonstrated in mammalian cells where both enzymes are localized in mitochondria (Liu and Phang 2012), has therefore been hypothesized to play a role also in plants. However, as long as P5CDH is active in mitochondria, it is very difficult to distinguish whether a glutamate-proline or a P5C-proline cycle is operative in vivo. Hyperactivity of either cycle, for instance, following the exogenous supply of proline, enhances ROS production in the mitochondrion. Consistently, p5cdh mutants showed hypersensitivity to exogenous proline (Deuschle et  al. 2004). Metabolic cycling between glutamate and proline or P5C and proline could potentially benefit the cell and play a critical role for plant survival under stress through maintenance of the cellular redox balance, regulation of the NAD(P)H/NAD(P)+ ratio, and enhancement of the oxidative pentose phosphate pathway (Hayat et  al. 2012; Kavi Kishor and Sreenivasulu 2014; Lv et  al. 2011; Miller et al. 2009b). Moreover, an unbalanced activity of ProDH and P5CDH could lead to direct electron transfer to O2 and production of ROS or unspecific damage to mitochondrial components by reaction with P5C (Liang et al. 2013). At low levels, ROS act as a signal for reinstating metabolic homeostasis during stress situations (Türkan and Demiral 2009), whereas at high levels ROS can play a role in the hypersensitive reaction to pathogens (see Sect. 5.3). Conclusive evidence for the occurrence of a P5C transporter in the mitochondrial membrane has not been obtained, yet, and in arabidopsis p5cs1-p5cs2 double mutants, arginine and ornithine could not substitute glutamate as precursor for proline (Funck et  al. 2012; Mattioli et al. 2012). Therefore, the P5C-proline cycle has still to be regarded as a hypothesis in plants, and further work is required to confirm its occurrence and physiological role. However, an increasing number of data point at the activation of proline metabolism, more than the resulting homeostatic level of the free amino acid inside the cell, as the determinant for an effective stress response of the plant (e.g. Signorelli et al. 2016; Forlani et al. 2018).

5.3  Proline Catabolism and ROS Generation Under Stress Under this perspective, some early experimental evidence about the expression of the catabolic pathway in rust-infected plants may be reconsidered and suggest a role for proline metabolism also in the plant response to biotic stress conditions. Early induction of the gene coding for P5CDH was shown in several crops following penetration of virulent, but not of avirulent fungal strains (Ayliffe et  al. 2002; Mitchell et al. 2006). Moreover, induction of proline oxidation was reported in arabidopsis during incompatible plant-pathogen interactions (Cecchini et  al. 2011). Therefore, the possibility that proline metabolism may be part of the process ­leading

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to programmed cell death (PCD) during the hypersensitive defence reaction was proposed (Senthil-Kumar and Mysore 2012). However, it is still unclear which may be the active molecule, whether proline itself, P5C, or ROS produced during proline catabolism. The early activation of ProDH during pathogen attack was accompanied by an increase in P5CR but not in P5CDH transcripts, apparently with few changes occurring in proline and P5C levels (Cecchini et al. 2011). Therefore, also in this case, the whole picture strengthened the possible occurrence of the P5C-­ proline cycle, leading to ROS production. Enhanced proline oxidation in the mitochondrion leads to sustained ROS generation (Cecchini et  al. 2011; Servet et  al. 2012), which in turn act as second messengers in various signalling cascades and induce the expression of defence pathways conferring tolerance to either abiotic or biotic stress conditions (Miller et al. 2011; Ben Rejeb et al. 2014). Indeed, the analysis of wild-type arabidopsis plants and p5cdh mutants showed that the absence of P5CDH does not reduce ROS production, cell death, or pathogen resistance and suggested that the enzyme does not act synergistically with ProDH in the potentiation of such defence responses (Monteoliva et al. 2014). The whole picture is made even more complex by the presence of another pathway yielding P5C (and possibly proline), the mitochondrial catabolism of arginine via ornithine (Fig. 3). Upon treatment with exogenous proline or pathogen infection, arabidopsis wild-type and p5cdh plants consecutively induced the expression of ProDH and Pro biosynthetic genes, but while the former seemed to induce both routes, p5cdh mutant plants may primarily activate the ornithine route and sustain ProDH induction without reducing the Pro content but rather increasing it (Rizzi et al. 2015). Whatever the way to fuel the P5C-proline cycle, the concurrent induction of P5CDH could make the difference between compatible and incompatible plant-pathogen interactions. In incompatible interactions, low levels of P5CDH activity increases the rate of proline-P5C interconversion, which in turn leads to increased ROS production by ProDH and (directly or indirectly) to PCD. In the former, on the contrary, high levels of P5CDH lower substrate availability for the cycle, delaying PCD induction and allowing a systemic spread of the pathogen. In any case, the exact mechanisms underlying such a role of proline metabolism under biotic stress conditions still await full elucidation.

6  Effect of Proline Metabolism on Antioxidant Enzymes Notwithstanding the role of proline catabolism in generating ROS in the mitochondrion, different reports have shown that proline accumulation correlates with an enhanced antioxidant enzymatic activity (Hoque et al. 2007, 2008; Islam et al. 2009; Kaushal et al. 2011). This effect has been mainly inferred from the capacity of proline to act as a protectant for enzymes (Ben Rejeb et al. 2014; Szabados and Savouré 2010) or from the transient ROS signals induced by proline catabolism, which result in increased expression of antioxidant enzymes (Ben Rejeb et al. 2014; Zarse et al. 2012). As mentioned before, proline accumulation was suggested to be dependent

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on Rboh activity (Ben Rejeb et al. 2015). This enzyme produces superoxide in the apoplast, which is then converted to H2O2, and it is considered to be one of the main sources of ROS accumulation under stress and to mediate a systemic ROS signal throughout the plant (Miller et  al. 2009b). Thus, it is not surprising that proline accumulation and induction of the levels of antioxidant enzymes occur in parallel, while at present it is unclear whether there is a direct effect of proline on the expression of antioxidant enzymes. In the arabidopsis mutants p5cs1–2 and p5cs1–4, unable to accumulate proline, some of the antioxidant enzymes showed higher activity (CAT, glutathione peroxidase), whereas others showed lower activity (SOD, APX, and glutathione reductase) (Szekely et al. 2008). These opposite effects questioned how important the endogenous proline accumulation is for the overexpression-­ protection of antioxidant enzymes in vivo. Moreover, when the transcriptomic data of one of these mutants (p5cs1–4) was analysed and compared to wild-type plants, none of the genes coding for the aforementioned antioxidant enzymes were differentially expressed under both stress and control conditions (Shinde et  al. 2016). This suggests that proline anabolism does not contribute to the regulation of antioxidant response; however, it does not exclude the effect of its catabolism. To establish whether proline catabolism regulates antioxidant response in plants, the study of the expression of antioxidant enzymes under stress conditions in wild-type and pdh mutant lines would be beneficial.

7  Proline as a Source of C and N During Recovery Because of the high consumption of reducing power needed for its synthesis, the oxidation of proline to glutamate and the subsequent channelling of the latter into the TCA cycle can yield as many as 30 ATP equivalents (Atkinson 1977). Proline accumulation may therefore represent an efficient method for energy storage. Consistently, honeybees and other nectar-foraging insects preferentially utilize proline as a fuel during the initial phases of flight (Micheu et al. 2000), and experimental evidence supports the preference of bees and butterflies for nectars or sugar solutions enriched with proline (Bertazzini et al. 2010). In plants, proline oxidation was shown to be required to sustain growth even at low external water potential, since high ProDH expression was maintained in the root apex and shoot meristem under stress rather than being repressed (Sharma et al. 2011). A fortiori, the use of proline to fuel cell metabolism and as a source of organic nitrogen and carbon to resume growth should be highly valuable after coming back to non-stressful conditions. In fact, a rapid reactivation of ProDH transcription to high levels has been reported in many cases during recovery (Yoshiba et al. 1997). However, modulation of proline metabolism during recovery and its role in plant survival are still largely unexplored. Some data showed that the post-drought response is dependent on drought severity, suggesting that sustained synthesis and accumulation of proline can promote plant damage reparability by up-regulating antioxidant activity also during the recovery from stress (An et al. 2013). If not required, as following the

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exogenous supply in the absence of stress, proline is promptly utilized, and its intracellular concentration rapidly lowers to homeostatic levels (e.g. Forlani et  al. 2015a). Therefore, there is a need to distinguish between different cases, in order to avoid that proline may be oxidized when its accumulation is functional to withstand stress conditions. This goal could be accomplished by differential signals regulating ProDH transcription. Additionally, recent results in arabidopsis described the identification of a mitochondrial protein, Drought and Freezing Responsive gene 1 (DFR1), involved in the inhibition of proline degradation during drought and cold stresses. Two alternatively spliced isoforms of DFR1 were detected that are strongly induced by stress and specifically interact with ProDH and P5CDH, thereby inhibiting their activities (Ren et al. 2018).

8  Conclusions Over the last decades, many studies have shown that proline accumulation in plants correlates with greater stress tolerance. The broad variety of cis-responsive elements present in the promoter of the plant P5CS1 gene explains why proline accumulation is a conserved response observed in a wide range of conditions. Furthermore, the fact that a master regulator of light-dependent processes such as HY5 mediates P5CS1 expression shows that proline biosynthesis is important to be coordinated with other light-dependent processes. However, the main mechanism by which proline accumulation contributes to stress tolerance remains largely elusive. It is unlikely that proline accumulation exerts its protective function just by preventing water withdrawal from the plant cells, as in many cases its contribution maximally reached 15% of the required osmotic adjustment. Instead, proline can act as a kosmotropic agent, stabilizing proteins and membranes under unfavourable conditions. More fundamental research needs to be done to understand the kosmotropic properties of proline. Recent findings showed that proline accumulation is also dependent on Rboh activity, suggesting that ROS signalling is involved in the regulation of its accumulation. This response could be important if proline accumulation attenuates oxidative damage. Currently, the most likely ways in which this can be achieved is directly by proline, protecting the antioxidant enzymes from denaturation, or by its catabolism, inducing the antioxidant response. Yet, recent findings showed that proline is unable to directly protect against most ROS and RNS. In addition, proline can contribute to regulation of the NAD(P)H/NAD(P)+ ratio, and the activation of a P5C-proline (or glutamate-proline) cycle in plants appears as an effective stress response, in which cytosolic reducing equivalents can be converted into mitochondrial reducing equivalents to fuel the respiratory chain. Regarding biotic stress, proline catabolism was suggested to lead to programmed cell death during the hypersensitive defence reaction. Overall, the latest research in the field has contributed to limit some speculations about the role of proline and its metabolism during abiotic and biotic stress in plants,

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but also several new questions have arisen. Thus, future research on proline ­metabolism in stressed plants needs to be supported to finally understand its molecular and physiological function under stress.

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Exogenous Proline-Mediated Abiotic Stress Tolerance in Plants: Possible Mechanisms Mohamed Zouari, Ameni Ben Hassena, Lina Trabelsi, Bechir Ben Rouina, Raphaël Decou, and Pascal Labrousse

1  Introduction Plants are constantly exposed to abiotic stresses such as drought, salinity, metal toxicity and extreme temperatures. One of the stress responses in plants is the stimulated production of reactive oxygen species (ROS) such as superoxide (O2.), hydroxyl radical (OH.) and hydrogen peroxide (H2O2) (Hayat et al. 2012; You and Chan 2015). ROS overproduction directly damages cellular biomolecules such as proteins, amino acids, purine nucleotides and nucleic acids and causes the peroxidation of the membrane lipids (Osman 2015; Choudhury et  al. 2017). Cells have developed and adapted different mechanisms to maintain low intracellular ROS level. These ROS are scavenged by antioxidative metabolites like glutathione (GSH), ascorbic acid (AsA), α-tocopherol (vitamin E) as well as antioxidative enzymes such as catalase (CAT), ascorbate peroxidase (APX) and superoxide dismutase (SOD) (Gill and Tuteja 2010; de Freitas et al. 2018). In addition to these antioxidants, osmotic regulators like proline also protect plant cell against abiotic stress. They are characterized by low molecular weight and high solubility. Proline accumulation is known to occur under water deficit, salinity, extreme temperature and heavy metal (Ashraf and Foolad 2007; Hayat et al. 2012; Hossain et al. 2014; Aslam et  al. 2017, De Freitas et  al. 2019). In addition to act as an osmolyte for osmotic adjustment, proline contributes to the stabilization of subcellular structures M. Zouari · L. Trabelsi · B. B. Rouina Laboratory of Improvement of Olive Productivity and Fruit Trees, Olive Tree Institute of Sfax, University of Sfax, Sfax, Tunisia A. B. Hassena Laboratory of Amelioration and Protection of Olive Genetic Resources, Olive Tree Institute of Sfax, University of Sfax, Sfax, Tunisia R. Decou · P. Labrousse (*) University of Limoges, Limoges, France e-mail: [email protected] © Springer Nature Switzerland AG 2019 M. A. Hossain et al. (eds.), Osmoprotectant-Mediated Abiotic Stress Tolerance in Plants, https://doi.org/10.1007/978-3-030-27423-8_4

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(membranes and proteins), to the scavenging of free radicals, and to buffering of cellular redox potential under stress conditions (Heuer 2010; Hossain et al. 2014). It may also act as protein-compatible hydrotrope, alleviating cytoplasmic acidosis and maintaining appropriate NADP+/NADPH ratios compatible with metabolism (Hare and Cress 1997; Gholami Zali and Ehsanzadeh 2018). In many plant species, proline accumulation under abiotic stress has been correlated with stress tolerance, and its concentration has been shown to be generally higher in tolerant plants than in salt-sensitive plants (Hayat et  al. 2012). The level of proline accumulation in plants varies from species to species and can be 100 times greater than in control situation. Thus, the exogenous use of proline is considered as a simple technique to provoke abiotic stress tolerance in plants.

2  Proline and Proline Metabolism in Plants Proline, abbreviated as Pro or P, is a nonessential proteinogenic amino acid with formula C5H9NO2 and a molecular mass of 115.13. Proline is encoded by the codon CCU, CCC, CCA and CCG and is the only proteinogenic amino acid including a secondary amine group called an imine leading to name proline an imino acid (Bhagavan and Ha 2015). The fusion of the three-carbon R-group of proline to the alpha-nitrogen group confers to this compound a rotationally constrained rigid ring structure and thus an exceptional conformational rigidity. Accumulation of proline under abiotic stress can be mediated by the increase in proline synthesis or a decrease in proline degradation. A diagrammatic representation of proline metabolic pathway and interconnection with polyamine (PA) and gamma-aminobutyric acid (GABA) metabolic pathways is presented in Fig.  1. Proline could be synthesized by two pathways, and, even if glutamate pathway is predominant, ornithine pathway also occurs. The glutamate pathway accounts for major proline accumulation during osmotic stress. Proline is synthesized from glutamic acid via glutamate semialdehyde (GSA) and Δ1-pyrroline-5-carboxylate (P5C). The glutamate to GSA reaction is catalyzed by Δ1-pyrroline-5-carboxylate synthetase (P5CS, E.C. 2.7.2.11). GSA is spontaneously converted to P5C, and Δ1-­ pyrroline-­5-carboxylate reductase (P5CR, E.C. 1.5.1.2) catalyze the transformation of P5C to proline. Proline catabolism occurs in mitochondria in several steps involving proline dehydrogenase (PDH, E.C. 1.5.5.2) producing P5C from proline and P5C dehydrogenase (P5CDH, E.C. 1.2.1.88) converting P5C to glutamate. As previously said, proline can be also synthesized from ornithine in an alternative pathway. Ornithine (Orn) is transaminated to P5C (and GSA) by ornithine delta-­ aminotransferase (δOAT, E.C. 2.6.1.13), a mitochondrial located enzyme (Hayat et al. 2012; Hossain et al. 2014). P5C is then converted into proline by P5CDH. It has been suggested that the ornithine pathway is important during seedling development and in some plants for stress-induced proline accumulation. Proline biosynthesis occurs in the cytosol and in the chloroplasts, while proline degradation takes place in mitochondria (de Freitas et al. 2019). Indeed, ­biosynthetic

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Arg

Fig. 1  Proline metabolic pathway in higher plants and possible interconnection with gammaaminobutyric acid and polyamines pathways. (Adapted from Pál et al. 2018, Huang et al. 2008, Szabados and Savouré 2010, Signorelli et al. 2015). Proline biosynthetic pathways appear with green arrows, catabolic pathway appears with red arrow, and the ornithine pathway is represented with blue arrow. ADP adenosine diphosphate, Ar arginase, Arg arginine, ATP adenosine triphosphate, FAD flavin adenine dinucleotide, FADH2 flavin adenine dinucleotide reduced, GABA gamma-aminobutyric acid, GSA glutamate-semialdehyde, GluDC glutamate decarboxylase, KG apha-ketoglutarate, NADP+ nicotinamide adenine dinucleotide phosphate, NADPH nicotinamide adenine dinucleotide phosphate reduced, OAT ornithine-delta-aminotransferase, Orn ornithine, P5C pyrroline-5-carboxylate, P5CR pyrroline-5-carboxylate reductase, P5CS pyrroline-­ 5carboxylate synthetase, P5CDH pyrroline-5-carboxylate dehydrogenase, PDH proline dehydrogenase, Put putrescine, Pyr pyrroline, PyrDH pyrolline dehydrogenase, SA succinic acid, Spd spermidine, SSA succinic semiadlehyde

enzymes are preferentially located in cytosol (PCS and PCR), whereas enzymes of proline catabolism are preferentially located in the mitochondria (PDH, PCDH, and OAT) (Szbados and Savouré 2010). This compartmentalization of proline metabolism suggests the occurrence of intracellular proline transport between the cytosol, the chloroplast and the mitochondria. If some proline carriers have been identified like mitochondrial proline uniporter and proline/glutamate antiporter, the involvement of basic amino acid transporters is also needed to transfer arginine and ornithine through mitochondrial membrane. Moreover, the preferential localization of proline catabolic enzyme in the mitochondria and the involvement of glutamate and alphaketoglutarate (KG) in the ornithine pathway suggest the interconnection with Krebs cycle (or tricarboxylic acid cycle) (Rana et al. 2017). Through glutamate and pyrroline, proline pathway could also be connected to GABA. Indeed, Δ1-­pyrroline is converted to GABA thanks to pyrroline dehydrogenase (PyrDH, E.C. 1.2.1.19) even if GABA is mainly produced from glutamate by glutamate decarboxylase (GluDC, E.C. 4.1.1.15). GABA accumulation occurs during several stresses leading to attri-

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Photo period

Light BR

Mitochondria Water stress

P5CDH

Cytosol

Chloroplast

Glutamate

Salt stress s

Ca2

P5CS

+

P5CS

GSA

GSA

GSA Light

P5C

P5C

P5C

BR H 2O 2 ABA Water stress

P5CR Re hydration

P5CR

PDH

Proline

Fig. 2  Possible regulation ways of proline metabolic pathway in higher plants by abiotic factors. (Adapted from Szabados and Savouré 2010). ABA abscisic acid, BR brassinolides, GSA glutamate-­ semialdehyde, KG alpha-ketoglutarate, P5C pyrroline-5-carboxylate, P5CR pyrroline-5-­ carboxylate reductase, P5CS pyrroline-5-carboxylate synthetase, P5CDH pyrroline-5-carboxylate dehydrogenase, PDH proline dehydrogenase

bute to this molecule several protective roles (like proline, GABA could be involved in osmoregulation, cell signaling, and protection against oxidative stress, cytosolic pH regulation). Recently, Signorelli et al. (2015) proposed an alternative pathway to connect proline to GABA via pyrroline through nonenzymatic reactions that would explain the simultaneous accumulation of GABA and proline under oxidative stress. Moreover, Pál et al. (2018) suggested the existence of an interconnection between proline pathways and polyamine (putrescine, spermidine) pathways. Indeed, putrescine is synthetized by ornithine decarboxylase (E.C. 4.1.1.17) from ornithine or indirectly by arginine decarboxylase (E.C. 4.1.1.19) from arginine via agmatine. Thus, complex interactions between proline, polyamines, GABA synthesis pathways, and ROS balance exist, and new connections must be probably deciphered in the future as abscisic acid plays also an important role in these stress responses. Proline metabolism is regulated by multiple factors (Fig. 2), and the regulation processes are still poorly known. Proline biosynthesis is stimulated during dehydration while its catabolism is reduced. At the contrary, the process is reversed during rehydration. Proline biosynthesis is stimulated by light and osmotic stress, whereas proline catabolism is stimulated in dark and during stress relief. Proline accumulation is also reported to be repressed by brassinosteroïds, whereas it was stimulated during salt stress. Under stress, proline metabolism is regulated by multiple and complex pathways that can drastically influence cell death and survival of the organism. Indeed, the coupling of proline metabolic pathways with the mitochondrial and chloroplastic electron transport chain (through NADPH/NADP+, NADH/NAD+, FADH2/FAD+)

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Table 1  Summary of proline functions in plant at different organization levels from cell to whole plant Organization level Cell wall

Proline compounds and pathways PRPs

Plasma membrane

RPRPs HRGPs

Cell

PRPs HRGPs

Callus

HRGPs HyPRPs

Phloem Embryo Leaf Floral buds Pollen and style

PRPs Proline biosynthesis impairment Normal proline level PRPs Proline accumulation HRGPs

Floral nectars Flower

Proline accumulation Normal proline level PRPs

Fruit Seed/grain

Proline accumulation Normal proline levels

Plant

PRPs Proline transporters Proline biosynthesis impairment

Function Wall component Drought stress Roots: sensitivity to ABA Links between plasma membrane and cytoskeleton Cell elongation Root hair development Cell wall assembly Cell wall remodeling Intercellular communications Somatic embryogenesis Germination of somatic embryos Cell elongation Size increase Expression during drought stress Embryo death Impaired seed development Flavor compounds Style structural integrity Bud break Pollen tube growth Style growth Pollinators attraction Flavor compounds Flower development Cotton fiber development Enhanced fermentability (grapevine) Seed germination Flavor compounds Abscission, senescence Development, abiotic stress tolerance Xylogenesis Reduced protein synthesis Cyclin genes downregulated

Adapted from Kavi Kishor et al. (2015) ABA abscisic acid, HRGPs hydroxyproline-rich glycoproteins, HyPRP hybrid proline-rich proteins, PRPs proline-rich proteins

induces an opportunity to balance the redox state by regulating the generation of ROS.  For example, Zhang and Becker (2015) indicated that proline metabolism may influence ROS signaling pathways to delay the senescence. Proline functions in plants are complex, are not entirely deciphered, and depend on the organization level (Table 1). Proline is a main element of the cell wall matrix

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NADPH/NADP+

Redox balance ROS

Osmoprotection

Proline

Translation

Photosynthesis enzymes GST, CAT, APX N2 assimilation in Fabaceae

Proteins

(Proline rich proteins)

Cell wall

Signaling

Mitochondrial functions (ROS, PCD)

Plant development (embryo, root growth, flowering)

Fig. 3  Proline role in plant functioning. (Adapted from Szabados and Savouré 2010, Verdoy et al. 2006)

through proteins like hydroxyproline-rich glycoproteins (HRGP) or proline-rich proteins (PRPs), thus giving to proline a key role in the plant development (Fig. 3). For example, proline is vital for proper seed development and for producing viable seeds. In in vitro culture, proline via HRGPs is necessary to embryo for their regeneration and their germination during somatic embryogenesis. HRGPs are also necessary to pollen tube and style development. Proline is a key actor in root elongation and in flower initiation but also in the further reproductive tissue development (Kavi Kishior et al. 2015). Thus, proline is not only involved in protein synthesis but regulates also key functions like osmotic adjustment or protein protection. It should be noted that a positive correlation probably exists between proline and glycine betaine, another molecule playing a highly beneficial role in plants exposed to stress (Murmu et al. 2017). Through its involvement in cell wall synthesis, root growth, embryo formation, and germination, proline becomes therefore a major stakeholder during all the plant life cycle.

3  Genetic Features of Proline Metabolism and Regulation As described above, the proline metabolism occurs through two pathways interconnecting various organelles in plants (Fig.  1). This metabolism appears conserved between prokaryote and eukaryote organisms, and various genes are involved in the

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Table 2  Genetics features of genes involved in the main network of proline biosynthesis Gene name P5CS1

P5CS2 P5CS3 P5CR P5CDH PDH1

PDH2 δOAT

E.C Plant number A. 2.7.2.11 thaliana

L. regale A. 1.5.1.2 thaliana A. 1.2.1.88 thaliana A. 1.5.5.2 thaliana

A. 2.6.1.13 thaliana

Gene/protein Exons Chrom. length (bp/aa) (number) 2 2154/717 20

3 ? 5

2181/726 2139/712 831/276

20 ? 7

Response to abiotic stress in plants Salinity/drought /oxidative stress /Light/phosphate starvation/cold/heat NaCl (weak)/cold/H2O2 Salinity/drought Salinity/drought/heat/cold

5

1671/556

16

Salinity/cold/drought/dark

3

1500/499

4

5 5

1431/476 1428/475

4 10

Salinity/drought/ hypo-osmolarity/ phosphate starvation/ABA Salinity/sucrose Salinity/drought/H2O2

biosynthesis of the different enzymes (Table 2). P5CS, the eukaryotic key fusion enzyme exhibiting the two conserved domains glutamate 5-kinase (GK, EC: 2.7.2.11; N-terminal) and γ-glutamyl phosphate reductase (GPR, EC: 1.2.1.41; C-terminal) (Pérez-Arellano et al. 2010; Fichman et al. 2015), is synthetized by two duplicated P5CS genes in most plants (P5CS1 and P5CS2). From several studies, P5CS revealed to play distinct roles according to the stress, in an organ-specific manner and following cell spatiotemporal expression patterns (thoroughly reviewed by Rai and Penna 2013, Amini et  al. 2015, and Rana et  al. 2017). For example, P5CS1 is mediated by hyperosmotic stress and regulated by abscisic acid, while P5CS2 appears as a constitutive and ubiquitous gene in plants (Savouré et al. 1997; Székely et  al. 2008; Verslues and Sharma 2010). Recently, a third P5CS gene (P5CS3) was found in the dicot Medicago truncatula (Kim and Nam 2013) and in the monocot Lilium regale (Wei et al. 2016). These genes contribute also to proline accumulation and abiotic stress tolerance. At the contrary, the second reduction step leading to proline from P5C is managed by only one gene of the plant genome. However, two P5CR isoforms were identified from pea and spinach allowing a lingering doubt on the exact number of P5CR genes in these plants (Murahama et al. 2001; Lehmann et al. 2010). In addition, although P5CS represent a rate-limiting step, the absence of a functional P5CR prevents both routes for proline biosynthesis what raise the P5CR gene to a paramount converging point of the two anabolic pathways. Therefore, the unique P5CR supposed fine transcriptional regulation although a post-translational regulation is highly suggested even more evident (Forlani et al. 2015; Anwar et al. 2018). As mentioned above, δOAT and P5CDH constitute another pathway for proline metabolism although δOAT is involved in the anabolism route contrary to P5CDH that corresponds more precisely to the proline

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catabolism pathway (cf. Fig. 1). Whatever, both genes are described for having only one copy in the nuclear plant genomes, and AtP5CDH exhibits a ubiquitous low basal level but can be upregulated by proline as shown in Fig. 2 (Deuschle et al. 2001). Concurrently, catabolism of proline to Glu is performed through PDH and P5CDH gene transcription. PDH is represented by two copies in the Arabidopsis thaliana genome (forming two isoforms, PDH1/PDH2), and their suppression leads to Pro accumulation. Indeed, as for P5CDH, proline cellular level insures the post-­ transcriptional regulation on PDH (Verbruggen and Hermans 2008). However, the two protein isoforms were shown to be differentially expressed (Funck et al. 2010). Overall, many transcription factors (TFs) revealed to be involved in the regulation of the proline metabolism genes. Several TFs gene families like MYC/MYB, bZIP, AP2/EREBP, RAV, PHR1, PHL, etc. participate to the abiotic stress tolerance in plants as already demonstrated or reported (Aleksza et al. 2017; Fichman et al. 2015; Roychoudhury et al. 2015; Anwar et al. 2018). In addition, various binding sites were predicted or demonstrated like the ACTCAT cis-acting element of the PDH1 promoter (Satoh et al. 2004; Weltmeier et al. 2006) or the HD-HOX, bZIP-­ DOF, AP2/EREBP, and P1BS-binding sites of AtP5CS1, AtP5CS2, AtP5CR, and AtOAT promoters (Fichman et al. 2015). Owing to the loss of crop productivity and the role of proline in the plant tolerance to abiotic stresses, engineering strategies using plant mutants for proline anabolism/catabolism allow to improve the knowledge on the molecular factors modulating these biological pathways (cf. reviews of Kavi Kishor et  al. 2015, Singh et al. 2017, and Hasanuzzaman et al. 2019). Biotechnologies could therefore give substantial advantage for developing new food crop cultivars tolerant to multiple abiotic stresses. Moreover, parallel to plant molecular enhancements, researchers proposed another tool for a higher crop productivity as described hereinafter.

4  A  pplication of Exogenous Proline on Plants Grown Under Abiotic Stresses 4.1  E  ffect of Exogenous Proline Application on Plant Water Status In an analysis of the beneficial effect of exogenous proline in plants exposed to abiotic stress (Table  3 and Fig.  4), it is important to consider the role of proline supplementation in plant water status. Drought stress is known to induce a decline in water content in plants tissues. For example, in cowpea (Vigna unguiculata L.) grown under three levels of water deficit (60, 40, and 20% of soil water holding capacity), Merwad et al. (2018) reported that water stress induced a significant decrease of leaf relative water content (LRWC). In

(0.4, 0.6, 0.8, 1.0, and 1.2 mM)

30 mM

5 mM

30 mm

4 mM

Foliar spray

Foliar spray

Foliar spray

Foliar spray

Foliar spray

Drought stress

Drought stress

Salt stress (75 mM) Salt stress (saline soil with EC = 1.84, 6.03, and 8.97 dS m−1)

Pea

Maize

Common bean

Sorghum

0.8 mM was the best concentration Improved growth, physiological, ionic, and biochemical attributes (biomass, photosynthetic rate, transpiration rate, and antioxidant enzyme activities) Decreased membrane damage and regulated proline levels Increased the activities of antioxidant enzymes and concentrations of carotenoids, ascorbic acid, and endogenous proline, increased the concentrations of P and K+, decreased Na+ ion concentrations, enhanced the growth Increased the content of seed sugar, oil, protein, moisture, fiber, and ash, increased the oil oleic and linoleic acid contents, increased the concentrations of antioxidant compounds in the seed oil, enhanced oil DPPH. free radical scavenging activity Increased the yield and soluble protein concentration, increase nonenzymatic antioxidant defense system, enhanced the production and translocation of assimilates from source to sink

Alleviated oxidative damages by enhancing the antioxidant and glyoxalase systems

Rice

Chili

Response Increased photosynthetic rate and total yield

Species Lettuce

Osman (2015)

Ali et al. (2013)

De Freitas et al. (2019) Abdelhamid et al. (2013)

Butt et al. (2016)

Hasanuzzaman et al. (2014)

Reference Orsini et al. (2018)

(continued)

5 mM

Foliar spray

Type and level of stress Salt stress (15 mM) Salt stress (150 and 300 mM NaCl) Salt stress (50 mM)

Table 3  Improvement in growth and regulation of various physiological and biochemical processes in different plant species by exogenous application of proline under abiotic stress

Concentration 5 μM

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Mode of application Foliar spray

Exogenous Proline-Mediated Abiotic Stress Tolerance in Plants: Possible Mechanisms

Concentration 80 mM

10 mM

60 mM

2, 4, and 6 mM

25 and 50 mM

1, 10, and 100 mM

Mode of application Foliar spray

Foliar spray

Foliar spray

Foliar spray

Rooting medium

Rooting medium

Table 3 (continued)

Cold stress (4 °C)

Salt stress (100 and 200 mM)

Water deficit

Heat stress (45 °C) Salt stress (100 mM)/nickel stress (100 μM)

Type and level of stress Heavy metal stress (400 μM, CuSO4.5H2O)

Larch, sitka spruce, and oak

Olive

Cowpea

Pea and ray grass

Barley

Species Wheat

Response Reduced the generation of reactive oxygen species and enhanced accumulation of proline and protein contents, enhanced the plant height and the shoot and root fresh and dry weight, enhanced the photosynthetic capacity Increased the tolerance of photosystem II through protection of the oxygen evolving complex (OEC) Improved growth, total chlorophyll content, photosynthetic attributes, RWC, and membrane stability index (MSI), increased activity of enzymatic antioxidants, enhanced osmolytes, lipid peroxidation, and polyamine metabolism Improved growth criteria, yield characteristics, contents of chlorophylls a and b, total carotenoids, shoot and seed nutrients, RWC, membrane stability index, activity of leaf antioxidant enzymes, and content of leaf proline 50 mM was the best concentration Increased photosynthetic activity, LRWC, chlorophyll and carotenoid, and starch contents, reduced the Na+ content in leaves and roots of stressed plants Increased growth rate and reduced K+ leakage

Gleeson et al. (2004)

Ben Ahmed et al. (2011)

Merwad et al. (2018)

Oukarroum et al. (2012) Shahid et al. (2014)

Reference Noreen et al. (2018)

108 M. Zouari et al.

20 mM

Rooting medium Rooting medium

Rooting medium

25, 50, and 100 μM

Rooting medium

20 mM

10 and 20 mM

10 μM

Rooting medium

Heat stress (30/25 °C, 35/30 °C, 40/35 °C, and 45/40 °C) Heavy metal stress (1, 2, 4, and 6 ppm selenium in hydroponic medium) Salt stress (200 mM) Cadmium stress (10 and 30 mg Cd kg−1 soil) Cadmium stress (10 and 30 mg Cd kg−1 soil) Olive

Date palm

Enhanced photosynthetic activity, nutritional status, plant growth, and oil content of olive fruit

50 μM was the best concentration Increased the endogenous proline content and enhanced the growth, increased the enzymatic and nonenzymatic antioxidants, stimulated components of the ascorbate-glutathione cycle, reduced lipid peroxidation and H2O2 content Increased fresh mass and the activities of enzymatic antioxidant Alleviated the oxidative damage, increased the activity of antioxidant enzymes in roots and leaves

Mung bean

Tobacco

Reduced oxidative injury by elevating enzymatic and nonenzymatic antioxidants, improved chlorophyll content and LRWC, enhanced the activities of enzymes of carbon metabolism

Chickpea

Zouari et al. (2016b)

Zouari et al. (2016a)

Hoque et al. (2007)

Aggarwal et al. (2011)

Kaushal et al. (2011)

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Abiotic stresses Drought

Heat/Cold

Salinity

Heavy metals

Secondary stress Osmotic stresses

Ionic stress

Oxydative stress

Adaptive strategies

Osmolyte

Antioxidant

Proline Glycine betaine Solubles sugar

Enzymatic antioxidant (SOD, APX, CAT...) Non-enzymatic antioxidant (glutathione, α-tocopherol, ascorbic acid...)

Stress tolerance

Fig. 4  Plant responses to abiotic stresses

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this study, the decline in LRWC due to water deficit stress can be explained by the decrease in the ability of osmotic adjustment due to the reduced absorption of nutrients, especially K+. The same authors reported that, when proline (6  mM) was applied as foliar spray treatments, LRWC increased. This enhancement was attributed to the significant accumulation of proline in cowpea that proves the important adjusting role of this osmolyte under unfavorable conditions. Its contribution to osmotic adjustment is considered as a mechanism to maintain water relations and postpone dehydration under osmotic stress. In addition, Iqbal (2018) observed that under drought conditions, the exogenous application of proline increased its endogenous level that decreased the water potential in cells to a level lower to the one in soil. This may facilitate the uptake of water by roots and therefore maintain the turgor pressure within cells. Salinity stress affects also plant-water relations. De Freitas et al. (2019) studied the impact of proline supply to sorghum (Sorghum bicolor L.) exposed to salt stress (75  mM NaCl) and observed a significant increase in LRWC of stressed plants sprayed with 30 mM proline solution. The beneficial role of exogenous proline was also obtained in salt-stressed plants such as rice (Oryza sativa L.) (Hasanuzzaman et al. 2014) and searocket (Cakile maritima L.) (Messedi et al. 2016). Referring to these authors, lowering of leaf osmotic potential by proline supplementation might be the result of higher accumulation of endogenous proline, which enhances the osmoregulation ability of plants under salt stress conditions. The same authors suggest that exogenous proline supplementation can restore water use efficiency, leaf water status, production of free proline, and membrane damage during salinity stress. Proline can enhance water influx and decrease water efflux to restore water content in plant exposed to stress. Other environmental stress conditions like extreme temperatures similarly account for a significant reduction in plant water status (Kaushal et  al. 2011; Oukarroum et al. 2012). In these studies, exogenous proline application maintained the leaf water status, whereas it was reduced in non-treated plants. According to these authors, the maintained leaf water status in proline-treated plants may be attributed to higher accumulation of compatible solutes like proline that possibly improved the turgor content. Plant-water relations are also affected by heavy metal stress. Zouari et al. (2016a) showed that LRWC and water potential (WP) were decreased in the leaves of date palm (Phoenix dactylifera L.) exposed to cadmium stress. The same authors reported that exogenous supply of proline improved the water status of Cd-stressed plants and attributed this enhancement to the interactive effect of proline on osmotic adjustment. Similarly, Shahid et  al. (2014) demonstrated that proline application significantly mitigated the alteration of water status of pea (Pisum sativum L.) induced by the phytotoxic effect of nickel. According to Aggarwal et  al. (2011), exogenous application of proline (50  μM) increased its endogenous levels that antagonized the toxic effects of selenium by improving water status of bean (Phaseolus vulgaris L.) seedlings.

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4.2  E  ffect of Exogenous Proline Application on Nutrient Status Absorption of mineral elements is a key process for plants to survive and grow. However, it is well known that several abiotic stresses result in decreased nutrient uptake and consequently reduced mineral nutrients content in plant tissues. Several studies reported that the exogenous supplementation of proline can ameliorate the uptake and accumulation of inorganic nutrients in stressed plants. Ali et al. (2008) reported that maize plants (Zea mays L.) subjected to drought stress by maintaining moisture content at 60% field capacity presented a decrease in N, P, K+, Ca2+, and Mg2+ contents in the shoots and roots. In the same study, exogenously proline (applied at 30 and 60 mM) increased endogenous proline and promoted the uptake of all the macronutrients under water stress conditions. According to these authors, 30 mM proline concentration was more beneficial than 60 mM as this concentration appeared more effective to increase the transpiration rate. Leaf transpiration creates the water tension necessary to the root absorption of essential nutrients from the soil solution. Similar findings were reported by Merwad et al. (2018) who noticed lower nutrient contents (shoots and seed N, P, and K+ contents) in cowpea plant submitted to drought stress than in control ones. These authors reported that exogenous proline has maintained nutrient status by promoting the uptake of N, P, and K+ under water stress. Salt stress causes also ion imbalance. Abdelhamid et  al. (2013) reported that highly saline soil (EC = 8.97 dS m−1) resulted in an increase of Na+ and in a decrease of P and K+ content in bean plants. In the same study, spraying bean plants with 5 mM proline significantly increased the content of P and K+ and the K+/Na+ ratio and decreased Na+ levels in salt-affected plants. Butt et al. (2016) also found similar results. These authors grown two chili genotypes under 50 mM NaCl saline condition with and without various concentrations of proline (0.4, 0.6, 0.8, 1 and 1.2 mM) applied as a foliar spray and concluded that proline supply had increased the K+ concentration in leaves of stressed plants. In this study, authors reported that K+ efflux was significantly reduced by the application of proline and ionic homeostasis was maintained by enhancing the H+ATPase activity. In the same way, Sobahan et al. (2009) reported that exogenous proline application reduces the Na+-enhanced apoplastic flow to reduce Na+ uptake and transport by plants, suggesting that proline interact with macromolecules in the Na+ diffusion pathways. It has been demonstrated that heavy metal stress may result in disturbance of ionic homeostasis. According to Noreen et al. (2018), the uptake of Ca2+, Mg2+, and K+ ions by root, shoot, and leaf organs of wheat (Triticum aestivum L.) was reduced by copper stress. On the other hand, copper content substantially increased in root organs compared to shoot and leaf organs under copper stress environment. The foliar spray of proline increased the uptake of Ca2+, Mg2+, Na+, and K+ by root, shoot, and leaf organs, while the copper uptake was reduced in all parts. Ashraf and Foolad (2007) reported that ion uptake by plants was regulated by proline spray under stress. In young olive plants (Olea europaea L.) treated with 30 mg CdCl2

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kg−1 soil, Zouari et al. (2016b) demonstrated that the content of Ca2+, Mg2+, and K+ was strongly reduced, while Cd2+ content was increased in leaf and root tissues. According to these authors, this perturbation of mineral nutrient status could be due to competitions between Cd2+ and essential elements via common transporters. In the same study, exogenous addition of proline to growth medium resulted in increased Ca2+, Mg2+, and K+ contents and in reduced Cd2+ content.

4.3  E  ffect of Exogenous Proline on Photosynthetic Performance Abiotic stresses generally affect the plant performance and development by altering the photosynthetic machinery (Hayat et al. 2012). Salinity stress is one of the most common abiotic factors that inhibit crop growth and productivity by reducing the photosynthetic capacity of plants. De Freitas et al. (2019) reported that under NaCl stress, photosynthesis rate, stomatal conductance, transpiration rate, and internal CO2 concentration of sorghum were significantly decreased as compared to the control. Salt toxic effects on photosynthesis can be generated by stomatal factors, including restrictions for CO2 diffusion, and by non-­ stomatal limitations such as decreased Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) activity or damage on photosynthetic apparatus by photosystem II performance impairment. The same authors indicated that stressed plants treated with proline presented higher CO2 assimilation in comparison to proline-untreated stressed plants, a response closely related to increases in stomatal conductance and transpiration rate. These responses indicate that proline supplementation might play a key role for CO2 assimilation and photosynthesis recovery in plants against salt stress. Kaushal et al. (2011) studied the comportment of chickpea (Cicer arietinum L.) grown under heat stress and investigated the effects of exogenous proline on total chlorophyll content. Proline-treated plants improved their chlorophyll content by 18% at 40/35  °C and by 44% at 45/40  °C in comparison to untreated plants. According to these authors, proline application significantly reduced the decrease in chlorophyll contents due to heat stress, and such physiological enhancement could result from leaf water status improvement and in possibly reduced photooxidation. The same authors suggested that proline may play an important role in maintaining respiratory metabolism and membrane structure of cells and organelles like chloroplast. Hayat et al. (2012) and Hossain et al. (2014) reported that proline application under drought conditions may maintain the photosynthetic capacity not only through increasing stomatal conductance but also by protecting the subcellular structures such as the chloroplast ultrastructure, the electron transport complex II in mitochondria, as well as the activity of many enzymes like Rubisco which thereby improved the photosynthetic capacity. Referring to Hare and Cress (1997) and Gholami Zali and Ehsanzadeh (2018), proline biosynthesis is a reductive pathway that require

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NADPH (for the reduction of glutamate to P5C and P5C to proline) and generate NADP+ which can be used further as electron acceptor and dissipate electron pressure in thylakoid electron transport chain thus avoiding the photoinhibition and thereby the alteration of photosynthetic machinery.

4.4  E  ffect of Exogenous Proline on Antioxidant Defense System Plants naturally synthesize ROS as byproducts of cellular oxidative metabolism. The role of proline as ROS scavenger was firstly observed in vivo by Smirnoff and Cumbes (1989) on Arabidopsis P5CS insertion mutants. Then, Ashraf and Foolad (2007) confirmed that proline was an effective scavenger of hydroxyl (OH.) and peroxide ion. Hong et al. (2000) concluded that the role of proline as a free radical scavenger is more important in alleviating stress than its role as a simple osmolyte. Reduced lipid peroxidation and H2O2 contents, along with the upregulation of the antioxidant defense system, were reported in rice seedlings under salt stress conditions when treated with proline (Hasanuzzaman et  al. 2014; Wutipraditkul et  al. 2015). Similar patterns were observed also by Butt et al. (2016) in chili genotypes subjected to salt stress and treated with various concentrations of proline. The results proved that both genotypes can cope with salt stress conditions by reducing lipid peroxidation and through the modulation of antioxidant enzymes (SOD and CAT) with exogenous application of proline. It has been reported that proline activates defense mechanisms in response to salt stress, such as activation of antioxidant enzymes. Proline also plays an important role in stress-induced phenolic synthesis, which exhibit antioxidant activities. It has been suggested that proline synthesis stimulates biosynthesis of phenolics via shikimate and phenylpropanoid pathways (Shetty 1997). Drought stress induces a severe oxidative stress in pea leading to oxidative damages as the antioxidant defense system was unable to cope with this stress (Osman 2015). In this study foliar applied proline (4 mM) enhanced the tolerance of peas to oxidative damage by enhancing ROS detoxification systems. These findings suggest that proline has protective effects against drought-induced oxidative stress by reducing H2O2 content and by increasing the enzymatic antioxidant defense system (SOD, CAT, and APX). Ghaffari et al. (2019) noticed in sugar beet (Beta vulgaris L.) exposed to drought stress (50% water requirement of plant) that foliar proline applications (low, 5  mM; high, 10  mM) increased enzymatic antioxidant activities and then reduced levels of MDA (malondialdehyde) and H2O2. Referring to these authors, proline foliar application might induce the drought tolerance in plants by up-regulating the antioxidant enzymatic activities, quenching the ROS and improving cellular membrane stability. In regard to heat stress, Kaushal et al. (2011) reported that elevated temperature causes significant reduction in proline content and antioxidant enzymes and resulted

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in severe membrane lipid peroxidation in chickpea plants. In this respect, a synchronic increase in some components of the antioxidative system would be necessary in order to obtain an improvement in heat stress tolerance. In this connection, exogenous application of proline increased enzymatic (SOD and APX) and nonenzymatic antioxidants (AsA and GSH) to a significant level comparing with control. According to these authors, proline has been shown to function as a molecular chaperone able to protect protein integrity and enhance the activities of different enzymes.

4.5  E  ffect of Exogenous Proline on Growth and Yield Quantity and Quality Several studies reported that different abiotic stresses reduced cell division and cell expansion, resulting in substantial growth reduction. Inhibition of stem and leaf development negatively affects plant height and leaf area and consequently reduces photosynthesis and crop productivity (Dawood et al. 2014; Osman 2015; Zouari et al. 2016a). Proline regulates many aspects of growth and development, particularly under abiotic stresses. Transgenic rice overexpressing P5C genes presented increased root and shoot growth and increased biomass production under drought conditions. Transgenic plant accumulated more proline than the control (Su and Wu 2004). Therefore, it has been postulated that exogenous application of proline can effectively stimulate growth and yield attributes. Ali et  al. (2013) reported that foliar applied proline significantly increased the seed oil content of maize under well irrigated and water-deficit conditions. Furthermore, exogenous application of proline increased the oil oleic and linoleic acid contents. In a similar study, Teh et al. (2016) reported that proline supplementation significantly increased the plant height and the number of roots of rice under salt stress. More recently, Merwad et al. (2018) reported that foliar application of proline ameliorated growth criteria (shoot dry weight, plant height, leaf area, and number of branches per plant) and yield ­characteristics (dry seed weight, biological yield per plant, and 100-seed weight) of cowpea submitted to water stress. Amelioration of plant growth and yield attributes due to proline application might be due to (i) the improved synthesis of compatible solutes leading to better osmotic adjustment (Dawood et al. 2014); (ii) the enhanced accumulation of total soluble phenolics, thus protecting the tridimensional structure of proteins and enzymes (Ashraf and Foolad 2007; Rasheed et al. 2014); (iii) the improvement in chlorophyll contents (Zouari et  al. 2016b); (iv) the reduced oxidative damages (Shahid et al. 2014); (v) the increased antioxidant system activities (Osman 2015); (vi) the stabilization of biological membranes (lipids, protein, plasma membrane) (Hayat et al. 2012); (vii) the enhancement of Rubisco activity (Kaushal et al. (2011); and (viii) the improved photosynthesis (De Freitas et  al. 2019). The growth-­ promoting effect of proline application could be also attributed to its role in protein synthesis.

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5  Effective Concentrations of Exogenous Proline Exogenous application of proline to abiotic-stressed plants generally provides a stress preventing or recovering effect. Despite the beneficial effects of exogenous proline application, proline has toxic effects if over-accumulated and/or applied at excessive concentrations (Ashraf and Foolad 2007). Therefore, it is essential to determine optimal concentrations of proline that provide beneficial effects for each plant species. In maize plants, for example, it was determined that foliar applied proline at 30 mM mitigated the adverse effects of NaCl stress, but, at 60 mM, proline inhibited the growth of salt-stressed and non-stressed plants (Ali et al. 2008). In lettuce (Lactuca sativa L.), exogenous proline spraying at 10 μM was very effective in alleviating the effects of salt stress, while higher concentrations (15 μM) were not beneficial (Orsini et al. 2018). Butt et al. (2016) applied various concentrations of proline (0.4, 0.6, 0.8, 1.0, and 1.2 mM) as a foliar spray on chili seedlings submitted to salt stress. Among all proline concentrations, 0.8 mM proved to be the best concentration regarding growth, physiological, ionic, and biochemical attributes. Proline application in high concentrations has shown to present harmful effects, such as an inhibition of growth and cellular metabolism (Ashraf and Foolad 2007). Thus, in spite of its protective role, the toxicity effect of proline at high concentrations may be a problem. This toxicity could be due to the repression of genes involved in key functions of the plant metabolism like photosynthesis or synthesis of cell wall-associated proteins (Verbruggen and Hermans 2008). The available information from different studies suggest that optimal concentrations of proline may be species- or genotype-dependent, which need to be determined a priori before commercial application of exogenous proline to improve crop stress tolerance.

6  Conclusion and Future Perspectives Abiotic environmental stresses remain the major obstacle in plant growth, development, and global crop productivity. However, understanding the physiological and biochemical responses of plants to stress remains necessary to plant science researchers around the world. Scientists are constantly developing new strategies to improve plant stress physiology. In this regard, many studies have provided the notion that the exogenous application of proline provided better protection against different abiotic stresses such as salinity, drought, metal toxicity and extreme temperatures, etc. Under these stressful environmental conditions, exogenous applications of proline have been shown to: (i) Increase the endogenous levels of proline and compatible solute which provide protection to cells through osmotic adjustment. (ii) Help to maintain cellular ionic homeostasis.

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Proline application

Foliar spray

Root medium

Beneficial effects of proline

Osmotic adjustment

ROS Scavenging

Protection of cellular structures

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Fig. 5  Beneficial effects of exogenous proline application on plants under abiotic stresses

(iii) Act as an antioxidative defense which efficiently scavenge toxic ROS, confer detoxification processes, and reduce oxidative damages through stabilizing antioxidant enzymes. (iv) Affect plant-water relations by maintaining turgidity of cells under stress and increase the photosynthesis rate. (v) Enhance plant growth and final crop yield (Fig. 5). Deciphering proline metabolic pathways and their interconnections with TCA cycle, GABA, polyamine pathway, etc. is of major interest to develop future applications of proline-mediated stress abiotic tolerance. GMO crop fully benefiting from these future breakthroughs are probably not ready before several decades and will be probably not accepted by the public, as they are not biological and environmental friendly. In that sense, combination of proline, glycine betaine, and polyamine exogenous application could constitute a main key to help plant coping with many stresses induced through climate change and global warming even if the exact effects of these applications must be elucidated and their effect on soil microbiota clarified. For plants, climate change leads to increased drought stress, salinity, and heavy metal stresses linked with the fast growing of water reuse techniques occurring currently. Assisting plants in their adaptation to this changing environment is probably the key to maintain crop production at an acceptable level to insure human survival and “proline engineering” is certainly one of the possible solutions.

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Biosynthesis and Degradation of Glycine Betaine and Its Potential to Control Plant Growth and Development Elisa M. Valenzuela-Soto and Ciria G. Figueroa-Soto

1  Introduction Plant growth and development is affected by abiotic stress which results in important losses in plant yield and in the money spent in agriculture. The abiotic stress that has a strong impact on plant yield is the hydric stress (drought, salinity, and cold). Plants have developed strategies to contend with hydric stress, between them one of the most important is the synthesis and accumulation of osmolytes (Yancey et al. 1982). In plants, one of the most studied osmolytes is glycine betaine followed by proline and trehalose (Singh et al. 1972; Stewart and Lee 1974; Hare et al. 1998; Iordachescu and Imai 2008; Chen and Murata 2008; Paul et al. 2008; Krasenky and Jonak 2012). Glycine betaine (N,N,N-trimethyl glycine, GB) is a quaternary amine, isolated for the first time from sugar beet (Scheibler 1869). In mammals, GB participates in homocysteine/methionine cycle [Hcy/Met cycle] as methyl donor to homocysteine to produce methionine, reaction catalyzed by betaine homocysteine methyl transferase [BHMT] (du Vigneaud et al. 1946; Finkelstein and Martin 1984; Pajares and Pérez-Sala 2006). As a consequence of GB participation in the Hcy/Met cycle, a wide set of physiological roles of GB has been found (Craig 2004; Olthof and Verhoef 2005; Lawson-Yuen and Levy 2006; Lever and Slow 2010; Ueland 2011; Figueroa-Soto and Valenzuela-Soto 2018). Physiological functions of GB are as an osmolyte to contribute to maintaining cellular volume, as an osmoprotector to protect cells under stress, and/or as a source of methyl groups through transmethylation reactions (Takabe et  al. 2006; Craig 2004; Chen and Murata 2011). However, not all plants accumulate GB in response to stress; in fact, the vast majority of plants of agricultural importance are not accumulators of GB. For this reason, attempts have been made to genetically transform

E. M. Valenzuela-Soto (*) · C. G. Figueroa-Soto Centro de Investigación en Alimentación y Desarrollo A.C, Hermosillo, Sonora, México e-mail: [email protected] © Springer Nature Switzerland AG 2019 M. A. Hossain et al. (eds.), Osmoprotectant-Mediated Abiotic Stress Tolerance in Plants, https://doi.org/10.1007/978-3-030-27423-8_5

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those plants with the genes of GB synthesis enzymes (Takabe et al. 2006; Giri 2011; Chen and Murata 2011; Wani et al. 2013). In plants, two routes of GB synthesis have been proposed, one from choline and the other from glycine. The first route requires two choline oxidation steps catalyzed by the choline monooxygenase [CMO] and betaine aldehyde dehydrogenase [BADH]; the second route involves two methylation steps catalyzed by glycine sarcosine methyltransferase [GSMT] and sarcosine dimethylglycine transferase [SDMT] (Weretilnyk and Hanson 1989; Rathinasabapathi et al. 1997; Valenzuela-­Soto and Muñoz-Clares 1994; Nyyssola et al. 2000; Waditee et al. 2005; Chen et al. 2008). There are several studies about the GB accumulation in different plant species as a response to abiotic stress [drought, salinity, cold, heat, etc.] (Rhodes and Hanson 1993; Sakamoto and Murata 2002; Giri 2011; Chen and Murata 2011; Kurepin et al. 2015). These studies have demonstrated that GB plays a role in different processes in plant metabolism, e.g., there is evidence of GB direct and/or indirect participation in protein stability, protein synthesis, enzyme activity, photosynthesis, oxidative stress response, and plant growth and development (Chen et al. 2008; Khan et al. 2009; Giri 2011; Chen and Murata 2011; Wani et al. 2013). However, less is known about the enzymes that synthesize GB or transform it into other compounds, e.g., what are the structural and kinetic characteristics of that enzymes or how they are regulated. The aim of this review is to summarize the knowledge garnered about GB’s metabolism and how it impacts the growth and development of plants under abiotic stress conditions.

2  Glycine Betaine Metabolism 2.1  Synthesis Pathways In GB accumulator plants, it is synthesized from choline, choline is oxidized to betaine aldehyde by choline monooxygenase [E.C. 1.14.15.7], and betaine aldehyde dehydrogenase [BADH EC 1.2.1.8] catalyzes the betaine aldehyde oxidation to GB (Fig. 1a) (Rathinasabapathi et al. 1997; Ling et al. 2001; Hibino 2002; Wang and Showalter 2004; Park et  al. 2007; Muñoz-Clares and Valenzuela-Soto 2008). Extremely halophilic plants and microorganisms, also methanogenic organisms, synthesize GB from glycine; it is methylated by glycine sarcosine methyl transferase [GSMT] to N,N-dimethylglycine and sarcosine; furthermore, N,N-­ dimethylglycine is methylated by the sarcosine dimethylglycine transferase [SDMT] to GB; thus, both enzymes use S-adenosylmethionine (SAM) as methyl donor (Fig. 1b) (Nyyssola et al. 2000; Waditee et al. 2005). Choline is synthesized in the cytosol, and there are three described possible choline synthesis routes, all of them start with ethanolamine [EA], which can be N-methylated by SAM as free bases, phosphorylethanolamine bases, or ­phosphatidylethanolamine bases; each methylation step is catalyzed by phosphoethanolamine methyltransferase [PEAMT] (Fig.  2) (Hanson and Rhodes 1983;

Biosynthesis and Degradation of Glycine Betaine and Its Potential to Control Plant… NAD

a

Choline

SAM

Glycine betaine BADH

Glycine

SAH

SAM

GSMT/ SDMT

Glycine betaine+Homocysteine

SAM

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c

NADH

Betaine aldehyde CMO

b

+

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N,N-Dimethyl glycine

SAH

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Glycine betaine

Dimethyl glycine + Methionine BHMT

Fig. 1  Glycine betaine synthesis and degradation pathways. (a) GB synthesis in plants. (b) GB synthesis in extremely halophylic plants and microorganisms or metanogenics organisms. (c) GB catabolism in animals, some bacteria and in the cyanobacteria Aphanothece halophytica. CMO choline monooxygenase, BADH betaine aldehyde dehydrogenase, GSMT glycine sarcosine methyl transferase, and SDMT dimethylglycine transferase, BHMT betainehomocysteine methyltransferase

Fig. 2  Interplay between choline, GB, ethylene, and polyamine synthesis pathways. SAMS S-adenosylmethionine synthase, PEAMT phosphoethanolamine methyltransferase, CK choline kinase, CMO choline monooxygenase, BADH betaine aldehyde dehydrogenase, ACC aminocyclopropane carboxylic acid, ACCS aminocyclopropane carboxylic acid synthase, EA ethanolamine, PEA phosphoethanolamine, Pd-EA phosphatidylethanolamine. Pink circle, choline transporter; green ellipse, chloroplast; red arrows indicate inhibition of PEAMT by P-choline

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Datko and Mudd 1988; Nuccio et al. 1998, 2000). The three pathways can be used by plants; however, preference by one of them has been found, e.g., in Chenopodiaceae plants, choline comes from phosphoethanolamine (P-EA); in tobacco, choline originates from phosphatidyl-EA; in soy bean instead, the first step is the methylation of P-EA to phosphomonomethyl ethanolamine [P-MME] followed by a conversion to phosphatidylmonomethylethanolamine [Ptd-MME] via a cytidyl intermediate (Mudd and Datko 1989a; McNeil et al. 2000a). Later Ptd-MME is methylated to phosphatidyldimethylethanolamine [Ptd-DME], which is converted to Ptd-choline and later to P-choline (Hanson and Rhodes 1983; McNeil et al. 2000a, b; Nuccio et al. 2000). The last step in choline synthesis is the dephosphorylation of P-choline by choline phosphatase or choline kinase [CK] (Summers and Weretilnyk 1993; McNeil et  al. 2000b). Choline synthesis is regulated by P-choline and S-adenosylhomocysteine [SAH]; both are inhibitors of PEAMT activity (Fig.  2) (Mudd and Datko 1989b; Nuccio et al. 2000; Sahu and Shaw 2009). Choline is transported to the chloroplast and used as a substrate by CMO, the first enzyme in the GB synthesis. CMO is unique in plants and catalyzes the BA synthesis, its crystallographic structure has not been determined yet, the molecular mass of the monomer is ≈ 45 kDa, and it contains a Rieske-type [2Fe-2S] center and requires ferredoxin to be active (Rathinasabapathi et al. 1997). Hibino et al. (2002) found that Cys-181 is essential to the spinach CMO function, and as found in other oxygenases, a histidine [Hys-283] participates in the coordination of the [2Fe-2S] center. The genes coding CMO have been studied in plant accumulator species and plant non-accumulator species. CMO gene sequences from spinach and sugar beet share 78% identity between them, while the sequence of CMO from Arabidopsis shares 51% identity with that of spinach and sugar beet (Hibino et  al. 2002). Instead, Amaranthus tricolor CMO shares 69.4% and 69.5% identity with spinach and sugar beet CMOs and Atriplex prostrate, while rice shares 82.9% and 63% identity with deduced amino acid sequence of spinach and sugar beet, respectively (Ling et al. 2001; Wang and Showalter 2004; Luo 2007). Amino acid sequence analysis of the CMO from Amaranthus tricolor, Arabidopsis, barley, rice, sugar beet, and spinach showed that all of them contained consensus sequences for coordination of the Rieske-type [2Fe-2S] cluster, CXHX15–17CX2H, and for coordination of mononuclear non-heme Fe, G/DX3–4 DX2HX4–5H [X equal to any amino acid] (Russell et al. 1998; Rathinasabapathi et  al. 1997; Meng et  al. 2001; Ling et  al. 2001; Hibino et  al. 2002; Wang and Showalter 2004; Luo et al. 2007; Mitsuya et al. 2011). In addition, the modeling of Spinacia oleracea CMO showed that in the active site there is an aromatic box conformed by Tyr281, Tyr295, and Phe301 and by a Glu residue [Glu346] (Carrillo-­ Campos et al. 2018). Aromatic box is involved in the choline’s trimethylammonium group, while the side chain carboxyl group of Glu346 participates in an ionic interaction with that group (Carrillo-Campos et al. 2018). An analysis of the promoter of Amaranthus tricolor CMO gene allowed identifying a fragment of 410 pb upstream of the translation start codon that contains the sequence responsive to salt stress (Bhuiyan et al. 2007). In addition, Xu et al. (2018)

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found that the CMO gene from watermelon [Citrullus lanatus] suspension cells contained responsive elements to light, plant hormone-responsive cis-elements, and cis-elements responsive to biotic and abiotic stresses. To this date, it seems that plants that do not accumulate GB possess the CMO genes, but those genes were proposed as not functional, as it has been found in rice and maize (Peel et al. 2010; Luo et al. 2012). However, there is other possible explanation to that results, for a recent phylogenetic study in Amaranthaceae plants showed that plant CMO evolved to two kinds of CMO proteins grouped in two clades called CMO1 and CMO2 and CMO2 diverged from CMO1 (Carrillo-Campos et al. 2018). From 167 plant CMO sequences analyzed, Carrillo et al. (2018) found that CMO1 and CMO2 proteins share 30% identity, CMO1 proteins share 50% identity between them, and otherwise CMO2 proteins share more than 85% identity. CMO1 and CMO2 modelling results showed that neither the CMO1 active site nor the Glu346 as found in CMO2 has the aromatic box; this would explain why CMO1 does not catalyze the oxidation of choline to betaine aldehyde (Carrillo-Campos et al. 2018). In addition, the chloroplast signal peptide is not conserved in CMO1 amino acid sequences (Carrillo-Campos et al. 2018). The second step in GB synthesis is catalyzed by betaine aldehyde dehydrogenase [BADH]. Plant BADHs belong to ALDH superfamily, and they are grouped in the family ten [ALDH10] (Sophos and Vasiliou 2003). Within the ALDH10 family, there are proteins that use as substrate ω-aminoaldehydes [3-amino propionaldehyde or 4-aminobutyraldehyde] and ω-quaternary amino group [trimethylammonium] and as betaine aldehyde, trimethylaminobutiraldehyde or dimethylsulfoniopropionaldehyde (Trossat et  al. 1997; Vojtechová et  al. 1997; Ŝebela et al. 2000; Brauner et al. 2003; Livingstone et al. 2003; Oishi and Ebina 2005; Bradbury et al. 2008; Fujiwara et al. 2008). It has been proposed that BADH activity depends on only one amino acid residue at position 441 [SoBADH numbering] (Muñoz-Clares et  al. 2014). The ability to oxidize betaine aldehyde by the BADH is related to the presence of an Ala or Cys in the 441 position in the protein (Muñoz-Clares et al. 2014). The first plant BADH crystal structure obtained was from spinach, which showed that there are four aromatic residues Tyr160, Trp167, Trp285, and Trp456 at the active site (Díaz-Sanchez et al. 2012). By using in silico model building, kinetic studies, and site-directed mutagenesis of SoBADH, it was found that the aromatic ring of Tyr160 is of great importance for BA binding, followed by Trp285 and Trp167 (Díaz-Sanchez et  al. 2012). The position that occupies in the active site pocket Trp456 is determined by the conformation adopted by the side chain of the amino acid residue in position 441 [Ile, Ala or Cys], to allow or not the proper positioning of the trimethylammonium group of BA, so Ile size would push Trp456 to such a position that there would be no adequate space for the binding of trimethylammonium group (Díaz-Sanchez et  al. 2012; Muñoz-Clares et  al. 2014). Interestingly, a great number of BADH from GB accumulators’ plants possess an Ala or Cys in position 441 (Muñoz-Clares et al. 2014). ALDH10 isoenzymes evolved from the gene coding to an Ile in position 441 as a consequence of environmental pressure; however, all plants conserved isoenzymes

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with one of three amino acids in the 441 position (Ile, Ala, and/or Cys), which allow isoenzymes to perform other metabolic functions in plants (Muñoz-Clares et al. 2014). Different studies have demonstrated that BADH in plants is a homodimer of ≈ 120 kDa, except in wild amaranth and pea which are heterodimeric and homotetrameric, respectively (Weretilnyk and Hanson 1989; Valenzuela-Soto and Muñoz-­ Clares 1994; Figueroa-Soto and Valenzuela-Soto 2001; Ŝebela et  al. 2000; Livingstone et al. 2002; Oishi and Ebina 2005; Fujiwara et al. 2008). Plant BADHs show an acidic pI, an optimum pH ranging from 8.0 to 8.5, and they exhibit preference to use NAD+ as coenzyme (Weretilnyk and Hanson 1989; Trossat et al. 1997; Valenzuela-Soto and Muñoz-Clares 1994; Incharoensakdi et al. 2000; Hibino et al. 2001; Fujiwara et al. 2008). Similar to cis-acting regulatory elements described before for CMO, the BADH is also regulated at the genetic level. Analysis of the BADHs’ gene promoter sequence from Suaeda liaotungensis revealed regulatory elements, such as a TATA-­ box, a CAAT-box, a GC-motif, EIRE, MRE, WUNmotif, a heat shock element, ABRE, methyl jasmonate-responsive element, and ethylene-responsive element [ERE] (Zhang et al. 2008; Xu et al. 2018).

2.2  Glycine Betaine Degradation Routes In animals and some bacteria, GB is catabolized to methionine and glycine by betaine homocysteine methyl transferase [BHMT], it removes a methyl group from GB to produce dimethylglycine, and the methyl group is transferred to homocysteine for methionine synthesis (Fig. 1c) (Pajares and Perez-Salas 2006). A glycine betaine transmethylase was proposed in Rhizobium meliloti as the enzyme to convert GB to dimethylglycine (Smith et  al. 1988), whereas in the cyanobacteria Aphanothece halophytica, GB was catabolized by BHMT under hyperosmotic conditions (Incharoensakdi and Waditee 2000; Waditee and Incharoensakdi 2001). After a deep search about BHMT in plants, no information was found. This being the reason why is not possible to relate methionine synthesis with GB degradation. However, it is possible to speculate that BHMT has not been searched and therefore identified.

2.3  Cellular Compartment of Glycine Betaine Synthesis GB synthesis has been localized in chloroplasts, peroxisomes, and cytoplasm. It has been suggested that in dicotyledons GB synthesis takes place in the chloroplast, while in monocotyledons it occurs in the peroxisome (Nakamura et al. 1997; Mitsuya et al. 2011). BADH isoenzyme localization differs between plants, e.g., in spinach, one of them is targeted to chloroplast and the other to cytosol; in barley, one isoenzyme is directed to the peroxisome and the other to the cytosol; and in rice, both isoenzymes are targeted to the peroxisome, whereas in Avicennia marina, one of them is delivered to the chloroplast and the other to peroxisome (Weigel et al. 1986;

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Nakamura et al. 1997; Hibino et al. 2001; Nakamura et al. 2001; Shirasawa et al. 2006). BADH isoenzymes targeted to chloroplast or to peroxisome possess a short signal peptide [seven or three residues, respectively]; in barley, the signal peptide is located in the C-terminus, whereas in spinach it is in the N-terminus (Weretilnyk and Hanson 1990; Nakamura et al. 2001). To this date, it is known the BADHs with high BA affinity are located in the chloroplast (Weigel et  al. 1986; Hibino et al. 2001).

2.4  GB Synthesis in Plant Tissue In plants capable of synthesizing and accumulating GB, it has been found that GB is distributed throughout the whole plant under stress conditions (Yamada et  al. 2009). The leaf is the tissue with the highest content of GB, but it is influenced by the leaf age; in barley and sugar beet, it was found that GB is synthesized mainly in old leaves where CMO activity was detected (Nakamura et al. 1996; Hattori et al. 2009; Yamada et al. 2009). The root has the ability to synthesize GB; however, the expression of CMO and BADH is lower compared to the leaf (Bhuiyan et al. 2007; Yamada et  al. 2009). On the other hand, BADH was detected in old and young leaves and roots of sugar beet, so it is concluded that the synthesis of GB is limited by the availability of CMO (Bhuiyan et al. 2007; Fujiwara et al. 2008; Yamada et al. 2009). Since GB has been found in tissues that do not contain CMO activity, the mobilization of GB has been investigated. Two transporters have been found: one in sugar beet and another in barley called BvBet/ProT1 and HvProT2, respectively; they transport proline and GB with the highest affinity detected for GB (Yamada et  al. 2009; Fujiwara et  al. 2010). BvBet/Pro1 and HvProT2 were localized in plasma membrane: BvBet/Pro1 was more abundant in old than in young leaves, while HvProT2 is distributed in old leaves and roots (Yamada et al. 2009; Fujiwara et al. 2010).

3  G  B Synthesis and Control of Plant Growth and Development GB synthesis requires choline in any cellular compartment where it is carried out; at the date, the important aspects that limit the synthesis of GB are the availability of choline and the structural characteristics to carry out the union of the substrates and the catalysis of the CMO and BADH isoenzymes (Nuccio et al. 1998; Díaz-­ Sanchez et  al. 2012; Muñoz-Clares et  al. 2014; Carrillo-Campos et  al. 2018). Synthesis of P-choline is strongly favored in the cytosol; however, it depends on the dephosporylation of choline because only choline can be transported to chloroplast or vacuole (Bligny et al. 1989; McNeil et al. 2000a). On the other hand, choline produced is distributed between vacuole, chloroplast, and cytosol which limit the

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availability of choline to GB synthesis (Nuccio et al. 1998; McNeil et al. 2000a; Sahu and Shaw 2009). Considering that the concentration of choline is not limiting, the synthesis of GB would require a high concentration of SAM which would immediately cause a decrease in the synthesis of ethylene and polyamines (Ravanel et al. 1998; Sahu and Shaw 2009; Wang et al. 2010; Khan et al. 2014). In addition, SAM is required for chlorophyll synthesis, DNA replication, cell wall synthesis, etc.; therefore, GB synthesis cannot be very high, which corresponds with GB concentrations found in plants (Huang et al. 2000; Holstrom et al. 2000; Sakamoto and Murata 2001; Quan et al. 2004; Tabuchi et al. 2005; Wei et al. 2017). Tabuchi et al. (2005) suggested a co-regulation between the levels of S-adenosyl-L-methionine synthetase [SAMS] transcript with those of CMO and PEAMT; this would allow sustain active GB production without significantly diminishing the synthesis of other metabolites dependent on SAM. Plants capable of synthesizing and accumulating GB show tolerance to stress mainly to drought, salinity, and extreme temperature [cold and heat] stress; the growth and development of those plants are affected depending on the stage of development of the plant, as well as the type and species of the plant. It has been demonstrated that drought, salinity, and low and high temperature decrease root and shoot growth and development, but GB’s synthesizing plants manage to reduce the effect of stress on both parameters. The degree or level of protection varies between species and even between varieties of the same species. Under stress conditions, GB participates in maintaining of fundamental processes for growth and development such as (a) photosynthesis, energy production [ATP], and carbon skeletal, (b) conservation of the cell-reducing environment, and (c) enzyme functionality. Since GB can be present in all parts of the plant [either by synthesis or transport], its effect can occur in the entire plant. With all the information generated, it can be said that GB effects on plants under stress conditions are related to its ability to stabilize protein structure and regulate gene transcription and enzyme activities; those functions are the ways on how GB can play a role in plant growth and development. Photosynthesis is inhibited by heat, chilling, salinity, and drought stress; however, GB contributes to maintain the photosynthesis activity through the PSII damaged reparation increasing the expression of D1 protein and increasing its degradation when it is damaged (Fig. 3) (Onishi and Murata 2006; Murata et al. 2007; Yang et al. 2008; Fan et  al. 2012). In addition, PSII oxygen-evolving complex structure, Mn cluster, and PSII association with extrinsic polypeptides 18, 23, and 33  kDa are strongly stabilized by GB in plants under stress (Murata et al. 1992; Papageorgiou and Murata 1995; Allakhverdiev et al. 1996; Allakhverdiev et al. 1999). An adequate electron transport in the thylakoid maintains adequate levels of photosynthetic parameters as photosynthetic rate [A], intercellular CO2 [Ci], transpiration rate [E], stomatal conductance [gs], and maximal efficiency of PSII [Fv/Fm] (Fig. 3) (Zhao et al. 2007; Yang et al. 2008; Guha et al. 2010; Wei et al. 2017). The other face of photosynthesis is the CO2 fixation by the RUBISCO and the flux of carbon skeletal through the Calvin cycle enzymes. RUBISCO, Rubisco acti-

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Fig. 3  Schematic model of glycine betaine synthesis effects and changes induced to explain its mode of action. The scheme includes plant hormones involved in the induction of GB synthesis. Orange ellipse enclosed proteins involved in photosynthesis; proteins are enclosed in a yellow circle

vase, fructose biphosphatase [FBPase], fructose biphosphatase aldolase [FBPaldolase], and phosphoribulose kinase [PRKase] are activated by GB by stabilizing their structure under stress conditions (Fig. 3) (Makela et al. 2000; Yang et al. 2005; Murata et al. 2007; Konrad and Zvi 2008; Fan et al. 2012). Interestingly, Yang et al. (2005) found that under heat stress conditions, Rubisco activase is associated with thylakoid membrane, which is avoided by GB. A suitable CO2 fixation has been proposed by Murata et al. (2007) as an important factor to decrease the PSII damage, because suppression of CO2 fixation drives to oxidative stress which inhibits D1 protein synthesis and the repair of PSII. To contend with oxidative stress, transgenic plants or wild-type plants able to synthesize GB increase the expression of enzymes of antioxidant system; an increase in mRNA of the enzymes superoxide dismutase [SOD], catalase [CAT], ascorbate peroxidase [APX], glutathione reductase [GR], glutathione peroxidase [GPX], and dehydroascorbate reductase [DHAR] has been found in different plant species (Fig. 3) (Hoque et al. 2008; Islam et al. 2009; Fan et al. 2012; Hasanuzzaman et al. 2014; Zhang et al. 2016; Yao et al. 2018). Increases in antioxidant enzyme activity decrease the lipid peroxidation and protein carbonylation protecting cell survival (Hoque et al. 2008; Islam et al. 2009; Karabudak et al. 2014). Likewise, increases in the concentration of metabolites with antioxidant activity have been found, e.g., increases in glutathione reduced [GSH], ascorbate reduced [ASA], phe-

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nolic compounds, and flavonoids (Hoque et al. 2008; Islam et al. 2009; Ahmed et al. 2013; Wang et al. 2019). Changes in the activity of enzymes involved in Calvin cycle, antioxidant system (enzymatic and nonenzymatic), or proline synthesis are consequence of changes in their gene expression or changes in the enzyme activity induced by GB. In animals, GB induces changes in DNA methylation status and interacts with transcription factors to modify gene expression (Song et al. 2007; Zhang et al. 2013; Deminice et al. 2015; Idriss et al. 2017); in plants, there is no information about it. However, it is tempting to propose that something similar to what happens in animals may be happening in plants. Synthesis of ATP under stress conditions is less studied; Jin et al. (2015) found a high ATP/ADP ratio induced by GB in loquat fruit submitted to a low-temperature conditioning. It has been proposed that GB improves the lipid composition of cell membranes, that is, thylakoid membranes of wheat were protected by GB application, which provoked changes in the fat acid composition (Zhao et al. 2007; Tiang et al. 2017). Changes in lipid composition of thylakoid membranes increased the membrane fluidity, improving their function (Zhao et al. 2007; Tiang et al. 2017). Therefore, if GB maintains the functionality of thylakoid membranes and positively modulates photosynthesis, then there is a good proton gradient to carry out the ATP synthesis (Yang et al. 2008; Zhao et al. 2007; Guha et al. 2010; Ogbaba et al. 2014; Wei et al. 2017; Tiang et al. 2017). Drought, salinity, heat, and cold stress increase GB synthesis both in GB natural synthesizing and in transgenic plants; this GB increase has a strong impact in the plant growth. Several works have been demonstrated that GB increases the growth of root, shoot, hypocotyl, and plant measured as high, biomass [fresh weight or dry weight], or leaf area under stress conditions and relative of the control plant (Kishitani et al. 2000; Quan et al. 2004; Yang et al. 2005; Park et al. 2007; Yang et al. 2008; Guha et al. 2010; Goel et al. 2011; Fan et al. 2012; Karabudak et al. 2014; Ke et  al. 2016; Manaf 2016). The spiking time in transgenic maize plants under stress was less affected compared with wild-type plants (Quan et al. 2004). GB increased the number of anthers, pistils, and petals in transgenic Arabidopsis plants (Sulpice et al. 2003). In transgenic maize, the reproductive development is promoted by GB under drought stress (Quan et al. 2004). The percentage and time of germination of seeds of transgenic rice, tomato, and tobacco plants are promoted by GB under salt and drought stress (Park et  al. 2007; Kathuria et  al. 2009; Goel et  al. 2011; Li et  al. 2011). Plant productivity of plants under stress conditions is also promoted in those plants capable of synthesizing GB. Based on maintaining the growth and development of the plants synthesizing GB, the productivity of them is also positively affected. Sulpice et  al. (2003) found in transgenic Arabidopsis plants a greater ­number of flowers and seeds per plant, whereas in transgenic maize, Quan et  al. (2004) reported a greater number of seeds per plant and a greater weight per grain. Despite all the positive effects of GB on growth and development of plants, there is no evidence that GB is directly promoting growth, so it has been proposed that GB could be interacting with plant hormones like auxins and ABA, since they are involved in the control of growth (Kurepin et al. 2015). To date it has been found

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that ABA and methyl jasmonate increase the synthesis of GB (Fig. 3) (Ishitani et al. 1995; Jagendorf and Takabe 2001; Xing and Rajashekar 2001; Xu et  al. 2018). Salicylic acid seems to be playing a role of increasing the methionine content to support the SAM production used to GB synthesis and decreasing the ethylene production (Khan et  al. 2014). Barley and poplar transgenic plants overexpressing CodA gene showed increased expression levels of auxin-responsive IAA genes (Li et al. 2014; Ke et al. 2016).

4  Conclusion and Future Perspectives The growth of plants primarily requires sugars, protein synthesis, ATP, reducing power, and a reducing cellular environment, all of which are influenced directly or indirectly by GB. All those aspects are influenced by GB synthesis, transport, and accumulation, by which it has a positive influence in plant growth and development under stress conditions. The mechanism by which GB influences the expression of genes is much less known and is an important aspect to study. The capacity of GB to stabilize proteins and to induce their synthesis explains in part changes in the activity of enzymes studied up to now; however, it remains to be defined if GB interacts directly as activator or inhibitor of enzymes. A great advance has been reached in the knowledge of the impact that GB synthesis has on the growth and development of the plants, as well as in the structural and evolutionary characteristics of the enzymes that catalyze its synthesis. The role of plant hormones in the induction of GB synthesis also begins to be clearer, as well as the impact that the synthesis of GB has on the ethylene and polyamine synthesis pathways. There are still important aspects of the GB synthesis that need to be defined to increase agricultural productivity through plants with the ability to synthesize GB.  Photosynthesis requires all proteins involved in H2O hydrolysis and in the transport of electrons and protons to remain functional, just as the enzymes that participate in ATP synthesis and carbon skeletal synthesis, as well as the chloroplast and thylakoid membranes. However, GB synthesis requires the availability of choline whose synthesis demands a high content of SAM, methionine, and ethanolamine. All these points must be taken into account for the improvement or genetic engineering of plants that synthesize and accumulate GB.

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Exogenous Glycinebetaine-Mediated Modulation of Abiotic Stress Tolerance in Plants: Possible Mechanisms Tianpeng Zhang and Xinghong Yang

1  Introduction Because of their sessile character, in the natural environment, plants are most frequently subjected to various types of abiotic stresses, such as salinity, drought, flooding, extreme temperatures, nutrient deficiency, heavy metals and high light intensities, all of which frequently exhibit decreased vegetative growth and negative impacts on crop production and reproductive capabilities (Tuteja et  al. 2011; Kurepin et  al. 2015; Kumar et  al. 2017). One of the best-documented and most important abiotic stress-responsive mechanisms in plants is the biosynthesis and accumulation of compatible solutes (Kumar et al. 2017), such as proline, glycinebetaine, trehalose and polyols (Khan et al. 2009; Jewell et al. 2010; Giri 2011; Kumar and Khare 2015). Glycinebetaine (GB), a fully N-methyl-substituted derivative of glycine found in a large variety of microorganisms, higher plants, and animals, is one of the best-studied compatible solutes that enables plants to tolerate abiotic stress (e.g. Rhodes and Hanson 1993; Chen and Murata 2002, 2008, 2011; Takabe et al. 2006; Masood et al. 2016). GB belongs to a group of compounds collectively known as ‘compatible solutes’, small organic metabolites that are very soluble in water and nontoxic at high concentrations. Both the exogenous application and the genetically engineered biosynthesis of GB increase the tolerance of plants to abiotic stress (Chen and Murata 2002, 2008, 2011). The exogenous application of GB can improve the tolerance of numerous plant species to various types of abiotic stresses, and it can enhance subsequent growth and yield. The GB applied to roots is usually taken up and accumulated in the cytosol, and only a small amount is translocated to chloroplasts. When applied to leaves, GB is translocated to meristematic tissues, in particular, flower buds and shoot T. Zhang · X. Yang (*) College of Life Science, State Key Laboratory of Crop Biology, Shandong Key Laboratory of Crop Biology, Shandong Agricultural University, Taian, China e-mail: [email protected] © Springer Nature Switzerland AG 2019 M. A. Hossain et al. (eds.), Osmoprotectant-Mediated Abiotic Stress Tolerance in Plants, https://doi.org/10.1007/978-3-030-27423-8_6

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a­ pices, and then translocated to actively growing and expanding tissues (Mäkelä et al. 1996; Park et al. 2006). In plants, even if GB is applied to old or mature tissues, this solute reallocates to young actively growing tissues, where its protective functions are mainly required (Ladyman et al. 1980; Annunziata et al. 2019). Due to the beneficial effects of GB, numerous experiments on the exogenous application of this compatible compound on low accumulator and non-accumulator plant species have been performed (Annunziata et  al. 2019). In this chapter, we summarize and discuss the current understanding of the physiological and molecular mechanisms of exogenous GB, including the regulation of reactive oxygen species (ROS) scavenging and detoxification under stress, protection of the photosynthetic machinery, interactions and synergistic physiological effects of GB with plant hormones and metabolites and the induction of specific genes involved in stress tolerance. The future perspective of the exogenous application of GB is also discussed.

2  E  xogenous Glycinebetaine Enhances Abiotic Oxidative Stress Tolerance All forms of abiotic stress, including drought, salinity, heat, cold, nutrient deficiency, heavy metals, high light intensities and UV radiation, can cause an excessive accumulation of ROS, leading to various types of deterioration, irreparable dysfunction and cell death in plants (Ashraf 2009; Chen and Murata 2011; Ahmad et al. 2013; Kumar et al. 2017). At present, many studies have shown that the exogenous application of GB on plants enhances oxidative stress tolerance (e.g. Park et al. 2006; Hoque et al. 2007, 2008; Farooq et al. 2008a, b; Hossain et al. 2010, 2011a, b, 2014; Anjum et al. 2012; Hu et al. 2012; Hasanuzzaman et al. 2014; Yildirim et al. 2015; Kumar et al. 2017). Ma et al. (2004) found that exogenous GB application ameliorated the water status of and improved the antioxidant enzyme activities in water-stressed wheat (Triticum aestivum L.) seedlings. Moreover, in fine rice (Oryza sativa), the exogenous application of GB significantly enhanced drought tolerance by altering the level of ROS and malondialdehyde (MDA), increasing the activities of enzyme antioxidants and promoting seedling growth (Farooq et al. 2008b). Additionally, in two maize (Zea mays L.) cultivars, prolonged drought stress increased lipid peroxidation, whereas GB treatment significantly reduced oxidative damage, as indicated by lower MDA levels. Importantly, GB-treated plants maintained higher antioxidant enzyme activity than did non-GB-treated plants in the course of drought stress, which ultimately enhanced the growth and yield of maize (Anjum et al. 2012). Furthermore, Molla et al. (2014) also demonstrated that the exogenous application of GB resulted in a significant increase in the glutathione (GSH) content and maintenance of the high activities of glutathione S-transferase (GST) and glyoxalase I (Gly I) enzymes, with a simultaneous reduction in glutathione disulfide (GSSG) and hydrogen peroxide

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(H2O2) levels in lentil (Lens culinaris) seedlings compared to control plants under drought stress, indicating that exogenous GB application enhances drought stress tolerance by limiting H2O2 accumulation and increasing the activities of the antioxidant and glyoxalase systems (Molla et al. 2014). Additionally, the protective roles of GB in modulating cold-, heat- and salinity-­ induced oxidative stress tolerance have also been well documented in plants. Park et al. (2006) showed that after the exogenous application of GB on tomato (Solanum lycopersicum) plants, the level of catalase activity and expression of the catalase gene (CAT1) were higher, and the H2O2 levels were lower in GB-treated plants than in control plants during 2 days of chilling treatment, indicating that GB may participate in the induction of H2O2-detoxifying antioxidant systems, namely, enhanced catalase expression and catalase activity, when the plants were exposed to chilling stress (Park et  al. 2006). Moreover, seed treatments with GB in hybrid maize reduced membrane electrolyte leakage (EL) and maintained higher tissue water contents, antioxidant enzyme activities and carbohydrate metabolism (Farooq et al. 2008a). Furthermore, under cold stress, the exogenous application of GB showed a protective effect on tea buds by regulating the formation of methylglyoxal (MG) and lipid peroxidation and by activating or protecting some antioxidant and glyoxalase pathway enzymes (Kumar and Yadav 2009). Similar effects were also observed in plants under heat stress. Sorwong and Sakhonwasee (2015) reported that the foliar application of GB enhanced heat stress tolerance in marigold (Calendula officinalis) cultivars by reducing the levels of H2O2, superoxide and MDA, indicating that GB may be involved in the induction of ROS detoxification, thereby mitigating the effect of heat stress on marigolds. Similarly, salinity stress inhibited the growth and development of most plants as a result of the overproduction of ROS, whereas exogenous GB ameliorated the detrimental effect of salinity stress on plants. Hoque et al. (2007) revealed that exogenous GB enhances salinity-induced oxidative stress tolerance in cultured tobacco (Nicotiana tabacum) (BY-2) cells by modulating the activities of ascorbate-­ glutathione (AsA-GSH) cycle enzymes and GST, glutathione peroxidase (GPX) and glyoxalase system enzymes activities and reducing protein oxidation (Hoque et  al. 2008). In addition, compared with control plants, in mung bean seedlings under salinity stress, the exogenous application of GB resulted in a conspicuous increase in the GSH content and the maintenance of a high glutathione redox state and higher activities of correlative enzymes involved in the ROS and methylglyoxal (MG) detoxification system, with a simultaneous decrease in the GSSG content and the levels of H2O2 and lipid peroxidation, suggesting that GB provides a protective action against salt-induced oxidative damage by activating antioxidant defence and MG detoxification systems and reducing the levels of H2O2 and lipid peroxidation (Hossain and Fujita 2010). Moreover, Nawaz and Ashraf (2010) found that compared with control maize plants, in two maize genotypes, the exogenous application of GB, as a modulator of salt tolerance, prominently enhanced the activities of superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD). Furthermore, compared with control plants, in perennial ryegrass (Lolium perenne) under salinity stress, the exogenous application of GB enhances salinity stress tolerance by

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r­educing the content of EL, MDA, and proline and increasing the vertical shoot growth rate (VSGR), relative water content (RWC), relative transpiration rate (Tr), chlorophyll (Chl) content and activities of SOD, CAT and ascorbate peroxidase (APX) (Hu et al. 2012). Recently, Kotb and Elhamahmy (2014) showed that longterm exogenous GB application at a suitable concentration (50 mM) on bread wheat under saline soil conditions significantly increased enzymatic antioxidant activities, total chlorophylls, leaf osmotic potential and the K+ contents in leaves and grain, thereby alleviating the oxidative stress damage of salinity stress, reflected by improving the growth and productivity of bread wheat plants. In addition, Hasanuzzaman et  al. (2014) found that the exogenous application of GB on rice seedlings enhanced salinity-induced oxidative stress tolerance by the upregulation of the ROS and MG detoxification pathways. Similarly, in lettuce (Lactuca sativa L.) plants, exogenous foliar applications of GB mitigated the deleterious effects of salt stress by reducing membrane permeability and the MDA and H2O2 content (Yildirim et al. 2015). The protective roles of GB have also been reported in plants subjected to nitrogen deficiency and cadmium (Cd) stress. Under nitrogen stress conditions, the exogenous application of GB was beneficial for improving the endogenous nitrogen status (Bowman and Rohringer 1970) and thereby enhancing photosynthesis and activating the antioxidant defence system in plants (Ashraf and Foolad 2007; Hoque et  al. 2008). Additionally, the exogenous application of varying doses of GB on maize plants resulted in a significant decrease in lipid peroxidation and the intercellular CO2 concentration (Ci), while an increase in the content of leaf total nitrogen and endogenous GB, net photosynthetic rate (Pn), and SOD, CAT, phosphoenolpyruvate carboxylase (PEPCase) and ribulose-1,5-bisphosphate carboxylase (RuBPCase) activities were observed under nitrogen stress (Zhang et al. 2014). Cd is a highly toxic environmental pollutant that can produce excessive ROS, resulting in cellular damage through the oxidation of membrane lipids, proteins and nucleic acids (Flora 2009; De Maria et  al. 2013; Lou et  al. 2015). Nevertheless, many researchers have demonstrated that exogenous GB ameliorates the adverse effect of Cd stress on plants. Hossain et al. (2010) showed that compared to control plants, the exogenous application of GB on mung bean (Vigna radiata L.) seedlings enhanced Cd tolerance by decreasing H2O2 and MDA levels and enhancing the activities of the relative enzymes involved in ROS and MG detoxification systems. Moreover, Duman et  al. (2011) concluded that the use of exogenous GB on an aquatic plant (Lemna gibba L.) relieved the deleterious effects of Cd stress by reducing both ROS and MDA levels, as well as enhancing photosynthetic activity, endogenous proline accumulation and antioxidant enzyme activities. Furthermore, compared with control plants, exogenous applications of GB on perennial ryegrass resulted in alleviating the detrimental effect of Cd stress by elevating SOD, CAT and POD activities and higher stress-responsive gene expression (Lou et al. 2015). From the above reports, it has become clear that GB performs a pivotal function in maintaining ROS levels by modulating the activities of correlative enzymes involved in ROS scavenging and detoxification and the glyoxalase system under various abiotic stresses.

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3  E  xogenous Glycinebetaine Protects Photosynthetic Machinery Under Abiotic Stress One of the physiological processes greatly affected by abiotic stress in plants is photosynthesis, and within the photosynthetic machinery, photosystem II (PSII) is the most vulnerable and crucial component that bears the brunt of abiotic stress (Nishiyama et al. 2006; Takahashi and Murata 2008; Nishiyama and Murata 2014; Gururani et al. 2015). Under no-stress conditions, exogenous glycinebetaine can improve the growth, CO2 assimilation and PSII photochemistry of maize plants, and the enhanced CO2 assimilation rate may be explained by the increased stomatal conductance (Yang and Lu 2006). It is now evident that exogenous glycinebetaine can also play a pivotal role in protecting the photosynthetic machinery in plants under various stressful conditions, which is considered to be one of the major mechanisms of attaining relief from abiotic stress (Chen and Murata 2011; Masood et al. 2016; Kurepin et al. 2017). Exogenous GB application improves CO2 assimilation under drought stress (Mäkelä et al. 1998, 1999; Xing and Rajashekar 1999) and salinity stress (Mäkelä et al. 1998, 1999; Lopez et al. 2002). The exogenous application of GB increased the relative area of starch granules in salt-stressed tomato leaflets and the relative area of plastoglobuli in GB-treated tomato plants under drought stress (Mäkelä et al. 2000). Furthermore, the application of GB on spinach leaves alleviated the photodamage of photosystem I (PSI) submembrane particles by minimizing the alteration in photochemical activity and chlorophyll-protein complexes under cold stress conditions (Rajagopal and Carpentier 2003). Foliar-applied GB also prevented photoinhibition in wheat under freezing (Allard et al. 1998) and drought stresses (Ma et al. 2006). Yang and Lu (2005) observed that the exogenous application of GB on maize plants improved photosynthesis by improving stomatal conductance and PSII efficiency. Similarly, in salt-stressed wheat plants, the application of GB mitigated the adverse effects on photosynthetic capacity by favouring the net CO2 fixation rate, increasing stomatal conductance and protecting the photosynthetic pigments in wheat cultivars (Raza et al. 2006). In another study, the foliar application of GB increased chlorophyll content, gas exchange and photosynthesis, alleviated the deleterious effect of drought on Hill reaction activities and improved the modified lipid composition of the thylakoid membranes in drought-stressed wheat cultivars (Zhao et al. 2007). When tobacco is subjected to low-temperature stress, the exogenous application of GB to plant roots could protect violaxanthin de-epoxidase and enhance non-radiative energy dissipation (NPQ), thereby improving the function of the thylakoid membrane (Wang et al. 2008). Moreover, pretreating rice plants with GB maintained a higher net photosynthetic rate and CO2 assimilation rate compared with those of control plants during drought stress (Farooq et  al. 2008b). Analogously, foliar-applied GB maintained water-use efficiency and pigments and increased plant height and the net photosynthetic rate when rice plants were exposed to salt stress (Cha-um and Kirdmanee 2010). Under heat stress, Oukarroum et al. (2012) reported that the foliar a­ pplication

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of GB on barley (Hordeum vulgare L.) plants mitigated thermal stress by protecting the oxygen-evolving complex and increasing the energy connectivity between the PSII antennae to increase the stability of the system PSII, thereby reinforcing the heat tolerance in GB-treated plants. In salt-stressed canola plants, foliar-­applied GB improved the water-use efficiency, photosynthetic CO2 fixation and stomatal conductance while protecting the oxygen-evolving centre of PSII and maintaining the activity of PSII (Athar et al. 2015). Furthermore, Gupta and Thind (2015) found that the exogenous application of GB on bread wheat plants prominently improved their photosynthetic performance due to more utilization of glutathione and high levels of ascorbic acid in wheat flag leaves under drought stress, indicating the role of nonenzymatic antioxidants in sustaining photosynthetic efficiency and yield stability under prolonged field drought stress conditions. Stepien et al. (2016) clearly demonstrated that the foliar application of exogenous GB could significantly mitigate the adverse effects of aluminium (Al) stress in cucumber (Cucumis sativus L. cv. Wisconsin) seedlings by protecting the photosynthetic apparatus components, leading to improved electron transport, gas exchange and enzymatic CO2 fixation. The exogenous application of GB plays a role in protecting the photosynthetic machinery in plants via improving the CO2 assimilation rate and chlorophyll content, as well as ameliorating the negative effect of photodamage and maintaining thylakoid membrane stabilization during various types of abiotic stresses.

4  I nteractions of Exogenous Glycinebetaine with Plant Hormones and Metabolites Under Abiotic Stress During the life span of plants, plant hormones are synthesized in very minute quantities, and these compounds regulate the development and growth of plants and play pivotal roles under various types of abiotic stresses (Masood et al. 2012, 2016; Khan et al. 2013, 2015; Khan and Khan 2014; Asgher et al. 2014, 2015). Many scientists have suggested that the interactions of exogenous GB, plant hormones, and metabolites can be beneficial to plants in abiotic stress tolerance (Yang et  al. 2012; Aldesuquy et al. 2012; Yildirim et al. 2015; Gupta and Thind 2019). The foliar application of either GB or abscisic acid (ABA) on creeping bentgrass (Agrostis stolonifera) and Kentucky bluegrass (Poa pratensis) similarly suppresses membrane EL and the accumulation of MDA and increases the activities of APX, POD, and SOD during prolonged periods of drought or salinity stress, indicating that the foliar application of ABA or GB could mitigate physiological damage in turfgrass under drought or salt stress (Yang et al. 2012). Similarly, in a recent study of wheat under water deficit conditions, the application of either salicylic acid (SA) or GB similarly increased grain yield; nevertheless, their co-application was more pronounced than the application of either applied alone due to the repairing effect of the provided chemicals on the growth and metabolism of wheat plants under drought stress (Aldesuquy et al. 2012).

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Yildirim et al. (2015) reported that compared to control plants, the foliar application of GB on lettuce plants mitigated the deleterious effect of salt stress by alleviating stomatal conductance, water status, plant nutrient uptake and soluble sugar content and elevating the concentrations of gibberellin (GA), SA and indole acetic acid (IAA) . Additionally, another study demonstrated that the foliar application of 100 mM GB on 19 wheat genotypes resulted in a higher total soluble sugar content under drought stress, whereas the starch content was reduced in GB-treated plants during anthesis. Furthermore, GB application also led to a decline in the activity of leaf sucrose phosphate synthase and sucrose synthase at both tillering and anthesis stages, confirming that exogenous applications of GB could alter the levels of the various sugar components that coordinate the drought response of selected wheat genotypes, resulting in grain yield benefit under prolonged field drought stress (Gupta and Thind 2019).

5  E  xogenous Glycinebetaine Induces Specific Gene Expression GB in the micromolar range, either after the uptake of exogenous GB in plants or as a result of its genetically engineered synthesis, can confer tolerance to several types of stresses (Einset et al. 2007; Chen and Murata 2008, 2011). Allard et al. (1998) demonstrated that the exogenous application of GB enhanced the freezing tolerance of wheat plants. Immunoblot analysis revealed that WCOR410, a low-temperature-inducible protein, was accumulated in the presence of GB, and the ultimate level depended on the concentration of GB.  Similarly, northern blotting analysis also illustrated that GB treatment resulted in the induction of a subset of low-temperature-responsive genes, such as WCOR410 and WCOR413, indicating that GB elevated the freezing tolerance of plants by inducing the expression of low-temperature-responsive genes. In tomato plants, after the exogenous application of GB, the level of catalase activity and expression of the catalase gene (CAT1) were higher than those in control plants during 2 days of chilling treatment, suggesting that GB may increase catalase expression and catalase activity when the plants were exposed to chilling stress (Park et al. 2006). Additionally, the exogenous application of GB on both the leaves and roots of Arabidopsis thaliana resulted in the upregulated expression of the genes in roots, including those for membrane-­ trafficking components, NADP-dependent ferric reductase, transcription factors and ROS-scavenging enzymes, suggesting that GB may confer chilling tolerance to plants by activating the expression of a number of stress-tolerance genes (Einset et al. 2007, 2008). Furthermore, compared to controls, exogenous GB application on tomato seeds under high-temperature stress resulted in elevated levels of heat-­ shock genes, such as MT-sHSP, HSP70 and HSC70, and accumulated HSP70 protein (Li et al. 2011).

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Consequently, based on previous research on the concentrations of GB and testimony of its effects on gene expression, it is reasonable to postulate that, at least in part, the effects of GB might be ascribed to the induction and activation of the expression of stress-tolerance genes (Einset et  al. 2007; Chen and Murata 2008, 2011). Further studies on the identification of GB-inducible genes and the functions of their products will advance our understanding of the GB-enhanced tolerance in plants under abiotic stress.

6  Conclusion and Future Perspectives The exogenous application of GB can improve the tolerance of numerous plant species to various types of abiotic stresses, and due to its multiple functions, the possible mechanisms of the exogenous GB-induced tolerance of plants to various types of abiotic stresses include but are not limited to (i) the regulation of ROS scavenging and detoxification under stress, (ii) the protection of the photosynthetic machinery, (iii) interactions with plant hormones and metabolites and (iv) the induction of specific genes whose products are involved in stress tolerance. Although research on the improvement of plant resistance by GB has made great progress, more in-depth studies are needed to reveal subtler regulatory roles for GB in modulating abiotic stress tolerance. For instance, why specific genes are direct targets of GB and whether GB could modulate the tolerance of plants under biotic stress, as well as how to use GB more effectively to develop crops with enhanced tolerance to multiple environmental stresses in the field. Acknowledgements  This work was supported by the National Natural Science Foundation of China (31470341, 31870216) and the State Key Basic Research and Development Plan of China (2015CB150105).

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Rajagopal S, Carpentier R (2003) Retardation of photo-induced changes in photosystem I submembrane particles by glycinebetaine and sucrose. Photosynth Res 78:77–85 Raza SH, Athar H, Ashraf M (2006) Influence of exogenously applied glycinebetaine on the photosynthetic capacity of two differently adapted wheat cultivars under salt stress. Pak J  Bot 38:341–351 Rhodes D, Hanson AD (1993) Quaternary ammonium and tertiary sulfonium compounds in higher plants. Annu Rev Plant Physiol Mol Biol 44:357–384 Sorwong A, Sakhonwasee S (2015) Foliar application of glycinebetaine mitigates the effect of heat stress in three marigold (Tagetes erecta) cultivars. Hort J 84:161–171 Stepien P, Gediga K, Piszcz U, Karmowska K (2016) Effects of the exogenous glycinebetaine on photosynthetic apparatus in cucumber leaves challenging Al stress. In Proceedings of the 18th International Conference on Heavy Metals in the Environment Takabe T, Rai V, Hibino T (2006) Metabolic engineering of glycinebetaine. In: Rai A, Takabe T (eds) Abiotic stress tolerance in plants: toward the improvement of global environment and food. Springer, Dordrecht, pp 137–151 Takahashi S, Murata N (2008) How do environmental stresses accelerate photoinhibition? Trends Plant Sci 13:178–182 Tuteja N, Gill SS, Tuteja R (2011) Plant responses to abiotic stresses: shedding light on salt drought cold heavy metal stress. In: Tuteja N, Gill SS, Tuteja R (eds) Omics and plant abiotic stress tolerance. Bentham Science Publishers Ltd., Beijing, pp 39–64 Wang C, Ma XL, Hui Z, Wang W (2008) Glycine betaine improves thylakoid membrane function of tobacco leaves under low-temperature stress. Photosynthetica 46:400–409 Xing W, Rajashekar CB (1999) Alleviation of water stress in beans by exogenous glycine betaine. Plant Sci 148:185–195 Yang XH, Lu CM (2005) Photosynthesis is improved by exogenous glycinebetaine in salt-stressed maize plants. Physiol Plant 124:343–352 Yang XH, Lu CM (2006) Effects of exogenous glycinebetaine on growth, CO2 assimilation, and photosystem II photochemistry of maize plants. Physiol Plant 127:593–602 Yang Z, Yu J, Merewitz E, Huang B (2012) Differential effects of abscisic acid and glycine betaine on physiological responses to drought and salinity stress for two perennial grass species. J Am Soc Hortic Sci 137:96–106 Yildirim E, Ekinci M, Turan M, Dursun A, Kul R, Parlakova F (2015) Roles of glycinebetaine in mitigating deleterious effect of salt stress on lettuce (Lactuca sativa L.). Arch Agron Soil Sci 61:1673–1689 Zhang LX, Lai JH, Gao M, Ashraf M (2014) Exogenous glycinebetaine and humic acid improve growth, nitrogen status, photosynthesis, and antioxidant defense system and confer tolerance to nitrogen stress in maize seedlings. J Plant Interact 9:159–166 Zhao XX, Ma QQ, Liang C, Fang Y, Wang YQ, Wang W (2007) Effect of glycinebetaine on function of thylakoid membranes in wheat flag leaves under drought stress. Biol Plant 51:584–588

Roles of Endogenous Glycinebetaine in Plant Abiotic Stress Responses Pirjo S. A. Mäkelä, Kari Jokinen, and Kristiina Himanen

1  Introduction Abiotic stresses, the most common of which are water deficit (Boyer 1982) followed by water logging, high and low temperature, and salinity, annually restrict not only plant growth but also global crop yield. It has been estimated that during the period 1961–2014, drought and heat spells caused a global production loss of US$ 237 billion (Mehrabi and Ramankutty 2017). According to an IPCC report in 2017, occurrences and damages caused by weather extremes will increase in the future due to climate change. The impact of global warming differs regionally, and it is envisaged that developing countries will be affected to a greater extent, resulting in increased food insecurity (Rosenzweig and Parry 1994). Changes in ambient temperature occur more rapidly than changes in stress factors such as water deficit and salinity. Furthermore, temperature extremes aggravate the adverse effects of other stresses, including water deficit and salinity, on crop production and quality. For example, heat stress adversely affects grain quality and final crop yield in 40% of the global irrigated wheat growing area (Fischer and Byerlee 1991). Cold stress, although seasonal, has some similarities to water deficit. As water freezes, it creates concentrated solutions of solutes, thereby subjecting plants to a shortage of liquid water (Sakai and Larcher 1987). Global agricultural land area is approximately 4.86 billion ha (FAO 2019). It is estimated that less than 10% of the world’s agricultural land may be free of major environmental stresses (Dudal 1976). As much as 45% of agricultural land is subject to different kinds of water deficit, and 38% of the world’s human population resides

P. S. A. Mäkelä (*) · K. Himanen Department of Agricultural Sciences, University of Helsinki, Helsinki, Finland e-mail: [email protected] K. Jokinen Luke Natural Resources Institute Finland, Helsinki, Finland © Springer Nature Switzerland AG 2019 M. A. Hossain et al. (eds.), Osmoprotectant-Mediated Abiotic Stress Tolerance in Plants, https://doi.org/10.1007/978-3-030-27423-8_7

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in those areas (Bot et al. 2000). In relation, the proportion of irrigated field area is approximately 20%, concentrating mostly in Asia (271 Mha) (FAO 2019). In 2015, approximately 510 Mha of total land area, and 19.5% of irrigated agricultural land, was considered saline (FAO and ITPS 2015). Each year a further 2 million ha (about 1%) of the world’s agricultural land deteriorates due to salinity, leading to reduced or no crop productivity (reviewed in Ashraf and Foolad 2007). Apart from irrigation, other major contributors to the increasing area of saline soils are poor management practices, low precipitation, high surface evaporation, and weathering of native rocks. However, secondary salinization causes further problems as productive agricultural land is becoming unsuitable for cultivation due to low quality of irrigation water (Munns 2010). To minimize the effects of abiotic stresses on crop yield, solutions have been actively sought and investigated. These include improving crop tolerance by means of crop management – for example, by the utilization of exogenous and endogenous compounds, including glycinebetaine (GB) – as well as by traditional and molecular plant breeding. Many of the traits resulting in increased abiotic stress tolerance are an interplay of several genes, which make them difficult to modify via traditional and modern plant breeding. Moreover, different abiotic stress factors may provoke osmotic stress, oxidative stress, and protein denaturation in plants. These lead to similar cellular adaptive responses in plants, such as accumulation of compatible solutes, induction of stress proteins, and acceleration of reactive oxygen species (ROS)-scavenging systems (Zhu 2002). Further complexity is associated with phenology as well as species- and cultivar-specific responses to abiotic stresses. Exposure to a single abiotic stress factor can lead to plants obtaining tolerance against a wide range of future abiotic stress events, which is referred to as priming, acclimation, conditioning, hardening, or cross-stress tolerance (Li and Gong 2011; Walter et al. 2013; Antoniou et al. 2016). This involves a memory phase that separates the primary stress event from the following stress events (Bäurle 2016). During the primary stress phase, changes take place at the physiological, biochemical, molecular, and epigenetic levels. These changes can be transient or maintained throughout the lifetime of a plant and, in some cases, can even be inherited by subsequent generations, for example, in seeds (Mauch-Mani et al. 2017). Over the last 10 years, significant steps have been taken in understanding the biology of osmolytes and especially GB in plants. New associations and insights between GB, genes, and ROS and plant hormones, for example, have been discovered. This chapter provides an update on the most recent research related to osmolytes with special emphasis on endogenous GB and on the transgenesis approach for GB.

2  Osmoprotectants in Plants Under Stress Conditions Identifying the mechanisms involved in plant adaptation to multiple abiotic stresses such as drought, salinity, nutrient imbalances, extreme temperatures, and light is essential for breeding new crop varieties. In addition, understanding the role of

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f­ actors resulting in increased plant abiotic stress tolerance may assist in developing novel management practices. In this respect, the early dispersion of stress signals, the successive activation of stress-responsive pathways, and finally the responses of plant yield formation are of primary interest to plant biologists, breeders, and agronomists. Within the last few years, several comprehensive reviews on plant stress and the roles of osmoprotectants in improving plant stress tolerance have been published (Singh et al. 2015; Verma et al. 2016; Zhu 2016; Hossain et al. 2018). Here we summarize the increasing amount of literature on osmoprotection in relation to plant stress tolerance. In response to different stresses, plants have developed several mechanisms that involve changes at the morphological, physiological, and molecular level. The sensing of various stresses initiates several complex signaling pathways in plants (Hossain et al. 2018 and cited literature). At first, plants recognize the external stress by using multiple sensors present in the plasma membrane or cell wall. Early signaling events usually include changes to intracellular calcium (Ca2+) concentration followed by an increase in secondary messengers, like reactive nitrogen species (such as nitric oxide), ROS (such as hydrogen peroxide), reactive carbonyl species (such as methylglyoxal), cytosolic calcium ions (Ca2+), hydrogen sulfide, and kinases. In addition, groups of plant hormones (auxins, gibberellins, cytokinins, abscisic acid, ethylene, salicylic acid, jasmonates, brassinosteroids, and strigolactones) participate in plant defense responses (Kurepin et al. 2015; Verma et al. 2016; Xu et al. 2018). Their signaling pathways are interconnected to assist the generation of an efficient stress response. Currently, the fundamental molecules in plant cells and tissues for the acquisition of stress tolerance are considered to be plant hormones. The compounds collaborate with each other to regulate gene expression, resulting in the modification of membrane rigidity and fluidity, changes in the levels of ROS and methylglyoxal detoxifying enzymatic and nonenzymatic antioxidants, and an increase in the synthesis of osmolytes and stress-related proteins. The complex set of responses at the cellular level is also considered to lead to the cross-stress tolerance discussed recently by Hossain et al. (2018). To improve plant tolerance to abiotic stresses such as excess light, water deficit, extreme environmental temperatures, or salinity, the osmotic potential of plant cells must increase. This occurs by the enhancement of cell solutes (reviewed in Singh et al. 2015, Stadmiller et al. 2017), which can be inorganic or organic. In general, inorganic solutes are energetically less expensive but may interfere with metabolism. Organic solutes are energetically more expensive but usually have only minor or no effect on metabolism. In addition, salts in the soil negatively affect water absorption by roots and may result in ion toxicity due to the accumulation of sodium (Na+) and chloride (Cl−) ions in the plant. Under stress conditions, a significant enhancement of extracellular salt concentration results in water efflux, which decreases cell volume and increases the concentration of macromolecules inside the cytoplasm. Accordingly, an increase of common solutes alone, such as organic acids and inorganic ions, may lead to ionic and nutritional imbalance and may prevent the activity of important plant enzymes. Therefore, the localization of common solutes

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is mainly in the vacuoles, where their increased concentration does not lower the metabolic activity of the cell. In contrast to common solutes, plants can produce different types of compatible organic solutes in response to various stresses (Burg and Ferraris 2008, Singh et al. 2015 and cited literature). In many cases, these solutes seem to accumulate in low concentrations when considered from the whole-plant perspective. However, they typically accumulate in the cytoplasm with high concentrations and do not adversely affect metabolic activity in the cell. Compatible solutes are highly soluble compounds, usually nontoxic at high cellular concentrations, and typically have low molecular weight. Compatible solutes protect plant cells and tissues from stress through several ways. These include contributing to cellular osmotic adjustment, protecting membrane integrity, stabilizing enzymes and proteins, and the detoxification of ROS (Burg and Ferraris 2008 and cited literature, Stadmiller et al. 2017, Hossain et al. 2018 and cited literature). Some compatible solutes can also act as antioxidants. Moreover, they may play a role in stress tolerance by regulating gene replication and transcription (reviewed in Giri 2011 and Hossain et al. 2018). Because some compatible solutes also protect cellular components from dehydration injury, they are called osmoprotectants. Recently, Singh et  al. (2015) categorized osmoprotectants into three different groups: osmoprotectants containing ammonium compounds (polyamines, GB, β-alanine betaine, dimethylsulfonio propionate, and choline-O-sulfate), osmoprotectants containing sugars and sugar alcohols (trehalose, fructan, mannitol, D-ononitol, and sorbitol), and osmoprotectants containing amino acids (proline and ectoine). The specific role of different osmoprotectants in plant metabolism and stress tolerance has recently been reviewed by Singh et al. (2015) and Hossain et al. (2018). The majority of osmoprotectants avoid participation in biochemical reactions and are stored in the cytosol. In addition to the conventional osmoprotective role of the compatible solutes, osmoprotectants also detoxify the adverse impacts of stress (e.g., from salinity, water deficit, and cold stress) through two different mechanisms. The first mechanism improves the antioxidant defense system, whereas the second one improves the sustainability of ion homeostasis (reviewed in Singh et al. 2015). In terms of the antioxidant defense system, several studies (Singh et al. 2015; Hossain et al. 2018; Wei et al. 2017; Razavi et al. 2018; Rady et al. 2018) have indicated that under various stress circumstances, osmoprotectants such as polyamines, GB, sugar alcohols, and proline upregulate antioxidant enzyme activities and increase the concentration of nonenzymatic antioxidants to reduce the adverse effects of oxidative stress. Well-known antioxidant enzymes include superoxide dismutase, peroxidase, catalase, and ascorbate peroxidase and some other nonenzymatic low-molecular-weight antioxidants, like glutathione, ascorbate, and carotenoids. Both enzymes and antioxidants have the capability of providing protection via reducing the toxicity of ROS.  In a series of detoxifying mechanisms, plants enhance the production of the metalloenzyme superoxide dismutase, which is responsible for the conversion of superoxide to hydrogen peroxide. The b­ reakdown

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of hydrogen peroxide is then catalyzed by CAT and peroxidases. The modulation of the glyoxalase (Gly 1 and Gly 2) and antioxidant defense systems by heat, cold, or osmo-priming has also shown the importance of osmoprotectants for induced crossstress tolerance. Accordingly, osmoprotectants are promising compounds for improving crop abiotic stress tolerance through the enhancement of the antioxidant system. During stress caused by salinity and water deficit, the sustainability of ion homeostasis is affected by the accumulation of osmoprotectants providing osmotic adjustment via specific ion exchange activity (Singh et al. 2015 and cited literature, Wei et al. 2017). Under salinity stress, the most common effect is a reduction of plant growth due to specific ion toxicity, such as from Na+ and Cl−. This also reduces the uptake of essential nutrients like phosphorus (P), potassium (K+), nitrogen (N), and calcium (Ca). The toxic ions negatively impact intracellular K+ influx, reducing the uptake of K+ by cells. Some osmoprotectants may maintain low cytoplasmic Na+ concentration in the cell by decreasing K+ efflux and increasing Na+ efflux, resulting in an optimal K+/Na+ ratio. In addition, osmoprotectants may increase efflux of Na+ from the roots to the environment, leading to less Na+ transfer to plant leaves. Thus, it has been proposed that some osmoprotectants also regulate ion channels and transporters in plants (Wei et al. 2017).

3  E  ndogenous Glycinebetaine and Plant Abiotic Stress Responses GB is usually classified as an osmolyte, an osmoprotectant, and a compatible solute. GB could also be regarded as a biostimulant, i.e., a non-fertilizer compound applied in low concentrations that promotes either plant growth, abiotic stress tolerance, or crop quality. Osmolytes and osmoprotectants have gained increased attention over the last two decades. A search in Google Scholar for articles related to GB found 338 published before 1979 and 25,800 published in the decade up to February 2019 (Fig. 1). GB (2-N,N,N-trimethylammonio acetate or N,N′,N″-trimethylglycine), earlier known as lycine or oxyneurine, is a quaternary amine derived from glycine with an average molecular mass of 117.15 (Fig. 2). Due to its zwitterionic nature, it is highly soluble and has low viscosity (Yancey et al. 1982; Yancey 2005). GB is a nontoxic, colorless, tasteless, and odorless compound that accumulates in many plant species, especially in halophytes, when grown under abiotic stresses (see comprehensive list of plant species available in Paleg and Aspinall (1981)). In higher plants, GB is synthesized as a result of the two-step oxidation of choline (Cromwell and Rennie 1954). The first step is catalyzed by choline monooxygenase (CMO), and the second step is mediated by betaine aldehyde dehydrogenase (BADH). The gene expression of CMO and BADH is induced by salinity, water deficit, and temperature stresses in various organisms (for a review, see Hashemi et al. (2018)). Under osmotic stress, changes of turgor may initiate the signal trans-

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Fig. 1  The number of scientific articles containing the word “glycinebetaine” published in different decades based on a search in Google Scholar

Fig. 2  Chemical structure of GB. GB has a zwitterionic nature as it possesses both negative (−) and positive (+) charges

duction (Xu et  al. 2018 and cited literature). Accordingly, under abiotic stresses, increased ion concentration (e.g., Ca2+ and Na+) can be detected by mitogen-­ activated protein kinase (MAPK), phospholipase D, and some proteins bound to the plasma membrane. MAPK signaling pathways transduce the stress signals which subsequently activate BADH and ROS-scavenging enzymes, such as peroxidase, catalase, superoxide dismutase, ascorbate peroxidase, and lipoxygenase. Finally, BADH accelerates the oxidation of betaine aldehyde to glycinebetaine. Within 24 h, GB is translocated via the phloem throughout the plant, especially to the youngest and developing plant parts (Mäkelä et al. 1996). BADH gene expression can also be regulated by abscisic acid (ABA) (Kurepin et al. 2015 and cited literature). Kurepin et al. (2015) suggested that the close interaction and synergistic physiological effects of GB and ABA, resulting in increased freezing tolerance and a dwarf phenotype, are the major factors leading to effective cold acclimation of higher plants. However, Xu et  al. (2018) concluded that the expression of BADH may also be ABA-independent. Instead, they proposed that jasmonate biosynthesis plays a dominant role in the activation of BADH and CMO under osmotic stress.

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3.1  Endogenous Glycinebetaine and Osmotic Stress Soil salinity is among the main abiotic stresses restricting crop production, and thus major efforts have been made to improve the salinity tolerance of crops. At first, the effect of soil salinity on plants is comparable to water deficit due to low water potential, and the effects of ion-specific toxicity only appear later, in the second phase (Munns 2010). Accumulation of osmolytes, such as GB, allows additional water uptake and therefore buffers the immediate effects of water deficit. While some crops, especially Amaranthaceae and Poaceae, accumulate GB, in the majority of cases the accumulated concentrations for the whole plant might not be physiologically significant. Red beet (Beta vulgaris L.) is salt tolerant and one of the crops which accumulate GB as a response to increasing cell Na+ concentration, among other triggers (Subbarao et al. 2001). In red beet subjected to salt stress, the leaf water content did not vary markedly even though the Na concentration increased up to 400 mol m−3 in the leaves and leaf osmotic potential increased. This was due to a simultaneous increase in GB concentration, contributing 50–60% to the leaf osmotic potential in the cytoplasm. Increasing GB concentration also correlates with maintenance of photosynthesis and chlorophyll fluorescence (Subbarao et al. 2001). According to Leigh et al. (1981), in red beet 26–84% of GB is localized in the cytoplasm, and the concentration in the cytoplasm varies between 46 and 467  mol  m−3, whereas the concentration in the vacuole ranges between 2.7 and 17.8 mol m−3. Furthermore, Robinson and Jones (1986) showed that in salt-stressed spinach (Spinacia oleracea L.), at least 40% of GB is localized in chloroplasts, contributing 36% of the leaf osmotic potential. Thus, when GB concentration is calculated according to cytoplasm volume, its physiological role becomes significant. Grumet and Hanson (1986) stated that GB has a marked role in osmoregulation of barley (Hordeum vulgare L.) by maintaining osmotic potential. Later, it was found that GB is the main compatible solute accumulating specifically in young barley leaves (Hattori et al. 2009). GB synthesis is localized in the vascular tissues of leaves and in the pericycle of roots. This is based on the finding that signal transcripts of BBD2 gene increased in the vascular parenchyma cells of leaves and in the root pericycle. BBD2, more abundant in barley, has a 2000-fold affinity for betaine aldehyde in comparison to BBD1. In durum wheat (Triticum durum Desf.), GB is one of the major osmolytes accumulating under prolonged salinity, accumulating especially in young leaves (Carillo et al. 2008). Interestingly, GB accumulation has been shown to correlate positively with glutamate synthase activity in young leaves, though it was independent of nitrogen nutrition of the plant. According to Khan et al. (2012), GB accumulation in salt-stressed bread wheat (Triticum aestivum L.) is linked to both increased salt tolerance and ethylene evolution. These changes are related to the maintenance of photosynthesis fluorescence and lower hydrogen peroxide content. Accumulation of GB can also be cultivar or genotype specific. In cereals, the species and cultivar differences in GB accumulation are marked. For example, some

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genotypes of sorghum (Sorghum bicolor (L.) Moench) and maize (Zea mays L.) accumulate GB, whereas others do not (Grote et  al. 1994; Saneoka et  al. 1995). However, even cereal cultivars that do not accumulate detectable concentrations of GB have active BADH and BADH protein in leaves (Ishitani et al. 1993). Peel et al. (2010) compared the GB metabolism in GB-accumulating and non-accumulating maize and sorghum. They concluded that GB deficiency in non-accumulating cereals could result either due to limited availability of choline or lack of choline transporter. The presence of genotypic differences in GB accumulation may explain at least partly the occurrence of stress-tolerant and stress-susceptible genotypes within individual plant species. Some legumes, including mung bean (Vigna radiata (L.) R. Wilczek), also accumulate GB as a response to abiotic stresses. Misra and Gupta (2005) showed a salt-­ tolerant mung bean cultivar accumulating a higher concentration of GB under salt treatment in comparison to a salt-sensitive cultivar. Similarly, chlorophyll remained higher in the salt-tolerant cultivar. Khan et al. (2014) found that under salinity, GB accumulation in mung beans was induced by salicylic acid, which increased methionine production and suppressed ethylene production, opposite to the results of their barley study (Khan et al. 2012). When salicylic acid inhibits ethylene production, the metabolite of methionine and precursor of ethylene, s-adenosyl methionine, donates a methyl group to GB synthesis and promotes GB synthesis.

3.2  Endogenous Glycinebetaine and Temperature Stress Yang et al. (1996) tested the high temperature (45 °C) tolerance of near-isogenic maize lines which differ in their ability to accumulate GB. The leaves of GB accumulators had less membrane damage, and the temperature threshold difference between the lines was 2 °C. Furthermore, the GB accumulators showed better thermostability of the PSII electron chain. These results indicate that GB might play a role in the protection of plasma membranes. At the other extreme, Kishitani et al. (1994) studied the role of GB on the freezing tolerance of barley leaves by using near-isogenic lines whose ability to accumulate GB ranges from 10 to 90 μmol g−1 DM. After acclimation at 5 °C and freezing at −5 °C, the youngest leaves with the highest GB concentration survived, whereas the oldest leaves with the lowest concentration of GB died. Thus, it was concluded that GB plays a marked role in cold acclimation against freezing injury in young barley leaves. Cooling is a useful storage method commonly employed to prolong postharvest life of plant produce. It reduces postharvest decay of tissues during transportation to distant markets and assures the availability of good quality produce to consumers for an extended period. However, many fruits and vegetables are chilling sensitive and highly vulnerable to chilling injury during cold storage at low temperatures, e.g., below 8 °C. The severe development of chilling injury decreases produce quality, for example, in appearance, texture, flavor, and nutrition. Unfavorable chilling

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temperature directly promotes membrane phase transition from fluid liquid crystalline to rigid solid gel, leading to a decline in the membrane selective permeability. In addition, chilling temperature as an oxidative stress factor indirectly promotes ROS accumulation, resulting in the peroxidation of unsaturated fatty acids in plant membranes. Recent reports, summarized here, indicate that GB is a useful molecule for reducing chilling injuries in several fruits. The mechanisms seem to be similar to those found in whole-plant studies and in their response to common stresses. Jin et al. (2015) studied the influence of low-temperature conditioning treatment (at 10 °C for 6 days) on chilling injury, GB concentration, and energy metabolism in loquat fruit (Eriobotrya japonica (Thunb.) Lindl) stored at 1 °C. Their results indicate that low-temperature conditioning treatment significantly reduces chilling injury, ion leakage, and malondialdehyde content in loquat fruit. BADH activity and endogenous GB content in loquats treated with low-temperature conditioning were significantly higher than in control fruit. Moreover, low-temperature conditioning treatment induced activities of energy metabolism-associated enzymes, including H+-adenosine triphosphatase, Ca2+-adenosine triphosphatase, succinic dehydrogenase, and cytochrome c oxidase. The low-temperature conditioning treatment clearly triggered higher levels of ATP content and energy charge, and together these results show that low-temperature conditioning may alleviate chilling injury and improve chilling tolerance of loquat fruit by enhancing endogenous GB accumulation and energy status. Yao et al. (2018) suggested that GB can ameliorate the chilling injury in zucchini (Cucurbita pepo L.) fruit. The effects of GB treatment were associated with an accumulation of proline and a reduction in lipid peroxidation. In addition, GB-treated fruit also showed lower levels of palmitic acid and stearic acid, and lower lipoxygenase and plant phospholipase D activities, but higher activity levels of enzymes related to proline metabolism. The gene expression and antioxidant enzyme activities of superoxide dismutase, catalase, and ascorbate peroxidase in GB-treated fruit were significantly higher than that of control fruit. Thus, GB could alleviate chilling injury in cold-stored zucchini fruit through improved antioxidant enzymatic mechanisms in addition to the involvement of fatty acid metabolism. Recently, Razavi et al. (2018) reported that in hawthorn (Crataegus monogyna Jacq.) fruits, GB applied by immersion for 15  min at 20  °C resulted in a steady increase of endogenous GB accumulation during storage at 1 °C for 20 days. This accumulation was then associated with delayed fruit pitting development. They also found that higher endogenous GB accumulation correlated with higher activity of antioxidant enzymes, such as superoxide dismutase, catalase, and ascorbate peroxidase, leading to lower buildup of hydrogen peroxide. In addition, fruits treated with GB exhibited significantly higher content of phenols, flavonoids, and anthocyanins, which was due to the higher activity of phenylalanine ammonia lyase enzyme. Furthermore, the observed higher ascorbic acid accumulation in GB-treated fruits resulted in higher 1,1-diphenyl-2-picrylhydrazyl-scavenging capacity during storage at 1 °C for 20 days. The authors propose that GB treatment is a useful strategy for attenuating chilling injury of hawthorn fruit due to lower ROS accumulation. Moreover, the application of GB could be favorable in terms of maintaining nutri-

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tional quality of hawthorn fruit because it increases the level of antioxidant molecules, beneficial for human health. Wang et al. (2019) also showed that GB could enhance the chilling tolerance of peach (Prunus persica (L.) Batsch) fruits through the regulation of phenolic and sugar metabolism, leading to the maintenance of high levels of individual phenolic and sucrose content.

4  G  lycinebetaine and Transgenesis Approaches to Improve Plant Stress Tolerance Plants cope with abiotic stresses by activating response pathways that result in redirection of resources from growth toward resistance. Abiotic stress tolerance is often manifested in the accumulation of protective enzymes and metabolites. Primary metabolites are conserved molecules required for normal growth and development, while secondary metabolites are related more to signaling and are more diverse among different species. Understanding metabolic fluxes in plant cells in response to many environmental factors requires genome-wide systems approaches. Plant metabolomics addresses the biochemistry and molecular mechanisms of plant responses to cope with osmotic stress. It combines sample separation by liquid or gas chromatography and the detection of metabolites based on their ion mass and charge. In general, metabolomic analysis is less dependent on genomic information than many other molecular omics studies, such as transcriptomics or proteomics. Therefore, this technology is accessible for a wide range of species. With regard to the accumulation of osmolytes, such as GB, plant species are recognized as GB accumulators or non-accumulators. Transgenesis has introduced the GB pathway into many non-accumulator species and increased GB levels in GB-accumulating species. In this chapter, we summarize the current understanding of the challenges in genetically engineering GB accumulation in plants.

4.1  Transgenesis for Improved GB Levels In plants, biosynthesis of GB is a simple two-step reaction cascade involving choline oxidation reaction by CMO followed by oxidation of the resulting BADH. In Escherichia coli, the BetA and BetB enzymes mediate these two reactions. The COD (Arthrobacter globiformis) and COX (Arthrobacter pascens) pathways represent prokaryotic choline oxidases that mediate direct conversion of choline to GB (Sakamoto and Murata 2001). Despite these straightforward reaction cascades, transgenesis approaches have proven challenging to optimize for obtaining physiologically relevant GB osmolyte levels. Transgenesis approaches in plant species lacking a functional GB biosynthesis pathway have utilized both prokaryotic and eukaryotic genes. Utilizing genes from a prokaryotic origin reduces considerations of translational and posttranslational modifications. Standard overexpression of one

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of the biosynthetic enzymes aims to increase levels of gene expression in the cell. Overexpression vectors usually harbor a 35S promoter and terminators together with antibiotic selection. Physiologically relevant levels for GB to act as an osmotic regulator range between tens of μM to hundreds of μM (Annunziata et al. 2019). GB accumulation at the level of 5 μmol g−1 DM, or down to 1 μmol g−1 FW, has also been suggested as promoting stress resistance as summarized in Khan et al. (2009) and Chen and Murata (2011). As stated earlier, this activity depends on the compartmentation of GB in cells. In tobacco, overexpression of E. coli BetA (CDH) alone or together with BetB (BADH) conferred the transgenic plants with increased resistance to salt stress compared to wild-type plants (Holmström et al. 2000). Overexpression resulted in functional enzymes and enhanced the plant’s ability to process betaine aldehyde, the toxic intermediate of the GB synthesis pathway. The GB levels, however, remained at a low level (40–80 nmol g−1 FW), suggesting that the stress-protective effect was not due to osmoregulation. Mild accumulation of GB might still be adequate to protect protein complexes and membranes, for example, in chloroplasts. Cotton cv. Luyuan890 has been engineered to constitutively overexpress the betA gene from E. coli (Lv et  al. 2007). In wild-type plants, the GB levels were already physiologically relevant, with high levels of approximately 100 μmol g−1 DM. The betA transgenic lines accumulated GB at over 130 μmol g−1 DM, and their drought resistance and physiological performance were analyzed. Four out of five of the lines were shown to perform better for maintenance of osmotic potential and relative water content. In overexpression approaches, codA from Arthrobacter globiformis has been most popular, although the resulting GB levels usually remain moderate (Khan et al. 2009; Chen and Murata 2011). In tomato (Solanum lycopersicum L.) transgenesis, codA from Arthrobacter globiformis was used to mediate direct choline conversion to GB, in contrast to two-step biosynthesis (Wei et  al. 2017; Khan et  al. 2009). Overexpression in tomato cv. Moneymaker resulted in L1, L2, and L3 lines with minor increases in GB accumulation of up to 2 μmol g−1 DM. Following NaCl treatment, GB accumulation reached 5–6 μmol g−1 DM and was shown during stress to increase photosynthetic rate and antioxidant enzyme activity and to reduce ROS accumulation (Wei et al. 2017). Changes in Na+/K+ ion balances were observed in the transgenic lines, resulting from increased Na+ exclusion and decreased K+ efflux. These effects were mediated through ion channel gene expression. It is proposed that GB could promote salt tolerance through regulation of the respective channels and transporters. In addition, GB may enhance antioxidant enzyme activities and thereby alleviate ROS responses and damage to photosynthesis in the leaves. Salt stress is known to impair photosynthesis, and it has been suggested that the positive impact of GB on photosynthesis results from better osmotic adjustment and prevention of stomatal closure (Lv et al. 2007). A second study on the tomato cv. Moneymaker codA transgenic lines (codA Arthrobacter globiformis) with relatively low GB accumulation (up to 2.5 μmol g−1 FW) addressed the role of GB in abiotic stress resulting from phosphate starvation (Li et al. 2019). The transgenics were able to maintain Pi/H+ co-transport, and the

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gene expression of the PHO regulon was also modified, and photosynthetic rates remained high. In the transgenic lines, growth was enhanced as indicated by increased fresh weight and shoot and root size, while stress responses such as anthocyanin accumulation were lower compared to wild type. Here, moderate GB accumulation mediated physiological and biochemical changes so that environmental adaptation processes were impacted. GB biosynthesis by COD/COX results in side product hydrogen peroxide accumulation, which operates in redox sensing, signaling, and regulation in eukaryotic cells (Sies 2017). In Arabidopsis (Arabidopsis thaliana L.) transformed with the codA gene for choline oxidase, accumulation of steady-state hydrogen peroxide was detected at the level of 960 nmol g−1 FW compared to 750 nmol g−1 FW in wild type (Hayashi et al. 1997; Sakamoto and Murata 2001). Part of the observed effects from COD/COX transgenesis thus might be due to such alternative responses. Transgenic wheat line (T6) has been generated to overexpress the Atriplex hortensis L. BADH gene in the shi4185 line. In the wild-type wheat line, GB concentration is already at a high level of 75 μmol g−1; BADH overexpression caused this to increase to 100 μmol g−1 DM (Wang et al. 2010). A similar increase was seen in the wild type after drought treatment. In the study, drought, heat, and their combination were tested in the wild-type and overexpressing line. The responses in the T6 line appeared milder compared to wild type for most of the parameters measured for the three replicates. The heat stress effects on transpiration and stomatal conductance deviated from drought and combination responses. Interestingly, most transgenic plants can utilize exogenously applied choline, and GB levels remain stress-inducible in transgenic lines even if transgenes are driven by a constitutive 35S promoter (Lv et al. 2007). This suggests that GB biosynthesis is further promoted by the stress condition. This regulation can be at the transcript level or at the post-translational level. Conversely, this also suggests that transgenesis approaches have not addressed all the components involved. In transgenesis of non-accumulators that lack all functional GB biosynthesis enzymes, overexpression of only one component often leaves the GB accumulation levels moderate. Unbalanced expression of biosynthetic enzymes from the GB pathway can create different cellular and metabolic imbalances (Hare et al. 1998; Gage et al. 2003; Chen and Murata 2011). For example, BADH is not a substrate-specific enzyme and has been associated with diverse aldehydes (Trossat et al. 1997; Muñoz-­ Clares et  al. 2014). The alternative reaction cascades of the GB biosynthesis enzymes can result in competition between substrates and cause side effects, for example, in polyamine metabolism, possibly resulting in new phenotypes (Trossat et al. 1997). Taken together, transgenesis of only one gene from a biosynthetic pathway is usually not enough to achieve the intended outcome. Limiting factors for GB biosynthesis can be the availability of choline, activity of the biosynthetic enzymes and their specificities toward the substrates, as well as the subcellular localization of the enzymes and their respective substrates (Huang et  al. 2000; Nuccio et  al. 1998, 2000; Kumar et al. 2004; Muñoz-Clares et al. 2014; Carrillo-Campos et al. 2018). Modifications to the single-gene overexpression approaches are represented by

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gene stacking, a transgenesis method in which combinations of constructs harbor more than one gene and can be transferred under one selection (Zorrilla-López et al. 2013). In principle, gene stacking would allow transferring all the limiting factors from a biosynthesis pathway in one or consecutive events. Hence, gene stacking could solve some of the bottlenecks in transgenesis for GB accumulation.

4.2  Considerations for CMO and BADH Isoenzymes Significant sequence-specific differences have been discovered in the GB biosynthesis isoenzymes. Phylogenetic studies show that all land plant species have genes encoding for CMO enzymes (Carrillo-Campos et  al. 2018). The CMO genes are present in two clades, CMO1 and CMO2, whereby CMO2 has diverged from the CMO1 after genome duplication. CMO2-type enzymes have evolved at a fast rate and are present in GB-accumulating plant species, such as spinach (Fig.  3). Homology modeling and docking simulations have shown that the CMO2 active site has three aromatic residues and a glutamate that allow efficient interaction with the substrate, choline. The four critical amino acids of CMO2 that confer substrate specificity for choline are indicated in Fig. 3. Such binding capacity toward choline is lacking from the CMO1-type isoenzymes, the isozymes that prevail in GB non-­ accumulators. Spinach also has CMO1-type enzymes that don’t utilize choline but act as oxygenases on different substrates. It would be interesting to verify which spinach CMO form was used in the transgenesis approaches that resulted in low GB accumulation (Shirasawa et al. 2006). Functional isoenzyme differences have also been discovered for the second step of GB biosynthesis, in the BADH isozymes (Muñoz-Clares et  al. 2014). BADH isoenzymes belong to the family 10 of aldehyde dehydrogenases, but only certain ALDH10 enzymes appear to have BADH activity on BAL. Phylogenetic analysis has shown that in spinach, a GB accumulator, the BADH enzyme has a particular amino acid at position 441 (alanine A441), while GB non-accumulators, such as Arabidopsis, have isoleucine at this position (Fig. 4). The amino acid in position 441 (painted gray in Fig. 3) appears to determine if enzymes are able to oxidize BAL into GB. These structure functional discoveries in GB biosynthesis enzymes are likely to influence the success of future transgenesis approaches for enhancing GB production in plants.

4.3  C  hloroplast Targeted Transgenesis for Optimized GB Production Endogenous GB biosynthesis is compartmentalized within the chloroplast. Targeting GB accumulation directly in the chloroplast can facilitate correct enzyme conformation in the correct subcellular compartment. Chloroplast genetic engineering has

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Spinacia_CMO2 Spinacia_CMO1 Arabidopsis_CMO1

MMAASASATTMLLKYPTTVCG-IPNPSSNNNNDPSNNIASIPQNTTNPTLKSRTPNKITT MS------------ITHSIT---QNPLTNHVTLQSFGNNFIPK-------IERFPNRIHQ MMTT----------LTATVPEFLPPSLKSTRGYFNSHSEFGVS-------ISKFSRRRFH * :: .. . . .: .:

59 38 43

Spinacia_CMO2 Spinacia_CMO1 Arabidopsis_CMO1

NAVAAPSFPSLTTTTPSSIQSLVHEFDPQIPPEDAHTPPSSWYTEPAFYSHELERIFYKG APIKLTKC-LSNSSSIQSTHKIAHEFDPNIPIEEAQTPPCSWYSDPEFYSHEIDRVFYSG NPTR--------VFAVSDISKLVTEFDPKIPLERASTPPSSWYTDPQFYSFELDRVFYGG : .. .:. ****:** * * ***.***::* ***.*::*:** *

119 97 95

Spinacia_CMO2 Spinacia_CMO1 Arabidopsis_CMO1

WQVAGISDQIKEPNQYFTGSLGNVEYLVSRDGEGKVHAFHNVCTHRASILACGSGKKSCF WRVVGCVDQIKNAHDYFTGRLGNVEYVICRDGVGKIHAFHNVCRHHASILAYGSGRKTCF WQAVGYSDQIKESRDFFTGRLGDVDFVVCRDENGKIHAFHNVCSHHASILASGNGRKSCF *:..* ****: .::*** **:*::::.** **:******* *:***** *.*:*:**

179 157 155

Spinacia_CMO2 Spinacia_CMO1 Arabidopsis_CMO1

VCPYHGWVYGMDGSLAKASKAKPEQNLDPKELGLVPLKVAVWGPFVLISLDRSLEEG--VCPYHGWTYGLEGNLLKAPRITGLRNFNPKEYGLVPINVATWGPFVVVNLSSSEEE---V VCLYHGWTYSLSGSLVKATRMSGIQNFSLSEMGLKPLRVAVWGPFVLLKVTAATSRKGEV ** ****.*.:.*.* ** : . :*:. .* ** *:.**.*****::.: : ..

236 214 215

Spinacia_CMO2 Spinacia_CMO1 Arabidopsis_CMO1

---GDVGTEWLGTSAEDVKAHAFDPSLQFIHRSELPMESNWKIFSDNYLDSSYHVPYAHK -DYGNMENDWLGGSADLLSINGVDTSLSYICRREYTLECNWKVFCDNYLDGGYHVPYAHK ETDELVASEWLGTSVGRLSQGGVDSPLSYICRREYTIDCNWKVFCDNYLDGGYHVPYAHK : .:*** *. :. ..* *.:* * * ::.***:*.*****..********

293 273 275

Spinacia_CMO2 Spinacia_CMO1 Arabidopsis_CMO1

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352 333 335

Spinacia_CMO2 Spinacia_CMO1 Arabidopsis_CMO1

TTMHIHPLGPRKCKLVVDYYIENSMLDDKDYIEKGIAINDNVQREDVVLCESVQRGLETP DTNLVIPLGPRKCQVVFDYFLDASLKDDKAFIERSLKDSEEVQIEDIMLCEGVQRGLESP DTNLVLPLGPRKCKVVFDYFLDPSLKDDEAFIKRSLEESDRVQMEDVMLCESVQRGLESQ * : *******::*.**::: *: **: :*::.: .:.** **::***.******:

412 393 395

Spinacia_CMO2 Spinacia_CMO1 Arabidopsis_CMO1

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439 426 422

Fig. 3  Spinacia CMO1 (XP_021866412.1) and CMO2 (ABN43460.1) amino acid sequence alignment with Arabidopsis CMO1 amino acid sequence by Clustal Omega tool (Madeira et al. 2019). Spinacia CMO2 functional motives for choline oxidation as indicated by Carrillo-Campos et al. (2018) are underlined and painted in grey. Stars under sequences indicate shared amino acids, single and double dot indicates semi- and conservative amino acids, respectively, no sign indicates non-conservative amino acid

many benefits over nuclear transgenesis (Kumar et  al. 2004). Direct chloroplast genome transgenesis and efficient transgene expression have been achieved with appropriate regulatory sequences for both selection and the gene of interest. Homologous recombination in the chloroplast genome requires extensive flanking sequences around the gene of interest. The carrot (Daucus carota subsp. sativus (Hoffm.) Schübl. & G.  Martens)-specific transformation vector, pDD-DC-aadA/ badh, harbored aadA and badh sequences regulated by the 5’ribosome-binding site region of the bacteriophage T7 gene 10 leader to facilitate expression in green and non-green tissues. Similarly, the promoter sequence was designed to harbor binding sites for both plastid- and nuclear-encoded RNA polymerases. Transgenesis was performed by particle bombardment of a yellow carrot cell culture. The untransformed cell remained yellow in color while transformed cells turned green, allowing selection without a selectable marker. The method was completed with the successful regeneration of mature plants through somatic embryogenesis. Directing

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Spinacia_BADH MAFPIPARQLFIDGEWREPIKKNRIPVINPSTEEIIGDIPAATAEDVEVAVVAARRAFRR Arabidopsis_ALDH MAIPMPTRQLFIDGEWREPILKKRIPIVNPATEEVIGDIPAATTEDVDVAVNAARRALSR **:*:*:************* *:***::**:***:********:***:*** *****: *

60 60

Spinacia_BADH N---NWSATSGAHRATYLRAIAAKITEKKDHFVKLETIDSGKPFDEAVLDIDDVASCFEY Arabidopsis_ALDH NKGKDWAKAPGAVRAKYLRAIAAKVNERKTDLAKLEALDCGKPLDEAVWDMDDVAGCFEF * :*: : ** **.********:.*:* .:.***::*.***:**** *:****.***:

117 120

Spinacia_BADH FAGQAEALDGKQKAPVTLPMERFKSHVLRQPLGVVGLISPWNYPLLMATWKIAPALAAGC Arabidopsis_ALDH YADLAEGLDAKQKAPVSLPMESFKSYVLKQPLGVVGLITPWNYPLLMAVWKVAPSLAAGC :*. **.**.******:**** ***:**:*********:*********.**:**:*****

177 180

Spinacia_BADH TAVLKPSELASVTCLEFGEVCNEVGLPPGVLNILTGLGPDAGAPLVSHPDVDKIAFTGSS Arabidopsis_ALDH TAILKPSELASVTCLELADICREVGLPPGVLNVLTGFGSEAGAPLASHPGVDKIAFTGSF **:*************:.::*.**********:***:* :*****.***.*********

237 240

Spinacia_BADH ATGSKVMASAAQLVKPVTLELGGKSPIVVFEDVDIDKVVEWTIFGCFWTNGQIXCSATSR Arabidopsis_ALDH ATGSKVMTAAAQLVKPVSMELGGKSPLIVFDDVDLDKAAEWALFGCFWTNGQI-CSATSR *******::********::*******::**:***:**..**::********** ******

297 299

Spinacia_BADH LLVHESIAAEFVDKLVKWTKNIKISDPFEEGCRLGPVISKGQYDKIMKFISTAKSEGATI Arabidopsis_ALDH LLVHESIASEFIEKLVKWSKNIKISDPMEEGCRLGPVVSKGQYEKILKFISTAKSEGATI ********:**::*****:********:*********:*****:**:*************

357 359

Spinacia_BADH LYGGSRPEHLKKGYYIEPTIVTDISTSMQIWKEEVFGPVLCVKTFSSEDEAIALANDTEY Arabidopsis_ALDH LHGGSRPEHLEKGFFIEPTIITDVTTSMQIWREEVFGPVLCVKTFASEDEAIELANDSHY *:********:**::*****:**::******:*************:****** ****:.*

417 419

Spinacia_BADH GLAAAVFSNDLERCERITKALEVGAVWVNCSQPCFVQAPWGGIKRSGFGRELGEWGIQNY Arabidopsis_ALDH GLGAAVISNDTERCDRISEAFEAGIVWINCSQPCFTQAPWGGVKRSGFGRELGEWGLDNY **.***:*** ***:**::*:*.* **:*******.******:*************::**

477 479

Spinacia_BADH LNIKQVTQDISDEPWGWYKSPArabidopsis_ALDH LSVKQVTLYTSNDPWGWYKSPN *.:**** *::********

498 501

Fig. 4 Amino acid sequence alignment of Spinacia BADH protein (ACM67311.1) with Arabidopsis non-BAL form ALDH (10A8) enzyme. The critical amino acid (441A or 441C) for BADH BAL activity as shown by Muñoz-Clares et al. (2014) is underlined and painted in grey. Stars under sequences indicate shared amino acids, single and double dot indicates semi- and conservative amino acids, respectively, no sign indicates non-conservative amino acid

BADH gene overexpression in carrot chloroplasts resulted in the highest salt tolerance of up to 400 mM NaCl (Kumar et al. 2004). While enzyme activities and substrate specificities are fundamental for biosynthetic pathways, subcellular localization of the biosynthesis enzymes also plays a significant role. In GB accumulators, all functional biosynthesis enzymes are present in the correct subcellular localization, and the substrate choline is also available. Biosynthetic enzymes of GB are encoded in the plant genome while GB biosynthesis takes place in chloroplasts. The substrate choline is transported into the chloroplasts via nuclear pores. Successful transgenesis for enhanced GB production would thus require enhanced levels of both the transgene products and the substrate c­ holine (Nuccio et al. 2000; McNeil et al. 2000). Gene expression levels and organ-­specific expression patterns of biosynthetic enzymes can be regulated by promoter elements, but localization of the gene products is also affected by signaling peptides, called transit signals. Overexpression of biosynthetic enzyme-encoding genes can be accompanied by signal sequences to translocate the gene products into chloroplasts. The study of Nuccio et al. (2000) represents a rigorous effort to optimize the expression, localization, and posttranslational modifications of GB biosynthesis enzymes.

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In the study, a 100-fold higher CMO activity was achieved in tobacco chloroplasts, yet the levels of GB remained at a low level. The availability of the substrate, choline, was shown to be the limiting factor. Plants engineered to express CMO in chloroplasts failed to produce GB even at high gene expression levels, while transgenic lines expressing CMO in the cytoplasm accumulated significantly more GB. It was shown that poor choline transport into chloroplasts caused the lack of GB accumulation in the chloroplast-targeted CMO line. These studies are a reminder of the importance of assessing all the components along the pathway. To further promote choline availability for GB biosynthesis, choline biosynthesis could also be enhanced through transgenesis. Recently, a newly identified factor, GB1, was shown to promote GB accumulation at high levels in different maize cultivars (Castiglioni et al. 2018). Overexpression of this fatty acid hydroxylase superfamily protein was speculated to be involved in choline biosynthesis and/or transported into chloroplasts. Future work will confirm GB1 function, but the availability of choline clearly represents a critical limiting factor for GB accumulation.

5  Conclusions and Future Perspectives The amount of scientific literature related to GB is accumulating quickly, yet our knowledge of the mechanisms by which GB affects crop stress tolerance remain partly unknown. It is proposed that GB acts as a compatible solute in plants with two major roles. The first role of GB involves the regulation of osmotic balance via acting as a conventional osmolyte. The second one includes the maintenance of normal cell metabolism under stress conditions and thus acting on ROS scavenging, macromolecule protection, and carbon and N reserves. Some of the proposed effects of GB might be the result of alternative metabolic routes caused by imbalanced metabolic engineering. Integrated omics analysis combining transcriptomic, proteomic, and metabolomic studies on the transgenic lines could shed light on the complete picture of the GB accumulation profiles of the different transgenesis approaches. There are many limiting factors that seemingly influence GB accumulation in transgenic plants. Gene stacking as a transgenesis strategy could solve some of the bottlenecks in improving GB accumulation. The significant structure function discoveries in the GB biosynthesis isoenzymes are especially likely to drive the success of future GB transgenesis approaches in plants. It could also be considered whether marker-assisted selection could prove useful in the isoenzyme approach. In future, more attention should be paid to investigating the mechanisms by which GB affects plant growth and metabolism instead of simply testing new plant species.

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Biosynthesis and Degradation of Trehalose and Its Potential to Control Plant Growth, Development, and (A)biotic Stress Tolerance Le Cong Huyen Bao Tran Phan and Patrick Van Dijck

1  Trehalose Biosynthesis and Degradation Trehalose is a nonreducing disaccharide of two glucose monomers which are linked in a 1,1-glycosidic bond. There are three possible anomers of trehalose (α,β-1,1-; β,β-1,1-; and α,α-1,1-), but only the latter has been isolated from living organisms so far. This sugar can be synthesized by a wide range of organisms such as bacteria, fungi, nematodes, arthropods, and plants (Elbein 2003) but not by vertebrates. In 1913, trehalose was for the first time reported in plants (Selaginella lepidophylla) (Anselmino ad Gilg 1913). Interestingly, trehalose levels are negligible in most higher plants, except for some resurrection plant species, for example, S. lepidophylla, where its high trehalose content was correlated with high stress tolerance (Iturriaga 2000). More recently, the origin of this trehalose was questioned as S. lepidophylla hosts many endophytes, which could be the real source for the high trehalose levels (Pampurova and Van Dijck 2014; Pampurova et al. 2014). Due to the presence of trace amounts of trehalose in the majority of angiosperms, it was suggested that trehalose had an unimportant role in plants. However, as described below, it is now clear that trehalose and the intermediates in its biosynthesis have an important role in plants.

L. C. H. B. T. Phan VIB-KU Leuven Center for Microbiology, Heverlee, Belgium Laboratory of Molecular Cell Biology, KU Leuven, Leuven, Belgium Department of Biology, College of Natural Sciences, Can Tho University, Can Tho, Vietnam P. Van Dijck (*) VIB-KU Leuven Center for Microbiology, Heverlee, Belgium Laboratory of Molecular Cell Biology, KU Leuven, Leuven, Belgium e-mail: [email protected] © Springer Nature Switzerland AG 2019 M. A. Hossain et al. (eds.), Osmoprotectant-Mediated Abiotic Stress Tolerance in Plants, https://doi.org/10.1007/978-3-030-27423-8_8

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TPS/TPP

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Fig. 1  The trehalose biosynthesis pathways. Abbreviations: Uridine diphosphate glucose (UDP-­ glc), glucose-6-phosphate (G6P), trehalose-6-phosphate (T6P), adenosine diphosphate glucose (ADP-glc), glucose (glc), glucose-1-phosphate (G1P), trehalose-6-phosphate synthase (TPS), trehalose-­6-phosphate phosphatase (TPP), trehalose synthase (TreS), maltooligosyl trehalose synthase (TreY), maltooligosyl trehalose trehalohydrolase (TreZ), trehalose glycosyl transferring synthase (TreT), trehalose phosphorylase (TreP), trehalase (TRE). (Figure based on Fernandez et al. (2010))

There are at least five trehalose biosynthesis pathways in nature (Fig. 1) (Avonce 2006). The most common pathway (the TPS-TPP pathway) is found in a wide range of organisms from eubacteria, archaea, fungi, insects, and plants. In this pathway, trehalose-6-phosphate synthase (TPS) (OtsA in Escherichia coli) catalyzes the transfer of glucose from UDP-glucose to glucose-6-phosphate (G6P), to produce the intermediate trehalose-6-phosphate (T6P). Trehalose-6-phosphate phosphatase (TPP) (OtsB in E. coli) dephosphorylates T6P to trehalose (Elbein 2003). The second pathway (the TreZ-TreY pathway) is distributed in thermophilic archaea of the genus Sulfolobus. Trehalose biosynthesis in this pathway involves the conversion of maltooligosaccharides or starch to trehalose under the catalysis of maltooligosyl trehalose synthase (TreY) and maltooligosyl trehalose trehalohydrolase (TreZ) subsequently (Maruta 1996). The trehalose synthase (TreS) isomerizes the α1-α4 linkage of maltose into the α1-α1 linkage of trehalose. The TreS pathway was first reported in Pimelobacter sp. (Elbein 2003). In fungi such as Agaricus bisporus and protista such as Euglena gracilis, trehalose phosphorylase (TreP) catalyses the reversible synthesis of trehalose from glucose-1-phosphate and glucose (Wannet 1998; Avonce 2006). The TreT pathway involves trehalose glycosyl-transferring synthase (TreT) which catalyzes the conversion of ADP-glucose and glucose into trehalose. This pathway occurs in extremophiles such as Thermococcus litoralis and Thermotoga maritima (Qu 2004). Some species, such as the genus Mycobacterium, use multiple distinct pathways (the TPS-TPP, TreY-TreZ, and TreS pathways) to

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synthesize trehalose. This allows the accumulation of trehalose without substrate depletion in response to abiotic stresses (De Smet 2000). Trehalose is hydrolyzed into two glucose units by trehalase (TRE). This has been observed in many organisms from bacteria, fungi, plants, and animals, including mammals that do not produce trehalose (Elbein 2003). Some species contain various isoforms of trehalase. For example, there are three trehalase genes in Saccharomyces cerevisiae. Two cytosolic trehalases are encoded by the neutral trehalase genes Nth1 and Nth2, respectively, while an acid trehalase (Ath1) is functional in the extracellular space and vacuoles (Parrou 2005). In E. coli, a periplasmic trehalase is encoded by TreA, and TreF encodes a cytosolic trehalase (Schluepmann 2003). However, there is only one single gene (AtTRE1) that encodes a functional trehalase in Arabidopsis thaliana as the Attre1-1 knockout mutant showed no detectable trehalase activity.

2  G  ene Families Involved in Trehalose Metabolism and Functional Characterization of Those Genes in Plants In A. thaliana, based on genome sequencing, it has been revealed that there are 11 genes encoding TPS proteins and 10 genes encoding TPP proteins and only a single trehalase-encoding gene. The TPS and TPP genes were also detected in other plant genome sequences, including monocotyledonous and eudicotyledonous angiosperms, suggesting that trehalose synthesis occurs widely in the plant kingdom (Lunn 2014).

2.1  Plant TPS Proteins The A. thaliana TPS proteins are encoded by 11 genes (AtTPS1–AtTPS11) which are divided into class I and class II proteins. The class I proteins (AtTPS1–AtTPS4) with fused TPS and TPP domains are more complex than ScTPS1 (Fig. 2). It has been shown that donor (UDP-glc) and acceptor (G6P) binding sites are highly conserved in the TPS domain when doing alignment of AtTPS1, AtTPS2, and AtTPS4 with OtsA sequence. AtTPS3 is likely a pseudogene as it contains a premature translational stop codon (Vandesteene 2010). Among the A. thaliana class I proteins, only AtTPS1 exhibits an N-terminal extension. Truncation of this N-terminal region in AtTPS1 led to a sharp increase in plant TPS activity upon expression of the different alleles in yeast. This result suggests that the plant-specific N-terminal extension might function as an autoinhibitory domain which mediates TPS activity (Van Dijck 2002). This domain is present throughout the plant kingdom, from single-celled green organisms (e.g., Ostreococcus tauri), over moss plants (Physcomitrella patens), to higher plants, which indicates that it may have an important role in the regulation of trehalose metabolism, as it was con-

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OtsA OtsB

ScTPS1 S. cerevisiae ScTPS2

AtTPS1 Class I AtTPS2-4 A. thaliana AtTPS5-11

Class II

AtTPPA-J

Class III

Fig. 2  Domain structure of bacterial, yeast, and plant trehalose metabolism proteins. E. coli and S. cerevisiae have active TPS and TPP proteins. In plants, all TPS enzymes contain TPS- and TPP-­ like domains. An extra N-terminal domain (green) is present in AtTPS1. The class III proteins lack the TPS-like domain, and all TPP enzymes are active phosphatases. Three conserved HAD motifs in the TPP domain are indicated by black boxes. Different shades of colors imply different levels of similarity in sequences. (Figure based on Vandesteene et al. (2010))

served during plant evolution (Van Dijck 2002; Avonce 2010; Lunn 2007). However, the real function in planta needs to be further investigated (Lunn 2014). In contrast to AtTPS1, the other two class I proteins AtTPS2 and AtTPS4 lack the N-terminal extension region. Previously, AtTPS2 and AtTPS4 were considered as catalytically inactive proteins as they were unable to complement the growth defect of the yeast tps1Δ mutant on glucose (Vandesteene 2010). Nonetheless, it has been found recently that both AtTPS2 and AtTPS4 can rescue the growth defect on glucose when expressed in yeast tps1Δtps2Δ mutant. Moreover, expression of these genes in the double yeast mutant also resulted in the accumulation of high levels of T6P when grown on glucose (Delorge 2015). These results have shown that AtTPS2 and AtTPS4 are also active enzymes. Interestingly, AtTPS2 and AtTPS4 produced much more T6P when compared to AtTPS1, when expressed in this double yeast mutant. In addition, expression of AtTPS2 and AtTPS4 also resulted in the production of trehalose in this strain background. The reason for the higher amount of T6P and the production of trehalose has most probably something to do with the yeast Tps2. To synthesize trehalose, a trehalose biosynthesis complex, consisting of ScTps1, ScTps2, and two regulatory subunits encoded by either ScTSL1 or ScTPS3, is required (Trevisol 2014). It is not unlikely that the plant TPS proteins participate in the enzyme complex formation in yeast cells and that the plant TPS proteins occupy the space of ScTps1 and ScTps2, resulting in an optimized situation to produce T6P and trehalose, which may not be the case for ScTps2 is still present (in the

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Sctps1∆ mutant). In addition, AtTPS2 and AtTPS4 genes are specifically expressed in developing seeds and siliques, which suggests that they might serve particular functions in reproductive tissues (Schmid 2005). None of class I proteins can complement the heat stress-sensitive phenotype of the yeast tps2Δ mutant ­ (Vandesteene 2010). This result obtained was as expected since the TPP-like domain is very poorly conserved. Nevertheless, as mentioned above, expression of AtTPS2 and AtTPS4 does result in the biosynthesis of trehalose in the double yeast mutant, which indicates that when ScTps1 is present, the TPS complex formation may not be optimal. In contrast to A. thaliana which contains four class I TPS proteins, most plants only harbor one or two class I genes. For example, class I family in rice has only one gene, and in poplar, two genes were identified (PtTPS1 and PtTPS2) (Lunn 2007). In Poaceae, sorghum harbors a single class I gene, whereas maize contains two genes. Also, the moss P. patens contains two active class I proteins (Delorge et al., unpublished). The A. thaliana class II proteins (AtTPS5-AtTPS11) (Fig. 2) are mostly similar to ScTPS2, especially in the C-terminal TPP-like domain (Leyman 2001). As compared to the class I proteins, the TPS catalytic sites in the class II proteins are less well conserved (Lunn 2007). No TPS or TPP activity could be detected in class II proteins upon their expression in S. cerevisiae. As the T6P binding sites are very well conserved, but no TPP activity can be observed, one hypothesis that can be stipulated is that these class II proteins are sensors for T6P. This may be supported by the fact that these 7 genes have a conditional and tissue-specific expression. Their expression is regulated by hormones, light, and nutrient supply (Ramon 2009; Vandesteene 2010). The expression of class II TPSs might be regulated by the nutritional status of the plants/tissues as, for instance, expression of AtTPS8-10 is induced by carbon deprivation and suppressed by sugars, whereas the opposite has been shown for AtTPS5 (Osuna 2007; Schluepmann 2004). In addition, several of the encoded proteins are phosphorylated by SnRK1 and CDPK, allowing them to be bound by 14-3-3 proteins (Glinski and Weckwerth 2005; Harthill 2006). These findings further suggest that the class II proteins may play important regulatory functions rather than a metabolic control mechanism in Arabidopsis (Ramon 2009). Recently, several studies have been done to support this opinion. For example, the silencing of PvTPS9, which is the major transcript of the class II TPS genes (PvTPS4-PvTPS10) in root nodules of common bean (Phaseolus vulgaris), reduced the expression of PvTPS9 by approximately 85% in the PvTPS9-RNAi transgenic nodules (Barraza 2016). Moreover, other class II genes such as PvTPS4, PvTPS6, PvTPS7, and PvTPS10 were also downregulated in the transgenic root nodules. The reduction in expression of these transcripts was correlated with a significant decrease in trehalose levels in plant biomass. It was suggested that the accumulation of trehalose in the root nodules most probably originates from the symbiont, rather than from the host plant (Müller 2001b; Suárez 2008; López 2009; Vauclare 2010). Hence, the silencing of PvTPS9, which leads to variations in the expression of some other class II TPS genes, might interfere with the biosynthesis of trehalose in rhizobia, resulting in the decrease in trehalose levels in the PvTPS9-RNAi transgenic root nodules. As data have shown above, it indicates that PvTPS9 plays an important role

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in regulation of trehalose metabolism in root nodules and, consequently, in the whole plant (Barraza 2016). Another evidence of regulatory functions of the class II TPS proteins was presented in a study of Zang et al. (2011). There are ten class II TPS proteins (OsTPS2-11) in rice, and none of them exhibit the TPS or TPP activity when expressed in the yeast tps1∆ or tps2∆ mutants, respectively. By yeast two-­ hybrid analysis and bimolecular fluorescence complementation assay, strongly direct interactions of OsTPS1 and OsTPS5/OsTPS8 were detected. Furthermore, two possible isoforms of OsTPS1 (OsTPS1a and OsTPS1b) were identified by gel filtration assay. In addition, it was observed that either OsTPS1a or OsTPS1b and OsTPS8 are incorporated into the TPS complex(es). These results suggest that the class II TPS proteins, particularly OsTPS8  in rice, might regulate the activity of OsTPS1 by forming the TPS complex(es), in order to modulate the concentration of intracellular T6P to control plant growth and development.

2.2  Plant TPP Proteins The Arabidopsis TPP proteins consist of ten isoforms (AtTPPA–AtTPPJ) with three well-conserved HAD (L-2-haloacid dehalogenase) phosphatase motifs (Fig.  2) (Vandesteene 2012). Similar to A. thaliana, the rice and poplar genomes contain 10 TPP genes. Some other species have less TPP isoforms, for example, P. patens has two, S. moellendorffii has three, and O. taurus has only one TPP protein. These proteins are likely derived from the endosymbiotic ancestor of mitochondria because TPP homologues exist in proteobacteria such as Rhodoferax ferrireducens (Lunn 2007; Avonce 2010). In addition, collinearity analysis indicated that all the TPP proteins of Arabidopsis originate from whole-genome duplication (Vandesteene 2012). Beside the conserved HAD domain, the plant TPP isoforms also contain a highly variable N-terminal domain with uncharacterized function. This might be involved in subcellular protein localization (Lunn 2007). During heat stress, T6P is accumulated in the yeast tps2Δ mutant, resulting in a thermosensitive growth phenotype. When complemented by each of the ten A. thaliana TPP proteins, growth deficiency of the yeast tps2Δ mutant at 38.6 °C was rescued, which indicates that all ten proteins have TPP activity (Vandesteene 2012). Similar results were observed for the TPP homologues from rice (Oryza sativa), maize (Zea mays), and grapevine (Vitis vinifera) (Pramanik and Imai 2005; Satoh-Nagasawa 2006; Fernandez 2012). Moreover, expression of the TPP genes is tightly controlled in cell- and tissue-­specific manner, which suggests that TPP proteins strictly regulate T6P levels in plants (Vandesteene 2012).

2.3  Trehalase The plant TPS and TPP proteins are encoded by multiple genes, while only one single gene encodes for trehalase (TRE) enzyme in most plants. Interestingly, a high degree of natural variations was identified in the catalytic domain of A. thaliana

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TRE1 when doing SNP analysis in 81 sequenced accessions of Arabidopsis (Schluepmann 2012). There are a few exceptions in which Populus trichocarpa and P. patens display three and four trehalase genes, respectively (Lunn 2007; Van Houtte et al., our unpublished results). AtTRE1 expression is abundant in flowers and siliques, whereas much less expression was observed in roots and shoots (Müller 2001a; van Dijken 2004). AtTRE1 activity might be important for the development of flowers, siliques, and seeds as addition of validamycin A, a trehalose analog and competitive inhibitor of trehalase, caused defects in fruiting and a poor seed set in Arabidopsis plants (Müller 2001a). AtTRE1 can fulfill the function of ScAth1 in yeast ath1Δ cells as AtTRE1 complemented the growth defect of the yeast ath1Δ mutant on the trehalose-containing growth medium, adjusted at pH 4.8. This indicates that A. thaliana trehalase has an extracellular activity. There are five putative N-glycosylation sites in the AtTRE1 sequence, indicating that trehalase might be secreted. It has been shown that AtTRE1 is a plasma membrane-bound protein with its catalytic domain facing the apoplast (Frison 2007). AtTRE1 anchors to the plasma membrane through a putative N-terminally transmembrane domain (residues 46–63). When plants were infected by Plasmodiophora brassicae, trehalase activity was increased even before pathogen-­produced trehalose increased in the host plant. This indicates that trehalase is unlikely to be induced/activated by trehalose (Brodmann 2002). These findings suggest that trehalase might be a sensor to detect extracellular pathogen-secreted trehalose and thereby prevents an accumulation of trehalose which could interfere with metabolic signaling inside the cells (Schluepmann 2004; Brodmann 2002; Gravot 2011). However, the question of how the extracellular AtTRE1 regulates intracellular trehalose levels is still unclear. It is also possible that trehalase is a sensor for stress and that it may be involved in closing stomata to prevent pathogens from entering the plant. This hypothesis is supported by the fact that trehalase is specifically expressed in guard cells and that more trehalase expression results in fast closing of stomata (Van Houtte 2013).

3  R  ole of Trehalose-6-Phosphate/Trehalose in Plant Growth and Development Trehalose acts as a stress protectant which stabilizes proteins and lipid membranes upon various abiotic stresses such as dehydration, cold, heat, and oxidative stress (Elbein 2003). This disaccharide is stable at high temperature (up to 100 °C) and in a broad pH range for 24 h (Richards 2002). Anhydrobiotic organisms such as yeast cells, fungal spores, resurrection plants, nematodes, rotifers, and the cysts of brine shrimp generally have high amounts of trehalose. The survival ability of these creatures is strongly correlated with the synthesis of trehalose in the absence of water. However, in most plants, except in certain resurrection plants (e.g., Selaginella lepidophylla), trehalose is hardly detectable. Therefore, trehalose was not considered as an osmoprotectant in plants (Schluepmann 2004).

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Interestingly, trehalose accumulation is most likely toxic for plant growth by interfering with cell wall biosynthesis (O’Hara 2013; Veluthambi 1982a). In Cuscuta reflexa, inhibition of trehalase by validamycin A or trehalose supplementation was associated with a decrease in sucrose and starch contents, leading to growth inhibition (Veluthambi 1982b). A similar pattern was detected in soybean plantlets and Arabidopsis when plants were treated with validamycin A and/or addition of trehalose (Müller 1995, 2001a). Nonetheless, the growth defect caused by trehalose feeding was not observed in plant species with high trehalase activity (Veluthambi 1981). Trehalose-­6-­phosphate (T6P), a metabolic precursor of trehalose, has emerged as a signaling metabolite, regulating plant metabolism, growth, and development (Schluepmann 2004; Lunn 2006). Schluepmann et  al. (2004) figured out that trehalose-mediated growth inhibition is due to a rapid accumulation of T6P in seedlings of Arabidopsis. When levels of trehalose are high, it might lead to a reduction of T6P dephosphorylation, resulting in an increase in T6P contents. Remarkably, the growth inhibition of T6P was rescued when metabolizable sugars were added to the trehalose containing growth medium. The first reports where the effects of T6P/trehalose on plant development were described were released in 1997. Transgenic tobacco plants expressing a heterologous TPS gene with accumulated trehalose showed lancet-shaped leaves and stunted growth phenotypes (Romero 1997; Goddijn 1997). In addition, tobacco plants that expressed E. coli TPP with lower T6P levels displayed an increase in leaf size. Another example is that the cell shape phenotype-1 (csp-1) mutant displayed a dramatic cellular effect in the leaf epidermis, which resulted in an altered pavement cell morphology, arrested development, as well as changes in plant architecture such as reduced trichome branching, altered stem branching, and increased stomatal density (Chary 2008). Mapping of the csp-1 locus revealed that the mutated gene in the csp-­ 1 mutant encodes AtTPS6. As expected, AtTPS6 complemented the defects in morphology (e.g., pavement cells, trichomes) and overall growth phenotypes of the csp-1 mutant. These results indicate that the trehalose metabolism plays a role in plant development. Although T6P is a causative metabolite for the growth defect of plants, when the addition of exogenous trehalose (100 mM) led to an accumulation of T6P levels, causing the growth inhibition of Arabidopsis seedlings (Schluepmann 2004), T6P is also critical for normal growth, with a main defect in root growth (Schluepmann 2003). In A. thaliana, the null tps1 mutant is embryo lethal. The mutant failed to germinate. Addition of trehalose cannot rescue the growth deficiency of tps1 mutants (Eastmond 2002), but this defect can be rescued by the heterologous expression of E. coli TPS (Schluepmann 2003). These results suggest that T6P plays an important role in embryo development. Besides that, T6P affects the vegetative growth of plants. For instance, TILLING mutants with weak TPS1 alleles displayed delayed vegetative growth phenotypes. In addition, Arabidopsistps1 loss-of-­ function alleles exhibited a delay in flowering as compared to the wild type (Gómez 2010). Furthermore, Arabidopsis seedlings expressing heterologous TPP or TPH from E. coli contain low levels of T6P, leading to a failure in the use of supplied sugars. Transgenic plants with reduced T6P levels experienced growth inhibition

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Catabolism (e.g. respiration)

Anabolism (e.g. photosynthesis)

Sucrose

UDPG + Glc6P

TPS

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TPP T6P

Trehalose

SnRK1

Fig. 3  The interaction network of T6P, sucrose, and SnRK1. An increase in T6P level represents an accumulation in sucrose level. This potentially triggers reprogramming in the cell to reduce the levels of sucrose to the optimal concentration range, by activating catabolism processes (e.g., sucrose consumption, respiration, etc.) and/or inhibiting sucrose synthesis via anabolism. When sucrose levels go back to the desired range, T6P also decreases. SnRK1, a key metabolic sensor which maintains energy homeostasis for cell growth and survival, is inhibited by T6P. Dashed lines with arrows and bars show activation and inhibition with unknown molecular mechanism, respectively. Lines with arrows indicate metabolite conversions. (Figure based on Lunn et al. (2014))

when sugars were applied, while plants with enhanced T6P levels demonstrated the opposite effect (Schluepmann 2003). From these experiments it is clear that adequate T6P levels are required for carbon utilization during normal growth. When T6P levels are not in balance with sugar availability, growth is hampered. Addition of sucrose to the medium can rescue trehalose-dependent growth inhibition, which indicates that T6P improves plant growth when carbon supply is high (Schluepmann 2004). Surprisingly, T6P is not only related to regulating the use of sucrose, but the amounts of T6P are also elevated dramatically when sucrose is supplied (Lunn 2006). The intracellular T6P levels are inversely proportional to the amounts of hexose phosphates and UDP-glucose (UDPG), which are downstream products of sucrose breakdown (Pellny 2004; Schluepmann 2003). Hence, when sucrose is added, a constitutive expression of TPS1 happens, leading to the rapid accumulation of T6P in order to response to a rise in the pool size of G6P and UDPG (Paul 2008). Therefore, T6P amounts might reflect the levels of hexose phosphates, UDPG, and sucrose. The sucrose-T6P interaction network is discussed in Fig. 3. Another example of metabolic regulation by the trehalose pathway is an increase of starch synthesis upon trehalose feeding (Wingler 2000). It was found that T6P activates AGP-glucose pyrophosphorylase (AGPase), the key enzyme of starch synthesis (Kolbe 2005). A strong accumulation of starch by trehalose feeding is not only due to an increase in starch synthesis but also comes from an inhibition of starch breakdown through suppressing the expression of SEX1 and β-amylase (Ramon 2007). These observations support the idea that the trehalose pathway

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affects the plant growth via regulation of the flux from central metabolism metabolites to starch. Sucrose non-fermenting-related kinase-1 (SnRK1) is a key kinase involved in plant starvation signaling (Baena-González 2007). During low sugar conditions, KIN10 (a catalytic α-subunit of SnRK1) induces genes involved in catabolism, while sugar feeding triggers an elevation in the expression of genes that are related to biosynthetic processes (Baena-González and Sheen 2008). Interestingly, downregulation of genes involved in photosynthesis and incatabolic processes was observed in Arabidopsis seedlings with accumulated T6P levels. In contrast, genes associated with biosynthetic processes, which are normally downregulated by SnRK1, were upregulated by T6P. These results indicate that T6P inhibits the catalytic activity of SnRK1 (Zhang 2009) (Fig.  3). Moreover, it was revealed that SnRK1-overexpressing seedlings with low T6P levels show a glucose-­hypersensitive phenotype in rice (Oryza sativa) and A. thaliana (Cho 2012). These results are consistent with T6P inhibition of SnRK1 and the sugar hypersensitivity of seedlings with low T6P (Schluepmann 2003). However, the inhibition of SnRK1 by T6P may only occur in young, growing tissues such as seedlings, but not in mature leaves. T6P did not show any inhibitory effect on SnRK1 activity when the catalytic α-subunits of SnRK1 (KIN10 and KIN11) purified by anion-exchange chromatography were assayed. Nonetheless, when the supernatant of a seedling crude extract, from which KIN10 and KIN11 had been removed, was added back to the purified KIN10 and KIN11, the inhibition of SnRK1 by T6P was restored (Zhang 2009). This suggests that (an) unknown factor(s) that is (are) present in seedlings is (are) necessary for inhibition of SnRK1 by T6P. The factors could be (a) protein(s) because they show a heat-labile property as boiling destroys the activity. In senescing leaves of A. thaliana, there is a strong accumulation of T6P, in combination with an increase in sugar levels (Wingler 2012). Moreover, when sugar levels are high, the anthocyanin biosynthetic pathway is stimulated. Mature otsB-­ expressing plants with decreased T6P exhibited a reduction of anthocyanin synthesis and delayed senescence phenotype (Wingler 2012). Furthermore, expression of senescence-associated genes is lower in otsB-expressing plants. These observations support the fact that T6P plays a role in senescence. On the other hand, altered T6P metabolism in otsB-expressing plants led to less sensitivity to sugar supply. For instance, in wild-type and otsA-expressing plants, senescence was promoted when metabolizable sugars such as glucose, fructose, or sucrose are supplied, but this effect was delayed in transgenic plants with lower T6P. T6P also controls flowering time and inflorescence architecture. Deletion of AtTPS1 prevents floral transition (van Dijken 2004), but overexpression of TPS1 postponed the time of flowering (Avonce 2004). The latter phenotype was also detected in KIN10-overexpressing plants (Baena-González 2007), indicating that the effect of T6P on flowering time might be mediated by SnRK1. Tobacco plants (Nicotiana tabacum) expressing otsA produced more axillary shoots (Goddijn 1997), which suggests that T6P might control meristem activity. The role of T6P on meristem development was confirmed in maize. A mutation in

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the RA3 gene, encoding a functional TPP protein, led to changes in flowering architectures such as improved branching of the male and the female inflorescences (Satoh-Nagasawa 2006). It is likely that most proteins involved in the trehalose biosynthetic pathway might have evolved to function as signaling sensors rather than catalytic enzymes since a point mutation in AtTPS6, a protein with inactive TPS activity, increased the branching of inflorescences in A. thaliana (Chary 2008). Likewise, a direct interaction between class II TPS proteins (inactive OsTPS5 and OsTPS8) and the active class I TPS enzyme (OsTPS1) in rice could mediate endogenous T6P concentration in a feedback loop (Zang 2011). Therefore, proteins with no catalytic activity in the trehalose biosynthetic pathway may sense the intracellular T6P levels in response to sugar availability to control meristem development. Regulation of plant growth and development by T6P/trehalose is a noncontroversial fact. Thus, researches on trehalose metabolism in crop plants need to be studied more to unravel the fundamental molecular mechanisms involved in trehalose signaling, providing opportunities for improving crop yield.

4  R  ole of Trehalose-6-Phosphate/Trehalose in Abiotic Stress Tolerance During the whole life cycle, plants face multiple stresses which cause severe effects on their growth and development. Among the two major types of stresses, abiotic and biotic stresses, abiotic stresses including cold, heat, wind, drought, oxidation, radiation, etc. can change physiological processes, leading to disruption of important signaling pathways and finally complete tissue damage (Hirt and Shinozaki 2004). Recently, trehalose and its precursor T6P have been shown that they are involved in plant stress responses (Fernandez 2010). In order to understand how trehalose metabolism may react to abiotic stresses, several studies were performed under various stress conditions.

4.1  Salt Stress Salinity stress brings a big challenge to agriculture, and trehalose alters many processes that may improve plant survival upon salt stress. For instance, an increase in trehalose levels was observed in the roots of rice and Medicago truncatula during NaCl stress (Shima 2007; López 2008a, b; Garcia 1997). Moreover, treatment of trehalose prevented chlorophyll loss in leaf blades and facilitated aerenchyma formation in roots of rice seedlings grown in a medium containing 1% NaCl (Garcia 1997). Although exogenous trehalose feeding did not prevent plants from excess NaCl uptake, it reduced Na+ ion accumulation in the leaf blade of NaCl-stressed

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rice. The protective effect of trehalose on plants could be explained by protection of ion pumps which prevent an uptake of sodium to chloroplasts (Garcia 1997) since trehalose stabilize proteins and lipid bilayer integrity during stress conditions (Elbein 2003). This may allow plants to continue growing without loss of chlorophyll (Garcia 1997). In agreement with the above results, trehalase is downregulated at the transcription level in root nodules of Medicago truncatula under NaCl stress, allowing trehalose accumulation (López 2008b). Similarly, trehalose levels also increased in a range of wheat cultivars during NaCl stress, potentially due to increased TPS activity and decreased trehalase activity (El-Bashiti 2005). Likewise, there was a transient induction of OsTPP1 expression in roots and shoots when rice plants were treated with NaCl (Pramanik and Imai 2005). One should take into consideration the fact that although trehalose levels increased during salt stress, the levels were still too low to function as an osmoprotectant. Thus, it is likely that there is no direct protective effect of trehalose against salt stress. Maize metabolomic analyses revealed an increase in T6P levels in the leaves, kernels, and cobs at the silking stage when 75 mM NaCl was treated. Moreover, an accumulation of sucrose was also observed in the leaf, kernel, and cob tissue at silking, pollination, and 3 days after pollination under salt stress (Henry 2015). Together with these, the levels of many primary metabolites of sugar metabolism, glycolysis, and Krebs cycle were also affected upon NaCl stress. For instance, intermediates of sucrose synthesis, soluble sugars, and starch increased, whereas most of the other metabolites decreased. However, it still remains unclear whether a change in the T6P/sucrose ratios resulting from an increase in T6P at the very early developmental stage (the silking stage) represents a primary response to salt stress, and afterward cellular metabolism is shaped in salt-stressed plants.

4.2  Drought Stress Trehalose is massively present in some desiccation-tolerant resurrection plants such as S. lepidophylla, Myrothamnus flabellifolius, and Sporobolus spp. These plants can persist in metabolic stasis for several years until they are rewatered (Iturriaga 2000, 2006). In addition, sucrose is also present in very high levels in the resurrection plants. It was believed that trehalose and sucrose work together as osmoprotectants to stabilize membranes, proteins, and other cellular components during stasis (Drennan 1993). Nonetheless, it was reported that S. moellendorffii, a drought stress-sensitive relative, contains even higher trehalose levels than S. lepidophylla (Pampurova and Van Dijck 2014; Pampurova et al. 2014), which suggests that high trehalose levels possibly contribute to but are not sufficient for desiccation tolerance of S. lepidophylla. More recently, it was discovered that high levels of trehalose in S. lepidophylla could be originating from endophytes, which are endosymbionts living in this host (Pampurova and Van Dijck 2014; Pampurova et  al. 2014).

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Interestingly, in S. lepidophylla polyols, such as sorbitol and xylitol, are present much more abundantly in comparison with S. moellendorffii. Hence, the question of whether sorbitol or xylitol is linked to drought stress tolerance of S. lepidophylla needs to be addressed. In contrast to those desiccation-tolerant resurrection plants, trehalose levels are limited in crop plants. In drought-tolerant wheat and cotton varieties, dehydration induces a small rise in trehalose levels. This could be due to an induction of TPS expression or a reduction of trehalase activity (El-Bashiti 2005; Kosmas 2006). However, the question of how small changes in trehalose content potentially lead to protective effects remains to be established. It has been stated that water shortage can change seed chemical composition and reduce seed quality (Ali and Ashraf 2011). Obviously, maintenance of seed quality is an important aspect in agriculture for many crops. Seed oil extracted from maize is considered as one of the best oils in the world since it contains high levels of unsaturated fatty acids, such as oleic and linolenic acid, and antioxidants as flavonoids and phenolics. Nonetheless, dehydration reduces the yield of seed oil and the levels of unsaturated fatty acids (Ali and Ashraf 2011). Noticeably, negative impacts of drought stress on maize seeds were rescued when spraying a trehalose solution on the leaves. For instance, contents of oleic and linolenic acid were improved, and the oil antioxidant activity was increased when exogenous trehalose was applied (Ali 2012).

4.3  Temperature Stress Cold or heat stress triggers many changes in physiological and biochemical processes in plants. Those changes include up- or downregulation of many genes and proteins, alteration in metabolite contents, and modification of membrane components and conformation (Sanghera 2011). A. thaliana metabolomic analyses revealed that levels of trehalose and other compatible solutes are elevated upon both cold or heat stresses. It suggests that trehalose may act in combination with other solutes to create synergistic effects during thermotolerant responses (Kaplan 2004). Nevertheless, temperature stress also induces sucrose; thus it is important to clarify if thermal stress directly drives changes in trehalose levels or trehalose induction is just a secondary response caused by changes in sucrose levels. Another evidence for a role of trehalose metabolism in high temperature stress was provided. A yeast two-hybrid screening demonstrated that AtTPS5 interacts with MBF1c (multiprotein bridging factor 1c), a key regulator of thermotolerance in A. thaliana. AtTPS5 is also heat-inducible, and the tps5 null mutants were hypersensitive to high temperature (Suzuki 2008). On the other hand, trehalose metabolism also plays a role in low temperature tolerance. Cold stress stimulated expression

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of OsTPP1 and OsTPP2 genes in rice (Shima 2007; Pramanik and Imai 2005). Similarly, V. vinifera TPPA levels were induced upon chilling stress in grapevine (Fernandez 2012). Iordachescu and Imai (2008) demonstrated that an increase in trehalose and T6P levels results from an induction of AtTPPA during cold stress. Remarkably, T6P levels were positively correlated with sucrose contents in all plant tissues, which suggests that low temperature-induced sucrose accumulation might mediate an increase of T6P levels (Fernandez 2012).

4.4  Hypoxia Hypoxia and anoxia which are caused by flooding are significant problems for many plants. Transcription profiling revealed that genes encoding TPP proteins (e.g., AtTPPA, AtTPPB, and AtTPPJ) and trehalase protein were upregulated, whereas expression of AtTPS11 gene was decreased in A. thaliana in response to low-oxygen stress (Liu 2005). Similarly, upregulation of TPP genes upon hypoxia stress was also observed in poplar (Populus x canescens) (Christianson 2010). The changes in expression of trehalose metabolism genes might lead to a reduction in T6P levels; consequently, it possibly lessens its inhibition on glycolysis and assists the sugar influx into anaerobic respiration (Liu 2005). In agreement with this, a decrease in T6P contents was detected in wild-type A. thaliana plants under hypoxic stress (Thiel 2011).

4.5  Oxidative Stress An accumulation of ROS (reactive oxygen species) resulting from abiotic and biotic stresses has both positive and negative impacts for the plants. On one hand, ROS act as signaling molecules which regulate different processes such as pathogen defense and programmed cell death (Grant and Loake 2000; Dangl and Jones 2001). On the other hand, when ROS mount up to toxic levels, they have to be scavenged to prevent oxidative damage. To deal with this kind of stress, plants develop multiple defense strategies, including accumulation of sugars. Studies both in  vitro and in  vivo provided evidences that trehalose protects against hydroxyl radicals (Roitsch 1999; Couee 2006). Tolerance to methyl viologen-­ induced oxidative stress was increased in transgenic tobacco and tomato plants expressing the yeast TPS1 gene (Romero 2002; Cortina and Culiáñez-Macià 2005). In addition, trehalose also scavenges free radicals generated by heat stress in wheat (Luo 2008).

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5  R  ole of Trehalose-6-Phosphate/Trehalose in Biotic Stress Tolerance 5.1  Trehalose and Plant-Microorganism Symbiosis Trehalose production is a common feature of many microbes, including microbial pathogens and beneficial microbes. Hence, the plant needs to discriminate various potential sources of extracellular trehalose. Under symbiotic and beneficial interactions, defense responses in plants have to be inhibited (Lunn 2014). Symbiotic plant-microorganism interactions promote growth and productivity in both the host and microorganism, as well as enhances plant tolerance to stress (Fernandez 2010). Trehalose accumulates in root nodules when nodules of several legumes form symbioses with rhizobia (Salminen and Streeter 1986; Müller 1992; Farias-Rodriguez 1998; Domínguez-Ferreras 2009; Brechenmacher 2010). Addition of exogenous trehalose to the growth medium increased sucrose synthase and alkaline invertase activities in soybean, potentially enhancing the source of hexose sugars to the symbionts (Müller 1998). Inoculation of common bean (Phaseolus vulgaris) with the symbiotic bacterium Rhizobium etli, which was engineered to overexpress the E. coli trehalose-6-phosphate synthase (otsA), resulted in more trehalose production and formation of more nodules with higher nitrogenase activity. The result was improved plant yield in comparison with plants infected with a wild-­type strain (Suárez 2008). In agreement with this, deletion of the endogenous otsA gene in R. etli reduced the number of nodules, nitrogenase activity, and plant biomass. In addition, trehalose also plays an important role in mycorrhizal fungi-plant relationships. Trehalose represents a main carbon source in Amanita muscaria and Pisolithus microcarpus associated with poplar (Populus tremula x tremuloides) and Eucalyptus globulus roots, respectively (López 2007; Martin 1998). Upon formation of the ectomycorrhizal symbiosis, expression of trehalose metabolism-related genes in A. muscaria was induced (López 2008a, b), while an increase in carbon distribution to trehalose was seen in the mycelium when E. globulus interacts with P. tinctorius (Martin 1998).

5.2  Trehalose: An Elicitor of the Plant Defense Responses Several studies have presented that trehalose might act as an elicitor of the plant defense responses. Transcriptional profiling revealed that exogenous trehalose feeding induced plant defense-related genes such as WRKY6 and β-1,3-glucanase genes, encoding a defense-related transcription factor and a PR (pathogenesis-related) protein, respectively (Schluepmann 2004). Moreover, trehalose treatment also enhanced partial resistance against wheat powdery mildew caused by the pathogen Blumeria graminis (Renard-Merlier 2007). Surprisingly, trehalose feeding changes neither

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pathogen membrane lipid composition (Muchembled 2006) nor conidia germination (Renard-Merlier 2007). These findings support the elicitor hypothesis since trehalose does not seem to affect the pathogen in a direct way. Nonetheless, the concentration of trehalose used in those experiments was 15 g l−1, which is much higher than the physiological levels. Therefore the question of whether defenses were stimulated following osmotic stress or by true elicitation remains to be addressed.

5.3  Trehalose: A Signal of Pathogen Attack Extracellular trehalose is a significant sign of danger to the plant, and this trehalose could be originating from fungi, bacteria (Lunn 2014), insects (Singh 2011), or nematodes (Hofmann 2010). For example, aphid honeydew contains high levels of trehalose which shows a potential signal of aphid attack. An increase in trehalose levels was seen in A. thaliana leaves, resulting from infestation of the peach potato aphid, Myzus persicae. Interestingly, high levels of trehalose were also detected in the phloem sap of plants attacked by aphids, indicating that aphids induce the plant to produce high amounts of trehalose. This trehalose is likely to move through the plant and into the aphids via the phloem sap. However, answers to the questions of whether the accumulation of trehalose in A. thaliana is modulated by the aphids in order to produce a carbohydrate storage substance favorable to themselves or whether trehalose acts as a signaling compound inducing resistance mechanisms in plants are required for further clarification (Hodge 2013). A second example to show the role of trehalose during virulence was obtained in the plant pathogenic fungus Magnaporthe oryzae. Deletion of TPS1 in M. oryzae resulted in low trehalose levels, and this resulted in a reduction of its pathogenicity (Wilson 2007). Similarly, a mutant strain of Pseudomonas aeruginosa which cannot synthesize trehalose is unable to infect the host plant A. thaliana, but this strain still has full capacity to infect non-host plants (Djonovic et al. 2013). These studies demonstrate that trehalose is a virulence factor of those pathogens in order to infect plants. An accumulation of trehalose in infected organs of A. thaliana was observed when plants were infected by P. brassicae, the clubroot pathogen. During the interaction of P. brassicae with A. thaliana, it was shown that the PbTPS1 gene is also upregulated, indicating that the accumulated trehalose was produced by the pathogen. The infection results in a defense response in the plants as AtTRE1 was induced in roots and hypocotyls probably to limit the accumulation of trehalose, which might have negative effects on the plant’s metabolism. Thus, AtTRE1 potentially acts as a sensor of extracellular, pathogen-produced trehalose and also as a defense against excessive accumulation of trehalose (Gravot 2011). In summary, microorganism or insect-derived trehalose represents a virulence or signal molecule during the interactions between plants and those organisms. These interactions interfere significantly with the plant’s own trehalose metabolism. Yet, the mechanisms and physiological changes upon these responses are still unknown.

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Unraveling the precise mechanisms that take place during these interactions may offer potential ways to improve the resistance of crop plants against microbial pathogens and insect pests while also promoting beneficial interactions with bacterial and fungal symbionts.

6  Application of Trehalose-6-Phosphate/Trehalose Modifications in Plants Due to the well-known stress-protecting properties of trehalose in microorganisms and some resurrection plants, trehalose metabolism has emerged as a valuable pathway to target in order to improve abiotic stress tolerance in crop plants. In early studies, tobacco (N. tabacum) and potato (Solanum tuberosum) were engineered by introducing bacterial or fungal genes encoding trehalose-synthesizing enzymes in order to improve the drought stress tolerance (Romero 1997; Goddijn and van Dun 1999). The heterologous TPS/TPP-expressing transgenic plants showed enhanced stress tolerance; however, unexpectedly abnormal phenotypes are exhibited such as stunted growth, lancet-shaped leaves, early flowering, and delayed senescence (Goddijn 1997; Pellny 2004; Goddijn and van Dun 1999; Schluepmann 2003; Paul 2008). The morphological defects were obviously due to changes in the level of T6P resulting from TPS versus TPP overexpression as simultaneous overexpression of both enzymes did not result in abnormal phenotypes (Schluepmann 2003; Salazar 2009; Garg 2002). Enzymes showing both enzymatic activities have been described for Cytophaga hutchinsonii, and such enzymes may be of interest to engineer plants (Avonce 2010). Over the past decades, several approaches in improving stress tolerance in plants without morphological defects have been attempted, apart from the abovementioned expression of both enzymes. An alternative for this approach would be to use enzymes that have both a synthesis and phosphatase activity, and such an enzyme has been described in the bacterium C. hutchinsonii (Avonce 2010). As far as we know, transgenic plants that express such a bifunctional enzyme have not been described yet. Another possibility is the use of conditional expression of the yeast TPS1 gene. This was achieved in potato and tobacco, where transgenic lines that expressed the yeast ScTPS1 gene under the control of a drought-inducible promoter were more tolerant to drought stress. Interestingly, these transgenic plants did not show major phenotypic growth defects (Stiller 2008; Kondrák 2012; Karim 2007). Interestingly, Arabidopsis plants engineered to overexpress ScTPS1 showed growth aberrations, whereas the plants engineered by introducing the AtRbcS1A promoter together with a chloroplast transit peptide in front of the coding sequence of ScTPS1 enhanced drought tolerance without undesired side effects (Karim 2007). This finding indicates that the cytosolic accumulation of T6P disturbs sugar signaling, generating the negative effects on plant growth and development. Another example is overexpression of the endogenous AtTPS1 in A. thaliana which also resulted in enhanced drought stress tolerance without visible effects on the plant’s morphology,

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except for delay in flowering (Avonce 2004). A similar study was performed in rice as overexpression of OsTPS1 increased the tolerance to drought, cold, and salinity stresses in this case without any severe morphological alterations (Li 2011). In addition, sugarcane (Saccharum officinarum L.) engineered to overexpress a Grifola frondosa trehalose synthase (TSase) gene exhibited an improved tolerance to drought stress. The transgenic sugarcane lines had no obvious aberrations in morphology and growth (Zhang 2006). The results obtained suggest that engineering by introducing trehalose biosynthesis genes to enhance abiotic stress tolerance in monocots seems to have less disturbance in morphology and growth when compared to dicots. However, more studies in monocots and dicots need to be investigated to support this statement. Although overexpression of endogenous trehalose-metabolizing genes improved abiotic stress tolerance, only a small increase in trehalose levels was observed (Avonce 2004; Li 2011). This could be due to the presence of the N-terminal autoinhibitory domain of the plant TPS proteins (Van Dijck 2002) as transgenic Arabidopsis lines overexpressing the N-terminal truncated version of AtTPS1 resulted in similar abnormalities as the expression of microbial TPS genes, and such alleles show much higher TPS activity (Van Dijck 2002). The N-terminal domain clearly results in lower enzymatic activity of the TPS enzymes. In addition, the enzymatic activity upon overexpressing of endogenous TPS genes might be controlled by endogenous regulatory mechanisms, hence potentially reducing the metabolic disturbance. This also could be a possible explanation for the minor phenotypic changes in plants engineered to constitutively express endogenous genes when compared to the serious abnormal phenotypes in plants overexpressing heterologous TPS proteins. An alternative approach to improve drought stress tolerance is suppressing the endogenous trehalase (AtTRE1) in A. thaliana (Van Houtte 2013). However, both the tre1-1 knockout mutant and tre1-2 knockdown mutant with higher concentrations of trehalose were surprisingly more sensitive to drought stress than wild-type plants. In line with this, AtTRE1 overexpressing lines such as tre1-3OE and 35S::AtTRE1 with reduced trehalose levels showed improved drought tolerance. Overexpression of AtTRE1 not only affects trehalose levels but also lowers T6P levels. These results indicate that drought tolerance observed in plants overexpressing TPS or TPP was not due to the small increase in trehalose levels but the results of a regulatory mechanism. This leads to further doubt on the role of trehalose as an osmoprotectant in plants, except for resurrection species. Even in some resurrection plants, the high levels of trehalose may come from endophytes (Pampurova and Van Dijck 2014; Pampurova et al. 2014). One of the regulatory mechanisms of trehalose metabolism on drought stress tolerance may be its effect on stomatal conductance and water-use efficiency. AtTPPG and AtTRE1 are strongly expressed in guard cells of A. thaliana leaves (Vandesteene 2012; Van Houtte 2013). Likewise, the AtTPS1 enzyme has hardly been detected in proteomic analyses of different types of cells (Tanz 2013), with the exception of guard cells, where it can be measured (Zhao 2008). This suggests that the AtTPS1 enzyme is probably present at higher contents in guard cells as compared to other cell types or tissues in A. thaliana.

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Abscisic acid (ABA) is an important plant hormone that regulates stomatal closure during drought stress in order to minimize water loss from the leaves. The Attppg, Attre1-1, and Attre1-2 mutants were resistant to ABA as their stomata failed to close when exogenous ABA was added (Vandesteene 2012; Van Houtte 2013). These results indicate that AtTPPG and AtTRE1 are important for ABA-regulated stomatal closure. There is no doubt on the importance of trehalose metabolism in controlling ABA-mediated stomatal conductance. Nonetheless, the underlying signaling mechanisms remain unclear. Therefore, studies that give more insights about the link between ABA signaling and trehalose metabolism or sugar signaling, especially in guard cells, need to be further investigated.

7  Conclusions and Future Perspectives There is no doubt that trehalose metabolism plays an important role in plant growth, development, metabolism, and stress tolerance. The exact mechanisms by which trehalose metabolism is affecting all these characteristics are still unclear. Also the regulation of trehalose metabolism at the cell, tissue, and development level remains to be determined at the molecular level. It is clear that there is a tight regulation between biosynthesis and hydrolysis of T6P at the cellular level as imbalance in the level of T6P results in aberrant phenotypes. The same is probably true for the level of trehalose itself where a tight balance between biosynthesis and hydrolysis of this metabolite is important. Currently, how trehalase, of which the catalytic domain is proposed to be apoplastic, can affect intracellular trehalose levels remains to be investigated. One approach to improve our understanding of the role of trehalose metabolism enzymes on plant growth or stress tolerance is an investigation of A. thaliana natural variations that affect trehalose biosynthesis and degradation. In addition, an extensive –omics approach at the cellular/tissue level would be a good approach for elucidating a holistic view of trehalose metabolism in plants during developmental stages and under stress conditions. Results obtained from such analysis could provide us with useful information for future engineering of trehalose metabolism in order to improve yield and tolerance against multiple stresses in crop plants.

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Proline, Glycinebetaine, and Trehalose Uptake and Inter-Organ Transport in Plants Under Stress Suriyan Cha-um, Vandna Rai, and Teruhiro Takabe

1  Introduction Compatible solutes, proline, glycinebetaine, and trehalose are upregulated in higher plants under abiotic stress, playing a key role in physiological responses and enabling the plants to better tolerate the adverse effects of abiotic stresses. Biosynthetic pathways of proline, glycinebetaine, and trehalose are well known. In addition, the metabolic flux analysis has been established. However, the information on the uptake and inter-organ transport in plants are largely unknown. Here, we review the update information on the transport of these osmoprotectants under abiotic stresses. Proline is mainly synthesized from glutamate in cytosol and/or chloroplast, which is reduced to glutamate-semialdehyde (GSA) by the pyrroline-5-carboxylate synthetase (P5CS), spontaneously converted to pyrroline-5-carboxylate (P5C) and reduced to proline by P5C reductase (P5CR). As an alternative pathway, proline can be synthesized from ornithine, which is transaminated first by ornithine-delta-­ aminotransferase (OAT) producing GSA and P5C, which is then converted to proline. Glycinebetaine (GB) is synthesized from choline by two-step oxidation of choline with enzymes choline monooxygenase (CMO) and betaine aldehyde dehydrogenase (BADH). Glycinebetaine is mainly localized in cytosol and chloroplast. Biosynthesis of trehalose involves the enzyme trehalose-6-phosphate synthase (TPS), which produces trehalose-6-phosphate (T6P) from UDP-glucose and S. Cha-um National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Pathum Thani, Thailand V. Rai National Research Center on Plant Biotechnology, IARI, New Delhi, India T. Takabe (*) Research Institute, Meijo University, Nagoya, Japan e-mail: [email protected] © Springer Nature Switzerland AG 2019 M. A. Hossain et al. (eds.), Osmoprotectant-Mediated Abiotic Stress Tolerance in Plants, https://doi.org/10.1007/978-3-030-27423-8_9

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glucose-­6-phosphate, and then glucose-6-phosphate is converted to trehalose and phosphate by the trehalose-6-phosphate phosphatase (TPP). Trehalose is synthesized in cytosol. We described the uptake and inter-organ transport of exogenous applied these osmoprotectants for cellular homeostasis, including redox balance and energy status, and signaling molecule.

2  Proline Uptake in Plants Accumulation of proline occurs due to the increased synthesis of proline and the decreased degradation of proline under various stress conditions. Plant transporters mediating proline uptake across the plasma membrane have been identified both in the amino acid transporter (ATF)/amino acid/auxin permease (AAAP) family and in the amino acid–polyamine–choline (APC) family (Table 1) (Rentsch et al. 2007). In the ATF/AAAP family, transporters that recognize proline have been identified in several subfamilies, namely, in the amino acid permease (AAP) family, the lysine/ histidine transporter (LHT) family, and the proline transporter (ProT) family. AAPs mediate proton-coupled uptake of glutamate (aspartate) and neutral amino acids including proline (Frommer et al. 1993; Fischer et al. 1995, 2002; Okumoto et al. 2002; Lee et  al. 2007; Schmidt et  al. 2007). LHTs transport neutral amino acids (including proline) and acidic amino acids with high affinity (Chen and Bush 1997; Lee and Tegeder 2004; Hirner et al. 2006). In contrast to transporters of the AAP and LHT family, ProTs transport proline but no other proteinogenic amino acids (Rentsch et al. 1996). Moreover, ProTs from Arabidopsis (Arabidopsis thaliana), tomato (Solanum lycopersicum L.), and gray mangrove [Avicennia marina (Forsk.) Vierh.] also transport glycinebetaine, though only the latter is a glycinebetaine-­ accumulating species (Breitkreuz et al. 1999; Schwacke et al. 1999; Waditee et al. 2002; Grallath et al. 2005). Three Arabidopsis ProTs and a ProT1 of tomato transport the stress-induced compound γ-aminobutyric acid (GABA), while GABA was not a substrate for the gray mangrove proline transporters (AmTs) (Breitkreuz et al. 1999; Schwacke et al. 1999; Waditee et al. 2002; Grallath et al. 2005). The affinity of the AtProTs for GABA was much lower than for proline or glycinebetaine (4.5 mM compared to 0.5 and 0.2 mM, respectively; Grallath et al. 2005). Barley (Hordeum vulgare L.) HvProT recognized only L-proline efficiently (Igarashi et al. 2000; Ueda et al. 2001). These data on substrate selectivity of AAPs, LHTs, and ProTs show that both transporters in plants with low and high selectivity for proline exist, indicating a role in general transfer of nitrogen and in proline-specific functions, respectively. In yeast, the general amino acid permease Gap1p and the proline transporter Put4p together mediate the major part of proline uptake (Lasko and Brandriss 1981). Gap1p transports all proteinogenic amino acids with low affinity, but Put4p transports only GABA, alanine, glycine, and proline. Other amino acid permeases are

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Table 1  Plant transporters for proline Target Accession Km for proline (μM) Expression organs Plant species proteins number ATF/AAAP (amino acid transporter family/amino acid/auxin permease) gene family ProT proline transporter (substrates: proline, glycinebetainea) Arabidopsis AtProT1 At2g39890 427b All organsa b AtProT2 At3g55740 500 Root, leafq b AtProT3 At2g36590 999 Leafb c Tomato LeProT1 AF014808 1900 Pollenc d Barley HvProT AB073084 25 Rootr e HvProT2 AB545851 246 Root, leaf e f Rice OsProT AB022783 – All organ Gray AmT1 AB075902 430g Root, leaf g mangrove AmT2 AB075903 320g Root, leaf g Oil palm EgProT1 AB597035 –h Root, leaf h Sugar beet BvBet/ProT1 AB477096 2100i Root, leafi Salt bush AqBet/ProT1 AB597034 1860i Root, leafi Amaranthus AmtBet/ AB597033 555i Root, leafi ProT1 AAP amino acid permease (substrates: neutral amino acids and glutamate, aspartatea) Arabidopsis AtAAP1 At1g58360 601, 1900j Root, flower, endosperms,t,u,v l AtAAP2 At5g09220 140 Root, stem, leafs,t m AtAAP3 At1g77380 250 Rootw m AtAAP4 At5g63850 134 Leaf, stems m AtAAP5 At1g44100 500 Root, leaf, stem, flowers n AtAAP6 At5g49630 67 All organsx LHT (lysine/histidine) transporter (substrates: neutral and acidic amino acidsa) Arabidopsis AtLHT1 At5g49780 10° Root, leaf, stemy n AtLHT1 At1g24400 13 Flowerz APC (amino acid–polyamine–choline) family CAT cationic amino acid transporters Arabidopsis AtCAT1 At4g21120 3000p Root, flower, leafp Rentsch et al. 2007, bGrallath et al. 2005, cSchwacke et al. 1999, dUeda et al. 2001, eFujiwara et al. 2010, fIgarashi et al. 2000, gWaditee et al. 2002, hYamada et al. 2011a, iYamada et al. 2011b, jFrommer et al. 1993, kBoorer et al. 1996, lKwart et al. 1993, mFischer et al. 1998, nLee and Tegeder 2004, o Hirner et al. 2006, pFrommer et al. 1995, qRentsch et al. 1996, rUeda et al. 2007, sFischer et al. 1995, tHirner et al. 1998, uLee et al. 2007, vSanders et al. 2009, wOkumoto et al. 2004, xOkumoto et al. 2002, yChen and Bush 1997, zFoster et al. 2008

a

also known but may contribute to residual low-affinity proline uptake (Andreasson et al. 2004). In bacteria, the accumulation of compatible solutes is controlled by synthesis, uptake, and export, though uptake is preferred over biosynthesis provided that proline or glycinebetaine is available (Kempf and Bremer 1998; Roeβler and Müller

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2001). A range of secondary active transporters (e.g., the E. coli H+/proline symporter ProP) and binding protein-dependent ABC transporters (e.g., E. coli ProU) mediate the uptake of proline and/or glycinebetaine and related substrates (Csonka 1989; Wood et al. 2001). Under osmotic stress, these transporters may be regulated via both increased gene expression and higher activity (Wood et al. 2001), and some were additionally shown to function as osmosensors (Morbach and Kramer 2002; Wood 2006). In addition to osmolyte uptake systems, E. coli uses the PutP transporter for uptake of proline as a nitrogen and carbon source (Csonka 1989). PutP expression is repressed by the trifunctional PutA protein (combining PDH, P5CDH, and regulatory functions in a single protein) in the absence of proline and becomes activated once the PutA protein is recruited to the membrane during proline degradation (Tanner 2008; Zhou et al. 2008).

3  Proline Uptake and Inter-Organ Transport During Stress ProT1 was expressed in all organs of the plants. High-level expression of ProT1 was observed during flowering and seed set. In contrast, mRNA levels of ProT2 were observed throughout the plant, but their expression was strongly induced by water or salt stress. This suggests that ProT2 is involved in nitrogen distribution during drought stress unlike the members of amino acid permease gene family such as AtAAP1–6, the expressions of which are generally suppressed under similar conditions. High proline concentrations were reported in the phloem sap of drought-stressed alfalfa. Active expression of ProT1 and ProT2 was observed in roots. High expression of ProT2 in root tip of maize was reported during stress (Verslues and Sharp 1999). These facts suggest that proline synthesis occurs more in roots and transported to shoot tissues and root tip under salt-stress conditions, while transport of broad specificity amino acids is suppressed. It was reported that LeProT1 supplies proline to both mature and germinating pollen. LeProT1 transports proline and g-amino butyric acid with low affinity and glycinebetaine with high affinity. Igarashi et al. (2000) found that OsProT specifically transported L-proline in a transport assay. Andreasson et al. (2004) found that proline and the toxic proline analogue azetidine-2-carboxylic acid are efficiently imported into yeast cells by four amino acid permeases, including two nitrogen-­ regulated permeases. Four ProT-homologous genes (ClProT1–4) from Chrysanthemum lavandulifolium were shown to be expressed in different organs under various stresses (Zhang et al. 2014). The expression of ClProT2 was restricted to above-ground organs and induced by various stress conditions.

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4  Physiological Role of Plant Proline Transporters Since AAP and LHT families are not selective for proline, these transporters may play a role in general acquisition and allocation of nitrogen in plants. Indeed, Arabidopsis knockout mutants LHT1, AAP8, and AAP1 as well as overexpression of AAP1 revealed complex changes in amino acid levels (Rolletschek et al. 2005; Hirner et al. 2006; Schmidt et al. 2007; Weigelt et al. 2008; Sanders et al. 2009). By contrast, the selectivity of ProTs for proline and other compatible solutes indicates a specific role in proline homeostasis under stress and non-stress conditions. Under salt stress, proline accumulation is accompanied by an increased expression of Arabidopsis ProT2; mangrove AmT1, AmT2, and AmT3; as well as HvProT (Rentsch et al. 1996; Ueda et al. 2001; Waditee et al. 2002; Table 1). Likewise, the high transcript abundance of AtProT1 and LeProT1 in pollen and of AtProT3 in the epidermis correlated with an elevated proline content (Schwacke et  al. 1999). In spite of these correlations, only few reports show the direct role of ProTs in proline transport in planta. The Arabidopsis knockout mutants AtProT1, AtProT2, or AtProT3 did not reveal phenotypic differences or altered proline content in the absence or presence of abiotic stress, indicating compensation by other transporters or altered proline metabolism (Lehmann and Rentsch, personal communication). However, the overexpression of HvProT in Arabidopsis resulted in reduced biomass and decreased proline levels in shoots, an effect that could be compensated by exogenous supply of low concentrations of proline (Ueda et al. 2008). On the other hand, root cap-specific expression of HvProT in Arabidopsis resulted in higher proline levels in root tips and enhanced root elongation (Ueda et al. 2008), supporting a role of proline in organ development.

5  Intracellular Transport Proline biosynthesis takes place in the cytosol and probably in chloroplasts under stress conditions (Szabados and Savoure 2010). Thus, at least in the absence of stress, proline import into plastids is necessary. Furthermore, transfer into mitochondria is essential for proline catabolism. Whereas information on proline transport into or out of plastids is lacking, proline uptake into mitochondria has been demonstrated to be mediated by two transport systems, i.e., a proline uniporter as well as a proline/glutamate antiport system (Elthon et  al. 1984; di Martino et  al. 2006), though a reversible switch of the transport mode as shown for other mitochondrial carriers cannot yet be excluded (Krämer 1998).

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6  E  ndogenous Proline Enrichment Using Proline Exogenous Application for Abiotic Stress-Tolerant Traits Alternatively, proline accumulation in higher plants using exogenous application is a useful method to elevate the high level of proline in the cellular levels. Exogenous foliar proline can uptake and translocate to other organs, especially under stressful conditions to function as major osmolyts as abiotic tolerant strategies (Osman 2015; Zouari et al. 2016a, b; Kahlaoui et al. 2018). The exogenous proline application for abiotic stress-tolerant traits, i.e., salinity, heavy metals, drought, and oxidative constraints, is summarized in Table 2. Based on the literatures, proline can be applied in both root via culture media (Ozden et al. 2009; Nounjan et al. 2012; Zheng et al. 2015) and foliar spray in the aerial parts (Moustakas et al. 2011; Kahlaoui et al. 2018; Zouari et  al. 2016b). Therefore, the level of proline in each plant species depends on the plant species, exogenous supply rates, growth conditions (in vitro culture, pot experiment, and field trials), plant developmental stages, and their interaction. In addition, proline biosynthesis key enzymes, i.e., P5CS and P5CR, are regulated by both stress and proline exogenous application, leading to play a role as antioxidant activities under abiotic stresses (Nounjan et  al. 2012). However, real mechanism on gene(s) regulation relating to proline metabolism by final product application is still uncleared.

7  Trehalose Uptake in Plants Trehalose is a nonreducing disaccharide formed by two glucose molecules. In plants, this disaccharide has diverse functions and plays an essential role in various stages of development (Griffiths et al. 2016). In Saccharomyces cerevisiae, it was shown that increasing intracellular trehalose is sufficient to confer desiccation tolerance to yeast (Tapia et  al. 2015). Sugar transporters have essential roles in the appropriate distribution of carbohydrates throughout the plants (Mueckler 1994). They are typically categorized in two groups: (i) secondary active membrane transporters, which promote the uphill permeation of sugars driven by electrochemical gradients of Na+ or H+ ions across the cellular membranes, and (ii) facilitative sugar transporters, which enable sugars to flow across membranes down the concentration gradients (Wood and Trayhurn 2003). Trehalose transporters have been identified from yeasts and insects (Stambuk et al. 1998; Kikawada et al. 2007). Saccharomyces cerevisiae possesses the α-glucoside transporter AGT1, which promotes uptake of disaccharides, including trehalose, sucrose, and maltose, via an electrochemical proton gradient, which suggests that AGT1 belongs to the group of secondary active transporters (Han et al. 1995). AGT1 acts as an H+-dependent trehalose transporter for the uptake of low-level trehalose as a nutrient from culture medium under low pH conditions. Insects have a facilitative trehalose transporter, TRET1, which seems

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Table 2  Proline accumulation using exogenous proline application for abiotic tolerant traits Exogenous Plant Plant species proline Endogenous proline Target traits organs Rice cv. KDML105 10 mM 500 μmol g−1 FW Salt tolerance Leaf cvs. MR varieties cvs. BRRI varieties Maize cv. BHM-7 Tomato cv. Rio Grande Eurya emarginata Olive tree cv. Chemlali Pea cv. Sprinter Olive tree cv. Chemlali Tobacco cv. BY-2 Eggplant Date palm cv. Deglet nour Rapeseed

References Nounjan et al. (2012) Teh et al. (2015)

5–20 mM

270 μmol g−1

Salt tolerance Leaf

5 mM

6.5 μmol g−1 FW

Salt tolerance Leaf

Hasanuzzaman et al. (2014)

15 mM

6 μmol g−1 FW

Salt tolerance Leaf

Rohman et al. (2015)

10 mg L−1

85 mg g−1 DW 60 mg g−1 DW 6 μg g−1 FW

Salt tolerance Leaf Root Salt tolerance Leaf

Kahlaoui et al. (2018) Zheng et al. (2015)

10 mM

25–50 mM 4.99 μmol mg−1 FW Salt tolerance Leaf 3.92 μmol mg−1 FW Root 60 mM

2.77 mg g−1 DW 2.70 mg g−1 DW

Ahmed et al. (2010)

Nickle (Ni) Leaf Salt tolerance

Shahid et al. (2014)

10–20 mM 140 μmol g−1 FW 200 μmol g−1 FW

Cadmium

Leaf Root

Zouari et al. (2016a)

10 mM

132.5 mM

Cadmium

25 μM

3.3 μg g−1 FW

Arsenate

Cell Islam et al. (2009) culture Leaf Singh et al. (2015)

20 mM

50 mg g−1 FW 4.2 mg g−1 FW 170 μmol g−1

Cadmium

5–20 mM

Leaf Root Leaf

Zouari et al. (2016b) Jonytienė et al. (2012)

Drought tolerance Drought tolerance

Leaf Seed Leaf

Osman (2015)

Cold tolerance

Pea cv. Master B

4 mM

Arabidopsis

10 mM

5.1 mg g−1 FW 30.2 mg g−1 FW 900 μmol g−1 DW

Grapevine cv. Öküzgözü

20 mM

0.45 μmol g−1 FW

H2O2 oxidative stress

Leaf

Ozden et al. (2009)

Wild almond 8 species

10 mM

0.6 μmol g−1 FW

H2O2 oxidative stress

Leaf

Sorkheh et al. (2012)

Moustakas et al. (2011)

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to be responsible for the regulation of trehalose levels in the hemolymph (Kikawada et al. 2007; Kanamori et al. 2010). Secondary active transporters for trehalose in multicellular organisms, including insects, have not been reported. It is difficult to predict the characteristics of sugar transporters based solely on the amino acid sequences because only subtle difference exists between proton-dependent sugar transporters and facilitative sugar transporters in the major facilitator superfamily (MFS; Pao et al. 1998).

8  E  ndogenous Trehalose Using Exogenous Trehalose Application for Abiotic Stress-Tolerant Traits In the natural habitat based on evaluation, high level of trehalose accumulation in higher plant is a rare report. Transgenic plants regulating on trehalose-metabolizing enzymes derived from microbial organisms are evidently discovered by several scientists (Garg et al. 2002; Paul et al. 2017; Kosar et al. 2018). On the other hand, it has been reported that non-stressed and stressed plants pretreated with trehalose increased the endogenous level of trehalose, which indicated that trehalose is readily absorbed by the roots and easily transported to the aerial parts to be functioned as major defensive responses to several abiotic stresses, i.e., salt tolerance, drought tolerance, heavy metal tolerance, heat tolerance, and stress-responsive regulation (Table 3). Therefore, there are large distributions in the exogenous concentration of trehalose and endogenous content depending on plant species, different providing methods, the stressors, and plant developmental stages (Yang et al. 2014; Ma et al. 2013; Abdallah et al. 2016).

9  Glycinebetaine Accumulation in Plants Plants in taxonomically distant species can synthesize GB and accumulate larger amounts when they are exposed to abiotic stress conditions, such as drought (Wyn Jones and Storey 1981), salt, and cold stress (Rhodes and Hanson 1993). But many plants do not accumulate GB and accumulate other osmolytes. Sugar beet (Beta vulgaris L.), spinach (Spinacia oleracea L.), barley (Hordeum vulgare L.), wheat (Triticum aestivum L.), and sorghum [Sorghum bicolor (L.) Monech] are known as GB accumulators. GB does not function only as “compatible solute” – a substance compatible with the cellular metabolism that accumulates in the cytoplasm to balance external osmotic pressure. It can protect proteins against thermodynamic perturbation caused by dehydration and heat denaturation. GB also protects the inhibition of several enzyme activity induced by NaCl. GB protects sugar beet root membranes against heat destabilization and spinach thylakoids against freezing stress. The concentra-

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Table 3  Endogenous trehalose using exogenous trehalose application for abiotic tolerant traits Plant species Arabidopsis

Rice cv. Nakdong cv. BRRI dhan29 cvs. Giza varieties Wheat cv. Zhoumai 18

Exogenous trehalose 30 mM

Endogenous trehalose 0.29 mg g−1 FW

5 mM

5.5 μmol g−1 DW

25 mM

0.242 mg g−1 Drought FW tolerance 1.5 μmol g−1 FW Copper

10 mM 25 mM

210 nmol g−1 FW

50 mM

380 μg g−1 FW

1.5 mM Duckweed

0.5–2.0 mM

Target traits Stress responses Salt tolerance

Plant organs Cell culture Seedling

Seedling Seedling

Salt tolerance Seedling

Water tolerance 1658 μg g−1 DW Heat tolerance 11.8 μg g−1 DW Cadmium

Callus culture Seedling Frond

References Bae et al. (2005) Yang et al. (2014) Redillas et al. (2012) Mostafa et al. (2015) Abdallah et al. (2016) Ma et al. (2013) Luo et al. (2010) Duman et al. (2011)

tions of GB required to produce these protective effects are often high (>0.5  M) (Rhodes and Hanson 1993). GB is thought to be distributed exclusively in cytoplasm and chloroplasts in plant cells; however, clear experimental data have not been reported. The amount of GB in the chloroplasts of spinach leaves was estimated to be close to 50% of the total GB in plant cells (Rhodes and Hanson 1993). Concentrations of GB of up to 0.3 M (20 times as high as the concentration calculated if GB distributes uniformly in leaf tissue) have been reported for chloroplasts isolated from salinized spinach plants. Such concentrations could contribute to chloroplast osmotic adjustment, facilitating maintenance of chloroplast volume and photosynthetic activity at low leaf-water potentials. The GB concentration was below detection limit in vacuoles of Sea Blite [Suaeda maritima (L.) Dumort.] leaf cells. Major solutes in vacuole are considered to be inorganic ions in leaves and sucrose in roots (Bell et al. 1996).

10  Glycinebetaine Biosynthesis in Monocot and Dicot Plants In plant, GB is synthesized by two-step oxidations of choline. Choline is first oxidized to betaine aldehyde by choline monooxygenase (CMO). Betaine aldehyde is further oxidized by betaine aldehyde dehydrogenase (BADH) to GB.  The CMO enzyme in GB-accumulating dicotyledonous Amaranthaceae (Chenopodiaceae, Beta vulgaris L., spinach, Atriplex, etc.) is localized in chloroplast, whereas the localization of CMO in GB-accumulating monocot such as barley and wheat seems

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to be different. Peroxysome localization of barley CMO and BADH has been reported (Fujiwara et al. 2008; Mitsuya et al. 2011). In addition to the localization of CMO and BADH, the sequence of CMO should be carefully analyzed. Sequence homology did not give us the information on the gene of CMO. For example, CMO homolog gene(s) can be found in Arabidopsis genome which is a well-known GB non-accumulator. The Arabidopsis CMO homolog did not show the choline oxidation activity (Hibino et al. 2002). Therefore, the real function of CMO homolog genes in GB non-accumulator must be clarified in the future. The experimental design is straightforward and the obtained results are clear.

11  Regulation of Gene Expression It has been known that the gene expression of CMO and BADH is induced by salt and drought stress. Production of GB involves expression of not only the abovementioned genes but also many genes that encode enzymes such as AdoMet synthetase, AdoHcy hydrolase, and methionine synthase. Tabuchi et al. (2006) examined transcriptional regulation of the GB-related genes in leaf beet. The expression of GB-related genes in leaf tissue showed induction under salt stress. Their induction showed diurnal rhythms were reduced in a dark condition. Salinization of spinach plants increases PEAMT mRNA abundance and enzyme activity in leaves by about tenfold, consistent with the high demand in stressed plants for choline to support GB synthesis (Nuccio et al. 2000).

12  Translocation of GB in Sugar Beet Halotolerant plants often accumulate GB under non-stress condition. GB of 420 μmol g−1 FW accumulated in young leaves of B. vulgaris L. even under normal growth conditions, whereas levels in mature leaves, cotyledons, hypocotyls, and roots were low (Yamada et al. 2009). Under the same conditions, CMO accumulates exclusively in old leaves and is difficult to be detected in young leaves. Incubation of segments of mature and young leaves with deuterium-labeled choline revealed that the rate of deuterium-labeled GB synthesis in mature leaves was about ninefold higher than that in young leaves. The levels of CMO mRNA were higher in mature leaves than in young leaves. Thus, the differential accumulation of CMO was essentially controlled by transcription level. When deuterium-substituted GB was foliar-­ applied to one of the attached mature leaf, labeled GB were preferentially detected in young leaves and root, but not in other expanded leaves. These data indicate that GB is primarily synthesized in mature leaves and translocated into young leaves. In response to salt stress, GB levels increased in all tissues, but most significantly increased in young leaves with only small increase in the levels of CMO (Yamada

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et al. 2009). GB functions as a chemical chaperone to protect the inactivation of proteins under high salinity and high temperature. In actively developing cells, the levels of protein synthesis and translocation would be high. GB might facilitate folding and translocation, serving as a chemical chaperone. Another possibility is that GB might be utilized to maintain high cellular pressure. It is known that high cellular pressure is required for cell growth (Bourot et al. 2000). Higher osmolarity in young leaves than in mature leaves is compatible with this idea. In animal cells, it has been shown that GB plays a role in cell volume homeostasis. The high concentration of GB in young leaves with a little de novo synthesis of GB suggests the importance of GB transporters in GB accumulation. To date, many amino acid transporter genes have been identified in the plant genomes (Yamada et al. 2009; Lehmann et al. 2010). Proline transporters (ProTs) were first isolated from A. thaliana as highly selective transporters for proline. However, ProTs from tomato (LeProTs) were subsequently shown to transport GB as well as proline, although tomato is a GB non-accumulating plant. Hitherto, functional properties of ProTs have been reported on GB non-accumulating plants, A. thaliana (AtProT1–3), tomato (LeProT1–3), oil palm (EgProT1), and rice (OsProT1), and GB-accumulating plants, mangrove (AmBet/ProT1–2), sugar beet (BvBet/ProT1), Atriplex (AqBet/ ProT1), Amaranthus (AmtBet/ProT1), and barley (HvProT1–2) (Ueda et al. 2001; Waditee et al. 2002; Grallath et al. 2005; Yamada et al. 2009, 2011a, b; Fujiwara et al. 2010). Among them, the selectivity of rice ProT for GB remains uninvestigated, and the barley HvProT1 was reported to recognize proline, but not GB. All other transporters mediate transport of both GB and proline. The selectivity of ProTs for proline suggests their specific role in proline homeostasis under stressed and non-stressed conditions (Lehmann et al. 2010). Oil production from oil palm is adversely affected by drought and salt. Under drought and salt stress, proline content increases in oil palm. The proline transporter gene from oil palm (Elaeis guineensis Jacq.) showed high similarity to Bet/ProT genes from several plants, but the highest homology to rice ProT1 (Yamada et al. 2011a). Expression of EgProT1 in Escherichia coli mutant exhibited uptake activities for GB and choline as well as proline. Under salt-stressed conditions, exogenous applied GB was taken up into the root more rapidly than the control. Two betaine/proline transporters (AmT1, AmT2) were isolated from GB-accumulating mangrove Avicennia marina (Waditee et al. 2002). Am1 and Am2 could efficiently take up GB and proline with similar affinities (Km, 0.32–0.43 mM). The uptakes of GB and proline were significantly inhibited by mono- and ­dimethylglycine but only partially inhibited by betaine aldehyde, choline, and 4-­ aminobutyrate. Betaine/proline transporter genes were isolated from GB-accumulating plants, sugar beet, Amaranthus, and Atriplex, as well as GB nonaccumulating (Arabidopsis) plant (Yamada et al. 2011b). Using a yeast mutant deficient for uptake of proline and GB, it was shown that all these transporters exhibited higher affinity for GB than proline. The uptake of GB and proline was pH-dependent and inhibited by the proton uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP). These transporters exhibited a higher affinity for choline uptake rather than GB uptake. Uptake of choline by sugar beet BvBet/ProT1 was indepen-

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dent of the proton gradient, and the inhibition by CCCP was reduced compared with that for uptake of GB, suggesting different proton-binding properties between the transport of choline and GB. The physiological role of GB uptake by Bet/ProTs still remains obscure. A homologous gene BvBet/ProT1 was isolated from sugar beet. The fusion protein of green fluorescent protein and BvBet/ProT1 showed that BvBet/ProT1 was localized at the plasma membrane. Levels of mRNA for BvBet/ProT1 were much higher in mature leaves than in young leaves under normal and salt-stress conditions. In situ hybridization experiments revealed the localization of BvBet/ProT1 in phloem and xylem parenchyma cells (Yamada et al. 2011b). Since GB is transported from the mature leaves to young leaves and the accumulated levels of BvBet/ProT1 gene transcript were higher in mature leaves than in young leaves, BvBet/ProT1 might play a role in the efflux of GB from CMO-expressing cells.

13  Exogenous Application for Crop Production In many crop plants, the natural accumulation of GB is lower than sufficient to ameliorate the adverse effects of dehydration caused by various environmental stresses. Exogenous application of GB to low-accumulating or non-accumulating plants may help reduce adverse effects of environmental stresses (Mäkelä 2004; Ashraf and Foolad 2007). Exogenous applied GB is, at least, partially immediately taken up by plant tissues and is readily translocated to roots, meristems, and expanding leaves (Mäkelä et al. 2000). Because GB is metabolically quite inert in plant, it remains in the plant tissue for several weeks. There is now strong evidence that GB plays an important role in tolerance to abiotic stress. Exogenous application of GB to non-accumulator plants may be a possible alternative approach for tolerance against multiple abiotic stresses (Table 4). Foliar spray of GB improved salt and drought tolerance in tomato. Application of tomato plants in field condition with 3.36 Kg ha−1 GB during mid flowering period increased fruit yield 36% in salt stress, as compared to control (Mäkelä 2004). Salt stress decreased net photosynthetic rate of tomato plant, and application of GB increased the rate of photosynthesis of the salt-stressed plants to the same level as control plants, which was not applied with GB. The increase of net photosynthetic rate by GB application was also observed in control plants. Stomatal conductance was increased by GB treatment, resulting in efficient gas exchange and reduced photorespiration. GB application protected chloroplast ultrastructure and prevented the decrease of chlorophyll and RuBisCO activity under salt and drought stress conditions (Mäkelä et  al. 2000). Relative water content of total shoot of tomato decreased under drought stress, and GB had increasing effect (Rezaei et al. 2012). The positive effect of GB on drought stress tolerance was more efficient at v­ egetative growth stage (Hussain et al. 2008). Exogenous application of GB increases chilling tolerance in tomato plants (Park et al. 2006). GB levels in tomato plants which took up exogenous GB were high in meristematic tissues such as shoot apices and flower

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Table 4  Endogenous glycinebetaine using exogenous glycinebetaine supplementation for abiotic tolerant traits Plant species Rice cv. KDML105 cv. Super-basmati

Cotton cv. MNH886 cv. CIM-496

Exogenous GB

References

Leaf Leaf

Cha-um et al. (2006) Farooq et al. (2008)

Cadmium 70 μmol g−1 Drought 140 μmol g−1 18 μmol g−1 FW tolerance

Leaf Root Leaf

Farooq et al. (2016) Ahmad et al. (2014)

240 μmol g−1 DW 45 μmol g−1 DW 51.02 g plant−1

Chilling tolerance Drought tolerance Drought tolerance

Cell culture Leaf Leaf

Chen et al. (2000) Ali and Ashraf (2011) LiXin et al. (2009)

10.88 μmol g−1 FW

Chilling tolerance

Leaf

Park et al. (2006)

16.38 μmol g−1 DW 50–100 mM 800 μg g−1 FW

Drought tolerance Drought tolerance

Leaf Leaf

Hussain et al. (2008) Iqbal et al. (2011)

5.0 mM 100 mg L−1

Tomato cv. Moneymaker

10 mM

Sunflower cv. Hysun −33

100 mM

cv. HF9703, SN215953 Bean cv. Tendergreen

Plant organs

2–6 mM 90 μmol g−1 DW Salt 50–150 mM 17 μmol g−1 DW tolerance Drought tolerance

Maize cv. Black Mexican 2–5 mM 30 mM sweet 50 mg L−1 cv. Agaiti-2002, EV-1098 cv. S9, S911

Cvs. Glushan-98, Suncross Tobacco cv. DHJ5210, ZY100 Wheat 19 genotypes

Endogenous GB Target traits

80 mM

900 μmol g−1 DW

Drought tolerance

Leaf

Ma et al. (2007)

100 mM

7.5 μmol g−1 Drought DW tolerance 56.8 μg g−1 DW Drought tolerance

Leaf

Gupta et al. (2014) Zhao et al. (2007)

1 μmol g−1 DW

Leaf

10 mM

Drought tolerance

Leaf

Xing and Rajashekar (1999)

buds. In leaves, GB existed mainly in cytosol with at most 22% in chloroplasts. The transport of GB from cytosol to chloroplasts might be inefficient. However, GB-treated plants exhibited increased levels of photosystem II (PSII) activity compared with control plants. GB-treated plants had significantly greater catalase (CAT) activity and CAT1 gene expression, although their H2O2 levels remained unchanged. During chilling treatment, H2O2 level was lower, and catalase activity was higher in

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GB-treated plants than those in control plants. These results suggest that GB may enhance the induction of antioxidant mechanisms under chilling stress condition (Park et al. 2006). When 28-day-old rice seedlings were exposed to NaCl, relative water content in the leaves drastically decreased. This adverse effect was largely prevented when the seedlings were treated with 15 mM GB before exposure to NaCl stress (Harinasut et al. 1996). GB added in culture medium was taken up by the roots and accumulated in the leaves to reach a concentration of 5.0 μmol g−1 FW−1 before transfer to NaCl stress. This level of GB is comparable with those of barley, GB accumulator. The level of GB in rice seedlings did not changed significantly after transfer to NaCl stress. No apparent difference in relative water content was found between control and GB-treated rice seedling before transfer to salt stress condition, and changes in relative water content after transfer to salt stress was significantly different. Control rice seedlings showed large decrease in relative water content (RWC) after transfer to salt stress; the extent of the decrease in RWC was small in GB-treated seedlings. Photosynthetic capacity of control seedlings also drastically decreased whereas that of GB-treated seedlings was maintained near the value of non-stressed seedlings. Higher water use efficiency in GB-treated rice plants than that in control plants under salt stress condition was reported (Cha-um et  al. 2006). Foliar spray of 50 mM GB to a salt-sensitive cultivar of rice enhanced proline accumulation under salt stress condition and resulted in maintaining transpiration efficiency and high photosynthetic performance. The photosynthetic abilities in plants were positively related to seed fertility percentage and total seed grain weight after recovery from the stress. This performance was rather inhibited with the high dose of 200 mM GB. Under salt stress, GB-treated rice plants had significantly lower Na+ and higher K+ concentrations in the shoots, compared with untreated plants (Cha-um et  al. 2006). Moreover, salt-induced ultrastructural damages in chloroplasts and mitochondria were prevented by GB pretreatment. Although the inhibition of root growth by NaCl was not alleviated, formation of large vacuoles was observed in root cells by GB pretreatment. It was proposed that the mitigation of the NaCl-­ induced damages in the shoots is due to the enhanced sequestration of Na+ into the vacuoles in root cells by GB pretreatment. The foliar GB application (50–100 mM) in cv. PT1 rice demonstrated the increase in water use efficiency, photosynthetic abilities, and seed grain yield under salt stress (150  mM NaCl) (Cha-um and Kirdmanee 2010) as well as under drought stress (25% soil water content) (Cha-um et al. 2013). Studies with wheat showed contradictory results. There were reports that exogenous GB delayed the canopy senescence of wheat, but this was not associated with its grain yields after the exposure to drought stress. On the other hand, there are reports that GB had effect to mitigate adverse effect of drought stress in wheat. There are also reports for the protective effects of GB against NaCl stress and freezing stress. Drought stress reduced plant biomass, grain yield, and phosphate (P) and K+ contents in shoot and root, but foliar-applied GB mitigated the adverse effects of drought stress (Ashraf and Foolad 2007). It was reported that mitigation of decrease in photosynthetic rates was not prominent but transpiration rate was apparently

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decreased by GB application. The extent of the effect of GB was varied among wheat cultivars. Drought stress caused an increase in osmotic pressure in flag leaves of wheat. The drought-resistant cultivar had higher values of osmotic pressure than the sensitive one. Drought stress induced marked decrease in grain yield, but spray of 10 mM GB mitigated the effect of drought stress. The application of GB increased the osmotic pressure, the osmolyte concentrations, as well as the grain yield (Ma et  al. 2006). Among the organic osmolytes, proline and keto acid concentrations were positively correlated with the grain yield. The drought-resistant cultivar had a higher concentration of K+ than the sensitive one, and the application of GB caused an accumulation in K+ in both wheat cultivars. Because K+ has some role in stabilization of native proteins, the increase in K+ level may play a role in adaptation of wheat plants to drought stress conditions (Raza et al. 2014).

14  Genetic Engineering of GB in Plants Various genes have been used to generate transgenic plants that accumulate GB and exhibit enhanced tolerance to abiotic stress (Chen and Murata 2011). Most of the transgenic plants of GB non-accumulators accumulate only low concentration of GB compared with that in GB accumulators. However, these transgenic plants often exhibited increases in abiotic stress tolerance. Two known factors have been reported to limit the accumulation of GB in transgenic plants: the availability of endogenous choline and the transport of choline across the chloroplast envelope (Nuccio et al. 2000). Exogenous supply of choline showed significant increase in the level of GB accumulation in transgenic plants. Indeed, the activity of phosphoethanolamine N-methyltransferase (PEAMT) was 30 to 100 times lower in tobacco, a GB non-­ accumulator, compared with spinach, a GB accumulator. Furthermore, it was revealed that the import of choline, a precursor of GB, into chloroplasts limits GB synthesis. A halotolerant cyanobacterium Aphanothece halophytica synthesizes GB by a three-step methylation of glycine with two genes: ApGSMT encodes the methylation of glycine and sarcosine and ApDMT encodes the methylation of dimethylglycine to GB.  The Arabidopsis plants expressing the glycine methylation genes (ApGSMT+ApDMT) showed improved tolerances for various abiotic stresses more than the CMO-expressing plants (Waditee et  al. 2005). The reason for the ­accumulation of GB in various tissues of the glycine methylation genes-expressing plants might be the availability of substrates. Glycine and serine are readily interconvertible via the action of serine hydroxymethyltransferase (SHMT) and glycine decarboxylase enzymes. The GB level in the transformed plants expressing the glycine methylation genes increased at high salinity. The high accumulation of serine at high salinity is reported (Ho and Saito 2001). Supply of exogenous glycine to Arabidopsis transformants enhanced the accumulation levels of GB and stress tolerance. Furthermore, a transgenic plant expressing ApGSMT and ApDMT together with 3-phosphoglycerate dehydrogenase (PGDH) encoding serine biosynthesis

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gene could accumulate more GB than plants transformed with ApGSMT and ApDMT (Waditee et al. 2007). This demonstrates the importance of precursor supply for biosynthesis of GB.

15  Summary and Future Prospects As described above, uptake and inter-organ transport of proline, glycinebetaine, and trehalose play important roles for plant adaptation under various abiotic stresses. While many studies have indicated a positive relationship between accumulation of these compatible solutes and plant stress tolerance, the accumulation levels are still low. Further efforts are required to increase the information on the transport mechanisms of these solutes, especially on the localization of solutes and transporters as well as the regulation mechanisms of these compounds. The future research using the important plants both in laboratory and field would contribute to understand the mechanisms of transport and their effective utilization in crop production under stress environments.

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Transgenic Plants Overexpressing Trehalose Biosynthetic Genes and Abiotic Stress Tolerance in Plants Zsófia Bánfalvi

1  Introduction A common response to water deficit is the accumulation of osmoprotectants such as sugars and amino acids. High concentrations of disaccharide trehalose occur in many anhydrobiotic organisms that survive complete dehydration and can be found in a relatively large amount in some resurrection plants. Accumulation of sucrose and trehalose during desiccation is proposed to prevent protein denaturation and membrane fusion (Suprasanna et al. 2016). In the yeast Saccharomyces cerevisiae, stress or starvation can lead to variations in trehalose content to over 20% of the cell dry weight. The most common pathway to produce trehalose involves two enzymes: trehalose-6-phosphate synthase (TPS), which catalyses the synthesis of trehalose-6-­ phosphate (T6P) from glucose-6-phosphate (G6P) and UDP-glucose (UDPG), and trehalose-6-phosphate phosphatase (TPP), which dephosphorylates T6P to trehalose. This pathway is found in a great variety of species, like insects, Escherichia coli and Mycobacterium tuberculosis and some species of nematodes and plants (Eleutherio et  al. 2015). In higher plants, however, trehalose is generally present in a low abundance. To improve abiotic stress tolerance of plants, several different approaches were used to increase the trehalose content of plants.

2  Abiotic Stress Tolerance with Phenotypic Alterations To increase the trehalose content of tobacco, as a model plant, Holmström et  al. (1996) transformed and expressed the TPS1 gene of yeast by the Rubisco promoter in tobacco (Nicotiana tabacum). Leaves of transgenic plants contained 0.8–3.2 mg Z. Bánfalvi (*) NARIC, Agricultural Biotechnology Institute, Gödöllő, Hungary e-mail: [email protected] © Springer Nature Switzerland AG 2019 M. A. Hossain et al. (eds.), Osmoprotectant-Mediated Abiotic Stress Tolerance in Plants, https://doi.org/10.1007/978-3-030-27423-8_10

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trehalose per g dry weight in contrast to 0.06 mg/g in non-transformed plants. T6P was not found in plants, suggesting that a tobacco enzyme dephosphorylated T6P to trehalose. When detached leaves were air-dried, those from trehalose-producing plants lost water more slowly than control leaves. When seedlings were drought-­ stressed, the trehalose-producing plants recovered turgor and recommenced growth, while the controls died. Nevertheless, TPS1 expression decreased the growth rate of the plants by 30–50% under optimal growth conditions. Constitutive expression of yeast TPS1 from the CaMV35S promoter also improved the drought tolerance of tobacco and, however, resulted in the same phenotypical changes as its expression by the Rubisco promoter (Romero et al. 1997; Lee et al. 2003; Karim et al. 2007). In prokaryotes, trehalose is frequently used as a compatible solute to contend with osmotic stress and can be used as an external carbon source. In Escherichia coli, trehalose biosynthesis reactions are catalysed also by TPS and TPP, encoded by the otsA and otsB gene, respectively (Giaever et al. 1988). Constitutive expression of otsA or otsA and otsB, like that of yeast TPS1, resulted in drought-tolerant tobacco plants. Nevertheless, these transgenic plants were similar also in phenotype to those with constitutive expression of yeast TPS1 because they were characterised by stunted growth and lancet-shaped leaves (Pilon-Smits et al. 1998; Jun et al. 2005). The situation was similar in Chinese cabbage (Brassica rapa subsp. pekinensis) expressing otsA. Although the transgenic Chinese cabbage plants were drought, salt and heat tolerant, they showed stunted growth and aberrant root development (Park et al. 2003). Constitutive expression of TPS1 with plant, namely, Arabidopsis origin in tobacco, had a similar effect on growth of the plants. However, it was demonstrated that these plants were not even drought but also salt and temperature tolerant and showed better acclimation to cadmium and excess copper compared to non-­ transformed control (Almeida et al. 2005, 2007a; Martins et al. 2014). Constitutive expression of yeast TPS1 by either Rubisco (RbcS1) or CaMV35S promoter in Arabidopsis also increased the tolerance of plants to different stresses, like drought, salt, freezing and heat, but also had negative effect on growth and root development (Karim et al. 2007; Miranda et al. 2007). The situation was the same in potato (Solanum tuberosum) and tomato (Lycopersicon esculentum), in which expression of yeast TPS1 by the CaMV35S promoter improved drought, salt and oxidative stress tolerance but the tomato plants had thick shoots, rigid dark-green leaves and aberrant root development (Yeo et al. 2000; Cortina and Culianez-Macia 2005). It should be noted, however, that potato plants showing altered phenotype in vitro recovered when planted in soil (Yeo et al. 2000). To avoid the negative effects of constitutive yeast TPS1 expression, Zhao et al. (2000) and Stiller et al. (2008) expressed TPS1 from a drought-inducible promoter in tobacco and potato, respectively. Both transgenic tobaccos and potatoes demonstrated increased drought tolerance under drought stress. Despite the drought-­ inducible nature of the promoters, a few transgenic tobacco plants had some obvious morphological changes including dwarf and fine shoot, lancet-shaped leaves and vigorous auxiliary buds, while the potato plants were smaller than the non-­ transformed control plants. These results indicated that tobacco and potato are so

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sensitive to expression of yeast TPS1 that even a low, basic activity of an inducible promoter can lead to phenotypical changes. It was known that in the basidiomycete Grifola frondosa, trehalose is synthesised directly from D-glucose and α-glucose 1-phosphate catalysed by trehalose synthase (TSase). To avoid potential regulatory problems with the introduction of multiple genes and expecting a higher catalytic efficiency for trehalose synthesis, Zhang et al. (2005) expressed the G. frondosa TSase gene by the CaMV35S promoter for manipulating abiotic stress tolerance in tobacco. These transgenic plants, however, also had obvious morphological changes, including thick and deep-coloured leaves, but showed no growth inhibition. However, the morphological changes disappeared in T2 progenies. Results are summarised in Table 1.

3  Abiotic Stress Tolerance Without Phenotypic Alterations Unlike expression of yeast TPS1 from the drought-inducible promoters Prd29A and StDS2 (Zhao et  al. 2000; Stiller et  al. 2008), fusion of the gene to the drought-­ inducible Arabidopsis promoter RAB18 clearly retained its drought stress responsiveness in tobacco and led to drought-tolerant transgenic plants with normal growth phenotype (Karim et al. 2007). Expression of yeast TPS1 together with the yeast phosphatase TPP or in fusion with TPP not only improved the drought tolerance of plants but raised the morphological aberrations even when expressed by constitutive promoters both in tobacco and Arabidopsis (Karim et al. 2007; Miranda et al. 2007). Fusion of otsA with otsB had the same effect in rice (Oryza sativa subsp. indica) and tomato (Garg et al. 2002; Jang et al. 2003; Lyu et al. 2013). By fusing the TPS1 and TPP genes of Zygosaccharomyces rouxii, the drought tolerance of potato could be improved when expressing the fused genes by the CaMV35S promoter (Kwon et al. 2004). An alternative solution was the transformation of chloroplast by yeast TPS1 or targeting it into the chloroplast with a transit peptide in front of the TPS1 coding sequence. In this way, osmotic and drought stress-tolerant tobacco and Arabidopsis lines were isolated with constitutive expression of TPS1 in chloroplasts (Lee et al. 2003; Karim et al. 2007). A similar result was obtained when otsA-otsB was targeted into rice chloroplasts (Garg et al. 2002). Overexpression of trehalose biosynthetic genes from plants may also be a good solution to overcome the problem of phenotypic alterations elicited by the E. coli or yeast TPS genes. For example, transgenic Arabidopsis plants overexpressing their own TPS1 gene displayed a dehydration tolerance phenotype without any visible morphological alterations, except for delayed flowering (Avonce et  al. 2004). Another good example is the expression of TPS gene of Zostera marina in rice. Z. marina is a kind of seed plant growing in seawater during its whole life history. Expressing its TPS gene by the CaMV35S promoter in rice resulted in salt-tolerant transgenic lines (Zhao et al. 2013). Another example is the constitutive expression

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Table 1 Stress-tolerant transgenic plants overexpressing trehalose biosynthetic genes with phenotypical changes Species Nicotiana tabacum

Promoter RbcS1

Gene(s) TPS1 Yeast

CaMV35S TPS1 Yeast CaMV35S otsA, otsB E. coli Prd29A

TPS1 Yeast

CaMV35S TPS1 Yeast CaMV35S otsA E. coli CaMV35S TSase Grifola CaMV35S TPS1 Yeast

Arabidopsis thaliana

Solanum tuberosum

Tolerance Drought

Changes in phenotype 30–50% decreased growth rate Drought Stunted growth, lancet-shaped leaves Drought Stunted growth, more leaves and larger leaf area Drought Dwarf and fine shoot, lancet-shaped leaves Osmotic stress Stunted growth, sterility Drought, salt Stunted growth, lancet-shaped leaves Drought, salt Thick and deep-­ coloured leaves Drought Delayed growth

CaMV35S TPS1 Arabidopsis CaMV35S TPS1 Arabidopsis RbcS1A TPS1 Yeast

Drought, salt, temperature Cu, Cd

Stunted growth

Drought

CaMV35S TPS1 Yeast

Drought, salt, freezing, heat Drought

Retarded growth and root development Aberrant growth, colour and shape Dwarfish growth, lancet-shaped leaves, aberrant root (in vitro); recovered in soil Smaller plants

CaMV35S TPS1 Yeast

StDS2

TPS1 Yeast

Drought

Lycopersicon CaMV35S TPS1 Yeast esculentum

Drought, salt, oxidative stress

Brassica rapa

Drought, salt, heat

CaMV35S otsA E. coli

Stunted growth

Thick shoots, rigid dark-green leaves, aberrant root development Stunted growth and aberrant root development

Reference Holmström et al. (1996) Romero et al. (1997) Pilon-Smits et al. (1998) Zhao et al. (2000) Lee et al. (2003) Jun et al. (2005) Zhang et al. (2005) Karim et al. (2007) Almeida et al. (2005, 2007a) Martins et al. (2014) Karim et al. (2007) Miranda et al. (2007) Yeo et al. (2000)

Stiller et al. (2008) Cortina and Culianez-­ Macia (2005) Park et al. (2003)

of TPS1 gene of cassava (Manihot esculenta Crantz) in tobacco leading to improved drought stress tolerance (Han et al. 2016). Nevertheless, overexpression of a plant TPS is not a guarantee of success. For example, TPS11 gene of cotton (Gossypium hirsutum), which is heat, drought and salt inducible, increased not the chilling stress tolerance but the chilling stress sensitivity of transgenic Arabidopsis seeds probably via the expression changes of some chilling-related genes (Wang et al. 2016).

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In various microorganisms including green algae and various fungi, trehalose phosphorylase (TP) produces trehalose by glucose-1-phosphate and glucose. When the TP gene of Pleurotus sajor-caju (oyster mushroom, one of the most commonly cultivated edible mushrooms in the world) was introduced and constitutively expressed in tobacco, it became drought tolerant (Han et al. 2005). In some bacteria, the biosynthesis of trehalose is mediated by maltooligosyl trehalose synthase (MTS) and maltooligosyl trehalose trehalohydrolase (MTH). In this pathway, T6P is not generated as an intermediate. One nonpathogenic bacterium, Brevibacterium helvolum, is known to contain these two enzymes. Joo et al. (2014) generated transgenic rice plants that overexpressed the bifunctional in-frame fusion gene of MTS and MTH and found that transgenic rice overexpressing the fused genes is drought stress tolerant without growth aberrations. Cassava (Manihot esculenta) is a perennial dicotyledonous crop belonging to the family of Euphorbiaceae. Its starchy tuberous roots have global importance as food and feed. Cassava is highly tolerant to abiotic stress and can be grown on arid and marginal land. Recently, unexpected high levels of trehalose in all tested cassava varieties have been detected may be due to the constant expression of a key active TPS gene. Transgenic tobacco lines constitutively expressing this gene accumulated significant level of trehalose and possessed improved drought stress tolerance (Han et al. 2016). Rice possesses several TPS genes divided in two classes: class I genes encoding enzymes with TPS activity and class II genes encoding proteins with no TPS activity. TPS1 in rice belongs to class I. N-terminal truncated rice TPS1 lacking the region just before the TPS domain has increased TPS activity compared to the full length protein. When it was overexpressed in transgenic rice, the truncated protein enhanced the tolerance to drought, salt and cold. To investigate the function of class II TPS genes, TPS2, 4, 5, 8 and 9 were overexpressed in rice. All of these class II TPSs improved the tolerance of rice to cold and salinity stress, and some of them also improved the drought tolerance of transgenic rice seedlings (Li et al. 2011). Vishal et al. (2019) supported Li and co-workers’ (2011) finding because it showed that overexpression of rice TPS8 was adequate to confer enhanced salinity tolerance of rice without any yield penalty, suggesting its usefulness in rice genetic improvement. Trehalose is hydrolysed to glucose by trehalase. TRE1 encodes the Arabidopsis trehalase. TRE1-overexpressing Arabidopsis plants had decreased trehalose levels and interestingly recovered better after drought stress. Leaf detachment assays showed that TRE1-overexpressing plants have a better water-retaining capacity (Van Houtte et al. 2013). Transgenic rice plants overexpressing the trehalase gene showed remarkable increases in trehalase activity and dramatic decreases in trehalose abundance compared with the wild type. The TRE1 overexpressors did not have notable morphological alterations or growth defects but interestingly exhibited enhanced salt tolerance, suggesting the involvement of TRE1 in salt stress tolerance in rice (Islam et al. 2019). Results are summarised in Table 2.

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Table 2  Stress-tolerant transgenic plants overexpressing trehalose biosynthetic genes without phenotypical changes Species Nicotiana tabacum

Promoter CaMV35S

CaMV35S RbcS1A RAB18 CaMV35S Arabidopsis thaliana

CaMV35S RbcS1A

CaMV35S rd29A CaMV35S CaMV35S Solanum tuberosum Lycopersicon esculentum Oryza sativa

CaMV35S CaMV35S ABA-­ inducible RbcS

Ubi1 CaMV35S CaMV35S CaMV35S 101MTSH

Gene(s) TPS1 Yeast Chloroplast transformation TP Pleurotus TPS1, TPP Yeast TPS1 Yeast TPS1 Cassava TPS1 Arabidopsis TPS1 Yeast Chloroplast targeting TPS1-TPP Yeast TPS1-TPP Yeast TRE1 Arabidopsis TPS11 Cotton TPS1-TPP Zygosaccharomyces otsA-otsB E. coli otsA-otsB E. coli otsA-otsB E. coli Chloroplast targeting otsA-otsB E. coli TPP1 Rice TPS1 (class I) Rice TPS (class II) Rice MTS-MTH Brevibacterium

Tolerance Osmotic stress

Reference Lee et al. (2003)

Drought

Han et al. (2005) Karim et al. (2007)

Drought Drought Drought

Han et al. (2016) Drought Avonce et al. (2004) Drought, fast recovery of Karim et al. dehydrated leaves (2007) Drought, salt, freezing, heat Drought, salt, freezing, heat Drought

Miranda et al. (2007)

Drought, salt, cold Salt, cold

Jang et al. (2003) Ge et al. (2008)

Drought, salt, cold

Li et al. (2011)

Van Houtte et al. (2013) Chilling stress sensitivity Wang et al. (2016) Drought Kwon et al. (2004) Drought, salt, heat Lyu et al. (2013, 2018) Drought, salt, cold Garg et al. (2002) Drought, salt, cold

Salt, cold Drought

Joo et al. (2014) (continued)

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Table 2 (continued) Species

Promoter 105MTSH CaMV35S Ubi1 OsTPS8

Zea mays

rd29A MADS6

Gene(s) MTS-MTH Brevibacterium TPS Zostera TRE1 Rice TPS8 Rice TPS1 Yeast TPP Rice

Tolerance Drought

Reference

Salt

Zhao et al. (2013) Islam et al. (2019) Vishal et al. (2019) Liu et al. (2015)

Salt Salt Drought Drought (field)

Nuccio et al. (2015)

4  The Role of Stomata in Drought Tolerance The water status of a plant is mainly regulated by the opening and closing of stomata. To avoid desiccation and ultimate death, stomata typically close during periods of water stress. However, stomata are the main gateways not only for water transpiration but also for photosynthetic CO2 exchange. Drought stress is known to depress gas exchange characteristics to a varying extent thereby affecting overall photosynthetic capacity of most plants. As drought continues, the stomata closure occurs for longer periods during the day. This, in turn, leads to the reduced carbon assimilation rate and water loss, resulting in maintenance of the carbon assimilation at the cost of low water availability with a final outcome in reduction of growth, biomass and yield (Ashraf and Harris 2013). Just a few months after publication by Holmström et  al. (1996) on drought-­ tolerant, but growth-retarded tobacco plants expressing the yeast TPS1 gene, Gaff (1996) raised the possibility of relating the phenotype of transgenic plants to altered stomatal movement. According to Gaff’s hypothesis, the transgenic plants might pass into the slow water-loss phase at higher fresh weights than non-transgenic plants, and thus transgenic stomata commenced closing at milder drought stress. As a result, water was retained for a longer time. During plant cultivation then, CO2 supply for photosynthesis would be restricted more frequently by stomatal closure, causing slower growth in the transgenic plants. Although no publication on the stomatal movement of TPS1 transgenic tobacco plants generated by Holmström et al. (1996) appeared in the literature to support or disprove Gaff’s theory, the results obtained by Stiller et al. (2008) in potato and Liu et al. (2015) in maize demonstrate a correlation between the number of stomata, drought tolerance and stunted growth of plants. In the case of TPS1 transgenic potatoes, detached leaves showed an 8-hour delay in wilting and a 30–40% reduction in stomatal densities compared to the non-transformed control, while under optimal growth conditions, lower CO2 fixation was detected in the smaller transgenic than in the larger control plants. The stomata density on the leaf surface of the TPS1

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transgenic maize was 20% lower than that of the wild-type plants (Liu et al. 2015). Stomatal development in the Arabidopsis model includes a signalling pathway with TMM, MAPK3, MAPK6 and SDD1 that negatively regulates the basal pathway of stomata lineage (Casson and Hetherington 2010). The expression of TMM, MAPK3, MAPK6 and SDD1 was increased in TPS1 transgenic maize (Zea mays) plants, indicating that TPS1 can negatively regulate stomata development and reduce the number of stomata to improve drought tolerance (Liu et al. 2015). In vitro studies revealed that trehalase TRE1-overexpressing, drought-tolerant Arabidopsis plants are more sensitive towards abscisic acid (ABA)-dependent stomatal closure. This observation is further supported by the altered leaf temperatures seen in trehalase-modified plantlets during in  vivo drought stress studies (Van Houtte et al. 2013).

5  T  ranscriptional Changes Associated with Abiotic Stress Tolerance Detection of transcriptional changes in abiotic stress-tolerant plants based on alteration in a trehalose biosynthetic pathway goes back to 2004, when the findings by Avonce et al. suggested that TPS1 in Arabidopsis has a pivotal role in the regulation of glucose and ABA signalling during vegetative development. The transgenic Arabidopsis plants overexpressing the Arabidopsis TPS1 gene showed up-regulation of the ABI4 and CAB1 genes. In the presence of glucose, CAB1 expression remained high, whereas ABI4, HXK1 and ApL3 levels were downregulated in the TPS1 overexpressing lines. Analysis of TPS1 expression in HXK1-antisense or HXK1-sense transgenic lines suggested the possible involvement of TPS1 in the hexokinase-dependent glucose signalling pathway in Arabidopsis. To obtain a general picture on gene expression influenced by the trehalose pathway genes, microarray analyses of transgenic Arabidopsis seedlings constitutively expressing otsA to elevate T6P level and otsB to decrease T6P level were performed. Analysis of microarray data showed up-regulation by T6P of genes involved in biosynthetic reactions, such as genes for amino acid, protein, and nucleotide synthesis, the tricarboxylic acid cycle and mitochondrial electron transport, which are normally downregulated by SnRK1. In contrast, genes involved in photosynthesis and degradation processes, which are normally up-regulated by SnRK1, were downregulated by T6P (Zhang et al. 2009). SnRKs are evolutionarily conserved SNF1related kinase complexes. SnRK1 is a key regulator in adjusting cellular metabolism during starvation, stress conditions and growth-promoting conditions. A number of different transcription factors have been identified recently as direct SnRK1 targets in plants (Wurzinger et al. 2018). Thus, we can conclude that T6P inhibits SnRK1 to activate biosynthetic processes, at least in part, via transcription factors. Another interesting finding highlighting the intimate connection between TPS1 and SnRK1 was obtained by Antal et al. (2013) demonstrating that repression of the regulatory

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subunit of the SnRK1 complex attenuates growth aberrations caused by the yeast TPS1 expression in potato. These plants, however, behaved like wild type in detached leaf assays. It therefore seems likely that the yeast TPS1-triggered morphological changes are indispensable for increased drought tolerance in potato. Kondrák et  al. (2011) analysed the transcriptome of potato leaves with a low level of yeast TPS1 expression, which improved drought tolerance, but slower growth and less stomata than in the wild type (Stiller et  al. 2008). Even under optimal growth conditions, 74 and 25 genes were up- and downregulated, respectively, in the mature source leaves of TPS1-transgenic plants when compared with the wild type. All of the seven genes, which were assigned into carbon fixation and metabolism group, were up-regulated, while about 42% of the assigned genes were involved in transcriptional and post-transcriptional regulation. By comparing the effect of otsA in Arabidopsis (Zhang et  al. 2009) with that of yeast TPS1 in potato, expression of 22 genes was changed in both plants. However, 10 of these displayed an inverse regulation (Kondrák et  al. 2011). Under drought stress conditions, wild-type potato leaves had twice as many genes with altered expression than TPS1 transgenic leaves, but 112 genes were differentially expressed in both strains (Kondrák et al. 2012). In transgenic rice lines overexpressing their own TPS1, some stress-related genes were up-regulated, including WSI18 (water stress-inducible protein), RAB16C (responsive to ABA), HSP70 (heat shock protein), DHN6 (dehydrin), LEA14A (late embryogenesis abundant protein) and ELIP (early light-inducible protein). These results demonstrate that rice TPS1 may enhance the abiotic stress tolerance of plants by regulating the expression of stress-related genes (Li et al. 2011). The salt-tolerant rice TPS8 expressing rice lines is ABA sensitive. TPS8 affects the gene expression level of ABA-responsive genes (RAB21, RAB16C, Xd422) and SnRK2s (Vishal et al. 2019), another family of SnRKs involved mainly in maintaining osmotic homeostasis (Yang and Guo 2018). It was demonstrated that TPP1 also plays a role in the activation of stress response genes in rice, which might be the main reason for the enhanced tolerance to abiotic stress of TPP1 overexpression lines (Ge et al. 2008).

6  M  etabolic Changes Associated with Abiotic Stress Tolerance Expression of TPS genes with various origins increased the trehalose level to different extent in different plant species from 1 to 2 μg/g fresh weight (FW) to approximately 1 mg/g FW or 3.2 mg/g dry weight (DW) (Iordachescu and Imai 2008). In contrast, some resurrection plants are able to accumulate trehalose in concentrations as high as 36 mg/g DW under dehydration condition (Drennan et al. 1993). In the organisms naturally accumulating osmolyte, the osmolyte concentration is 13–23 mg/g FW (Chen and Murata 2002). Thus, it was concluded that the amount of trehalose found in tissues of TPS expressing plants is too low for it to act merely

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as an osmoprotectant. It may be true even for tobacco plants expressing the TSase gene of Grifola, which accumulated 2.1–2.6 mg/g FW trehalose that was 400-fold higher than that of transgenic tobacco plants co-transformed with otsA and otsB (Zhang et  al. 2005). According to a recent theory, trehalose may promote the autophagy that is associated with drought tolerance (Williams et  al. 2015). It is interesting to note that in the drought-tolerant crop cassava, high amounts of trehalose (0.23–1.29 mg/g FW) were detected and the trehalose level was highly correlated with dehydration stress tolerance of detached leaves of the varieties. This is, however, not a general feature of plants. The biosynthesis of trehalose in cassava may have been enhanced in the evolutionary history by growing in arid and marginal areas (Han et al. 2016). Enhanced TPS expression unavoidably coincides with enhanced T6P levels, which might be the cause of phenotypic alterations. Although T6P measurements were rarely performed in abiotic stress-tolerant transgenic plants, the lack of morphological aberrations of tobacco plants co-expressing TPS1 and TPP or Arabidopsis plants expressing a fusion protein of TPS1 and TPP indicated that growth aberrations and improved drought tolerance can be uncoupled (Karim et al. 2007; Miranda et  al. 2007). This was demonstrated also for potato and tomato (Kwon et al. 2004; Lyu et al. 2013). A possible explanation was that overproduction of both TPS1 and TPP enzymes should lead to more rapid removal of T6P and thus results in normal plants. The role of T6P in regulation of whole-plant carbohydrate allocation and utilisation is recently reviewed and showed that decreasing T6P promotes resource mobilisation enabling better performance under abiotic stress (Paul et al. 2018). As early as in 1998, differences in levels of carbohydrates were detected in tobacco expressing the otsA and otsB. Leaves of one of the stressed transgenic plants contained about threefold higher levels of each of the four non-structural carbohydrates starch, sucrose, glucose and fructose, than leaves of stressed wild-­ type plants (Pilon-Smits et al. 1998). In contrast, starch concentration of the stressed potato leaves was very low, and the contents of fructose, galactose and glucose were increased and decreased in the wild-type and TPS1 transgenic potato leaves, respectively, while the amounts of proline, inositol and raffinose were highly increased in both the wild-type and TPS1 transgenic leaves under drought conditions (Kondrák et al. 2012). Concerning the proline content, a similar result was obtained in TPS1-expressing rice plants in which cold stress treatment increased proline levels four to five times than those without the treatment both in wild-type and transgenic plants (Li et  al. 2011). Thus it is fairly likely that alterations in the trehalose synthesis pathway do not influence the proline synthesis. The general accumulation of proline in stressed plants is elicited in a trehalose independent pathway (Fichman et al. 2015). Salt-tolerant transgenic rice plants overexpressing the rice trehalase TRE1 showed remarkable increases in trehalase activity and dramatic decreases in trehalose abundance compared with the wild type, with little change in the levels of other soluble sugars, such as glucose, fructose and sucrose (Islam et  al. 2019). Together with the previous finding that trehalase is involved in drought stress

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tolerance in Arabidopsis (Van Houtte et al. 2013), these data suggest that activation of the entire trehalose metabolic pathway is one of the strategies by which plants can adapt to abiotic stresses in their environment. Similarly, the improvement of stress tolerance without trehalose accumulation was previously reported for TPP1-­ overexpressing plants (Ge et al. 2008).

7  Conclusions The expression of both trehalose biosynthesis and degradation genes are induced in plants in response to abiotic stresses (Iordachescu and Imai 2008) and overexpression of both trehalose biosynthesis and degradation genes improves abiotic stress tolerance (Tables 1 and 2). Thus accumulation of trehalose is not a prerequisite of better adaptation to stress conditions. In dicotyledonous plant species, however, constitutive overexpression of trehalose biosynthetic genes resulted in stunted growth and lancet-shaped leaves. In contrast, no phenotypic alterations were detected in rice plants, and while alterations in carbohydrate metabolism were demonstrated in dicotyledonous plants both at transcript at metabolite levels, the rice plants altered in trehalose biosynthesis were characterised by induction the transcription of ABA-regulated stress-related genes. One of the explanations relates this difference to the so-called T6P:sucrose nexus, which forms part of a homeostatic mechanism to maintain sucrose levels within a range and that is related to the role of T6P in regulation of SnRK1 activity (Yadav et al. 2014; Paul et al. 2018). It is possible that while, for example, in Arabidopsis, T6P level appears to consistently correlate with sucrose level, this correlation, like in maize kernels (Bledsoe et al. 2017), is much weaker in rice than in Arabidopsis. Therefore, unlike in Arabidopsis, the alteration of T6P:sucrose ratio by TPS expression does not influence the growth rate in rice. An alternative explanation for different influence of constitutive TPS expression on dicotyledonous and rice plants can be based on a new finding related to the function of TPS1 in yeast. It was demonstrated that TPS1 protein itself, not trehalose, was the main determinant for stress resistance and protection of cells from commitment to apoptosis during growth in yeast (Petitjean et  al. 2015, 2016). Immunocytochemical staining, followed by electron microscopy, showed that the yeast TPS1 protein in transgenic tobacco with growth alteration was markedly present in the vacuoles and the cell wall (Almeida et  al. 2007b). Chloroplast transformation or targeting of yeast TPS1 into the chloroplast raised the aberrant phenotype of plants (Lee et  al. 2003; Karim et  al. 2007). A similar result was obtained by fusing TPS1 with TPP (Kwon et al. 2004; Miranda et al. 2007) or otsA with otsB (Lyu et  al. 2013). These results indicate that growth aberrations and abiotic stress tolerance of plants can be uncoupled and suggest that it is probably due to the prevention of TPS1 admission into the vacuoles or cell wall. Nevertheless, further experiments are needed to clarify the role of TPS1 as a protein per se in

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abiotic stress tolerance of plants. Overexpression of an inactive variant of yeast TPS1 in Arabidopsis or tobacco would be a possible approach to clarify this point. Despite the fact that using inducible promoters or gene fusions the abiotic stress tolerance of different crops could be improved without phenotypic alterations (Table 2), there is no sign that these crops have been transferred to field cultivation. The only exception is transgenic maize that expresses rice TPP1 from the rice MADS6 promoter, which is active over the flowering period, and produces higher yields than wild type with or without drought conditions (Nuccio et  al. 2015). Studying this maize line, it was found that decreasing the level of T6P in reproductive tissues downregulates primary metabolism and up-regulates secondary metabolism (Oszvald et  al. 2018). Thus, we can conclude that modification of the trehalose pathway provides multiple possibilities in crop improvement as a tool to increase abiotic stress tolerance and cereal yields as well as to alter the metabolite composition of seeds and tubers.

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The Role of Proline, Glycinebetaine, and Trehalose in Stress-Responsive Gene Expression Merve Kahraman, Gulcin Sevim, and Melike Bor

1  Introduction Biosynthesis of osmoprotectant molecules such as proline, glycinebetaine, trehalose, polyols, poliamines, and sugars are among the most common protective mechanisms against stresses which affect the osmotic potential of the cells. Introducing or increasing the expression of genes related to the biosynthesis of osmoprotectant molecules was reported to be promising for accelerating stress tolerance in plants. There has been a huge amount of information regarding the contribution of these solutes to tolerance against drought and any other types of stress that cause osmotic effect; however, we still lack the knowledge on their exact mode of action. For instance, since the first resurrection plant was discovered, high concentration and rapid accumulation of trehalose were demonstrated to be a unique feature of these plants. However, drought-sensitive Selaginella sp. accumulated more trehalose than drought-tolerant Selaginella sp. (Pampurova and Van Dijck 2014; Bledsoe et  al. 2017) which indicated that even in the drought-tolerant plants, the role and contribution of trehalose or other osmoprotectant molecules to stress tolerance was not completely deciphered. Protective function of proline, glycinebetaine, and trehalose has been known since the 1990s with confirmation from transgenic studies in A. thaliana, tobacco, rice, and wheat (Liu and Zhu 1997; Sakamoto and Murata 1998, Bor and Ozdemir 2018). Several crop plants have been genetically engineered for proline-, glycinebetaine-, and trehalose-related genes which were reported to have improved tolerance to several environmental constraints. Among the pioneer investigations, overexpression of proline biosynthetic geneΔ-pyrroline-5-carboxylate synthase in A. thaliana and tobacco plants (Liu and Zhu 1997) and overexpression of choline oxidase

M. Kahraman · G. Sevim · M. Bor (*) University of Ege, Faculty of Science, Department of Biology, Bornova-Izmir, Turkey e-mail: [email protected] © Springer Nature Switzerland AG 2019 M. A. Hossain et al. (eds.), Osmoprotectant-Mediated Abiotic Stress Tolerance in Plants, https://doi.org/10.1007/978-3-030-27423-8_11

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(­glycinebetaine biosynthesis-related gene) in rice (Sakamoto and Murata 1998) might be given as examples of which resulted in increased salinity tolerance in relation to proline and glycinebetaine accumulation, respectively. A more recent transgenic approach was reported by Nuccio et al. (2015). Maize plants overexpressing a rice trehalose-6-phosphate phosphatase gene had better yield performance under drought stress conditions at field trials (Nuccio et al. 2015). The amino acid proline, is reported to be accumulated to high levels when plants encounter different type of stress conditions. Besides its function in growth and development, it acts as an osmoprotectant and redox-buffering agent with an antioxidant characteristic under abiotic stresses (Kishor and Sreenivasulu 2014). On the other hand, high levels of proline have detrimental effects in plant cells leading to cell death; therefore, keeping cellular proline content in balance was reported to be critical for plant survival (Szabados and Savoure 2010; Kishor and Sreenivasulu 2014). The second well-known osmoprotectant molecule, glycinebetaine (GB), is an N-methyl-substituted glycine derivative found in microorganisms, animals, and plants such as sugar beet, wheat, and spinach (Sakamoto and Murata 2002; Ahmad et al. 2013). Besides osmotic adjustment capacity, stabilization of macromolecules, protection of membrane integrity, and contribution to regulating reactive oxygen species (ROS) are among the major roles for GB under stress conditions (Chen and Murata 2011; Ahmad et al. 2013). The third and most studied osmoprotectant molecule, trehalose, is a non-reducing sugar which was reported to be responsible for osmoregulation and protection against environmental stresses in different organisms including plants (Houtte et  al. 2013). Unlike other osmotic solutes, trehalose concentrations in wild-type and genetically engineered plants were reported to be low, and cellular compartmentalization was important. Therefore, trehalose-­mediated improvement in abiotic stress responses was suggested to be related to the activation of stress-responsive genes and transcription factors rather than being as an osmoprotectant molecule (Lunn et  al. 2006; Zhang et al. 2009; Houtte et al. 2013). Contribution of proline, glycinebetaine, and trehalose to stress-responsive gene expression for increased tolerance has been investigated extensively (Table  1). Understanding which genes and especially which transcription factors are up- or downregulated by these molecules would be important not only for a better understanding of stress-coping mechanisms in plants but also for maintenance of better crop performance and yield through manipulation of these genes in cultivated plants. In this chapter, we summarized the latest information regarding the effects of proline, glycinebetaine, and trehalose on the expression of stress-responsive genes in plants. Among these three molecules, only glycinebetaine was reported to be compatible which had no toxic effects even at high levels. Keeping the balance in proline and trehalose contents of the cells need to be tightly regulated. Therefore, for these two molecules, we have given information both for the genes that they regulate and the genes which are related to their biosynthesis and hydrolysis.

The Role of Proline, Glycinebetaine, and Trehalose in Stress-Responsive Gene Expression 243 Table 1  Proline-, glycinebetaine-, and trehalose-induced transcription factors and their roles in plant development and stress responses Transcription Factors bZIP11 bZIP53 bZIP44 bZIP2 MYBCORE WRKY MYC2 AP2/ERF TSRF1 JERF1 JERF3 SpERF1 DREB21 ERF71 Glycinebetaine DREB2A NAC5 WRKY bZIP53 IAA9 bHLH-FRO2 NDPK2 Trehalose bZIP11 bZIP12 bZIP53 bZIP44 bZIP2 WRINKL1 HY5 ABI5 EEL KNOTTED1 LEAFY WUSCHEL ATAF1 MYBS1 CIPK15 SUSIBA2 WRYK6 AGL4 RNA polymerase σ 70-type initiation factor JUMANJI

Proline

Function in Plants ProDH-related sugar signaling (Verslues and Sharma 2010) Proline catabolism (Satoh et al. 2004) Upregulation of P5CS1 and P5CS2 (Su et al. 2011) Regulation of P5CS (Gao et al.2008; Zhang et al. 2013; Yang et al. 2016) ABA and proline signaling (Li et al. 2018)

CMO gene expression (Khattab et al. 2014) BADH gene expression (Liang et al. 2017). Chilling tolerance (Einset et al. 2007) Fruit development (Zhang et al. 2019) Dehydration response (Ahmad et al. 2013)

Fine-tuning of carbon and nitrogen metabolism (Garapati et al. 2015; Chen et al. 2016; Laser and Weiste 2018) Development and growth responses (Garapati et al. 2015; Tsai and Gazzattini 2014; Chen et al. 2016; Laser and Weiste 2018) Low energy signaling (Garapati et al. 2015; Laser and Weiste 2018) Sugar signaling (Sun et al. 2003; Bae et al. 2005; Kretzshmer et al. 2015; Zhai et al. 2018) Fatty acid signaling (Zhai et al. 2018) ABA signaling (Bae et al. 2005; Tsai and Gazzattini 2014) Meristem identity function (Tsai and Gazzattini 2014; Coelho et al. 2018) Autophagy (Garapati et al. 2015) Anaerobic germination tolerance (Kretzschmer et al. 2015) Starch mobilization (Kretzschmer et al. 2015) Leaf senescence (Bae et al. 2005) Floral morphogenesis (Bae et al. 2005; Coelho et al. 2018) Plastid genome transcription, chromatin modification, transcriptional repression (Kondrak et al. 2012) Floral transition and shoot development (Coelho et al. 2018)

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2  Proline Proline is an amino acid which serves as an osmoprotectant and protective molecule at drought, salt, and other stress conditions. Although the accumulation of proline is a well-known response in stress-tolerant plants, the mode of action is still unclear (Ghars et al. 2012). Different roles have been attributed to proline such as scavenging of the hydroxyl radical, interacting with enzymes responsible for stress tolerance, protecting protein structure and enzyme activity, maintaining pH and redox balance, and supplementation of carbon, nitrogen, and energy (Hare et  al. 1999; Szabados and Savoure 2010; Ghars et al. 2012). Biosynthesis of proline occurs in two different pathways which include glutamate and ornithine. Glutamate pathway is the predominant route with two steps: first, glutamate is phosphorylated and reduced to Δ-pyrroline-5-carboxylate (P5C) by PC5 synthase enzyme (P5CS), and then it is reduced to proline by P5C reductase (P5CR) enzyme (Kim and Nam 2013). The second pathway is related to the activity of ornithine δ-aminotransferase (OAT) which also produces P5C that contributes to proline (Szabados and Savoure 2010; Liang et al. 2013). Recent findings have proved that proline has a significant role in osmotic adjustment, stabilization of cellular structures, and protection of photosynthetic apparatus. The translation start site of proline metabolism-related genes has putative cis-regulatory elements (CREs) site which interacts with several general transcription factors such as HD-HOX, AP2/EREBP, MYB, WRKY, and bZIP (Fichman et al. 2015). Therefore, regulation of proline content might be important not only for proline biosynthesis and catabolism but also for the control of the expression of different stress-responsive transcription factors and genes (Table 1). Accordingly, proline inhibited stomatal closure while promoted Ca+2 uptake in contrast to other amino acids such as histidine, methionine, aspartic acid, glutamic acid, and alanine (Rai and Sharma.1991; Rana and Rai 1996; Hayat et al. 2012). However, high levels of proline lead to impairment in the destabilization of DNA helix and susceptibility to S1 nuclease activity (Rajendrakumar et al. 1997; Szabados and Savoure 2010). To date, all of the defined stress response and tolerance processes in plants are regulated by complex signaling networks and have multigenic characteristics. Since proline is a common stress-responsive and adaptive molecule, it would be a good candidate for manipulating stress responses and tolerance mechanisms. Understanding which genes and especially which transcription factors are induced by proline will be beneficial for providing solutions to agricultural practices under changing environmental conditions. Enhanced proline accumulation at stress conditions was reported to be parallel to increased transcriptional activation of P5CS and P5CR genes while ornithine route seemed to have a less impact (Fig. 1). There are two P5CS enzymes in A. thaliana; one is chloroplastic and the other is cytosolic (Liang et al. 2013). P5CS1 is reported to be responsible for stress-induced proline biosynthesis, while the second one is required for developmental processes (Strizhov et al. 1997; Mattioli et al. 2009). P5CS1 transcription and proline accumulation are induced by cooperation of Ca+2-dependent calmodulin with MYB2

The Role of Proline, Glycinebetaine, and Trehalose in Stress-Responsive Gene Expression 245 ABIOTIC & BIOTIC STRESS

HR Ca+2 Level

Salicylic acid related-pathway

mETC NADPH oxidase NADPH

NADP

P5CDH L-GLUTAMATE

FADH2 FAD

P5CR

ProDH

GSA

P5C

L-PROLINE

P5C

pH maintanence

P5CS1 P5CS2 GSA

L-GLUTAMATE

OAT

Carbon & Nitrogen Sources

ORNITHINE BIOSYNTHESIS

CATABOLiSM Membrane trafficking

Ion channels & Proline transporters

Anoxidant-related genes

Toxicity

H2O2

Lipid seconder messenger

ABA signalling pathway

Phospholipase D & C

Fig. 1  Model of proline-related and proline-regulated processes in the plant metabolism. Proline biosynthesis and catabolism-related enzymes contribute to different physiological processes in development and stress responses. P5CS pyrroline-5-carboxylate synthase, P5CDH pyrroline-­5-­ carboxylate dehydrogenase, P5CR pyrroline-5-carboxylate reductase, ProDH proline dehydrogenase, HR hypersensitive response

transcription factor (Yoo et al. 2005). P5CS2 affected development of reproductive organs, and this was proposed to be related to flowering regulator CONSTANS genes (Samach et al. 2000). Expression level of P5CS, which encodes the enzyme that catalyzes the rate-­ limiting step in proline biosynthesis, was increased in response to salinity and drought. In addition, transcript level of P5CR encoding gene was also found out to be upregulated in the leaves of A. thaliana and in the roots of soybean and pea under osmotic stress (Delauney and Verma 1990; Williamson and Slocum 1992; Verbruggen et al. 1993; Liang et al. 2013). Transcription of P5CS is tightly regulated by proline levels by feedback inhibition (Zhang et al. 1995; Liang et al. 2013). On the other hand, proline levels are determined by the activities of proline dehydrogenase (proDH), P5CR, and pyrroline-5-carboxylate dehydrogenase (P5CDH) which are transcriptionally regulated and alter ROS-mediated signaling processes (Liang et al. 2013). The analyses of the promoter regions of several stress marker genes by bioinformatics tools have revealed that many of them had at least one proline-responsive element (PRE) in their promoter regions although their expressions were not affected by proline (Sharma and Verslues 2010). For example, a bZIP transcription

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factor which has a proline binding element was related to the induction of proDH by exogenous proline treatment. However, at stress conditions, the presence of neither proline nor ABA did not alter proDH expression (Sharma and Verslues 2010). Accordingly, bHLH-related two G-BOX motifs were found at Oryza sativa P5CS promoters. Overexpression of bHLH leads to enhanced osmotic and cold stress tolerance with increased proline levels (Liu et al. 2014, 2015; Jin et al. 2016). Similarly, OsP5CS2 and OsP5CR promoters had CACG NAC-core motif in their promoter regions, and overexpression of NAC genes increased drought and salt tolerance in relation to proline accumulation (Liu et  al. 2013; Hong et  al. 2016). Moreover, P5CS expression can be negatively regulated by different proteins such as annexins. These proteins are light-dependent Ca+2 and phospholipid binding proteins, and annexin mutants have increased P5CS expression which leads to drought and salt tolerance (Huh et al. 2010). Exogenous treatment of plants with proline or proline precursors affected the expression of different stress-related genes which resulted in tolerance against not only to abiotic stresses but to biotic stress. A recent confirmation was reported by Wang et al. (2017). Amino acid permease 1 (AAP1)-mediated proline uptake has improved salt stress tolerance in A. thaliana (Wang et al. 2017). When treated with the precursor of proline, P5C increased HR-like responses against pathogens by the activation of AvrB and AvrRpt2 genes (Funck et  al. 2008). Chen et  al. (2011) reported that proline affected calcium-mediated production of H2O2 and increased NDR1 expression-activated SA signaling pathway which lead to pathogenesis-­ related (PR) gene expression. In abiotic stress responses, exogenous proline was reported to be also responsible for protection of plants; however, there are controversial results which indicated the negative impact of proline on growth and metabolic processes. A. thaliana plants treated with proline at salt stress conditions had growth inhibition and accelerated senescence (Yamada et  al. 2005). Antioxidant enzymes Cu/ZnSOD and MnSOD encoding genes were upregulated in rice plants when treated with proline under salinity; however, in the absence of NaCl, the expression of these genes was suppressed (Nounjan et al. 2012). In the light of these findings, regulation of biosynthesis and catabolism of proline within the plant cells seemed to be more effective than exogenous proline treatment.

3  Glycinebetaine Glycinebetaine (GB) is the most common and best-known compatible solute that is found in several organisms including bacteria and plants (Castiglioni et al. 2018). GB is biosynthesized by two pathways; the most common route is via the oxidation of choline, while the other one is a bacteria-specific glycine methylation pathway (Fig. 2). In plants, choline is oxidized to betaine aldehyde by a ferredoxin-­dependent choline monooxygenase (CMO) which is then converted to GB by the activity of betaine aldehyde dehydrogenase (BADH) (Nuccio et al. 1998; Ahmad et al. 2013). Plants are divided into two classes: GB accumulators and non-accumulators

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Plants CMO BADH CHOLINE

Escherichia coli CDH BADH

GLYCINE BETAINE

Artrobacter globiformis COD/COX 2O2 + H2

GLYCINE

2O2

Acnopolispora halophilia GSMT SDMT SDMT

Fig. 2  Biosynthesis of glycinebetaine from different precursor molecules in different organisms. CMO choline monooxygenase, COD choline oxidase, BADH betaine aldehyde dehydrogenase, CDH choline dehydrogenase, GSMT glycine sarcosine methyltransferase, SDMT sarcosine dimethylglycine methyltransferase

according to their ability for GB biosynthesis. Accumulator plants such as sugar beet, spinach, and mangrove have the ability to well-adapt to drought and salinity conditions (Bor et  al. 2003; Ahmad et  al. 2013). Under osmotic stress-imposing conditions, even exogenous GB treatment was found out to have a protective role in plants; therefore, engineering non-accumulator plants for genes related to GB biosynthesis was proposed to be important for increasing yield of crop plants (Castiglioni et al. 2018; Bor and Ozdemir 2018). Crop plants such as rice, carrot, tomato, and potato are non-accumulators of GB, and in the recent years, transgenic studies for GB were accelerated for increasing crop biomass and yield (Ahmad et  al. 2013). In GB-synthesizing transgenic rice plants, more than 165 genes were upregulated and 76 genes were downregulated (Kathuria et al. 2009; Ahmad et al. 2013). Within the upregulated genes, 50 of them were related to the alleviation of various stress effects, and 115 of them were involved in regulation of gene expression, membrane transport, growth and development, signal transduction, and metabolism (Kathuria et al. 2009). GB functions at

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important processes such as osmoprotection, destabilization of DNA, refolding and thermal stabilization of proteins, maintenance of membrane integrity, and protection of enzymes (rubisco, rubisco activase, malate dehydrogenase, etc.) which are all remarkable components of plant tolerance to abiotic stresses (Chen and Murata 2011; Ahmad et al. 2013). Wei et al. (2017) reported that the activity of ion channels and transporters was regulated by GB which provided high potassium and low sodium levels conferring to salt tolerance in transgenic tomato plants. On the other hand, codA-and BADH-transgenic tomato plants had differential regulation of cell wall invertase, protein kinase, sucrose transporter, cyclin-dependent kinase, auxin transcription factor, and miniature zinc-finger protein (IMA) encoding genes which might be responsible for flower and fruit development (Wei et al. 2017). A generalized scheme for the processes and contribution of these genes to overall plant metabolism and stress responses was given in Fig. 3. In the case of stress-coping mechanisms, the possibility of different interactions between GB and stress-related metabolites was proposed (Fig.  3). For instance, maize plants treated with a nitric oxide (NO) inhibitor (Nω-nitro-L-arginine methyl ester; L-NAME) had reduced BADH gene expression which leads to low GB levels (Phillips et al. 2018). NO is known to contribute to ROS detoxification, regulation of antioxidant enzymes, and compatible solutes during abiotic and biotic stresses OSMOPROTECTION Proline Trehalose Carotenoids

DEFENSE MECHANISM Synthesis of antioxidant APX enzymes CAT SOD

Choline availibility IMPROVING PLANT GROWTH

*Biomass *Yield *Growth of reproductive organs

ROS

LIGHT

e-

PQ

2O2

CHOLINE

COD

PSII

CMO BADH

GLYCINE BETAIN

PC

H2

Transgenic plants

H2 O 2

CYT COMPLEX

D1

O2

PSI

O2

Repairing process of PSII

TRANSCRIPTIONAL CHANGES Synergistic Effect Stress-related TFs *Ethylene *ABA *Salicylic Acid Inhibition of LOX and PLD levels

CO2 RUBİSCO

Calvin Cycle

Protecting of CO2 Assimilation

Chaperon-mediate protein disaggregation

Interaction with chaperon-like ASR1

Fig. 3  The direct and indirect contribution of glycinebetaine metabolism to stress-coping processes in plants. SOD superoxide dismutase, CAT catalase, APX ascorbate peroxidase, CMO choline monooxygenase, COD choline oxidase, BADH betaine aldehyde dehydrogenase, ABA abscisic acid, PLD phospholipase D, LOX lipoxygenase

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(Uchida et al. 2002; Zhang et al. 2006; Guo et al. 2009; Phillips et al. 2018). Several metabolic routes are affected by GB accumulation and/or exogenous GB treatment. As indicated before, GB served not only by protecting proteins and enzymes but also by triggering transcription of stress-responsive genes or their transcription factors. Antioxidant enzymes, fatty acid metabolism-related enzymes such as lipoxygenase (LOX) and phospholipase-D (PLD) are among the most important enzymes which are regulated by GB levels.

4  Trehalose Trehalose is synthesized from uridine diphosphate glucose (UDP-Glc) and glucose-­ 6-­ phosphate (G6P) via trehalose-6-phosphate synthase (TPS) enzyme which dephosphorylated to a more effective form, trehalose-6-phosphate (T6P) by the activity of trehalose-6-phosphate phosphatase (Figueroa et al. 2016). In A. thaliana, T6P proposed to act as a signaling molecule in the regulation of sucrose level in order to provide optimal level of sucrose within the cell (Fig. 4). Oryza sativa TPS overexpressing lines, trehalose, and proline levels were highly induced with or without stress treatment. Expression of stress-related genes such as ELIP, HSP70, CRP, DHN6, LEA14A, and WS118 were increased up to twofold in these plants as compared to wild-type plants (Li et al. 2011). Increased level of T6P was related to the activation of nitrate reductase (NR) and phosphoenolpyruvate carboxylase (PEPC) through posttranslational modifications (Figueroa et al. 2016). Protein kinases, protein phosphatases, and other enzymes involved in these modifications were proposed to be the potential targets of T6P (Fig. 4). Trehalose was reported to serve as a compatible solute for the stabilization of membranes and biomolecules (Fernandez et al. 2010). In plant cells, trehalose is synthesized at low levels as compared to other compatible solutes such as proline, glycinebetaine, mannitol, etc. Hence, its being a common compatible solute is still under debate. High levels of trehalose were detected only in resurrection plants and in specific organs upon stress exposure (Avonce et  al. 2004; Schluepmann et  al. 2003; Grennan 2007; El-Bashiti et al. 2005; Garg et al. 2002; Fernandez et al. 2010). Since trehalose and T6P levels are usually very low in plants, they were proposed to have regulatory or sensing roles for source-sink relationship. Trehalose pathway might be a facilitator between the cellular compartments via regulation of different transcription factors under different environmental stresses (Table  1 and Fig.  5). T6P was thought to be a negative-feedback regulator for the adjustment of sucrose levels by interaction with SnRK1 (Bledsoe et al. 2017). T6P-sucrose interaction is adjusted according to developmental stage, tissue and cell type, and various environmental factors such as low temperature stress (Figueroa et al. 2016). Various studies indicated the importance of trehalose metabolism at transcriptional, translational, and posttranslational levels for controlling and regulating stress responses in plants (Table  1). In plant cells, sucrose:T6P ratio affects important metabolic processes in multiple ways via induction or repression of several

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TRE1 UDPHEXOSE-P

PHOTOASSIMILATES

SnRK1 AMINO ACID

TFs

ORGANIC ACID

SUCROSE

TCA Cycle HEXOSE-P STARCH AGPase ADP-glc

TRANSPORT OF SUGAR

SUC1 SWEET1

CO O2

PEPC NR

Glycolysis CARBON META METABOLISM & NITROGEN FIXATION

ABA related stomatal funcon

POST-TRANSLATIONAL MODIFICATION *PHOSPHORILATION PHOSPHORILATION *MONOUBIQUITINATION

Fig. 4  The interaction of trehalose pathway with different metabolic processes. TPS trehalose phosphate synthase, TPP trehalose phosphate phosphatase, TRE trehalose, T6P trehalose-6-­ phosphate, ABI4 ABA insensitive 4; Glc-6P glucose-6-phosphate, AGPase ADP-glucose-­ pyrophosphorylase, PEPC phosphoenolpyruvate carboxylase, ABA abscisic acid, SnRK sucrose non-fermenting receptor kinase, FUS3 mitogen-activated kinase, bZIP11 basic leucine zipper transcription factor 11

s­ tress-­responsive transcription factors (Fig. 4). For instance, increased T6P levels resulted in the repression of SnRK1 which is a key transcriptional regulator that responds to carbon and energy supply (Nuccio et al. 2015). Therefore, T6P influences SnRK1-­upregulated genes negatively and SnRK1-downregulated genes positively. Another transcription factor bZIP11 which affects the regulation of carbohydrate metabolism is also regulated by T6P. The developmental phase transitions, carbohydrate, and amino acid metabolisms are regulated by bZIPs (Tsai and Gazzarini 2014). Accordingly, it has been suggested that OsTPP7 contributes to anaerobic germination tolerance by modulating local T6P:sucrose ratios in germinating tissues which lead to upregulation of MYBS1 and CIPK15 genes for regulating amylase activation for increased starch mobilization (Kretzshmer et al. 2015). Trehalase catalyzes the hydrolysis of trehalose into two glucose monomers which was reported to be important for osmotic regulation and stress responses (Lunn 2007; Avonce et al. 2010; Houtte et al. 2013). A. thaliana had one trehalase

The Role of Proline, Glycinebetaine, and Trehalose in Stress-Responsive Gene Expression 251

SUCROSE LEVELS

SnRK1 NUCLEUS

FUS3

SUCROSE:T6P

ABI4

BZIP11 DEVELOPMENTAL PHASE TRANSITION

CYTOSOL

TPS

Tre6P

UDP-Glc + Glc6P

CHLOROPLAST

TPP

AGPase

Tre6P

ABA

TRE TREHALOSE

2GLU

PEPC STARCH

MALATE

STOMATAL OPENING

PHOTOSYNTHETIC ACTIVITY

Fig. 5  Impact of T6P-mediated photoassimilates partitioning on the plant metabolism. TPS trehalose phosphate synthase, TPP trehalose phosphate phosphatase, TRE trehalose, T6P trehalose-­6-­ phosphate, SnRK sucrose non-fermenting receptor kinase, ABA abscisic acid, PEPC phosphoenolpyruvate carboxylase, NR nitrate reductase, SUC1 sucrose transporter 1

encoding gene, TRE1, that has a MYB4 binding site in its promoter region (Lunn 2007; Avonce et al. 2010; Houtte et al. 2013). Besides this, a W-box promoter motif was identified in the AtTRE1 promoter for MYB102 and WRKY transcription factors which are known to be involved in ABA signaling at dehydration and osmotic stress conditions (Houtte et al. 2013). Since both MYB4 and MYB102 are members of the R2R3-type MYB family, these transcription factors can induce AtTRE1 expression during developmental processes such as guard cell differentiation (Houtte et al. 2013). Genetic control of trehalase would be a good tool for adjusting endogenous trehalose levels; therefore, drought tolerance might be manipulated by regulation of AtTRE1 (Houtte et al. 2013). Increased trehalase activity affected the sensitivity of guard cells to exogenous ABA treatments; thus, AtTRE1 may be essential for the ABA-induced stoma closure. One confirmation was reported from a study with Attre1-1 and Attre1-2 mutants which were unable to close their stomata in response to the ABA treatments (Houtte et al. 2013). On the other hand, hydrolysis of trehalose would be essential for different developmental processes. AtTRE1 was strongly upregulated during senescence in A. thaliana which indicated the contribution of trehalose degradation during programmed cell death (Yamada et al. 2005).

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Although upregulation of trehalose biosynthesis and exogenous trehalose treatments both have protective and regulatory functions in various plants such as tomato, tobacco, and rice under drought, salt, and cold stresses, we are still far from explaining the exact mode of action of trehalose in plants. Despite increasing stress tolerance in plants, overexpression of trehalose pathway-related genes has frequently resulted in dwarfism, delay in flowering, and abnormalities in leaf and root morphologies (Li et  al. 2011). Exogenous trehalose treatment in rice resulted in reduced damage under salinity which was proposed to be related to preservation of root integrity, decreased Na+ accumulation, and regulation of the genes responsible for osmotic adjustment (Garcia et al. 1997; Bae et al. 2005; Fernandez et al. 2010).

5  Conclusion Understanding stress-coping mechanisms is among the hot topics of plant science not only for basic scientific curiosity but also for improving agricultural yield and performance. Plants have evolved sophisticated stress tolerance mechanisms against abiotic and biotic stresses of which can be introduced to crop plants by transgenic approaches. Stress tolerance is a complex network of gene activation and signaling transduction routes; therefore, manipulation of one metabolic process may lead to undesired or unsufficient effects. Among these mechanisms, accumulation of osmoprotectant solutes was found out to be the most effective and compatible one since most of the crop plants have at least one type of these molecules or their precursors. Studies presented in this chapter might give an idea for how the biosynthetic and catabolic routes of these three molecules might be manipulated by genetic approach for improvement of stress responses in plants. Different characteristics and features of these molecules and how they affected transcription of stress-responsive and stress-related genes were discussed in detail. All of these molecules have an impact and ameliorative effect on stress tolerance in plants, and one might consider carefully for choosing the best candidate. Proline, for being a component of free amino acid; glycinebetaine, for being the most compatible solute among these molecules; and trehalose, for being an unusual sugar molecule with ability to preserve water, are all promising for regulating and controlling stress tolerance processes in plants.

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The Role of Proline, Glycinebetaine, and Trehalose in Stress-Responsive Gene Expression 253 Avonce N, Wuyts J, Verschooten K, Vandesteene L, Van Dijck P (2010) The Cytophaga hutchinsonii ChTPSP: first characterized bifunctional TPS–TPP protein as putative ancestor of all eukaryotic trehalose biosynthesis proteins. Mol Biol Evol 27(2):359–369 Bae H, Herman E, Bailey B, Bae HJ, Sicher R (2005) Exogenous trehalose alters Arabidopsis transcripts involved in cell wall modification, abiotic stress, nitrogen metabolism, and plant defense. Physiol Plant 125:114–126 Bledsoe SW, Henry C, Griffiths CA, Paul MJ, Feil R, Lunn JE, Lagrimini LM (2017) The role of Tre6P and SnRK1 in maize early kernel development and events leading to stress-induced kernel abortion. BMC Plant Biol 17:74 Bor M, Ozdemir F (2018) Manipulating metabolic pathways for development of salt-tolerant crops. In: Kumar V, Wani S, Suprasanna P, Tran LS (eds) Salinity responses and tolerance in plants, vol 1. Springer, Cham Bor M, Ozdemir F, Turkan I (2003) The effect of salt stress on lipid peroxidation and antioxidants in leaves of sugar beet Beta vulgaris L. and wild beet Beta maritima L. Plant Sci 164:77–84 Castiglioni P, Bell E, Lund A, Rosenberg AF, Meghan GM, Hinchey BS, Bauer S, Nelson DE, Robert J, Bensen RJ (2018) Identification of GB1, a gene whose constitutive overexpression increases glycinebetaine content in maize and soybean. Plant Direct 2(2):1–7 Chen TH, Murata N (2011) Glycinebetaine protects plants against abiotic stress: mechanisms and biotechnological applications. Plant Cell Environ 34:1–20 Chen J, Zhang Y, Wang C, Lu W, Jin JB, Hua X (2011) Proline induces calcium-mediated oxidative burst and salicylic acid signaling. Amino Acids 40:1473–1484 Chen X, Yao Q, Gao X, Jiang C, Harberd NP, Fu X (2016) Shoot-to-root mobile transcription factor HY5 coordinates plant carbon and nitrogen acquisition. Curr Biol 26:640–646 Coelho CP, Huang P, Lee DY, Brutnell TP (2018) Making roots, shoots, and seeds: IDD gene family diversification in plants. Trends Plant Sci 23:66–78 Delauney AJ, Verma DPS (1990) A soybean gene encoding delta-1-pyrroline-5-carboxylate reductase was isolated by functional complementation in Escherichia-coli and is foundtobeosmoregulated. Mol Gen Genet 221:299–305 Dröge-Laser W, Weiste C (2018) The C/S1 bZIP network: a regulatory hub orchestrating plant energy homeostasis. Trends Plant Sci 23:422–433 Einset J, Nielsen E, Connolly EL, Bones A, Sparstad T, Winge P (2007) Membrane trafficking RabA4c involved in the effect of glycine betaine on recovery from chilling stress in Arabidopsis. Physiol Plant 130:511–518 El-Bashiti T, Hamamcı H, Oktem H, Yucel M (2005) Biochemical analysis of trehalose and its metabolizing enzymes in wheat under abiotic stress conditions. Plant Sci 169:47–54 Fernandez O, Béthencourt L, Quero A, Sangwan RS, Clément C (2010) Trehalose and plant stress responses: friend or foe? Trends Plant Sci 15:409–417 Fichman Y, Gerdes SY, Kovács H, Szabados L, Zilberstein A, Csonka LN (2015) Evolution of proline biosynthesis: enzymology, bioinformatics, genetics, and transcriptional regulation. Biol Rev 90:1065–1099 Figueroa CM, Feil R, Ishihara H, Watanabe M, Kölling K, Krause U, Höhne M, Encke B, Plaxton WC, Zeeman SC, Li Z, Schulze WX, Hoefgen R, Stitt M, Lunn JE (2016) Trehalose 6-phosphate coordinates organic and amino acid metabolism with carbon availability. Plant J 85:410–423 Funck D et al (2008) Ornithine-delta-aminotransferase is essential for arginine catabolism but not for proline biosynthesis. BMC Plant Biol 8:40 Gao S, Ouyang C, Wang S, Xu Y, Tang L, Chen F (2008) Effects of salt stress on growth, antioxidant enzyme and phenyalanine ammonia-lyase activities in Jatropha curcas L seedlings. Plant Soil Environ 54:374–381 Garapati P, Feil R, Lunn JE, Van Dijck P, Balazadeh S, Mueller-Roeber B (2015) Transcription factor Arabidopsis activating factor1 integrates carbon starvation responses with trehalose metabolism. Plant Physiol 169:379–390 Garg AK, Kim JK, Owens TG, Ranwala AP, Choi YD, Kochian LV, Wu RJ (2002) Trehalose accumulation in rice plants confers high tolerance levels to different abiotic stresses. Proc Natl Acad Sci U S A 99:15898–15903

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Seed Osmolyte Priming and Abiotic Stress Tolerance Danny Ginzburg and Joshua D. Klein

1  Introduction Seed priming is any technique in which seeds are imbibed in a solution prior to sowing in order to improve germination rates and uniformity and/or to confer abiotic and biotic stress tolerance. Priming can be performed with water alone (hydropriming) or with chemical or bioactive compounds (Paparella et al. 2015). The controlled seed rehydration induced by priming triggers metabolic processes associated with early states of germination such as restoration of cellular integrity, initiation of respiration and DNA repair functions, and increased activity of antioxidant enzymes and reactive oxygen species (ROS) scavenging. Priming with water, chemical, or biological solutions can enhance seedling or mature-plant tolerance to abiotic stresses. The method of introducing a priming compound into a seed (soaking, solid matrix priming, seed coating) can influence the efficacy of the treatment (Klein et al. 2017; Wang et al. 2016). Priming conditions – chemical concentrations/osmotic potentials, durations, and temperatures – can in turn affect the amount of material taken up by the seed (El-Araby and Hegazi 2004; Posmyk et al. 2008). With the correct priming conditions, a seed has a high enough water content to initiate germination-related processes, but low enough to prevent germination (radicle emergence) and to retain desiccation tolerance upon drying. The specific processes induced by priming and the degree of their expression may also vary depending on plant species, seed structure, and quality (Paparella et  al. 2015). Embryo location (external as in monocots or internal as in dicots) may influence the ability of a priming compound to affect seed germination and seedling growth, as can amount of endosperm (low in onions or tomatoes versus high in wheat or beans). The amount of mucilage generated on the seed coat (as in basil or rocket seeds) may also affect uptake and effectiveness of the priming compounds (Western 2012, and references therein). D. Ginzburg · J. D. Klein (*) Institute for Plant Science, ARO-Volcani Center, Rishon LeZion, Israel e-mail: [email protected] © Springer Nature Switzerland AG 2019 M. A. Hossain et al. (eds.), Osmoprotectant-Mediated Abiotic Stress Tolerance in Plants, https://doi.org/10.1007/978-3-030-27423-8_12

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We review here recent and foundational literature on methods and compounds used in seed priming to achieve tolerance to abiotic stress in emerging and growing plants, as well as proposed mechanisms of action and potential for future research and development.

2  A  biotic Stress in Plants: Phenomenon and Induced Tolerance Abiotic stress can affect all major aspects of plant development, from seed germination to growth, flowering, and seed development. The degree to which a given stress affects a crop depends both on the severity and on the crop’s tolerance or resistance to the stress. The effects of abiotic stress, and the plant’s responses, are specific to the type of stress (i.e., limited photosynthesis and nutrient availability under drought and salinity, suboptimal cellular respiration under heavy metal stress, and slowed metabolic activity in cold temperatures). Under all abiotic stresses, though, plants produce toxic levels of ROS capable of lipid peroxidation, protein denaturation, and DNA mutation (Jaspers and Kangasjärvi 2010). Although necessary for proper development at low concentrations, ROS concentrations accumulate to toxic levels under abiotic stress and become particularly damaging to organelles such as mitochondria and chloroplasts, which facilitate a high rate of electron flow (Gill and Tuteja 2010). A common response to abiotic stress is therefore the induction of antioxidative enzymes and metabolites to neutralize the damaging effects of ROS (Bernstein et al. 2010). The specific antioxidant compounds (enzymatic and nonenzymatic) upregulated during stress conditions and the degree of their expression are influenced by the plant developmental state, duration of stress, and subcellular localization of ROS accumulation (Reddy et al. 2004; Gill and Tuteja 2010). Another general characteristic of stress tolerance is maintaining osmotic homeostasis, which is critical for protecting membranes from desiccation and facilitating continued nutrient uptake from a potentially high-ionic root zone, such as during salinity or heavy metal stress. Maintaining osmotic homeostasis is achieved in general by increasing the concentration of osmolytic compounds, for example, total soluble sugars (Jisha and Puthur 2016b), and/or amino acids such as proline (Hayat et al. 2012). Abiotic stress tolerance is a complex biological trait expressed both physiologically and morphologically through altered gene expression, modified hormone levels, metabolite biosynthesis, and antioxidant activity (Zhang et al. 2014; Nguyen et al. 2018). Examples of stress tolerance responses include increasing root growth, modifying cellular relative water content via osmotic adjustment, and abscisic acid-induced stomatal closure to improve water use efficiency upon drought and salinity stress (Bartels and Sunkar 2005; Taiz and Zeiger 2006), and faster and more efficient utilization of storage compounds during chilling stress (Hussain et al. 2017).

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3  P  riming Compounds Conferring Abiotic Stress Tolerance in Plants Plant growth regulators (PGRs) are naturally occurring or synthetic compounds which modify developmental and/or metabolic processes by specifically affecting a plant’s natural hormone system (Rademacher 2015). Often bioactive at very small concentrations, PGRs are widely used to modify plant morphology, confer stress tolerance, improve yield, and increase harvesting efficiencies. In addition to the bioactive compound used, the method of PGR application and plant developmental stage is critical to achieve the desired effects. Under abiotic stress conditions, exogenous application of PGRs induces common stress tolerance responses such as increased antioxidant enzyme activity, leaf proline content, and relative water content. Examples include the brassinosteroid 28-homobrassinolide on Indian mustard (Brassica juncea) (Zhang et al. 2014), melatonin on fava beans (Vicia faba) (Dawood and El-Awadi 2015), salicylic acid on rice (Oryza sativa) (Wang et al. 2016), and gamma aminobutyric acid (GABA) on ryegrass (Lolium perenne) (Krishnan et al. 2013). While PGRs are usually applied as a foliar spray, they have proven to be valuable seed priming agents as well. Wheat seeds (Triticum aestivum) primed with benzyl aminopurine had increased α-amylase activity and soluble sugar concentrations during salt stress (Bajwa et al. 2018). Under drought conditions, rice grown from seeds primed with salicylic acid had increased antioxidant activity, osmolyte concentration, and cellular water potential (Farooq et al. 2009). Red cabbage seedlings (Brassica oleracea rubrum) grown from melatonin-primed seeds had higher germination rates, decreased levels of lipid peroxidation, and higher fresh weight when grown under high concentrations of copper (Posmyk et al. 2008). In addition to traditional PGRs, essential oils (EOs) and botanical extracts are increasingly used in place of synthetic compounds to protect against biotic and abiotic stresses. Thymol and carvacrol, monoterpenes derived from the essential oils of thyme and oregano, have antioxidant properties which support membrane integrity during abiotic or biotic stresses (De Azeredo et al. 2011; Ye et al. 2016). Seedlings grown from radish seeds (Raphanus raphanistrum) imbibed in carvacrol had increased pigment (carotenoid and anthocyanin) concentrations, antioxidant activity, and survival rate under drought conditions (Klein et al. 2017). Thymol-priming reduced the effects of salinity stress on pea seedlings (Pisum sativum) by increasing superoxide dismutase (SOD) activity (Kazemi 2013). Rice seeds primed with sunflower extract had increased root and shoot length and increased fresh weight when grown under high salinity (Farooq et al. 2011). Allelopathic sorghum extract priming increased total phenolics and soluble sugar concentrations and decreased Na+ content in wheat grown under high salinity (Bajwa et al. 2018). Given their bioactivity at small concentrations, optimal priming concentrations for PGRs, EOs, and botanical extracts need to be determined to avoid negatively affecting germination and seedling development (Fariduddin et al. 2003; Posmyk et al. 2008; Martino et al. 2010; Arteca 2013). Recently, priming with osmolytic compounds has received considerable interest by the scientific community. Some of

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the beneficial effects of priming with osmolytic compounds in various plant species against different abiotic stresses and at various stages of plant growth are shown in Table 1. Table 1  Selected references for seed priming with osmolytes and resulting abiotic stress tolerance Priming compound Proline

Plant species Vigna radiata

Oryza sativa L.

Mannitol

Cakile maritima L.

Medicago sativa L.

Cicer arietinum L.

Glycinebetaine Four (GB) turfgrass species Oryza sativa L.

Trehalose

Zea mays L.

Raphanus sativus L.

Concentration and duration of priming 5 mM and 150 mM proline; 6 hours

Positive effects of priming regulating stress tolerance Increased germination %, proline content, and hypocotyl growth, decreased lipid peroxidation under chilling stress 1, 5, and 10 mM Increased root and shoot proline; 12 hours length, chlorophyll concentration, and proline biosynthesis under salinity stress Increased RWC, GSH and 2% mannitol proline content, and SOD solution; activity, decreased MDA levels 12 hours under drought and salinity stresses 4% mannitol; Increased root and shoot 12 hours length, proline content, and antioxidant activity, decreased ion leakage under salinity stress 1–10% mannitol; Increased root and root fresh 24, 48, 72 hours and dry weight, amylase and sucrose synthase activities, and total leaf sugar content under osmotic stress Increased germination rate, 50, 100, 150, seedling fresh weight, and and water content under both 200 mM GB; osmotic and salinity stresses 24 hours Increased leaf GB content, 50, 100, and soluble sugar and antioxidant 150 mg/L GB; concentrations, increased 48 hours RWC, and decreased ion leakage under drought stress Decreased ion leakage and 10 mM lipid peroxidation, increased trehalose; leaf K/Na ratio under salinity 8 hours stress Increased root fresh weight, 25 and 50 mM GB content, antioxidant trehalose; content and activity, decreased 14 hours lipid peroxidation under drought stress

Reference Posmyk and Janas (2007)

Deivanai et al. (2011)

Ellouzi et al. (2017)

Amooaghaie (2011)

Kaur et al. (2005)

Zhang and Rue (2012)

Farooq et al. (2008)

Zeid (2009)

Shafiq et al. (2015)

(continued)

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Table 1 (continued) Priming compound Spermidine

Plant species Oryza sativa L.

Triticum aestivum L. Trifolium repens

GABA/BABA

Trifolium repens

Oryza sativa L.

Concentration and duration of priming 5 mM spermidine; 24 hours

Positive effects of priming regulating stress tolerance Increased phenolic, GB, soluble sugar and protein contents, α-amylase, and antioxidant activities under chilling stress Increased stomatal 5 mM conductance and grain yield, spermidine; decreased shoot [Na+] and 12 hours [Cl−] under salinity stress Increased α-amylase activity, 30 μM fructose and glucose spermidine; concentrations, increased 3 hours antioxidant activity, and decreased lipid peroxidation under osmotic stress 1 μM GABA; Increased root and shoot 2 hours length, fresh and dry weight, and dehydrin concentrations, decreased peroxidation levels under salinity stress 0–2.5 mM Increased pigment BABA; 12 hours concentration, PS I and II activities, antioxidant enzyme activity, and proline content, decreased MDA concentration under salinity and osmotic stresses

Reference Sheteiwy et al. (2017)

Iqbal and Ashraf (2005)

Li et al. (2014)

Cheng et al. (2018)

Jisha and Puthur (2016b)

4  P  ossible Mechanisms of Osmolyte Priming: Induced Abiotic Stress Tolerance The currently understood mechanisms of priming for abiotic stress tolerance are twofold. Seed imbibition and drying promote the seed to an advanced germinative state such that primed seeds germinate faster and at higher rates than non-primed seeds under adverse conditions, resulting in increased competitiveness for limited resources and overall yield increases (Chen and Arora 2013; Paparella et al. 2015). This advanced germinative state is the result of multiple induced processes such as repair and increased synthesis of metabolic machinery, cell-cycle components, aquaporin activity, and ion transporters. Seed imbibition additionally promotes increased gibberellic acid (GA) biosynthesis and abscisic acid (ABA) degradation, thus furthering the germinative state via reserve mobilization and endosperm weakening (Bewley et al. 2012; Chen and Arora 2013; Zhang et al. 2014). Seed imbibition with water alone also imparts a stress imprint or “memory” to the seed which remains after drying. Even under optimal priming conditions, and regardless of the protocol used, seed hydration and drying are inevitably damaging

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to a seed due to ROS production and membrane disruption upon imbibition and the likely decreased desiccation tolerance caused by drying (Chen and Arora 2013). Improper priming conditions, such as rapid water uptake, priming at low temperatures, and/or rapid drying, would therefore only exacerbate such damage (Bewley et al. 2012). This stress imprint is often connected to increased antioxidant activity and protective compounds in the seedling. Antioxidant enzyme activity has been correlated to stress tolerance in many plant species (Munns 2002; Ashraf and Ali 2008; Posmyk et al. 2008; Zhang et al. 2014; Hussain et al. 2017). Increased antioxidant activity can be attributed both to seed rehydration in general and to specific priming agents and conditions to induce stress (osmotic, salinity, heavy metal, etc.) such as seed microencapsulation (Murungu et  al. 2004) or priming temperature and duration (Bujalski and Nienow 1991). Based on their bioactivity and concentration, the specific priming compounds used may further influence seed germination and seedling growth beyond that of water alone (hydropriming) (Posmyk et al. 2008; Baier et al. 2019; Bajwa et al. 2018). The protective effects of seed priming against oxidative stress have been extensively reported. Primed seedlings exposed to abiotic stress had increased activities of ROS scavenging enzymes such as superoxide dismutase, catalase, and ascorbate peroxidase (Bailly et al. 2000; El-Araby and Hegazi 2004; Lei et al. 2005; Hussain et al. 2017; Latif et al. 2016). In all cases, seedlings from primed seeds had higher germination rates and decreased levels of lipid peroxidation. Priming also combats oxidative stress by increasing polyphenol and pigment biosynthesis (Bailly 2004; Nouman et al. 2012; Latif et al. 2016; Klein et al. 2017). In addition to increasing ROS scavenging, seed priming with osmolytes directly supports membrane integrity and protein structure during abiotic stress. This has been achieved by increasing the concentration of late embryogenesis abundant (LEA) proteins in polyethylene-glycol (PEG)-primed pepper (Cortez-Baheza et al. 2007), heat shock proteins in hydroprimed sugar beet (Catusse et  al. 2011), and protein folding and stabilization proteins in PEG-primed rapeseed (Kubala et  al. 2015). The ability of plants to break down starch into an adequate supply of soluble sugars for metabolism is crucial for survival and tolerance to abiotic stresses (Rosa et al. 2009; Zheng et al. 2016). Rice seedlings from KCl and CaCl2 primed seeds demonstrated increased starch hydrolysis and resulted in greater yield (Farooq et al. 2006). Under salinity stress, BABA-primed mung bean seedlings had increased soluble carbohydrate levels, contributing to increased photosynthetic pigment concentration and seedling fresh weight (Jisha and Puthur 2016a). An additional benefit of increased soluble sugar concentrations is to support cellular osmotic homeostasis (Jisha and Puthur 2016a), which, as previously noted, supports nutrient uptake and membrane integrity. Priming-induced mechanisms to increase osmolyte concentrations also include increasing proline, as noted in cauliflower (Latif et al. 2016), and total soluble protein concentrations in wheat (Bajwa et al. 2018).

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5  Conclusion and Future Perspectives The beneficial effects of seed priming have not been observed consistently. Most studies to date do not report beneficial residual effects of seed priming beyond the seedling stage or without early stress exposure. Protection induced by priming is often more pronounced if the stress is present at sowing, upon germination or shortly after emergence. Certain priming treatments are species-specific, perhaps based on the structure of the seeds and the amount of endosperm they contain that can “store” the priming compound (Dawood 2018; Zheng et al. 2016). The protective effects of a priming treatment, if expressed at all, do not always persist as the plant matures or if it grows in nonstressed conditions (Cayuela et al. 1996; Posmyk et al. 2008; Poonam et al. 2013; Jisha and Puthur 2016a; Savvides et al. 2016; Ye et al. 2016; Ellouzi et al. 2017). Some studies have reported a beneficial residual effect of seed priming beyond the seedling stage and/or without the immediate induction of stress such as maize seeds soaked in water (Murungu et al. 2004), or microencapsulated with the fungicide tebuconazole (Yang et  al. 2014), and melatonin-primed fava beans (Dawood and El-Awadi 2015). Although epigenetics have been invoked as a possible mode of action for the protection provided by seed priming (Savvides et  al. 2016), there is not yet evidence that plants that grow from primed seeds can themselves produce seeds with endogenous protection. There is no evidence that any of the osmolytes or other compounds thus far studied as priming materials are injurious to human health or to the environment at the concentrations used with seed treatments. However, fungal and bacterial populations on seeds can increase during extended treatment with osmolytes (Wright et al. 2003), even if the seeds are subsequently coated with antimicrobial compounds, but this is unlikely to affect human health in seeds that germinate. In extreme cases, ingesting plants with an induced overproduction of natural antioxidants can cause internal injury (Bast and Haenen 2002), although this is unlikely to occur as a result of seed priming. However, there is evidence that antioxidant activity measured in  vitro may not correlate with the actual activity in  vivo (Ndhlala et  al. 2010). Noninvasive methods of measuring both plant structures (Tardieu et al. 2017) and physiological compounds or mechanisms (Boughton et  al. 2016) which protect against abiotic stress must be further developed to allow in vivo measurement of the effects of priming treatments. This will provide both more accurate measurements of the true influence of seed treatments and may well provide an impetus to the development/discovery of more and better-targeted osmolytes and other compounds. The resulting improved plant resistance to abiotic stress will in turn enhance food security in an era of global uncertainty. We suggest three fronts for further investigations of seed priming for abiotic stress resistance: 1. Growing plants from treated seeds to maturity, so as to ensure that there are no negative effects of treatments on quality or yield, despite the fact that the seedling can withstand stress.

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2. Developing treatments with effects that persist if treated seeds are stored for one or more seasons after treatment. 3. Always testing seeds from at least two cultivars of the plant being investigated, to ensure that the treatment being developed will be suitable for as broad a genetic background as possible.

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Relationship Between Polyamines and Osmoprotectants in the Response to Salinity of the Legume-Rhizobia Symbiosis Miguel López-Gómez, Javier Hidalgo-Castellanos, Agustín J. Marín-Peña, and J. Antonio Herrera-Cervera

1  Salinity Salinity is an environmental factor produced by the accumulation of mineral salts in soils and waters. The dissolved salts form cationic electrolytes such as Na+, Ca2+, Mg2+, and K+ and anionic electrolytes such as Cl−, SO42−, HCO3−, CO32−, and NO3− (Manchanda and Garg 2008). Soil salinity negatively affects the normal development of plants, being one of the main environmental factors that limit agricultural productivity in arid and semiarid regions of the planet. It is estimated that about 45 million hectares of arable land, approximately 20% of the total, are affected by salinity (Munns 2009). This is a serious problem to reach the objective of increasing agricultural production by 70% by the year 2050 (FAO 2009), which is what would be needed to meet the demand of an expected world population close to 10 billion people. The causes of salinity are varied: primary salinity is produced by the erosion of rocks with a high content of soluble salts and also by the deposition of marine salts carried by the wind, rain, or tides, with sodium chloride being the main salt present (Rengasamy 2006). Secondary salinity is caused by anthropogenic activities, such as the misuse of fertilizers, excessive irrigation with low-quality water, drainage system deficient in crops, deforestation, monocultures, or excess urbanization of the fields, which alters the water balance of the soil causing salinization (Abiala et al. 2018). As a result, salinity affects the physicochemical properties of the soil, which leads to an adverse effect on the ecological balance of the affected area, which at the agrarian level results in a decrease in crop production as well as a degradation of the arable land.

M. López-Gómez (*) · J. Hidalgo-Castellanos · A. J. Marín-Peña · J. A. Herrera-Cervera Dpto. Fisiología Vegetal, Facultad de Ciencias, Universidad de Granada, Granada, Spain e-mail: [email protected] © Springer Nature Switzerland AG 2019 M. A. Hossain et al. (eds.), Osmoprotectant-Mediated Abiotic Stress Tolerance in Plants, https://doi.org/10.1007/978-3-030-27423-8_13

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2  Effects of Salinity on Plants Soil salinity results in plant growth limitation, development, and survival due to the saline stress induction with alterations in physiological and metabolic processes of plants depending on the severity of stress, duration, and tolerance of the plant (Hasanuzzaman et al. 2013). The main physiological effect of saline stress on vegetables is related with a reduction in photosynthesis and restricted water and nutrient uptake by the plant with a concomitant growth inhibition produced, initially, by an osmotic effect and then followed by an ionic toxicity and a nutritional imbalance due to the interference of saline ions with nutrients. Saline stress triggers oxidative damage (Huang et al. 2017), which causes alterations in proteins and membranes structure.

2.1  Osmotic Stress Elevated salt concentration in the rhizosphere provokes a reduction in the capacity of water absorption by the roots due to a reduction of the soil water potential, generating a hyperosmotic stress that entails several changes at the physiological level. Among these changes are a reduction in cell division and elongation, decrease in leaf area and photosynthetic capacity caused by alterations in electronic transport and inhibition of key enzymes of the Calvin cycle (Manchanda and Garg 2008), a disruption of the membranes, reduction of the ability to eliminate reactive oxygen species and stomatal closure due to the reduction of the turgor pressure of the stomatal cells (Munns and Tester 2008).

2.2  Ionic Stress Another harmful effect of salt stress is the excessive accumulation of ions from the dissolved salts, especially Na+ and Cl-, which causes an ionic imbalance in the plant. Ionic toxicity produces a nutritional imbalance when Na+ ions compete with essential ions such as Ca2+ and K+ (Tejera et al. 2006), which causes severe physiological and structural disorders, since K+ is essential for growth and development.

2.3  Oxidative Stress Saline stress induces the formation of reactive oxygen species (ROS) in the cell due to imbalances and breaks in the electron transport chain in chloroplasts and mitochondria. Among the ROS that accumulate are hydrogen peroxide (H2O2), hydroxyl

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radical (OH⋅), superoxide anion radical (O2−), and singlet oxygen (1O2). ROS produce oxidative damage in different cellular components such as DNA, proteins, and lipids, producing alterations in cellular functions (Foyer and Noctor 2005). At the protein level, these radicals are able to act on the side chains of amino acid residues, which completely change the structure and protein function. Fatty acids, especially membrane phospholipids, are very susceptible to oxidation, generating lipid peroxides. This causes the alteration of the fluidity and produces changes in the transporters and receptors of the membrane (Koca et al. 2007). In addition, ROS are able to break and alter the DNA chains, originating mutations (Møller et al. 2007).

3  Agrarian Importance and Legume-Rhizobia Symbiosis Legumes are plants belonging to the Fabaceae family with approximately 20,000 species distributed in about 700 genera of cosmopolitan distribution, with capacity to adapt to all kinds of ecological conditions thanks to a great variety of strategies (Smýkal et  al. 2015). This family contains three subfamilies  – Mimosoideae, Caesalpinoidae (tropical subtropical trees and shrubs), and Papilionoideae – formed by shrub and herbaceous species that include most of the cultivable species (Doyle and Luckow 2003). Within this subfamily are the legumes with greater agronomic interest such as groundnut (Arachis hypogaea), soybean (Glycine max), common bean (Phaseolus vulgaris), pea (Pisum sativum), and beans (Vicia faba), among others (Le et al. 2007). The legumes are the second family in agronomic importance after the grasses (Gepts et al. 2005) due to their ability to establish symbiosis with soil diazotrophic bacteria known as rhizobia and fix atmospheric nitrogen. This important characteristic makes legumes fundamental pieces in crop rotations, since they allow the addition of organic nitrogen to the soil (Mantri et al. 2013), reducing the need of chemical fertilizers, with a concomitant reduction in the use of energy and CO2 emissions associated with their production (Jensen et  al. 2012). In addition, legumes can improve saline soil fertility and help to reintroduce agriculture to these lands due to their capacity to grow on nitrogen-poor soils (Crespi and Gálvez 2000; Coba de la Peña and Pueyo 2012). The symbiosis legume-rhizobia results in the formation of nodules in the roots of legumes where the rhizobia differentiate into bacteroides, and the proper conditions for reduction of atmospheric nitrogen to ammonium are provided by the plant (Burris 1984). Under such conditions, bacteroides experiment a genetic reprograming that allow the synthesis of the nitrogenase complex, where the nitrogen fixation takes place, providing the plant with ammonia in exchange of carbon and energy supply (Udvardi and Poole 2013).

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4  Effect of Salt Stress on Legume-Rhizobia Symbiosis One of the most successful strategies of plants to cope with environmental changes is to establish symbiotic relationships with soil microorganisms. However, in spite of the capacity to establish symbiotic interactions with rhizobia, legumes are considered salinity sensitive species, since the establishment of the symbiosis and its efficiency in fixing nitrogen are very sensitive to this stress. Nevertheless, considerable variability in salinity tolerance among crop legumes has been reported (Bruning and Rozema 2013) showing grain legumes such as Phaseolus vulgaris, Cicer arietinum, and Vigna radiata high sensitivity to salinity (Abdel-Wahab et al. 2002), meanwhile Pisum sativum, Vicia faba, and Medicago sativa are moderately salt sensitive. Under these conditions is reduced the ability of rhizobia to infect the root and nitrogenase activity (Aranjuelo et al. 2014). This decrease in nitrogen fixation under saline stress may be related to limitation in the energetic substrate shortage to bacteroides following photosynthetic activity decline and nodule sucrose breakdown reduction (Lopez et  al. 2008). Sucrose is the main carbon source required by the nodule; therefore, sucrose synthase (SS) is a key enzyme for the functioning of nitrogenase (Gordon 1995) and has been shown to be sensitive to different stresses, among them salinity (López et al. 2008). In addition, Na+ toxicity reduces homeostasis and uptake of essential nutrients, including Ca, K, and P, which reduces the growth of the nodule and its proper functioning (Ben Salah et al. 2010).

5  Tolerance to Salinity in the Legume-Rhizobia Symbiosis Legumes in symbiosis have adopted physiological, morphological, and molecular changes to tolerate salt stress in the nodule and in the rest of the plant. These changes include the accumulation of osmoprotectants (such as proline and sugars) to counteract osmotic stress (López-Gómez et  al. 2011). These molecules are small and electrically neutral that are nontoxic at molar concentrations and stabilize proteins and membranes against the denaturing effect of high concentrations of salts. In dry or saline environments, osmoprotectants can therefore serve both to raise cellular osmotic pressure and to protect cell constituents (McNeil et al. 1999). Exclusion of toxic ions to balance nutrient acquisition (Tejera et al. 2006) and increment of antioxidant metabolites and enzymes to prevent accumulation of ROS and protect membranes, proteins, and DNA in the nodular tissues are also mechanisms involved in nodule salt stress responses (Rubio et al. 2009). Previous studies have highlighted the increment in the concentrations of disaccharide trehalose by the inhibition of its degrading enzyme, trehalase, as one of the strategies to cope with salt stress in root nodules of Medicago truncatula (Lopez et al. 2008), a fact that corroborates the high concentrations of trehalose found in nodules of Lotus japonicus during a saline stress (Lopez et al. 2006).

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Within the amino acids, proline shows the largest increase in the nodular tissue in plants of M. truncatula and L. japonicus (López-Gómez et al. 2011). An increase in proline has also been observed in nodules of P. vulgaris and M. sativa related to osmotic adjustment (Tejera García et al. 2007; Lopez-Gomez et al. 2014). Biosynthesis of antioxidants (Becana et al. 2010) and differential gene expression (Molina et al. 2011) are other responses described in root nodules under salt stress conditions. All these responses are orchestrated by specific hormones such as abscisic acid (ABA) (Palma et al. 2014) and salicylic acid (SA) (Palma et al. 2013). These endogenous molecules regulate, via synergistic and antagonistic actions, the expression of different but overlapping suites of genes, which is referred to as signaling crosstalk (Nishi et al. 2015).

6  Polyamines Polyamines (PAs) are hormonal compounds and growth regulators, with low molecular weight, aliphatic nature, and polycationic character at physiological pH, present in different types of organisms and particularly in plants, where they are involved in the regulation of various physiological processes related to growth and development as well as in responses to abiotic and biotic stresses (Pal et  al. 2015). Polyamines also participate in the responses to drought, high and low temperatures, wounds, ozone, heavy metals and oxidative stress (Wimalasekera et  al. 2011). During the last years, a special attention to the involvement of PAs in the response to salinity has been paid, since a relationship between the accumulation of this type of compounds and tolerance to salinity has been described (Minocha et al. 2014). The most common PAs in nature are putrescine (Put), spermidine (Spd), and spermine (Spm), as well as other diamines such as 1–3 diaminopropane (DAP) and cadaverine (Cad). There are also rare PAs that have a very limited distribution in nature, found mainly in prokaryotes (Terui et al. 2005). These consist of molecules derived or related to Spd and Spm, among which are homospermidine (HomSpd), homospermine (HomSpm), norspermidine (NorSpd), and other pentamines and hexamines (Sagor et al. 2013).

6.1  Polyamines Metabolism Common PAs, Put, Spd, and Spm are synthesized by the decarboxylation of arginine and ornithine by arginine and ornithine decarboxylases (ADC and ODC), respectively. The triamine and tetraamine Spd and Spm are derivatives from the diamine Put by the addition of aminopropyl groups donated by S-adenosylmethionine in reactions catalyzed by Spd synthase (SPSD) and Spm synthase (SPMS), respectively (Handa et al. 2018) (Fig. 1). However, the content of PAs can also be regulated by their degradation rates by the catabolic enzymes diamine oxidase (DAO)

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Urea Cycle

Arginine

Ornithine

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ADC

NH4+

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Agmatine

α -KG

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Krebs Cycle

∆¹-Pirroline

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PAO

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dcSAM

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Dap

PAO

Spm

4-Aminobutanal

PAO

+

3-(Aminopropil) 4-Aminobutanal

H2O2

Lys

Cad

LDC

Fig. 1  Polyamines (PAs) metabolism and interaction with the synthesis of osmoprotectants proline and γ-aminobutiric acid (GABA) and Krebs and glutamate cycles. Put (putrescine), Spd (spermidine), Spm (spermine), Cad (cadaverine), Glu (glutamate), Asp (aspartic acid), Lys (lysine), α-KG (α-ketoglutarate), DAO (diamine oxidase), PAO (polyamine oxidase), H2O2 (hydrogen peroxide), ADC (arginine decarboxylase), ODC (ornithine decarboxylase), SPDS (spermidine synthase), SPMS (spermine synthase), SAMDC (S-adenosyl methionine decarboxylase), LDC (lysine decarboxylase), OAT (ornithine aminotransferase), AIH (agmatine iminohydrolase), CPA (carbamoil putrescine aminohydrolase)

and polyamine oxidase (PAO). Spermidine and Spm are preferably oxidized by PAO activity, classified depending on whether they terminally oxidize PAs or catalyze their back conversion producing Put and Spd from Spd and Spm, respectively. In addition to contribute to the homeostasis of PAs, plant amine oxidases contribute to other physiological processes through the production of H2O2, which has a versatile role in plants as a signal molecule during abiotic and biotic stresses (Gupta et al. 2016), and γ-aminobutyric acid (GABA), also produced by cytosolic glutamate decarboxylase, and with important functions in response to biotic and abiotic stresses (Podlešáková et al. 2019).

6.2  Polyamines Against Salt Stress Numerous studies have shown the participation of PAs in salt stress tolerance in plants (Tang and Newton 2005; Jiménez-Bremont et  al. 2007; Li et  al. 2016; López-Gómez et al. 2017). The accumulation of PAs in response to salt stress is one of the strategies of plants to acquire tolerance to this stress; however, the levels of PAs change depending on several factors such as the species, the organ

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analyzed, their tolerance or sensitivity to the salinity stress, or the duration of it (Groppa and Benavides 2008). In plant cells, PAs occur as free bases but may also be covalently linked to small molecules such as hydroxycinnamic acids to form soluble conjugated PAs. The conjugation of PAs has a role in the regulation of free PAs levels, which may be related to the variations of PA levels in the responses to salt stress. The application of exogenous PAs has been shown to alleviate the effects of NaCl stress on various plants (Verma and Mishra 2005). Especially Spm and Spd resulted in increased reactive oxygen metabolism and photosynthesis, which improved plant growth and reduced the inhibitory effects of salt stress (Baniasadi et al. 2018). Implication of PAs in the protection against salinity is due to their capacity to stabilize macromolecules such as DNA, RNA, proteins and phospholipids, as well as their free radical scavenging activity (Hussain et  al. 2011). Some of these properties are shared with other compatible solutes such as proline; however, the concentration of stress-induced PAs is lower than proline and other compatible solutes such as sucrose and trehalose, which suggests that PAs do not behave as compatible solutes.

6.3  P  olyamines Interaction with Other Abiotic Stress Regulators Polyamines play critical roles in the adaptation of plants to stress conditions through an intricate crosstalk with other growth regulators and signaling molecules, which is confirmed by the fact that metabolic pathways that regulate the levels of PAs share common substrates with other molecules that also participate in stress responses, such as nitric oxide (NO), H2O2, GABA, ethylene, or proline; so it is difficult to discern the relationships between PAs and the involvement of other molecules in the abiotic stress responses (Shi et al. 2013). The alteration of PA metabolism has been used as a strategy to uncover its relationships with other molecules involved in defense against abiotic stresses (Duque et al. 2016). For instance, the inhibition of PA catabolism has been shown to reduce the level of osmoprotectants such as proline and GABA (Su and Bai 2008; Xing et al. 2007), suggesting that PAs might act as stress alleviators through the modulation of the levels of these amino acids (Fig.  2). However, the inhibition of PAO activity in Arabidopsis thaliana increased the tolerance to salinity and drought (Sagor et  al. 2016) by reducing ROS production and inducing stress responsive genes under stress conditions. Glutamate is a common precursor for the biosynthesis of proline and PAs (Fig. 1), and therefore considerable changes in the pool of PAs can be caused by a shift between syntheses of both compounds. All together suggests that PAs would be in the center of a complex regulation network in which the levels of osmoprotectants, such as proline and GABA, would depend on PAs metabolic alterations (Fig. 2). Additionally, production of signaling molecules such as H2O2 by PAs catabolism has been related with the induction of defensive responses of hypersensitivity

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Salt Stress

BRs

ABA

PAs

SA

Proline

H2O2

GABA

NO

Ethylene

Fig. 2  Schematic representation of polyamines interaction with hormones in the response to salt stress. PAs (polyamines), BRs (brasinosteroids), ABA (abscisic acid), SA (salicylic acid), GABA (γ-aminobutiric acid), NO (nitric oxide)

(Jasso-Robles et al. 2016), including the activation and signaling of abiotic stress responses (Moschou et al. 2008). Nitric oxide (NO) also participates in the PA signaling as it is produced in the course of PA metabolism and is a key signaling molecule that mediates a variety of physiological functions, including defense responses against abiotic stresses in plants (Diao et al. 2017). Polyamines play a positive role in NO production with an inverse correlation between NO and ethylene presence in abscission tissue (Parra-Lobato and Gomez-Jimenez 2011). The reduction of the expression level of de NCED2 gene, involved in the abscisic acid (ABA) synthesis, by a DAO inhibitor and osmotic stress, indicates a relationship between PA oxidation and ABA synthesis under osmotic stress conditions (Hatmi et al. 2018), which highlights the role of PAs in controlling abiotic stress responses through ABA responses (Fig. 2). Indeed, ABA pretreatment alleviates the negative effect of salinity in plants of M. sativa by increasing antioxidant responses and the level of Put derived PAs such as Spd and Spm (Palma et al. 2014). Exogenous ABA also increases the Put contents in chickpea, while ABA has been reported to trigger PAs synthesis through transcriptional regulation of genes encoding SPDS (Jiménez-Bremont et al. 2007). All these results suggest that ABA would be involved in the modulation of PAs metabolism at transcriptional level. By contrary, salicylic acid (SA), which participates in the signaling of salt stress in legumes (Palma et al. 2009), prevents the accumulation of PAs under salt stress conditions (Palma et al. 2013). This negative relationship has been related with the linkage between PAs and ethylene synthesis that is favored by SA through the common precursor S-adenosylmethionine (SAM). Another example of interaction is in

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mutants impaired on the biosynthetic pathways of both ethylene and PAs, which prevented premature cell death of xylem elements (Milhinhos and Miguel 2013). Brassinosteroids (BRs) are steroid hormones that regulate salt stress responses in plants (Bajguz 2011) in crosstalk with PAs (Zheng et al. 2016). Indeed, foliar treatment with epibrassinolide induces a protective effect in M. truncatula against salt stress by an increment in the Spm levels (López-Gómez et al. 2016). In addition, regulation of the nodule number in legumes has been shown to occur through an interrelation between PAs and BRs (Terakado-Tonooka and Fujihara 2008), which indicates a complex network in which different hormones and plant growth regulators, including PAs, would interact during the abiotic stress responses and the biotic interactions of plants (Fig. 2).

6.4  PAs in the Legume-Rhizobia Symbiosis The levels of PAs in plants depend on the species and the stage of development, being more abundant in growing tissues or in plants exposed to different stress conditions, including biotic interactions (Jiménez Bremont et al. 2014). In general, the levels of Put are higher than the rest of the PAs; however, in legumes, higher concentrations of Cad can be found (Tomar et al. 2013). Polyamines are also necessary for growth and division of microorganisms, including rhizobia (Becerra-Rivera et al. 2018), in which among the usual PAs (Put, Spd, and Spm), rare PAs considered structural analogues of the main PAs have also been identified. Therefore, PAs composition in the root nodules of legumes is the result of the mixture and cooperation of the plant and rhizobia metabolisms. In general, PAs in root nodules of legumes accumulate in values 5–10 times higher than any other organ of the plant, and the composition of these PAs can vary depending on the species of legume. Most of these PAs are specific to the nodule, since they are synthesized by the bacteroides (Fujihara 2009), including unusual PAs such as HomSpd, which is the most abundant in nodules of M. sativa and P. vulgaris (López-Gómez et al. 2014a, b). In nodules of P. vulgaris has also been found 4-aminobutilcadaverine (4-Abcad), a polyamine exclusive of the bacteroides, whose synthesis depends on the enzyme HomSpd synthase in the bacteroides (Lopez-Gomez et  al.  2016a). The presence of both uncommon PAs in nodules is possible by the supply of Cad from the plant cytosol to the bacteroides, since Cad is neither produced by the free-living bacteria nor the bacteroides (Fujihara 2009) (Fig. 3). During the establishment of the legume-rhizobia symbiosis, alterations in PAs levels have been described (Jimenez-Bremont et al. 2014; Terakado-Tonooka and Fujihara 2008), suggesting an active role of these molecules in the symbiotic interaction. The production of H2O2 by PAs catabolism has proved to be a requirement for the infection of roots in M. truncatula during the establishment of the symbiosis (Jamet et al. 2007) and the nodule functioning (Andrio et al. 2013). In this regard, the oxidation of PAs has been concluded to be involved in the establishment of the symbiosis between M. truncatula and S. meliloti, as shown by the reduction of the nodulation by the inhibitor of DAO, aminoguanidine (AG) (Hidalgo-Castellanos et al. Data not published).

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Arg

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+

Cad

SPDS

SPMS

Spm

Put dcSAM

HSS

LDC

Spd

SAMDC

Lys

Plant cytosol

Put

HomSpd

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Bacteroid

Fig. 3  Schematic representation of polyamines biosynthesis in root nodules of legumes. ADC, (arginine decarboxylase), Arg (arginine), ABCad (aminobutylcadaverine), Cad (cadaverine), Homspd (homospermidine), HSS (homospermidine synthase), LDC (lysine decarboxylase), Lys (lysine), ODC (ornithine decarboxylase), Orn (ornithine), Put (putrescine), SAM (S-adenosylmethionine), SAMDC (S-adenosylmethionine decarboxylase), Spd (spermidine), SPDS (spermidine synthase), Spm (spermine), SPMS (spermine synthase). (Adapted from Plant and Soil (2016) 404: 413–425)

H2O2

PAs NO

Infection

Fig. 4  Schematic representation for the role of signal molecules induced by polyamines (PAs) in the legume–rhizobia interaction. NO (nitric oxide), H2O2 (hydrogen peroxide)

NO has been shown to be required for an optimal establishment of the M. truncatula-­S. meliloti symbiosis and has been suggested that it could have functions in bacterial infection as well as in nodule development (del Giudice et  al. 2011). Interaction between PAs metabolism and NO has been shown by the exogenous application of Spd and Spm that induced the generation of NO by the nitrate reductase pathway (Diao et al. 2017). Therefore, PAs metabolism has an active role in the legume-rhizobia symbiosis providing signal molecules necessary for the infection threads formation and nodule organogenesis (Fig. 4). In addition, PA concentrations participate in the control of root nodule number and biomass in crosstalk with BRs, as demonstrated by the inhibition of Spd synthesis in a supernodulating genotype of soybean, that restored

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the wild-type nodule number and the fact that exogenous application of BRs restored the Spd levels and reduced the nodule number in this supernodulating mutant (Terakado et al. 2006). The involvement of PAs in nodule organogenesis has also been shown in nodules of L. japonicus, where the expression of genes involved in the synthesis of PAs is induced during nodule development and declines with aging (Efrose et al. 2008). Indeed, significant linear correlations between the total concentrations of free PAs in nodules and nitrogenase activity and leghaemoglobin content have been reported in field-grown bean (Lahiri et al. 2004), suggesting their implication in other functions related to nitrogen fixation.

6.5  P  olyamines in the Salt Stress Response in the Legume-­Rhizobia Symbiosis Nodule-specific PAs such as HomSpd, Cad, or AbCad are involved in mechanisms of tolerance to salinity in the legume-rhizobia symbiosis (López-Gómez et  al. 2014a, 2016). Specifically, the concentration of the bacteroidal-produced polyamine AbCad augmented under salinity stress in nodules of P. vulgaris, suggesting the modification of the bacteroidal metabolism toward the synthesis of this compound as a strategy to cope with salt stress. HomSpd synthase seems to be the enzyme responsible for this nodular metabolic response to salinity, based on the reduction of the nodulation observed in plants of P. vulgaris inoculated with a mutant strain of Rhizobium tropici impaired in the synthesis of HomSpd (Rt hss::Ω, Spr) (Lopez-Gomez et al. 2016). In addition, this enzyme is involved in the adaptation to salt stress conditions in the free living bacteria, since the reduction of the growth by the salinity observed in the mutant strain (Rt hss::Ω, Spr) was restored by the exogenous addition of HomSpd to the growth medium. The involvement of PAs in the tolerance to salinity in the symbiosis M. truncatula-­Sinorhizobium meliloti has been shown by the exogenous addition of Spd and Spm to the growth medium (López-Gómez et al. 2017). In this work, a reduction of the oxidative stress was detected, and in addition, evidence of the crosstalk between PAs and BRs was reported based on the induction of the expression of genes involved in BRs biosynthesis by exogenous PAs. Indeed, a similar result was induced by foliar treatment with BRs which induced an increment in the Spd levels in leaves and restored growth under salt stress conditions (LópezGómez et al. 2016b).

7  Conclusion The involvement of PAs in the response to salinity has acquired a special attention since a relationship between the accumulation of this type of compounds and tolerance to salinity has been described. Additionally, PAs share common metabolic

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pathways with other molecules that also participate in salt stress responses, such as GABA or proline, which suggest that PAs are in the center of a complex regulation network in which the levels of these osmoprotectants depend on PAs metabolic alterations. Additionally, the production of signaling molecules such as H2O2 by PAs catabolism are also involved in salt stress responses. The symbiotic interaction between legumes and soil nitrogen-fixing bacteria reduces the need to use chemical fertilizers but is a salt-sensitive process, which make of special interest the improvement of the tolerance to salinity of this symbiotic interaction. PAs metabolism has an active role in the legume-rhizobia symbiosis, providing signal molecules necessary for the infection threads formation and nodule organogenesis. In addition, PAs composition in the root nodules of legumes is the result of the cooperation of the plant and rhizobia metabolism with some of the nodule-specific PAs synthesized by the bacteroides. Nodule-specific PAs are involved in mechanisms of tolerance to salinity in the legume-rhizobia symbiosis, suggesting the modification of the bacteroidal metabolism toward the synthesis of this compound as a strategy to cope with salt stress. Therefore, the gain of knowledge in the alterations of the metabolism of PAs in the legume-rhizobia symbiosis and its interaction with other molecules involved in salt stress tolerance is of great interest to improve the ability to fix atmospheric nitrogen of legumes and to reduce the need to use chemical fertilizers.

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Engineering Polyamine Metabolic Pathways for Abiotic Stress Tolerance in Plants Susana de Sousa Araújo, André Luis Wendt dos Santos, and Ana Sofia Duque

1  Introduction Polyamines (PAs) are low-molecular-weight polycationic nitrogenous compounds that are ubiquitously distributed in eukaryotic and prokaryotic cells (Liu et  al. 2015a) and may even be found in plant RNA viruses and plant tumors (Chen et al. 2019). In plants, the most abundant PAs include putrescine (Put, 1,4-diaminobutane), spermidine (Spd, N-(3-Aminopropyl)-1,4-diaminobutane), spermine (Spm, N,N’-Bis(3-aminopropyl)-1,4-diaminobutane), and its structural isomer thermospermine (tSpm) (Saha et  al. 2015). Cadaverine (Cad, 1,5-Diaminopentane), a diamine less known compared to major PAs, is commonly found in plants belonging to the families Gramineae, Leguminosae, and Solanaceae (Kakkar and Sawhney 2002; Lutts et al. 2013; Saha et al. 2015). They are synthesized from decarboxylation of amino acid precursors including arginine, ornithine, methionine, and lysine (Falahi et al. 2018). PAs are a class of plant biomolecules that have been implicated in several plant growth and development processes, which include the promotion of cell division, responses to biotic and abiotic stresses, rhizogenesis, senescence, flower development, N:C balance, fruit ripening, and embryogenesis (Baron and Stasolla 2008; Duque et al. 2016; de Oliveira et al. 2017, 2018). The polycationic structure of PAs, at physiological pH, mediates their biological activity, since they are able to electrostatically bind negatively charged macromolecules such as DNA, proteins, membrane phospholipids, and pectic polysaccharides via amine and imine groups S. de Sousa Araújo · A. S. Duque (*) Laboratory of Plant Cell Biotechnology (BCV), Instituto de Tecnologia Química e Biológica António Xavier (Green-it Unit), Universidade Nova de Lisboa, Oeiras, Portugal e-mail: [email protected] A. L. W. dos Santos Laboratory of Plant Cellular Biology (BIOCEL), Instituto de Biociências (IB), Universidade de São Paulo (USP), São Paulo, Brazil © Springer Nature Switzerland AG 2019 M. A. Hossain et al. (eds.), Osmoprotectant-Mediated Abiotic Stress Tolerance in Plants, https://doi.org/10.1007/978-3-030-27423-8_14

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(Martin-Tanguy 2001; Liu et al. 2015a). Consequently, these electrostatic interactions can affect DNA replication and gene transcription, protein conformational structure, cellular structures integrity, and membrane permeability and therefore ion homeostasis (Liu et al. 2015a; Mattoo et al. 2014). Furthermore, the presence of cationic amine and imine groups also suggests that PAs may act as free radical scavengers and/or as signaling molecules that trigger enzymatic and nonenzymatic antioxidative components during exposition to biotic and abiotic stresses in plants (Liu et al. 2015a; Saha et al. 2015). PAs can exist as free amines present either inside or outside the plant cells (free cations or involved in ionic interactions), as soluble bound or soluble conjugated in association with low molecular mass compounds (coumaroyl, ferulic, phenolics, caffeic, or hydrocinnamic acids), and insoluble bound or insoluble conjugated amines associated with high molecular mass compounds (lipids, nucleic acids, and proteins) and cell wall (Yadav and Rajam 1997; Bouchereau et al. 2000; Wuddineh et al. 2018). PA homeostasis, which refers to the adjustments made on cellular levels of Put, Spd, and Spm, is tightly regulated in plants. Intracellular PA levels are regulated not only by fine-tuning of their biosynthesis and catabolism or turnover but also through their interconversion, conversion into secondary metabolites, and allocation to other tissues/organs (Wuddineh et al. 2018). Unlike mammals, plants can withstand the accumulation of large amounts of PAs (up to mmolar concentration) as they can buffer this excess by binding PAs to TCA-soluble conjugates or by storing them in the vacuole (Serafini-Fracassini and Del Duca 2008; Wuddineh et al. 2018). However, high cellular accumulation of PAs as well as high rate of PAs catabolism may be prejudicial to plant cells (Wuddineh et al. 2018). In plants, PA endogenous levels are influenced by different factors such as species, stress tolerance capacity, stress types and conditions, and the physiological status of the examined tissue/organs (Liu et al. 2015a). In this context, numerous review articles have described the effects of PA modulation in plants from an agricultural and biotechnology point of view (Tiburcio and Alcázar 2018; Anwar et al. 2018; He et al. 2018; Seifi and Shelp 2019). This is well reflected on the growing number of research studies and articles addressing these aspects. A non-exhaustive survey conducted in the PubMed repository of the National Center for Biotechnology Information NCBI (http://www.ncbi.nlm.nih.gov/ pubmed) retrieved 947 research articles dedicated to PA research in the context of plant sciences (6th March 2019). Of those, 799 articles were published during the period of 1998–2018. The evolution in the number of polyamine articles scored in the two last decades is depicted in Fig. 1a. One of the most interesting features is that the number of articles on plant PAs almost duplicated since 2008. Potentially, this aspect was supported by the development of new high-throughput methodologies to characterize the global gene expression or metabolite accumulation (omics) with reduced cost for user and the release of several plant genome sequences that supports the development of new research efforts on the topic. Another interesting outcome of the survey made is that the majority of the PAs studies have been conducted in non-model species (Fig. 1b), which may likely reflect a translational application of the early results obtained with models as Arabidopsis thaliana L. and Nicotiana

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Fig. 1  Query of research scientific papers on plant polyamines found at NCBI between 1998 and 2018. The search for best match results was conducted using the following advanced search settings: (Polyamines[Title/Abstract]) AND (plant[Title/Abstract]) NOT “review” (Publication Type). Review manuscripts were not included. The search was conducted on the 6th of March 2018. (a) Evolution on the total number of scientific research papers published per year. (b) Evolution on the number of paper published in main model species (Arabidopsis and tobacco) compared to the ones published on other plant species. This query was made by searching the name of the species on the title

tabacum L. As one example, among the 76 articles on plants PAs published during the year of 2018, 69 describes research conducted in other species beside the refereed models. This reflects the interest of the scientific community to elucidate the different roles of these important biomolecules in plants. Indeed, based on the overall analysis of Fig. 1, it is tempting to claim that the number of scientific PA articles is expected to increase in forthcoming years. This book chapter focuses on the contributions that the modulation of the PAs content has brought to improve our knowledge on the role that these interesting biomolecules have on plant physiology, development, and response to environment. The recent development of plants with altered PAs contents will be discussed in a context of crop improvement and food and feed safety, under the current scenario of climate changes.

2  Polyamine Metabolism and Regulation 2.1  Biosynthetic Pathways In animals and fungi, Put is synthesized primarily through l-ornithine decarboxylation via the catalytic action of ornithine decarboxylase (ODC, EC 4.1.1.17). In plants, Put synthesis involves the ODC pathway and the l-arginine decarboxylation via arginine decarboxylase (ADC, EC 4.1.1.19) (Flemetakis et al. 2004), except in

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the model plant A. thaliana, in whose genome the ODC gene was found to be missing (Mattoo et al. 2014). The ODC pathway is a single step reaction, in which l-ornithine is directly converted to Put. In plants, Put biosynthesis via ADC pathway involves the production of the intermediate agmatine (Agm), followed by two successive steps catalyzed by agmatine iminohydrolase (AIH, EC 3.5.3.12) and N-carbamoylputrescine amidohydrolase (CPA, EC 3.5.1.53) (Fig. 2). In addition, plants can use ADC and arginase/agmatinase (ARGAH) as a third route for Put synthesis (Patel et al. 2017). Put is then converted into Spd by spermidine synthase (SPDS; EC 2.5.1.16), after addition of an aminopropyl group donated by decarboxylated S-adenosylmethionine (dcSAM). DcSAM is produced from l-methionine via two sequential reactions that are catalyzed by methionine adenosyltransferase (MAT, EC 2.5.1.6) and S-adenosylmethionine decarboxylase (SAMDC, EC 4.1.1.50), respectively. Spd is then converted into Spm or thermospermine (tSpm), using dcSAM as an aminopropyl donor, in a reaction catalyzed by Spm synthase (SPMS, EC 2.5.1.22) and thermospermine synthase (ACL5, EC 2.5.1.79), respectively (Liu et al. 2015a). Cadaverine (Cad), a diamine less known as compared to major PAs (Put, Spd, and Spm), is formed directly from l-lysine decarboxylation via lysine decarboxylase (LDC, EC 4.1.1.18) (Kakkar and Sawhney 2002) (Fig. 2).

> Glutamate > α-Ketoglutarate

CADAVERINE

L-Arginine

ADC

Argininosuccinate

Aminocyclopropane Caboxylic Acid (ACC)

Glutamate Glu

Citruline

N-Carbamoylputrescine

SAMDC Decarboxylated S-Adenosylmethionine (dcSAM)

ACL5

L-Ornithine

ODC

DAO

PUTRESCINE

SPDS

PAO

SPERMIDINE

SPMS THERMOSPERMINE

Ethylene

Lysine

Agmane

Methionine

S-Adenosylmethionine (SAM)

LDC

PAO

y-Aminobutyric Acid (GABA)

Succinic semialdehyde

∆1-Pyrroline H2O2 + NH3 ∆1-Pyrroline

PAO

Succinate

TCA cycle

1,3-Diaminopropane

SPERMINE

PAO 1,3-Diaminopropane

Fig. 2  Schematic representations of polyamines metabolism and ethylene biosynthesis in plants. Blue arrows represent PA anabolic and back conversion pathways, green arrows denote PA catabolic pathways, and black arrows indicate PA interaction with other metabolic routes. Abbreviations: arginine decarboxylase (ADC), ornithine decarboxylase (ODC), spermidine synthase (SPDS), spermine synthase (SPMS), S-adenosylmethionine decarboxylase (SAMDC), lysine decarboxylase (LDC), flavin-containing polyamine oxidase (PAO), diamine oxidase (DAO), thermospermine synthase (ACL5). (Adapted from Alcázar et al. 2010a)

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2.2  Degradation Pathways PAs catabolic pathways occur either through direct terminal oxidative deamination/ acetylation or indirectly through PAs back-conversion pathway and subsequent oxidation. In plants, PAs catabolism is catalyzed by two classes of amine oxidases (AOs), classified according to their cofactor: diamine oxidases (DAO), copper-­ containing AOs (CuAOs, also known as primary AOs, EC 1.4.3.6), and FAD-­ dependent AOs (FAD-AOs), also known as polyamine oxidase (PAOs, EC 1.5.3.3) (Podlešáková et al. 2018; Fig. 2). Both DAO and PAO are localized in the cytoplasm and cell wall and are involved in production of hydrogen peroxide (H2O2) required for cell wall stiffening (Saha et  al. 2015). CuAOs catalyze the oxidation of the diamines Put and Cad at the primary amino groups. In this reaction, Put is converted to 4-aminobutanal (ABAL, that spontaneously cyclizes to Δ1-pyrroline, PYRR), H2O2, and ammonia (NH3) (Tiburcio et al. 1997; Podlešáková et al. 2018; Fig. 2). Following the oxidation of Put, PYRR is catabolyzed into γ-aminobutyric acid (GABA) by pyrroline dehydrogenase (PDH), which is ultimately converted into succinate, a component of the Krebs cycle (Gill and Tuteja 2010; Michaeli and Fromm 2015). CuAOs can also catalyze the oxidation of Spd and Spm, although with a lower affinity (Wuddineh et al. 2018). Plant PAOs catabolize primarily Spd, Spm/tSpm, and their derivatives (Alcázar et al. 2010a). Furthermore, PAOs are able to back-convert Spm to Spd and Spd to Put (Fig. 2), in a two-step reaction with an acetylation (spermidine/spermine N-1 acetyl transferase (SSATs)) followed by an oxidation (PAOs) with the production of 3-acetamidopropanal and H2O2 (Moschou et al. 2008).

2.3  P  A Signaling Pathways and Interconnection with Other Metabolic Pathways Although early studies claimed a just protective role for PAs, nowadays PAs are known to be involved in a complex signaling system and have a key role in the regulation of biotic and abiotic stress tolerance (Pál et al. 2015; Romero et al. 2018). Oxygen and nitrogen reactive species (ROS and RNS, respectively) are considered one of the major links between polyamines and other metabolic pathways in the response to stresses. Indeed, H2O2 and nitric oxide (NO) that are produced during polyamine metabolism may be implicated in the transmission of signals that influence gene expression via an increase in the cytoplasmic calcium (Ca2+) level (Pál et al. 2015). Other links of PAs metabolism with other metabolic pathways are found on the amino acids (proline and GABA), alkaloids, and ethylene metabolisms. All together, these pathways represent an important way of assimilation and partitioning of carbon and nitrogen (producing other amino acids and signaling molecules) that play critical functions in responses to stress and developmental process in plants (Page et al. 2012; Minocha et al. 2014; Majumdar et al. 2016).

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PAs are biochemically related to NO through arginine, a common precursor in their biosynthetic routes, suggesting that alteration in NO homeostasis can affect PAs bioavailability and vice versa (Filippou et al. 2013; Tanou et al. 2014). Moreover, NO has been shown to be produced from PAs through a still uncharacterized mechanism (Tun et al. 2006; Silveira et al. 2006). It has been demonstrated that the addition of Spd and Spm to the culture medium may induce the formation of NO in A. thaliana roots (Tun et  al. 2006) and in embryogenic cultures of Ocotea catharinensis Mez (Santa-Catarina et al. 2007) and Araucaria angustifolia (Bertol.) Kuntze (Silveira et al. 2006). These pioneering studies showed the first evidence of the relationship between PAs and NO in plants. Furthermore, the relationship between PAs and NO has provided new perspectives for the study of the biosynthesis and catabolism of PAs and production of ROS mediated by the enzymes copper amine oxidase1 (CuAO1) and polyamine oxidases (PAO) (Wimalasekera et  al. 2011). The overlapping roles between PAs and NO raise the question of how both molecules may act in coordination during plant development and stress resistance (Tun et  al. 2006; Silveira et  al. 2006). In Cucumis sativus L. (cucumber), it was demonstrated that PAs have a regulatory effect on NO biosynthesis related to the drought stress response (Arasimowicz-Jelonek et  al. 2009). More recently, Diao et  al. (2016) investigated the effects of Put and Spd on NO generation and the potential role of Spd-induced NO in the tolerance of tomato (Solanum lycopersicum L.) seedlings to chilling stress. Spd increased NO release via the nitric oxide synthase (NOS)-like and nitrate reductase (NR) enzymatic pathways in the tomato seedlings, whereas Put had no such effect (Diao et  al. 2016). One of the most interesting findings of these studies is that H2O2 might also act as an upstream signal to stimulate NO production, highlighting again the important crosstalk between PAs, ROS, and NRS in the modulation of abiotic stress responses in plants. Many potential links between PAs and hormones exist in processes related to plant growth and environmental stress (Wimalasekera et al. 2011). PA biosynthetic genes, such as those encoding ADC, SAMDC, and SPDS, have been shown to be induced under drought stress or following ABA treatment, and this is accompanied by an increase in the endogenous PAs (Liu et al. 2015a). Moreover, to reinforce the fact that PAs biosynthesis may be regulated by ABA, several stress-responsive elements, such as drought-responsive (DRE), low temperature-responsive (LTR), and ABA-responsive elements (ABRE and/or ABRE-related motifs), are present in the promoters of the polyamine biosynthetic genes (Alcázar et al. 2006). Plants with overexpression or downregulation of PAs implicated genes have been essential tools to provide new clarifications on the existing links between PAs and plant hormones. Plants with altered PA levels often presented abnormal phenotypic alterations as stem elongation or branching, root growth, leaf morphology, and flowering delay (Kumar et  al. 1996; Masgrau et al. 1997; Hanzawa et al. 2000; Alcázar et al. 2005). Some of these phenotypic alterations were found very similar to the ones observed in mutant plants with defective hormone biosynthetic pathways (Hanzawa et al. 2000), being this a driving force for investigating the crosstalk between PAs and hormones. Alcázar et al. (2005) demonstrated that transgenic Arabidopsis plants with increased levels of Adc2 transcript and elevated Put content showed dwarfism and late flowering, a

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phenotype that was rescued by gibberellin A3 (GA3) application. The data obtained by these authors in these transgenic plants showed that Put accumulation downregulated the expression of several GA oxidases as AtGA20ox1, AtGA3ox1, and AtGA3ox3 implicated in GA biosynthesis. Nevertheless, it remains to be fully understood the molecular mechanism by which Put might act as an endogenous signal regulating the expression of the dioxygenase genes in GA metabolism in a way similar to GAs. The metabolic pathway of PAs is also linked to ethylene, another important plant hormone, since both share a common precursor, the S-adenosylmethionine (SAM) (Fig. 2). A recent report on grain filling in wheat (Triticum aestivum L.) under severe water stress showed that an increase in Spd levels may affect the rates of ethylene biosynthesis (Yang et  al. 2017). Indeed, severe stress decreased Spd levels but increased the 1-aminocylopropane-1-carboxylic acid (ACC) concentration which is an enzyme implicated in ethylene biosynthesis. This indicated that Spd and ACC exhibited an antagonistic relationship. Interestingly, the competition for the SAM pool between PAs and ethylene biosynthesis might explain its antagonistic effects during fruit ripening and plant senescence (Tiburcio et al. 1997). While ethylene is described as a ripening and senescence promoter, PAs are described as promoters of growth and act as anti-senescence regulators (Mattoo et al. 2014).

3  Involvement of Polyamines in Stress Responses In the early 1990s, a possible role for polyamines in plant responses to abiotic stress was proposed by Flores (1991) based on studies indicating that plants subjected to osmotic stress showed a rapid increase in putrescine levels due to the activation of the arginine decarboxylase (ADC) enzyme and by the significant increase of its transcript level (Flores and Galston 1982). In the model A. thaliana, the Adc2 expression was correlated with free Put accumulation under salinity and dehydration (Urano et  al. 2003, 2004). Several studies demonstrated that PA levels increased under a number of environmental stress conditions, including drought, high salinity, and low and high temperatures (Borrell et al. 1996; Kasinathan and Wingler 2002; Do et al. 2014; Liu et al. 2007; Ikbal et al. 2014; Zapata et al. 2017; Falahi et al. 2018). It was also reported that stress-tolerant plants accumulate higher levels of polyamines in response to several stresses, comparatively to sensitive plants (e.g., Chattopadhyay et al. 1997, 2002). There are also numerous references regarding the advantage of exogenous PAs application, at different concentrations, in an attempt to study stress effects and improve stress tolerance in several species. A recent review by Khare et al. (2018) considered the exogenous polyamine application has a convenient and effective approach to alleviate salt stress and eventually improve crop productivity under high salinity and presented several examples for exogenous PAs effect in various plant species. Others of many examples are further described. Exogenous Put application was shown to prevent abiotic stress damage and increase stress tolerance in Conyza bonariensis L. and wheat (Ye et al. 1998), in Glycine max

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(L.) Merr. (Nayyar et al. 2005), in Oryza sativa L. (Ndayiragije and Lutts 2006), in Medicago sativa L. (Zeid and Shedeed 2006), and in cucumber (Duan et al. 2008). Wheat showed enhanced thermotolerance by the application of Put at pre-anthesis stage (Kumar et al. 2014), better growth and productivity under lead stress (Rady et al. 2016), and the beneficial of Put pretreatment when subject to cadmium stress (Tajti et al. 2018). In field-grown plants of Thymus vulgaris L., the foliar application of Put could acts as elicitor to trigger physiological processes and induce valuable metabolites biosynthesis, which may compensate the negative impacts of drought stress (Mohammadi et  al. 2018). The supplementation of Spd can alleviate salt stress effects in sorghum (Sorghum bicolor L.) (Yin et al. 2016); mitigate saline– alkaline stress in tomato (Solanum lycopersicum L.) at physiological and proteomic levels (Zhang et al. 2015); reduce grape (Vitis vinifera L.) berries’ chilling injury during storage at low temperature, increasing postharvest life (Champa et al. 2015); and also alleviate oxidative damage in rice caused by submergence stress (Liu et al. 2015b). Also in rice, exogenously applied PAs (Put, Spd, and Spm) as seed priming and foliar spray increased drought tolerance, being foliar application more effective and Spm the most efficient PA in improving leaf water status, photosynthesis, and membrane properties (Farooq et  al. 2009). Additionally, Spm application in rice worked as antioxidant by inhibition of ROS and malondialdehyde (MDA) accumulation and enhanced plant growth (Farooq et al. 2009; Radhakrishnan and Lee 2013). The foliar application of Spm alleviated the negative effects of water stress in maize (Zea mays L.) plants by increasing the activity of antioxidant enzymes (Talaat et al. 2015). In a more recent study, application of Spm alleviated water stress-induced oxidative stress of Rosa damascene Mill. by improving the growth characters, relative water content (RWC), chlorophyll content, and stomatal conductance (Hassan et al. 2018). As a conclusion, the application of exogenous PAs, mainly Put, Spd, and Spm, benefits several plant species by enhanced drought, salt, flooding, heat and cold tolerance, heavy metal, ozone, and copper, among others. Without ceasing to attribute meaning to these studies, there are also other important works encompassing plant genetic engineering strategies. Transgenic approaches have been used with the advantage of endogenous manipulation of PAs levels, focused toward the agricultural use of PAs capacity for enhancing abiotic stress tolerance and also to further study the regulatory mechanisms controlling plant cellular polyamine levels.

4  Changes in Polyamine Metabolism by Genetic Engineering 4.1  A Brief Historical Overview With the cloning of several genes coding for enzymes of the polyamine biosynthetic pathway, it has become possible to manipulate polyamine biosynthesis using transgenic approaches (for cloned genes available, see Kumar et al. 1997, Liu et al.

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2007, Pathak et al. 2014). Studies using genes or coding sequences (cDNAs) for enzymes of the PA biosynthetic pathways have become frequent. A first discovery using transgenic approaches was that Put levels were increased in tobacco roots as a result of expressing yeast Odc (Hamill et al. 1990). The expression of a mammalian Odc in transgenic carrot (Daucus carota L.) and tobacco also resulted in a significant increase in cellular Put levels (DeScenso and Minocha 1993; Bastola and Minocha 1995). Burtin and Michael (1997) and Capell et al. (1998) reported that the ectopic expression of the Avena sativa L. Adc in tobacco and rice, respectively, resulted an increase in Agm and Put. Regarding the plant performance, Roy and Wu (2001) presented first evidences that transgenic rice plants, with enhanced Adc expression, showed increased biomass under saline conditions, compared to control plants. By the same time Hanfrey et al. (2001) reported that in A. thaliana, the gene coding for ODC enzyme was not present, and the corresponding enzyme activity could not be detected. In this way, Arabidopsis depended only in the ADC pathway for Put biosynthesis. Based on the relative frequencies of Adc and Odc sequences in large EST collections in many plant species, including Glycine max, Medicago truncatula Gaertn., and Lotus japonicus (Regel) K. Larsen, the ADC pathway was considered the primary source of putrescine in plants (Flemetakis et al. 2004); and consequently, the focus of the transgenic approaches favored the use of the Adc gene. Other genes additionally used for transgenic approaches included the ones involved in Spd and Spm synthesis (Spds and Spms); in the decarboxylation of S-adenosylmethionine (Samdc), related with PAs catabolic pathways (coding for DAO and PAO); and lately transcription factors positively involved with PAs biosynthesis (see Table 1). In addition to the choice of genes to be used in the transformation, it is also necessary to take into account the choice of the promoter used to regulate their expression. The promoters that have been most commonly employed include the cauliflower mosaic virus (CaMV) 35S promoter (used for dicot crops) and the actin 1 promoter (Act-1) (used for monocot crops) (Grover et al. 2003). The use of inducible promoters, that allow the expression of a transgene only when it is required, was also a strategy used for PAs studies (an example is the use of ABA-inducible promoter in rice (Roy and Wu 2001, 2002)). The methodologies used to change PA levels have also take into account if the purpose is to have the gene expression upregulated, by sense overexpression of the transgene, or downregulated, by the antisense or RNA interference (RNAi) techniques (review in Duque et al. 2013). Early studies were accomplished in model species like Arabidopsis, tobacco, or plants with agricultural value as rice. Some examples include the heterologous expression of mouse Odc in tobacco, driven by CaMV 35S promoter, which resulted in a significant increase in Put and Spd (two- to threefold) and conferred increased tolerance to salt stress (Kumria and Rajam 2002). Nevertheless, transgenic plants showed altered in vitro growth and development, an aspect that was correlated with the supraoptimal Put levels. Transgenic tobacco expressing human Samdc (driven by CaMv 35S) showed increased Put and Spd levels; tolerance to multiple abiotic and biotic stresses, including salinity and drought; as well as resistance against Fusarium and Verticillium wilts (Waie and Rajam 2003). The introduction of Datura stramonium L. spermidine synthase (SPDS) cDNA into tobacco has led to the

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Table 1  Examples of genetic engineering of plants toward the accumulation or reduction of polyamines levels and the subsequent responses to environmental stresses Transformed plant Nicotiana tabacum Nicotiana tabacum Solanum lycopersicum Oryza sativa

Effects on PA metabolism and Gene/origin/promoter responses to environmental stresses Odc, mouse, CaMV Increased Put and Spd, increased 35S tolerance to salt stress; altered in vitro plant growth correlated with Put levels Samdc, human, Increased Put and Spd, tolerance to CaMV 35S salinity and drought; resistance against Fusarium and Verticillium wilts Samdc, yeast, E8 Increased Spd and Spm, enhanced promoter phytonutrient content and fruit quality Higher Put, Spd, and Spm levels; increased drought tolerance

Arabidopsis thaliana

Adc, Datura stramonium, Ubi-1promoter Spds, Cucurbita ficifolia, CaMV 35S

Ipomoea batatas

Spds, C. ficifolia, CaMV 35S

Solanum melongena

Adc, Avena sativa, CaMV 35S

Oryza sativa

Samdc, Datura stramonium, CaMV 35S

Arabidopsis thaliana

Adc1, Adc2, A. thaliana, CaMV 35S

Pyrus communis

Spds, Malus Higher Spd accumulation, strong sylvestris, CaMV35S tolerance to salt, osmotic stress, and heavy metals (CuSO4, CdCl2, PbCl2, and ZnCl2). Additional tolerance to AlCl3 Increment in endogenous Put levels; Adc, A. sativa, transgenic lines more resistant to both pRD29A stress-­ cold and dehydration stresses inducible promoter

Arabidopsis thaliana

Increased SPDS activity, increased Spd; enhanced tolerance to various stresses including chilling, freezing, salinity, hyperosmosis, drought, and paraquat toxicity Increased Spd; improved number of storage roots; increased tolerance to chilling- and heat-mediated damage to photosynthesis and enhanced tolerance to paraquat Increased Put, Spd, and Spm levels; multiple abiotic stress resistance (salinity, drought, low and high temperature, and cadmium), and fungal resistance Increased Spd and Spm levels, normal levels of Put. Plants showed drought symptoms, however, demonstrate a more robust recovery from stress Increased Put; improved freezing, and drought tolerance

References Kumria and Rajam (2002) Waie and Rajam (2003) Mehta et al. (2002); Mattoo et al. (2006) Capell et al. (2004) Kasukabe et al. (2004)

Kasukabe et al. (2006)

Prabhavathi and Rajam (2007)

Peremarti et al. (2009)

Altabella et al. (2009); Alcázar et al. (2010b) Wen et al. (2008, 2009, 2010, 2011)

Alet et al. (2011) (continued)

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Table 1 (continued) Transformed plant Solanum lycopersicum

Gene/origin/promoter Sams, S. lycopersicum, CaMV35S

Lotus tenuis

Adc, A. sativa, pRD29A

Nicotiana tabacum

Myb, Poncirus trifoliate, CaMV35S

Gossypium hirsutum

Myb, Gossypium barbadense, virus-induced gene silencing (VIGS) Myb, G. barbadense, CaMV35S

Nicotiana tabacum

Solanum lycopersicum

Odc, mouse, 2A11 fruit-specific promoter

Medicago truncatula

Adc, A. sativa, 2XCaMV35S

Citrus sinensis

Pao, C. sinensis, CaMV35S

Effects on PA metabolism and responses to environmental stresses Transgenic plants showed lower concentrations of Put, but enhanced Spd and Spm. Improved tomato fruit setting and yield and increased tolerance to alkali stress. Higher photosynthetic capacity and lower oxidative stress Increased Put and improved cellular hydration and increased root growth. Put also controls the level of ABA by modulating ABA synthesis at the transcriptional level Higher mRNA levels of two ADC genes, higher levels of PAs (Put, Spd, and Spm); enhanced dehydration tolerance, and lower levels of ROS and MDA Decreased tolerance to drought stress; decrease in proline and antioxidant enzyme, and increase in MDA Improved survival and reduced water loss in plants under drought stress; enhanced proline and antioxidant enzymes, increased transcript levels of transcript levels of ADC1 and SAMDC Transgenic plants with enhanced levels of Put, Spd, and Spm; concomitant reduction in ethylene levels, respiration rate, and physiological loss of water. Tomato plants with enhanced fruit quality Transgenic plants with elevated Put and Spd, higher leaf RWC, and increased photosynthetic parameters during drought stress; higher seed yield upon stress recovery Transgenic plants with increased PAO activity, concomitant with a marked decrease of Spm and Spd, and elevation of H2O2; transgenic seeds germinate better under salt stress, but by contrast vegetative growth and root elongation are inhibited

References Gong et al. (2014)

Espasandin et al. (2014)

Sun et al. (2014)

Chen et al. (2015)

 Chen et al. (2015)

Pandey et al. (2015)

Duque et al. (2016)

Wang and Liu (2016)

(continued)

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Table 1 (continued) Transformed plant Arabidopsis thaliana

Eremochloa ophiuroides

Nicotiana tabacum

Lotus tenuis

Effects on PA metabolism and Gene/origin/promoter responses to environmental stresses Pao, G. hirsutum, Decreased Spm and increased Put in CaMV35S transgenic lines; discrepancies on salt resistance during germination and seedling stage Samdc, Cynodon Transgenic plants with higher levels of dactylon, CaMV35S PAO activity; improved cold tolerance through the involvement of H2O2 and NO signaling Overexpression of MYB21 plays a Myb21, Pyrus positive role in drought tolerance; betulaefolia, possible modulation of polyamine CaMV35S biosynthesis by regulating the ADC expression Adc, A. sativa, Improved tolerance to salt stress in pRD29A transgenic lines, smaller reduction in shoot biomass, and a slight increase in root growth in response to stress; increased osmotic adjustment via proline production

References Cheng et al. (2017)

Luo et al. (2017)

Li et al. (2017)

Espasandin et al. (2018)

increased tolerance against multiple abiotic stresses and also provided evidences that spermidine synthase was not a limiting step in the biosynthesis of polyamines (Franceschetti et al. 2004). Transgenic rice expressing heterologous Samdc, driven by an ABA-inducible promoter, have showed increased Put and Spm levels (threeto fourfold), and transgenic seedlings showed increased growth under salinity conditions (Roy and Wu 2002). One the other side, the expression of Datura stramonium Adc gene, driven by the strong maize polyubiquitin-1 (Ubi-1) promoter, increased Put, Spd, and Spm levels and conferred drought tolerance to rice (Capell et al. 2004). The spermidine synthase cDNA from Cucurbita ficifolia Bouché was inserted into Arabidopsis (35S promoter), and this approach resulted in plants with increased tolerance against multiple abiotic stresses, including chilling, freezing, salinity, hyperosmosis, drought, and paraquat toxicity (Kasukabe et al. 2004). The constitutive expression of homologous Adc1 and Adc2 genes in Arabidopsis resulted in freezing and drought tolerance, respectively (Altabella et al. 2009; Alcázar et al. 2010b). As previously referred, from 2004 to 2006 onward, the majority of the PAs studies have been conducted in non-model species (see Fig.  1b), reflecting the interest of the scientific community on the possible use of these biomolecules on plant crop improvement toward abiotic stress. Several studies based on the engineering of PA biosynthesis for the production of stress-tolerant plants are summarized in Table 1 and will be further discussed on the following sections. A common feature observed in drought (here in the sense of water deficit), salinity, and cold/heat stress is the primary consequence of loss of water by the cells, resulting in a decrease in the osmotic potential. However, cell water loss in drought is due to water shortage in soil and/or in atmosphere; in salinity, stress is

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related to the decreases caused by the increased ion concentration; in cold stress, the cell water content diminished due to the difficulty of the soil available water to be transported to the living cells; and finally, with increased temperatures, the root moisture is lost to harmful levels (reviewed in Duque et al. 2013; Lamaoui et al. 2018). For the sake of structuring this chapter, we decided to focus each subsection in a specific stress response; it is however important to take into consideration that stresses are related and that in nature they do not occur normally as isolated phenomena.

4.2  Polyamines and Drought Stress Among abiotic stresses, drought affects foremost arable area and, in the current scenario of climate change and increased world population, is a major challenge for agriculture (reviewed in Duque et al. 2013; Araújo et al. 2015; Tardieu et al. 2018). Plant growth and productivity are greatly affected by drought, which causes a primary loss of cell water and, therefore, a decrease of cell osmotic potential (Duque et al. 2013). Plant reactions to water deficit can be divided in short-term and long-term responses. Physiological mechanisms primarily triggered act as short-term feedback: for example, an increase in transpiration rate tends to cause partial stomatal closure, thereby stabilizing transpiration, and a decrease in osmotic potential is rapidly compensated by rapid buildup and/or uptake of solutes to maintain turgor homeostasis (reviewed in Tardieu et al. 2018). Changes in photosynthetic parameters and in carbon metabolism are also promptly observed, with subsequent changes in the pool of sugars used for signaling cellular processes (Chaves et  al. 2009; Liu et al. 2013). Long-term water shortage negatively affects photosynthesis, leaf and root growth, and reproductive development, resulting in lower biomass accumulation, lower yield, and poor quality of the harvested plant parts (e.g., grains, biomass, and stalks) (reviewed in Araújo et al. 2015). Additionally, it is well documented that root growth is usually less affected by water potential changes than shoot growth; thus, an increased root/shoot ratio is commonly observed under water deficit (e.g., Xu et al. 2015; Purushothaman et al. 2017). In Arabidopsis, drought stress induces Adc2 expression (Alcázar et  al. 2005), and since most of the key genes involved in polyamine biosynthesis are duplicated and a functional ODC pathway is missing (Hanfrey et  al. 2001), the constitutive expression of homologous Adc2 gene was the choice for plant transformation (Alcázar et  al. 2010b). In this study, 4-week-old plant lines grown in soil were exposed to water deprivation by 2 weeks water withholding, followed by 7 days of recovery. Survival rates in transgenic lines were 18–75%, higher than the 12% wild-­ type (WT) survival rate. Measurements of leaf relative water content (RWC), stomata conductance (gs), and stomata aperture showed reduced water loss, reduced stomata conductance, and stomata closure in 35S::ADC2 drought-tolerant lines compared to WT (Alcázar et al. 2010b). Arabidopsis Adc2 overexpressing transgenic

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lines presented Put accumulation but unaltered Spd or Spm. Moreover, a linear correlation between total Put content and drought resistance was observed (Alcázar et al. 2005, 2010b). Espasandin et  al. (2014) studied the drought response of a transgenic Lotus tenuis cv INTA PAMPA line that expresses the oat Adc gene, driven by the stress-­ inducible Arabidopsis pRD29A promoter. The pRD29A promoter presents DRE (drought-responsive element) and ABRE (ABA responding element) cis acting elements, enabling induction under ABA and osmotic stress (Wu et al. 2008). Plants were grown for 6–8  weeks and subjected to continuous soil drying by water withholding until reaching Ψsoil  =  −2  MPa. Interestingly, the RWC of pRD29A::oatADC line under stress conditions did not change significantly with the decrease in soil water potential, when comparing to the WT line. In drought conditions, the transgenic line also presented higher stomatal conductance (gs) and transpiration (E) values (Espasandin et al. 2014). Put accumulation was considered sufficient to promote drought tolerance since Spm and Spd levels did not increase during the dehydration period. Additionally, the highest Put content in pRD29A::oatADC plants improved the cell’s water balance by adjusting the osmotic potential through the production of proline and by adapting the plant growth pattern through the redistribution of photo-assimilates in favor of root growth at the expense of leaf area (Espasandin et al. 2014). Nunes et al. (2008) evaluated the two main mechanisms of drought resistance, drought avoidance, and drought tolerance, in the model legume Medicago truncatula cv. Jemalong. Under mild stress conditions, when the soil water content (SWC) decreased to 1/2 of its maximum, M. truncatula plants maintained identical leaf RWC, net CO2 fixation rate, and photochemical and biochemical photosynthetic processes, suggesting that plants are able to avoid leaf dehydration. However, under severe water deficit (SWD), ribulose-1,5-bisphosphate (RuBP) regeneration and Rubisco carboxylation efficiency were both decreased, suggesting that nonstomatal limitations also occur in addition to mechanisms involving osmotic adjustment (reviewed in Araújo et  al. 2015). Based on the previous study, 11-week-old M. truncatula plants constructively expressing the oat Adc gene (35S::oatADC) were subjected to severe water deficit (SWD), by water withholding, until SWC reached values below 1/5, followed by 4 days of recovery (Duque et al. 2016). In the 35S::oatADC transformed line, higher leaf RWC was observed under SWD, compared to WT plants. Additionally, and independently of the light intensity, under SWD, the transgenic line stood out with increased photosynthetic parameters, namely, leaf internal CO2 concentration (Ci), net CO2 assimilation rate (A), transpiration (E), and stomatal conductance (gs). Elevated Put and Spd observed in the transgenic line could be responsible for preserving the guard cells turgor pressure (Duque et  al. 2016), which ultimately regulate stomatal aperture (Damour et  al. 2010). Moreover, 35S::oatADC transgenic plants that recovered from SWD had higher seed yield compared to control WT, suggesting a possible benefit of PA metabolism manipulation in preserving harvested grain yield in legumes exposed to drought stress.

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Works focusing on the overexpression of the SAMDC and SPDS enzymes were also conducted toward the possibility of enhancing plants’ drought tolerance. A heterologous S-adenosylmethionine decarboxylase (SAMDC) gene from Datura stramonium was used to generate transgenic rice plants with altered Spd and Spm levels, although maintaining Put titers (Peremarti et al. 2009). SAM is a common precursor for both the synthesis of PAs and ethylene; SAMDC is essential for the production of decarboxylated S-adenosylmethionine (dcSAM) and to provide aminopropyl groups to convert Put into Spd and Spm (see Fig. 2). Two-month-old WT and Ubi:DsSAMDC-transformed rice plants (Oryza sativa L. subsp. Japonica cv. EYI105) were watered during 60  days and after were subjected to 6  days treatment with 20% polyethylene glycol. The plants were allowed to recover for 20  days by replacing the PEG solution with water. Symptoms of dehydration (wilting and curling of the leaves) were clear after 3 days and became more severe after 6  days of PEG treatment, with WT and transgenic plants showing similar drought stress phenotypes. Peremarti et al. (2009) demonstrated that the transgenic plants expressing SAMDC produced normal levels of Put and presented similar drought syndromes when compared to WT. However, transgenic plants demonstrated a more robust recovery upon return to normal conditions. They suggested that, while plants with elevated Put are able to tolerate stress because of Put direct protective role in preventing the symptoms of dehydration, the higher PAs (Spd and Spm) could play an important in role in stress recovery. Moreover, Spd and Spm can also feedback to regulate the Put pool, with the result that normal metabolic and morphological phenotypes could be restored under stress (Peremarti et al. 2009). In a previous study, sweet potato (Ipomoea batatas cv. Kokei) was constructively transformed with Spds from Cucurbita ficifolia, and resulting transgenic plants showed twofold Spd content compared to the WT counterpart, both in leaves and storage roots (Kasukabe et al. 2006). Potted plants were exposed to drought stress and revealed a marked growth reduction in both vines and storage roots. However, transgenic plants were less affected than WT in terms of the number of storage roots and their fresh weights. Drought stress also decreased starch content in WT storage roots, but not in the transgenic plants, which showed a 1.5-fold higher starch yield. Authors concluded that transgenic FSPD1 sweet potatoes were more tolerant to drought stresses in the root zone than the WT plants (Kasukabe et al. 2006). Interestingly, MYB transcription factor genes from Gossypium barbadense L. (GbMyb5) and from Poncirus trifoliata L. Raf. (PtsrMyb) were found to confer drought tolerance in cotton and transgenic tobacco, Chen et al. (2015) and Sun et al. (2014), respectively. In the first work, GbMyb5 virus-induced gene silencing compromised the tolerance of cotton plantlets to drought stress by decreasing the recovery survival rate after the re-watering of stressed plants (a reduction of 90% to 50% was observed, when comparing transgenic and WT plants). A decrease in proline and antioxidant enzyme activities, and the opposite increase in MDA, was observed as a consequence of GbMyb5 silencing in stressed cotton (Chen et  al. 2015). Oppositely, the overexpression of GbMyb5 in tobacco revealed hypersensitivity to ABA, improved survival, and reduced water loss rates in plants under drought stress. Proline and antioxidant enzymes showed enhanced

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accumulation, while MDA was reduced in transgenic tobacco under drought stress. The transcript levels of the antioxidant genes SOD, CAT, and GST and of polyamine biosynthesis genes Adc1 and Samdc were also generally higher in GbMyb5 transgenic tobacco plants (Chen et  al. 2015). The overexpression of PtsrMyb in tobacco conferred enhanced dehydration tolerance (by diminishing water loss) and showed lower levels of ROS and MDA production. Transgenic tobacco lines presented higher mRNA levels of two Adc genes, before and after dehydration treatments, when compared to WT, and consequently higher levels of PAs (Sun et al. 2014). Additionally, several MYB-recognizing cis-acting elements are present on PtAdc gene promoters, and yeast-hybrid assays demonstrated that PtsrMYB interacts predominantly with two regions of the Adc promoter, indicating the PtAdc may be a target gene of PtsrMYB (Sun et al. 2014). Both works revealed that MYB transcription factors play a positive role in dehydration tolerance through the modulation of polyamine biosynthesis by the means of regulating the Adc gene expression (Sun et  al. 2014; Chen et  al. 2015). In this way, the identification of important transcriptional factors, capable to trigger the expression of the multiple genes encoding for enzymes of PA metabolic pathways, and their further use for PA modulation is a promising approach for future studies in this field.

4.3  Polyamines and Salinity Stress A recent Springer book entitled Salinity Responses and Tolerance in Plant provided an excellent update to the state of the art of the complex machinery of plant responses and adaptive mechanisms to cope with hyper soil salinity (Kumar et al. 2018). This book also includes transgenic approaches to develop salt-tolerant crops in this challenge era of climate changes. Salt stress is a major environmental limitation affecting crop performance and yield due to its osmotic and ionic effects (Negrão et al. 2017; Bor and Özdemir 2018). Manipulating metabolic pathways for higher osmotic and ionic tolerance would be more realistic to mitigate the negative impact of salt stress on crop plants, in opposition of targeting signaling or regulatory networks (Bor and Özdemir 2018). Among possible metabolic pathways to be engineered is the overexpression of polyamine biosynthesis-related genes, consensually accepted to contribute for salt stress tolerance in several species (Bor and Özdemir 2018; Khare et al. 2018). Khare et al. (2018) presented an overview of transgenic plants that, by the overexpression of PA biosynthesis-related genes, are able to perform better under salt stress (see Table  13.1 in Khare et  al. 2018 and references therein). Salinity effect on plants is similar to drought effect in an initial stage; however, in the long term the consequences of Na+ and Cl− leaf accumulation and hyperosmotic and hyper-ionic stresses enhance the severity and impact of this stress (Chaves et al. 2009; Bor and Özdemir 2018). Salinity directly affects stomata and mesophyll (gm) conductance as well as gene expression, resulting in alteration of photosynthetic metabolism (Chaves et al. 2009). The effects can be direct, with plants losing water

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from their tissues, with a rapid effect on cell expansion and cell divisions (Negrão et al. 2017), suffering a decrease in CO2 availability caused by diffusion limitations through the stomata and the mesophyll, being damaged by photosynthetic alterations, and leading to abscisic acid (ABA) accumulation, or can be related to secondary effects, specifically oxidative stress (Chaves et al. 2009). In experimental settings, one of the first observable responses after salinity imposition is a reduction in shoot growth (Negrão et al. 2017). Here we present some non-exhaustive examples of PA content modification toward salinity resistance given the actuality of the previously mentioned up-to-date revisions. Prabhavathi and Rajam (2007) reported an increase in Put but also in higher PAs, Spd, and Spm in eggplant (Solanum melongena L. cv. Pusa Purple Long) transformed with oat Adc (under the control of CaMV 35S promoter). Regarding the salinity tolerance assay, plant seeds were inoculated on MS basal medium containing 150 and 200 mM NaCl, and tolerance was accessed based on the percentage of seed germination according to Prabhavathi et al. (2002). Transgenic seeds germinated in 200 mM NaCl after 15 days of inoculation, and the seedlings grew well on salt-amended medium, while WT seeds failed to germinate. Seed survival was also tested with a sublethal 150 mM salt concentration; in this condition, transgenic seedling performed better, and their growth in terms of shoot length and fresh and dry weight was greater than in the WT seedlings. Additionally, in this study, authors also found that PA accumulation in transgenic eggplants resulted in increased tolerance to other abiotic stresses (drought, low and high temperature, and heavy metal) and also fungal resistance. Moreover, in addition to the ADC enzyme activity increase, the DAO activity was also augmented in transgenic plant lines (Prabhavathi and Rajam 2007). Espasandin et al. (2018) overexpressed the oat Adc gene in Lotus tenuis cv INTA PAMPA, using the stress-inducible pRD29A promoter (pRD29A::oatADC construct; previously used in Espasandin et al. 2014). In addition to the previous work regarding the contribution of ADC overexpression in drought tolerance (Espasandin et al. 2014) the authors also tested the improved tolerance to salt stress in 6-month-old WT and transgenic plants at vegetative stage, by gradually increasing salinity, that was applied by means of sodium chloride irrigation (NaCl from 0 to 0.3 mol.L−1) (Espasandin et al. 2018). Transgenic lines appeared healthier than the WT plants and showed a smaller reduction in shoot biomass and a slightly increase in root growth in response to the applied stress. ADC overexpression also increased osmotic adjustment by 5.8-fold, via proline production. Moreover, salinity treatment doubled the potassium uptake by transgenic ADC roots in stressed plants, with a concomitant decrease in the sodium accumulation, balancing the Na+/K+ ratio. The shoot/root ratio increased at the expense of roots in WT; contrastingly, the stressed transgenic plants showed lesser reduction in shoot biomass and a minor promotion in root growth. A detailed analysis of gene expression, of enzymatic activities, and of hormone metabolism suggested an important crosstalk between PAs and ABA in response to salinity, via the modulation of the ABA biosynthesis-related enzyme 9-cis-epoxycarotenoid dioxygenase (EC 1.13.11.51) at the transcriptional level (Espasandin et al. 2018).

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In Kasukabe et al. (2006) work, concerning sweet potato constructively transformed with Spds (previously discussed in Sect. 4.1 in respect to the drought tolerance), the response to high salinity was also evaluated. The establishment of a saline soil for plant growth was accomplished by the addition of 8 g NaCl, before planting, and by an extra addition of 4 g NaCl 45 days after planting (for 20 L of soil). Salt stress suppressed storage root growth; however, transgenic plants were less affected and produced significantly larger mass of storage roots and starches (1.2-fold higher fresh mass) than WT plants. Transgenic plants also showed increased tolerance to chilling- and heat-mediated damage to photosynthesis compared to the WT plants (Kasukabe et al. 2006). Overexpression of an apple (Malus sylvestris (L.) Mill.) spermidine synthase (SPDS) gene substantially increased the tolerance to multiple stresses by altering the PA levels in pear (Pyrus communis L.). A specific transgenic line, which revealed highest Spd accumulation, showed the strongest tolerance to salt (150 mM NaCl), osmotic stress (300 mM mannitol), and heavy metal (500 μM CuSO4). Additionally, this line showed the lowest growth inhibition and the least increased in the electrolyte leakage (EL) and in the production of thiobarbituric acid reactive substances (TBARS) under stress conditions (Wen et al. 2008). Wen et al. (2009) further studied the possibility of this MdSPDS1 pear transgenic lines to tolerate aluminum (Al) stress, a major cause of poor crop yields, particularly in countries with acidic soil prevalence. Overexpressing apple spermidine synthase (MdSPDS1) plants and WT plants was subjected to long-term stress with 30  μM AlCl3. The performance of transgenic line was superior in terms of shoot height increment (SHI), fresh weight increment (FWI), and observed morphological features, when compared to WT counterparts (Wen et al. 2009). Favorable to the better performance and survival of MdSPDS1 transgenic line was the observation that, upon this stress, there was an increase of the activities of superoxide dismutase (SOD), of glutathione reductase (GR), and of proline and MDA accumulation. Moreover, Ca2+ concentration and some co-factor metals of SOD were higher in transgenic than in WT, after the imposed stress. These antioxidant parameters were closely related to the Spd levels, evidencing that Spd is implicated in Al stress tolerance via ameliorating oxidative status as well as by affecting mineral element balance (Wen et al. 2009). Wen et al. (2010) also found that the transgenic MdSPDS1 line had better performance than WT line when subjected to heavy metals stress (using CdCl2, PbCl2, ZnCl2, or a combination of those). Results were based on either shoot height increment or fresh weight and morphological changes upon heavy metal stress (Wen et al. 2010). The importance of PAs in stress responses can also be evaluated by using approaches of antisense inhibition of polyamine biosynthesis-related genes. An example was the transformation of P. communis with a construct containing an apple Spds gene (MdSPDS1) in antisense orientation (Wen et  al. 2011). In this work, the antioxidant system was not effectively induced under both salt and cadmium stress in the antisense transgenic line, when compared to WT, as could be perceived by the determination of glutathione (GSH) content, glutathione reductase (GR) activity, SOD, and evaluating the proline accumulation. Moreover, under stress the antisense line showed greater accumulation of MDA; an important

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indicator of lipid peroxidation. These results provide evidences for the important role of PAs in both salt and cadmium stress tolerance, in which the PAs act, to some extent, by influencing the antioxidant system (Wen et al. 2011). Attempts to change the PA concentrations by modulation of its catabolic pathway were also accomplished, and here we present an example for genetic engineering with the model Arabidopsis. Cotton (Gossypium hirsutum L.) PAO3 cDNA (genome-­ wide identified from a released genome database) was selected for Arabidopsis transformation (35S::GhPAO3 construct) (Cheng et al. 2017). To compare the stress tolerance of WT and GhPAO3 transgenic line, 2-week-old Arabidopsis seedlings were treated with 300 mM NaCl. Overall, and during seed development, the WT plants performed better than the transgenic lines under NaCl treatment. Interestingly, WT and GhPAO3 Arabidopsis plants showed an opposite performance in soil under 300 mM NaCl and 100 mM NaCl treatment (Cheng et al. 2017). In the transgenic 35S::GhPAO3 line, the Spm content was significantly decreased, whereas the Put content was enhanced, which indicates a potential role of GhPAO3 in the back-­conversion of Spd and Spm to Put. Hydrogen peroxide analysis in transgenic and WT indicated that H2O2 was mainly produced from polyamine oxidation in this process. The discrepancies in salt stress tolerance that were observed in germination and during seedling development (which might be controlled by the H2O2 concentration threshold) suggested an enhance resistance to NaCl stress in GhPAO3 plants at a certain level; whereas unusually higher H2O2 concentrations may become harmful for Arabidopsis development and be implicated in programed cell death (Cheng et al. 2017).

4.4  Polyamines and Tolerance to Extreme Temperatures In the current scenario of climate change, plants are being challenged with frequent episodes of extreme weather events that include, among other aspects, spells of very high temperatures which are strongly connected to drought episodes (Rosenzweig et al. 2001). But not only high temperatures compromise serious plant growth, and suboptimal temperatures have been considered also a major threat to agricultural production in temperate regions. Despite most of the temperate plants acquiring chilling and freezing tolerance upon prior exposure to sublethal cold stress commonly called cold acclimation, still many important crops with agronomic relevance are incapable of cold acclimation (Yadav 2010). Many plant crops are sensitive to supraoptimal temperatures that lead to disruption of cellular and physiological homeostasis, a phenomena also known as heat stress (Xu et al. 2017). The impacts of heat stress on plant performance are well described and associated with alterations on photosynthesis, assimilating partitioning, growth, and development (Bokszczanin et al. 2013). Importantly, heat stress strongly negatively affects reproductive success on the majority of the plants species, which commonly translates into yield losses in agricultural settings (Asseng et al. 2011).

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Approaches to improve tolerance to extreme temperatures through the PA content modification embrace some case-stories of success. Tomato is one of the most important horticultural crops worldwide, and the damaging effects of heat stress in tomato production are very well documented (Sato et  al. 2000, 2001; Xu et  al. 2017). Thus, the identification and development of tomato cultivars better adapted to growth, and set fruits under supraoptimal temperature conditions are still an important need of the agricultural sector. In this context, Cheng and collaborators (2009) have generated transgenic plants of tomato variety “zhongshu No.6” constitutively overexpressing the Saccharomyces cerevisiae Samdc. In average, 1.7to 2.4-fold higher levels of Spd and Spm were found in transgenic lines compared to WT under high temperature stress (38  °C/30  °C). Associated with this PA accumulation, a markedly increased antioxidant enzyme activity and decreased MDA contents were also noticed in transgenic plants. Heat stress increased lipid peroxidation in the nontransformed line, an aspect that was not observed in transgenic lines. The authors concluded that PA accumulation generated a heat tolerance mechanism, supported the PA antioxidant activity, and alleviated the membrane damage caused by ROS during heat stress (Cheng et al. 2009). Another interesting aspect, likely beyond the scope of this aspect, is that the overexpression of Samdc in tomato modulated the nutrient contents of the fruits (Kolotilin et al. 2011), which can have added value for the consumers or impact in product shelf-life. The physiological impact of low temperatures is also well described in plants. Low temperature reduces water availability for the plant, decreases membrane fluidity, and causes an imbalance between the light energy absorbed by photosystems and the energy consumed by metabolic reactions, compromising plant growth and survival (Ruelland et  al. 2009). Not all plants or crops are well adapted to low temperatures or chilling, being this related mainly to their tropical or subtropical origin (Lyons 1973). One of those examples is the centipede grass (Eremochloa ophiuroides (Munro) Hack.), a warm-season (C4) perennial grass native to South and Central China (Hanna 1995). Centipede grass is widely used as turf and has gained growing interest as a low-input plant for soil conservation in many countries prone to erosion. In environments where frosts are not severe, centipede grass is green throughout the winter, but its growing period is from spring through late autumn, becoming brown or dormant in winter (Islam and Hirata 2005). Among the different research efforts conducted to improve cold tolerance in this grass, we highlight here one that included the overexpression of PA biosynthetic genes. Recently, Luo and collaborators (2017) overexpressed in centipede grass a Samdc gene from Bermuda grass (Cynodon dactylon (L.) Pers.) and investigated their effects in response to cold. In resulting SAMDC transgenic plants, Spd levels were in average 2.3- to 2.9-fold higher, while Spm levels were 1.6- to 1.8-fold higher, in comparison with the nontransformed plants. Freezing tolerance of transgenic and nontransgenic lines was evaluated by determination of the temperature that resulted in 50% lethality (LT50) and assessment of survival rate. Under non-acclimated conditions, transgenic lines showed a lower level of LT50 (−5.2  °C), when compared to the −3.2  °C observed in nontransformed plants, suggesting a better tolerance to freezing. After a progressive freezing treatment, the

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survival rate of transgenic lines was higher (59–61%) comparing to those observed in nontransformed ones (40%) under non-acclimated conditions. Biochemical studies provided evidences about the mechanisms triggered by PA accumulation under cold. As one example, transgenic plants showed higher catalase and SOD activities and higher PAO activity and H2O2 levels upon cold. These results supported the assumption that the elevated cold tolerance was associated with PAO-catalyzed production of H2O2, a key signaling molecule that led to nitrate reductase (NR)derived NO production (Lu et al. 2014) and triggered antioxidant enzyme activities in transgenic plants (Luo et al. 2017). Importantly, this work provided new evidences of the prevailing crosstalk between PAs, ROS, and NRS in the modulation of cold stress responses in plants. The knowledge about the crosstalk between PAs and hormones as ethylene was essential to derive or test new strategies to increase plants’ tolerance to cold stress. One of these approaches has as key player a cold-induced transcription factor, the ethylene responsive factor (ERF), which has been isolated from Medicago falcata L. (Pang et al. 2009). M. falcata is a forage legume well known by its enhanced cold tolerance, when compared to other species of the same genera (Lesins and Lesins 1979). The role of MfERF1 on cold acclimation has been further assessed in tobacco (Zhuo et  al. 2018). These authors found that the constitutive overexpression of MfErf1 resulted in an increased tolerance to freezing and chilling in transgenic tobacco plants. Indeed, a freezing treatment at −3 °C for 6 h. was lethal for most of nontransformed plants, whereas 47–50% of transgenic plants were able to survive. One of the most interesting aspects of this work is that the overexpression of MfErf1 upregulated, among others, the expression of PA genes implicated in Spd and Spm synthesis (SAMDC1, SAMDC2, SPDS1, SPDS2, and SPMS) and PA catabolism (PAO) in comparison with WT plants, which is an indirect approach to modulate PA metabolism. Nevertheless, no significant accumulation of Spd and Spm was noticed in transgenic plants, likely as result of the increased PAO activity, which contributed for maintaining PAs at nonlethal levels. Among other findings, these results suggested that MfERF1 conferred cold tolerance by promoting polyamine turnover, antioxidant protection, and proline accumulation (Zhuo et  al. 2018). The use of transcription factors, such as MfERF1, that trigger the expression of several protective genes like the ones responsible for PA biosynthesis constitutes a very promissory and still unexploited approach to enhance cold tolerance in plants. In this section, we have reviewed several successful approaches to improve heat and cold tolerance in plants through the modulation of the PA metabolism. Interestingly, most of the approaches targeted the constitutive overexpression of SAMDC, an enzyme essential for the addition of aminopropyl groups, needed for the conversion of Put to Spd and Spd to Spm. Most of the studies provided clear evidences of the link between PAs, ROS, NRS, and hormones to enhance plants adaptation to extreme temperatures. Still, there is an open window for the development of new strategies based on the modulation of PA contents and turnover. One promissory could be related to the identification of transcription factors capable to trigger the expression of the multiple genes encoding for enzymes PA metabolism.

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4.5  O  xidative Stress: A Common Mechanism in Response to Stress All kinds of abiotic stresses previously referred trigger a generalized stress response denominated oxidative stress, due to the accumulation of ROS, and can seriously affect leaf photosynthetic machinery (Chaves and Oliveira 2004). Oxidative stress can be generally defined as a physiological state in which oxidation is superior to its opposite mechanism, the reduction (Harshavardhan et al. 2018). In respect to the antioxidant activity of PAs, the research data is contradictory; on the one hand, PAs have been suggested to protect cells against ROS, and on the other hand, their catabolism generates ROS. PA catabolism produces H2O2, a signaling molecule that not only acts by promoting an antioxidative defense response upon stress but can also act as a peroxidation agent (Groppa and Benavides 2008; Gupta et al. 2016) (Fig. 2). In a study using poplar cells in culture (isogenic cell lines of Populus nigra × Populus maximowiczii), the effect of increased Put accumulation was found to negatively impact their oxidative state owing to the enhanced turnover of Put (Mohapatra et al. 2009). Gill and Tuteja (2010) suggested that while increase Put accumulation could have a protective role against ROS in plants, an enhanced Put turnover can in reality make them more vulnerable to increased oxidative damage. Contrastingly, the higher polyamines, Spd, and Spm are believed to be most efficient antioxidants and considered as scavengers of oxyradicals (He et  al. 2008; Channarayappa and Biradar 2018). The work by Cheng et  al. (2017), previously described in Sect. 4.2, concerning GhPAO3 Arabidopsis transgenic plants, is a clear example of the duality of the balance process between consumption and production of H2O2. Another interesting work is the transformation of Citrus sinensis (L.) Osbeck with the homologous CsPAO4 by Wang and Liu (2016). These transgenic plants showed prominent increase in PAO activity, concomitantly with a marked decrease of Spm and Spd and elevation of H2O2. While transgenic seeds germinate better when compared with WT under salt stress, vegetative growth and root elongation are further inhibited (with the maintenance of stressed conditions), accompanied with higher accumulation of H2O2 and a more noticeable programmed cell death (PCD).

5  Conclusions Polyamines are a class of plant biomolecules that have been implicated in plant growth and development processes and are also very important in plant responses to biotic and abiotic stresses. PAs’ endogenous levels are influenced by different factors such as plant species, stress tolerance capacity, stress types and conditions, and the physiological status of the studied tissue or organ. The major forms of PAs

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are Put, Spd, and Spm; although plants also synthesized a variety of other related compounds. PA intracellular levels are regulated by anabolic and catabolic processes as well as by polyamine conjugation with hydroxycinnamic acids, fatty acids, and macromolecules. Moreover, polyamines metabolic pathways are closely interconnected with other metabolic pathways, such as the glutamate cycle, proline, urea, γ-aminobutiric acid (GABA), and TCA cycle. PA metabolic pathway is also closely related to ethylene by the means of sharing a common precursor, the S-adenosylmethionine (SAM). Several studies also provided evidences of the link between PAs, ROS, NRS, and hormones (besides ethylene), being a driving force for further studies on this subject. One interesting fact is that the number of articles on plant PAs almost duplicated since 2008. This may be due to the development of new high-throughput methodologies to characterize the global gene expression or metabolite accumulation (omics) with reduced cost for user, together with the release of several plant genome sequences. Another interesting fact is that the majority of the PA studies have been conducted into nonmodel species, which may likely reflect a translational application of the early results obtained with models for further use in important agricultural crops. In this chapter, we presented non-­ exhaustive examples of endogenous PA content modification toward the tolerance/ resistance to abiotic stresses, by the means of plant genetic engineering. We focus each subsection in a specific stress, drought, salinity, and tolerance to extreme temperatures; however, PA manipulation usually confers tolerance to multiple abiotic stresses. Moreover, stress phenomena are related, and in nature they typically do occur as isolated events. PA biosynthetic genes usually used for transgenic approaches included the ones involved in Put, Spd, and Spm synthesis (Adc, Spds, and Spms); in the decarboxylation of SAM (Samdc), related with PA catabolic pathways (coding for DAO and PAO); and lately, in transcription factors positively involved with PA biosynthesis. The identification and further study of transcription factors capable to trigger the expression of multiple genes encoding for enzymes of PA metabolism constitutes a promising path for future studies. Despite the complexity and intricate network of the mechanisms involved in the PA synthesis and regulation, the increased number of recent works, as well as the application of patents related to PAs and stress tolerance, corroborates the continued interest of the scientific community in elucidating the different roles of these important biomolecules in plants. Acknowledgments  Financial support from FCT (Fundação para a Ciência e Tecnologia, Lisbon, Portugal) is acknowledged through the research unit “GREEN-it: Bioresources for Sustainability” (UID/Multi/04551/2013) and through ASD and SSA PhD holders DL57 research contracts. ALWS is supported by FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo) and Young Investigators Grants 15/21075-4 and 17/01284-3. ALWS thanks Dra. Eny IS Floh (Department of Botany, University of São Paulo) for her valuable collaboration and pioneering studies with polyamines in Brazil.

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Fructan Metabolism in Plant Growth and Development and Stress Tolerance Alejandro del Pozo, Ana María Méndez-Espinoza, and Alejandra Yáñez

1  Introduction The basic components for biomass accumulation derive from the assimilation of carbon into carbohydrates via photosynthesis, and these are used for the synthesis of other compounds like organic acids, amino acids and lipids (Medrano and Flexas 2000; Geigenberger et al. 2005). Most plants store sucrose or starch as reserve carbohydrates, whereas about 10–15% of flowering plant species store fructans (Hendry 1993; Vijn and Smeekens 1999; Van den Ende et al. 2004). Among the families that accumulate fructans are dicots of the Asteraceae, Campanulaceae and Boraginaceae and monocots of the Liliaceae and Poaceae (Versluys et al. 2017a). Fructans are abundant in plants from temperate and arid zones (with frost and drought), but are almost absent in tropical and aquatic environments (Hendry 1993). The accumulation of fructans in plant organs has been related to adaptation to abiotic stresses such as freezing and drought (Livingston et al. 2009). In temperate cereals, such as wheat (Triticum aestivum) and barley (Hordeum vulgare), carbohydrates are stored in the stem and leaf sheaths as water-soluble carbohydrates (WSCs). These are composed predominantly by fructans, followed by sucrose and, to a lesser extent, glucose and fructose (Virgona and Barlow 1991; Chalmers et  al. 2005). These stem WSCs contribute to grain growth and filling, particularly under water-deficit conditions when leaf

A. del Pozo (*) · A. M. Méndez-Espinoza Centro de Mejoramiento Genético y Fenómica Vegetal, Facultad de Ciencias Agrarias, Universidad de Talca, Talca, Chile e-mail: [email protected] A. Yáñez Facultad de Ciencias Agrarias y Forestales, Universidad Católica del Maule, Curicó, Chile © Springer Nature Switzerland AG 2019 M. A. Hossain et al. (eds.), Osmoprotectant-Mediated Abiotic Stress Tolerance in Plants, https://doi.org/10.1007/978-3-030-27423-8_15

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p­ hotosynthesis and the production of assimilates destined for grain filling are ­inhibited (Blum et al. 1994; Blum 1998; Dreccer et al. 2009). In this chapter we describe briefly the WSCs and examine fructan metabolism, function and dynamics in plants, particularly in temperate cereals. Also, we have analysed the role of fructans in stress tolerance and the expression of genes involved in fructan accumulation and remobilization in temperate cereals.

2  Water-Soluble Carbohydrates WSCs play a central role in the metabolism of plants as carbon and energy sources in cells. Their levels are continuously adjusted as a result of the balance between supply and demand of carbon at the whole plant level. Plants accumulate WSCs in roots (de Roover et al. 2000; Jiang and Huang 2001), stems (Bogeat-Triboulot et al. 2007; Méndez et  al. 2011; del Pozo et  al. 2012, 2016), leaves (Kim et  al. 2000; Teulat et  al. 2001; Cramer et  al. 2007) and flowers and fruits (Liu et  al. 2004; Mercier et al. 2009). The metabolism of sugars is enormously dynamic and varies with the stage of development of plants and in response to the environment (Rolland et al. 2006; Ramel et al. 2009; Martínez-Vilalta et al. 2016). In most plants, the primary products of the photosynthetic assimilation of carbon in the leaves are sucrose and starch (Zeeman et al. 2007), and the rate of synthesis of sucrose in the cytosol is coordinated with the rate of CO2 fixation and the synthesis of starch in the chloroplast (Lunn 2007). On the one hand, sucrose is a form of carbon storage that often accumulates in plants exposed to cold or drought (Pérez et al. 2001; Xue et al. 2008a; Méndez et al. 2011), acting as a compatible solute for protecting cell integrity (Xue et  al. 2008a). It also acts as a signalling molecule, modulating the expression of genes involved in metabolism and development (Koch 2004; Osuna et al. 2007). Starch, on the other hand, is the main storage carbohydrate in higher plants, and it is deposited in granules in the chloroplasts of photosynthetic tissues, which represent a transitory storage of carbon that is mobilized during the night to maintain respiration, export of sucrose and growth in the dark (Scofield et al. 2009). Alongside sucrose and starch, there is a third reserve of carbohydrate in plants: fructans, which are the main storage carbohydrates in cereals like wheat and barley (Pollock and Cairns 1991). Fructans are linear or branched polymers of fructose derived from sucrose (Lasseur et al. 2006). They consist of a sucrose molecule with additional fructose linked in the β-(2-1) and/or β-(2-6) positions (Gadegaard et al. 2008). They can be found in grains and in vegetative organs – stems, leaves and roots – depending on the plant’s state of development and its surrounding environmental conditions including light intensity, temperature and water availability, as well as the nutritional state of the plant (Morcuende et al. 2004; Yang et al. 2004; Morcuende et al. 2005; Verspreet et al. 2013).

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3  Fructan Metabolism and Functions Fructans are linear or branched polymers of fructose derived from sucrose (Lasseur et  al. 2006). Five structural classes of fructans have been distinguished: inulin, levan, mixed levan, inulin neoseries and levan neoseries (Halford et  al. 2011). Fructans are synthesized from sucrose in the vacuole by a group of fructosyltransferases belonging to a family of 32 plant glycoside hydrolase enzymes (Van den Ende et al. 2011); they differ in length (degree of polymerization), branching, the types of junctions between adjacent fructose molecules and the position of glucose residues (Halford et al. 2011). The incorporation of a fructose molecule into one of the three primary alcohol groups of sucrose gives rise to one of the three basic trisaccharides, 1-kestose, 6-kestose or 6G-kestose (neokestose), which are the precursors of longer fructans with a higher degree of  polymerization. The linkage between the C2 of one fructose and the primary alcoholic group of C1 or C6 of another gives rise to the β-(2-1) (inulin) or β-(2-6) (levan) fructan types, respectively (Chalmers et al. 2005). Inulins are predominantly linear chains of two to 60 fructan units and one glucose unit, and this class is present mainly in dicots species (van Laere and van Den Ende 2002). The levan-type consists mainly of linear fructans containing β-(2-6) linkages found mainly in bacteria and grasses (van Laere and van Den Ende 2002). Graminan-type fructans are branched polymers containing a mixture of β-(2-­1) and β-(2-6) linkages, which are present in cereals like wheat and barley (Veenstra et al. 2017). Fructan biosynthesis is mediated by four fructosyltransferase (FT) enzymes (Xue et al. 2008b). First, the sucrose:sucrose 1-fructosyltransferase (1-SST) allows the transfer of fructose from one donor sucrose to another molecule of sucrose to generate 1-kestose and glucose (Vijn and Smeekens 1999; Kawakami and Yoshida 2005). Second, sucrose:fructan 6-fructosyltransferase (6-SFT) transfers one molecule of fructose from a donor sucrose to 1-kestose to form 1,6-kestotetraose; this also facilitates the elongation of fructan molecules by fructosyl transfer from sucrose to fructans with β-(2-6) linkages (Duchateau et al. 1995; Rao et al. 2011; Yue et al. 2015). Fructan:fructan 1-fructosyltransferase (1-FFT) catalyses the elongation of the fructan chain (Kawakami and Yoshida 2005; Van den Ende et al. 2005). Finally, fructan:fructan 6G-fructosyltransferase (6G-FFT) transfers the fructose from a fructan molecule to the glucosyl residue of another fructan or sucrose molecule and facilitates the subsequent elongation of the chain with β-(2-1) or β-(2-6) linked, allowing the respective formation of the inulin neoseries or levan neoseries fructan types (Chalmers et al. 2005; Hou et al. 2018). The degradation of fructans is catalysed by fructan exohydrolase (FEH) enzymes, which release terminal fructose units (Van den Ende et al. 2004). Different isoforms of FEH have been isolated, which include fructan 1-exohydrolases (1-FEH) and fructan 6-exohydrolases (6-FEH), and these hydrolyse fructans with β-(2-1) and β-(2-6) linkages, respectively (Van den Ende et al. 2004). Fructan exohydrolases are

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regulated at the transcriptional level, so their transcript levels are related to variations in the fructan content (Xue et al. 2008b). The accumulation and remobilization of fructans in plants involve the concerted action of FT and FEH, which are closely and coordinately regulated alongside invertases (Joudi et al. 2012). There is evidence that the regulation of fructan synthesis in barley leaves is independent of hexokinase, and it seems to depend on sucrose sensitization (Müller et al. 2000). It has been proposed that the existence of a threshold concentration of sucrose is necessary for the induction of fructan synthesis, as evidenced by certain studies with cut leaves of Lolium temulentum (Cairns and Pollock 1988) and barley leaves incubated in low illumination (Simmen et al. 1993) and/or incubated with sucrose in the dark (Wagner 1986) to induce the biosynthesis of fructans. Abscisic acid (ABA) is important for the induction of FEHs in wheat (Ruuska et  al. 2008). However, sucrose could be an inhibitor of FEH enzyme activity in chicory, so this sugar could be an inhibitor of fructan hydrolysis during mobilization (Verhaest et  al. 2007). Additionally, phosphate decreases sucrose phosphate synthase activity and reduces sucrose levels, which is the substrate for fructan biosynthesis. The principal function of fructans is to reduce the gap between the availability of photosynthates and the demand for them. Along with their role as a carbohydrate reserve, fructans contribute to regrowth (Morvan-Bertrand et al. 2001) and osmotic regulation during floral opening (Le Roy et al. 2007), they confer tolerance to cold and drought (Pilon-Smits et al. 1995; de Roover et al. 2000; Kawakami et al. 2008; Livingston et al. 2009; Salinas et al. 2016), and they contribute to the maintenance of osmotic potential through the stabilization of cell membranes (Hincha et al. 2007). Indeed, transgenic plants of tobacco and sugar beet that accumulate fructans show a high tolerance to drought (Pilon-Smits et al. 1995; Pilon-Smits et al. 1999). It has also been suggested that they could counteract oxidative stress (Peshev et al. 2013). Fructans and FEH enzymes have been observed in the apoplast (Van den Ende et  al. 2005) in response to stress (Livingston and Henson 1998; Yoshida and Kawakami 2013), where they can be degraded by the FEHs, generating sucrose, fructose or other oligosaccharides that could affect membrane stabilization (Livingston and Henson 1998). The production of apoplastic fructans is one of the possible mechanisms that the plant uses to respond to cell rupture, with at least part of the polysaccharide inserted into the lipid head group region of the membrane (Livingston et al. 2009). In other words, fructans play a role in the plant immune system (Versluys et al. 2017b). In addition, inulin-type fructans increase the nutritional value of the grain in cereals because they are a component of the dietary fibre that is easily fermented by colon microbiota (Verspreet et al. 2015). Fructans accumulate in the vegetative tissues of many species including temperate forage grasses and cereals. The study conducted by Pollock and Cairns (1991) in excised leaves of the grass Lolium temulentum showed that sucrose accumulation preceded the synthesis of trisaccharides and more complex fructan molecules, suggesting that the synthesis of fructans begins when the carbohydrate supply exceeds the demand of the plant. The analysis of WSCs in stems of wheat plants showed

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that fructans were the predominant WSCs, followed by sucrose, fructose and ­glucose (Fig. 1), and they can represent up to 85% of the WSCs in the internodes (Turner et al. 2008). In temperate cereals, stem WSCs are accumulated from stem elongation to the early phase of grain filling (Ehdaie et al. 2006; Dreccer et al. 2009). Usually the lower internodes of the stem contain larger quantities of fructans (Virgona and Barlow 1991; Ruuska et al. 2006; Xue et al. 2008a; Joudi et al. 2012; Khoshro et al. 2014), which suggests that the synthesis of fructans was induced in the stems when the availability of carbon was higher than the demand for other assimilate (Ruuska et al. 2008). McIntyre et al. (2011) suggested that the assimilation of nitrogen into amino acids is an important factor that modulates WSC levels in stems of wheat.

4  Role of Fructans in Stress Tolerance in Temperate Cereals The accumulation of WSCs in the stem is influenced by environmental factors (Blum et al. 1994; Ehdaie et al. 2006; Ruuska et al. 2006) such as drought (Xue et al. 2008a; Dreccer et al. 2009; del Pozo et al. 2016), low temperatures (Pérez et al. 2001; Del Viso et al. 2009), reduction in the size of the sink (Martínez-Carrasco et al. 1993), deficiency in nitrogen (Wang et al. 2000; Shiomi et al. 2006), high CO2 (Pérez et al. 2005; Aranjuelo et al. 2011) and biotic stresses such as plant disease (Dreccer et al. 2009; Yue et al. 2015). In the absence of stress, fructans accumulate in stems until approximately 2 weeks post-anthesis (Dreccer et al. 2009); later they are degraded

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and partially remobilized to the grain for the synthesis of starch in late stages of grain filling (Fig. 1d). However, under unfavourable environmental conditions, stem fructans can be degraded in the early stages of grain filling to effectively compensate for the decrease in photosynthates and thus help maintain grain filling (Li et al. 2013). In grasses and cereals, grain filling depends on carbohydrates that originate from two fundamental sources: (1) newly synthesized carbohydrates that are transported directly to the grains from photosynthesis in the leaves and the spike and (2) assimilates stored and distributed to reserve organs from the vegetative tissues (Gebbing et al. 1999; Yang and Zhang 2006; Ehdaie et al. 2008). In spite of the decline in photosynthesis during water-deficit conditions, the concentration of carbohydrates in the different organs of the plant increases, which suggests a decoupling between the supply (photosynthesis) and the carbon demand (growth) that leads to an improvement in the carbon status of the plant (Chaves et al. 2009; Hummel et al. 2010; Müller et al. 2011). An increase in carbohydrate content in plants subjected to water deficit has been shown in several plant species, in different parts of the plant and for different forms of carbon (soluble and structural) (Müller et al. 2011). In wheat, water deficit increased the accumulation of stem glucose, fructose, sucrose and fructans after anthesis, compared to plants grown under well-watered conditions (Fig. 1d). In Mediterranean climate regions, crops are exposed to a progressive water deficit during flowering and grain filling stages, leading to what has been called ‘terminal drought stress’ (Monneveux et al. 2006; Dolferus et al. 2013; del Pozo et al. 2016). The water deficit during these phenological stages reduces leaf photosynthesis and the production of photosynthetic assimilates that are directly transferred to the grain (Monneveux et al. 2006). As a consequence, large changes in stem weight as well as stem WSC concentration and content occur between anthesis and maturity, particularly under water-limited conditions (Fig. 2). It has been estimated that the stem reserves contribute up to 74% and 57% of the kernel weight of barley and wheat, respectively, when the crops are under drought stress after anthesis (Gallagher et al. 1976). Under water-deficit conditions around anthesis, the stem WSC concentration and content has been shown to increase compared to well-watered plants (Ehdaie et  al. 2006; del Pozo et  al. 2016 for wheat; Méndez et  al. 2011; McIntyre et  al. 2012; del Pozo et al. 2012 for barley). A positive and significant relationship has been found between the stem WSCs remobilized from anthesis to maturity and the grain yield of 225 wheat genotypes, when plants were grown in rainfed (water stress) conditions, but not under well-watered conditions (Fig.  3). Other studies have reported positive correlations between WSCs and grain yield in both waterdeficit and well-­watered conditions (Foulkes et  al. 2007; McIntyre et  al. 2012). Also, the stem WSCs can contribute significantly to the final kernel weight (Schnyder 1993; Gebbing et al. 1999; del Pozo et al. 2016), and positive correlations between WSC content around anthesis and kernel weight at maturity in wheat genotypes support this view (Ruuska et al. 2006; Dreccer et al. 2009; Xue et al. 2008a; del Pozo et al. 2016). Therefore, high WSC concentrations are considered a potentially useful feature for grain weight improvement and productivity in environments where production may be limited by water availability (Blum 1998; Ruuska et al. 2006; Foulkes et al. 2007).

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5  G  enetic Viability and Expression of Genes Involved in Fructan Accumulation and Remobilisation in Cereals The variation in the stem WSCs is one of the phenotypic traits that subsequently affects kernel weight and grain yield under water-deficit conditions (Li et al. 2015). In wheat, there is wide genetic variability in the accumulation of WSCs in the stems, a characteristic that presents a high heritability (Ruuska et al. 2006; Ehdaie et al. 2006; del Pozo et al. 2016). The genotypic differences in the concentration of WSCs during flowering are attributed mainly to fructans (Ruuska et al. 2006; Xue et al. 2008b; Yue et  al. 2015). Differences in the composition of fructans according to their degree of polymerization could possibly depend on the availability of sucrose

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for the accumulation of reserves and the capacity of the particular fructans to be transformed (Ruuska et  al. 2006). It has been shown that the main difference between the types of fructans that accumulate in the short and long term is the abundance of branches between the fructosyl residues, with β-(2-1) linkages predominating initially and β-(2-6) linkages occurring secondarily (Carpita et al. 1991), and in the second case fructans with high degree of polarization also accumulate (Bancal et al. 1992). The stem WSCs are complex traits associated with multiple quantitative trait loci (QTLs), each one having a small individual effect (McIntyre et al. 2012), but with a relatively high heritability (Ruuska et al. 2006). The identification of QTLs related to stem WSCs in bread wheat has been carried out in biparental populations (e.g. recombinant inbred lines (RILs) or double haploid lines (DHLs)), as well as diverse panels of advanced lines and cultivars. For example, studies conducted in DHLs under two water regimes identified seven additive QTLs for the interaction between WSCs and the environment, in chromosomes 1A, 1D, 2D, 4A, 6B and 7B, with different additive effects and contribution percentages (Yang et  al. 2007). Another study in DHLs of wheat grown over different environments and seasons reported five QTLs for WSCs (Snape et al. 2007). In a RIL population, Salem et al. (2007) identified three QTLs associated with the mobilization of WSCs and the maintenance of grain size. In three mapping populations (Cranbrook/Halberd, Sunco/ Tasman and CD87/Katepwa), Rebetzke et al. (2008) identified four to eight significant QTLs in each population. Further, in a population derived from the cross between durum (Triticum turgidum ssp. durum) and emmer wheat (Triticum turgidum ssp. dicoccoides), 15 and 22 QTLs were reported under well-watered and water-deficit conditions, respectively (Peleg et al. 2009). Finally, in a collection of 166 wheat cultivars planted in four environments, and using 18,207 SNP markers, 52 significant marker-trait associations were identified on all wheat chromosomes except for 2A, 2D, 4D, 5B, 6A and 6D (Dong et al. 2016). The fructosyltransferase enzymes necessary for the biosynthesis of fructans in higher plants are 1-SST, 1-FFT and 6-SFT (Xue et al. 2008a; Xiang et al. 2010; Hou et al. 2018). Three of these genes (1-SST, 1-FFT and 6-SFT) have been cloned and characterized in wheat (Kawakami et al. 2005; Xue et al. 2008a). The synthesis of fructans occurs in the vacuole of cell stems from sucrose synthesized in the cytosol and moved into vacuole through sucrose transporters. The process involves the synthesis of β-(2-6)-linked fructans by the consecutive action of the enzymes 1-SST and 6-SFT, and β-(2-1)-linked fructans by the activity of the enzymes 1-SST and 1-FFT (Xue et al. 2008a). The predominance of β-(2-6) linkage in the fructose polymers of the stem seems to be related to degradation of the branches by the simultaneous action of fructan exohydrolases during the biosynthesis of fructans (Van den Ende et al. 2003). An analysis of transcript levels of carbohydrate metabolic enzymes of 16 recombinant inbred lines of wheat, differing in stem WSCs, which were grown under water deficit in field conditions, revealed that the expression of the 1-SST and 6-SFT genes was positively correlated with stem WSC and fructan concentrations and negatively correlated with the expression of sucrose synthase and soluble acid invertase genes (Xue et  al. 2008a). Under terminal drought stress, the transcript

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levels of 1-SST and 6-SFT in stems and roots of wheat increased significantly at 7, 14 and 21 days after anthesis, but this was not the case for the 1-FFT gene (Bagherikia et al. 2019). The strong positive correlation between the expression of the 1-SST and 6-SFT genes and stem WSC concentrations suggests that both enzymes are related to genotypic variation in fructan accumulation (Xue et al. 2008a; Khoshro et al. 2014) and explained the predominance of fructans with β-(2-6) linkages in the stems (Carpita et al. 1991; Bancal et al. 1992; Bancal and Triboï 1993). Comparisons between drought tolerant and susceptible genotypes of wheat have revealed differences in the expression of genes involved in fructan metabolism in response to water deficit. For instance, the drought-tolerant genotype had higher up-regulation of the fructan 1-fructosyltransferase B (1-FFTB) and fructan 1-­exohydrolase w2 (1-FEHw2) genes, whereas the susceptible genotype presented an up-regulation of the 6-SFT and fructan 1-exohydrolase w3 (1-FEHw3) genes (Yañez et al. 2017). In another study conducted in two wheat varieties, the drought-­ tolerant genotype presented higher expression levels of the genes involved in fructan synthesis (1-SST-A1, 1-SST-A2, 1-SST-D, 6-SFT, FFT-A and FFT-B) at 10–20 days after anthesis, whereas the expression levels of the 1-FEH-W3, 6-FEH and INV3 genes were higher at 30 days after anthesis (Hou et al. 2018). The mobilization of fructans from the stem to the grain was accompanied by an increase in the activity of 1-FEH (Van den Ende et al. 2003; Kawakami et al. 2005; Khoshro et al. 2014) and the transcripts levels of the enzyme (Zhang et al. 2009). Therefore, the 1-FEH-w3 and 6-FEH genes seem to play an important role in the metabolism of fructans and could be used to improve the mobilization of fructans and increase kernel weight. Abscisic acid is also important for the induction of FEH in wheat (Ruuska et al. 2008). In addition, other genes have been reported to be involved in the metabolism of fructans: the SPS gene (encoding the enzyme that transforms sucrose 6-P into D-fructose 6-P and vice versa), SPP (encoding the enzyme that transforms sucrose 6-P into sucrose), INV (which transforms sucrose into fructose), SUT1 and SUT2 (transporter genes that move sucrose from the cytosol to the apoplast and vice versa), all of which are related to a high fructan content and remobilization under terminal drought stress (Bagherikia et al. 2019).

6  Conclusion and Future Perspectives Fructans are an important carbohydrate reserves in plants, particularly in temperate cereals. Remobilization of fructans from the stem to the grain during grain filling contributes to the kernel weight and grain yield of barley and wheat. Under terminal drought conditions, the contribution of stem reserves to grain growth is of paramount importance. A large genetic variability in traits related to stem reserves has been reported in wheat, and QTLs have been associated with these traits. Genotypes with greater remobilization of fructans under drought stress are more productive. Genes related to fructan metabolism and other candidate genes could be used in breeding programmes for selecting drought stress-tolerant cultivars.

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Index

A ABA responding element (ABRE), 292, 300 Abiotic stresses, 1 and biotic, 288, 291, 308 degradation genes, 235 drought stress, 106 exogenous GB-mediated modulation, 148 photosynthetic machinery, 145–146 plant hormones, 146–147 types, 141, 142 exogenous Put application, 293 extreme temperatures, 111 metabolic changes, 233–235 on nutrient status, 112, 113 osmolyte priming, 261, 262 PAs, ROS and NRS, 292 without phenotypic alterations, 227, 229, 236 with phenotypic alterations, 225–227 physiological and biochemical processes, 106–109 in plants, 258 plant-water relations, 111 polyamines, 293 priming compounds, 259–261 salinity stress, 111 seed priming, 263 transcriptional changes, 232, 233 trehalose biosynthesis, 235 Abiotic stress-tolerant traits, 206 Abscisic acid (ABA), 51, 146, 158, 193, 232, 261, 273, 276, 303, 322 AdoHcy hydrolase, 210 AdoMet synthetase, 210 Agmatine iminohydrolase (AIH), 274 AGP-glucose pyrophosphorylase (AGPase), 183

Agrobacterium rhizogenes, 57 ALDH10 isoenzymes, 127 Amino acid/auxin permease (AAAP), 48 Amino acid permease (AAP), 48, 202 Amino acid–polyamine–choline (APC) family, 202 Amino acid proline, 42 4-Aminobutanal (ABAL), 291 1-Aminocylopropane-1-carboxylic acid (ACC), 293 Anhydrobiotic organisms, 181 Anthocyanin, 259 Antioxidant defense system, 114, 115 Antioxidant enzymes, 88, 89 Antioxidants activity, 258, 259 overproduction, 263 and ROS scavenging, 257 stress imprint, 262 Antioxidative defence, 143, 144 Arginine decarboxylase (ADC), 289, 290, 292, 293, 295, 303 B Bacterial P2CRs, 43 Bacteroides, 271, 272, 277, 280 Betaine aldehyde dehydrogenase (BADH), 14, 20, 23, 24, 157, 201, 209, 210, 246–248 GB synthesis, 124, 127 Betaine homocysteine methyl transferase (BHMT), 128 Betaines, 3 Bioinformatic, 5, 30

© Springer Nature Switzerland AG 2019 M. A. Hossain et al. (eds.), Osmoprotectant-Mediated Abiotic Stress Tolerance in Plants, https://doi.org/10.1007/978-3-030-27423-8

335

336 Biomass accumulation, 319 Biotic stresses, 1 Brassinosteroids (BRs), 276, 277 C Cadaverine (Cad), 273, 274, 277–279 Cadmium (Cd), 144 Calvin cycle, 270 Carbamoil putrescine aminohydrolase (CPA), 274 Carbohydrates, 15–16 Carbonyl cyanide m-chlorophenylhydrazone (CCCP), 211 Carotenoid, 259 Cereals, 319 fructans, role, 320, 322 grain filling, 324 Chaperones, 2, 4 Choline monooxygenase (CMO), 157, 209, 210, 246–248 cis-acting regulatory elements, 128 CMO1 and CMO2, 127 Cys-181, 126 GB synthesis, 124 gene sequences, 126 SAMS transcript, 130 Chromatin remodelers, 2 Compatible solutes, 141, 246, 248, 249, 252 CPA (N-carbamoylputrescine amidohydrolase) gene, 15 Cyanobacteria, 41 D Decarboxylated S-adenosylmethionine (dcSAM), 290, 301 DEOP database, 2 DHS (deoxyhypusine synthase) gene, 15 Diamine oxidase (DAO), 273, 274, 276, 277, 291 1,4-Diaminobutane, 287 1,5-Diaminopentane, 287 1-3 Diaminopropane (DAP), 273 DNA Data Bank of Japan (DDBJ), 5 Drought, 1, 295, 298, 305, 309 stress, 106, 292–294, 299–302 tolerance, 231, 232, 294, 298 Drought-responsive element (DRE), 292, 300 Durum wheat, 159 E Ectoine, 3 Embryogenesis, 61, 62

Index Endogenous GB abiotic stresses, 153 ambient temperature, 153 cellular adaptive responses, 154 chloroplast targeted transgenesis, 165, 168 CMO and BADH isoenzymes, 165 crop yield, 154 global agricultural land area, 153 heat and cold stress, 153 metabolic routes, 168 osmoprotectants (see Osmoprotectants) osmotic stress, 159, 160 plant abiotic stress responses, 157, 158 primary stress phase, 154 ROS and plant hormones, 154 saline soils, 154 and temperature stress, 160–162 transgene approaches, 162 transgenesis, 162–165 transgenic plants, 168 Energy, 41 Environmental stresses, 1, 2 Enzymes FEH, 321, 322, 328 glycoside hydrolase, 321 Ethylene biosynthesis, 290 European Nucleotide Archive (ENA), 5 Exogenous application, GB abiotic oxidative stress tolerance, 142 in cultured tobacco, 143 in fine rice, 142 lentil seedlings, 143 maize, 142 in marigold, 143 in mung bean seedlings, 143 nitrogen deficiency and Cd stress, 144 in perennial ryegrass, 143 on rice seedlings, 144 on tea buds, 143 on tomato, 143 water-stressed wheat seedlings, 142 gene expression, 147–148 photosynthetic machinery, 145–146 plant hormones and metabolites, 146–147 plant resistance, 148 ROS scavenging and detoxification, 148 Exogenous proline under abiotic stress (see Abiotic stress) antioxidant defense system, 114, 115 concentrations, 116 growth and yield quantity and quality, 115 photosynthetic performance, 113, 114 Extracellular trehalose, 190 Extreme temperatures, 305–307, 309

Index F FAD-dependent AOs (FAD-AOs), 291 Fructan exohydrolase (FEH), 321 Fructans, 3, 27 ABA, 322 accumulation, 319, 322 biosynthesis, 321 degradation, 321 expression of genes, 327, 328 and FEH enzymes, 322 genetic variability, 326 inulins, 321 levan-type, 321 metabolism, 328 mobilization, 328 principal function, 322 remobilization, 322 in stress tolerance, 323–326 structural classes, 321 in temperate cereals, 323 vegetative tissues, 322 WSCs, 320 Fructosyltransferase (FT), 321, 327 G Gamma aminobutyric acid (GABA), 48, 202, 259, 274, 276, 291, 309 GenBank, 5 Gene expression fructans, roles, 324, 327, 328 GB, exogenous application, 147–148 proline metabolism, 49, 50, 53, 54 Genetically modified (GM) plants, 22–23, 25–28 Genomic-scale approach, 5 Gibberellic acid (GA), 261 Glucose-6-phosphate (G6P), 225, 249 Glutamate (Glu), 274 Glutamate-semialdehyde (GSA), 11, 44, 46, 201 Glutathione reductase (GR), 304 Glycine betaine (GB), 6, 9, 14, 20, 22–24, 154, 156–168, 201–204 abiotic and biotic stresses, 248 accumulation in plants, 208, 209 bacteria-specific glycine methylation pathway, 246 beneficial effects, 142 biosynthesis, 209, 210 codA-and BADH-transgenic tomato, 248 as compatible solutes, 141 crop plants, 247 description, 141 exogenous application, 141, 212, 214, 215 (see also Exogenous application, GB)

337 exogenous treatment, 247 gene expression, 210 genetic engineering, 215, 216 inter-organ transport, 216 metabolism BHMT, 128 cellular components, 128 cellular volume maintenance, 123 CMO and BADH, 124 direct and/or indirect participation, 124 Hcy/Met cycle, 123 plant growth and development, 123, 129, 130, 132, 133 plant tissue, 129 quaternary amine, 123 synthesis pathway, 124, 126–128 stress-related metabolites, 248 stress-responsive genes, 249 translocation, 210, 212 Grain filling, 324 Graminan-type fructans, 321 H Heat shock proteins, 2, 27 Hexamines, 273 High temperatures, 1 Histone modifiers, 2 Homospermidine (HomSpd), 273 Homospermidine synthase (HSS), 278 Homospermine (HomSpm), 273 Hydrogen peroxide (H2O2), 278 Hydropriming, 257, 262 Hypoxia, 188 I Indole acetic acid (IAA), 147 In silico genome mapping, 19 analysis, 17 cysteine, 17 GB, 17 grasses, 18 legumes and soybean, 17, 18 myo-inositol, 17 plant species, mapping, 17 proline, 17 as transgenes amino acid proline, 20 carbohydrates, 26, 27 GB, 20–24 sugar alcohols, 28 trehalose, 17

338 Inulins, 321 Ionic stress, 270 K KEGG (Kyoto Encyclopedia of Genes and Genomes), 6 L Late embryogenesis abundant (LEA), 262 Legume-rhizobia symbiosis agrarian, 271 PAs, 277–279 salinity, 272, 273 salt stress, 272, 279 Lipoxygenase (LOX), 249 Low temperature-responsive (LTR), 292 Lysine decarboxylase (LDC), 274, 278 Lysine histidine transporter (LHT), 48, 202 M Malondialdehyde (MDA), 294 Maltooligosyl trehalose synthase (MTS), 229–231 Maltooligosyl trehalose trehalohydrolase (MTH), 229, 230 Marker-assisted selection (MAS), 30 MEDLINE, 20 Metabolism, 247–249 Metabolites, 235, 236 MetaCyc databases (Metabolic Pathway Database), 6 Methionine synthase, 210 Methylglyoxal (MG) detoxification system, 143 Mitogen-activated protein kinase (MAPK), 158 Mung bean, 160 Myo-inositol, 16, 17, 29 N Nitrogen fixation, 271, 272, 279 Non-photosynthetic organisms, 41 Norspermidine (NorSpd), 273 O Oligosaccharides, 3 Ornithine aminotransferase (OAT), 274 Ornithine cyclodeaminase (OCD), 42, 43, 57, 58 Ornithine decarboxylase (ODC), 278, 289, 290, 295, 299

Index Ornithine-delta-aminotransferase (OAT), 43, 44, 46, 48, 53, 57, 201 Osmolytes chemical/molecular chaperones, 4 potential pathways, 6 protective, 3 Osmoprotectant degradation, 7 Osmoprotectant-related genes analysis, in plant species, 8 and associated pathways, 6–8 drought stress, 11 experimental assays, 8 expression amino acid proline, 11–14 carbohydrates, 15, 16 GB, 14 polyamines (PA), 14–15 sugar alcohols, 16–17 salinity stress, 8 in silico genome mapping, 17–20 stress application methods, 11 Osmoprotectants, 201, 202, 272, 275, 280 abiotic stresses, 155 antioxidant defense system, 156 classes, 2 as compatible solutes, 2 definition, 2 external stress, 155 groups, 156 metalloenzyme superoxide dismutase, 156 optimal K+/Na+ ratio, 157 organic solutes, 156 osmotic stress, 4 plant abiotic stress tolerance, 155 plant hormones, 155 plant stress tolerance, 155 plants under abiotic stress, 3, 4 salinity and water deficit, 157 transcriptomic study, 4–6 water efflux, 155 Osmoprotection cellular water potential (ΨW), 77 compatible osmolyte, 77, 79 enzymes and membranes, 79, 81 free proline concentration, 77 (see also ROS scavenging) Osmoprotective osmolytes, 2, 5 Osmotic adjustment, 242, 244, 252 Osmotic stress, 3, 4, 270 Oxidative pentose phosphate pathway (OPPP), 57, 58 Oxidative stress, 308

Index P P5C dehydrogenase (P5CDH), 43, 46, 48, 50, 53, 55 P5C-proline cycle, 86, 87 P5C reductase (P5CR), 244 P5C synthetase (P5CS), 44, 45, 47, 49, 52, 244 Pentamines, 273 Phosphoenolpyruvate carboxylase (PEPC), 249–251 Phosphoethanolamine N-methyltransferase (PEAMT), 215 3-Phosphoglycerate dehydrogenase (PGDH), 215 Phospholipase-D (PLD), 249 Photosynthesis, 41, 145 Photosynthetic machinery under abiotic stress, exogenous GB, 145–146 Photosystem II (PSII), 145, 146 Plant growth and development, T6P/trehalose, 181–185 Plant growth regulators (PGRs), 259 Plant hormones exogenous GB, 146, 148 Plant osmoprotectants, 3 See also Osmoprotectants Plants under abiotic stress, 3 under stress, 1 Plasmodesmata, 49 Polyamine oxidase (PAO), 274, 275 Polyamines (PAs), 3, 7, 287–295, 298–309 abiotic stress regulators, 275–277 ADC, ODC and OAT genes, 14 biosynthesis, 278 CPA and DHS genes, 15 functions, 14 legume-rhizobia symbiosis, 277–279 metabolism, 273, 274 in nature, 273 in plants, 273 putrescine (Put), 14 and salinity stress, 302–305 salt stress, 274, 275 spermidine (Spd), 14 and transgenic plants, 24, 26 Polyamines metabolism, 290 Polyols, 16 Proline, 3, 22 abiotic stress responses, 246 abscisic acid, 102 accumulation, 244 biosynthesis, 100, 102 biosynthesis enzymes, 49–53

339 Ca+2-dependent calmodulin, 244 catabolic enzymes, 53–55 catabolism, 100 Cu/ZnSOD and MnSOD encoding genes, 246 to drought stress, 11 endogenous, 206 exogenous treatment, 246 GABA synthesis, 101 glutamate pathway, 244 inter-organ transport, 204, 216 intracellular transport, 205 metabolism and regulation, 104, 106 nonessential proteinogenic amino acid, 100 osmotic adjustment, 244 OsP5CS2 and OsP5CR promoters, 246 P5CS and P5CR genes, 244, 245 pathways, 100 PDH expression, 11 physiological role, 205 in plant at organization levels, 103, 104 ROS signaling pathways, 103 to salt stress, 11 stress marker genes, 245 stress response and tolerance processes, 244 stress-responsive transcription factors, 244 uptake in plants, 202, 204 Proline accumulation ABA production, 76 ABRE, 77 antioxidant enzymes, 88, 89 C and N source, 89, 90 CRE, 77 de novo biosynthesis, 74 HY5, 76 hydrogen peroxide (H2O2), 76 mitochondrial oxidation, 74 osmoprotection (see Osmoprotection) P5CR, 75 photosynthetic tissues, 74 plant response, abiotic stresses, 74 preliminary evidence, 73 ProDH, 76 putative cis-regulatory elements, 74 (see also Redox state) role, 73 ROS, 76 stress-induced, plant species, 74, 75 Proline biosynthetic enzymes OCD, 42, 43 P2CRs, 43 P5C, 44

340 Proline degradation enzymes P5C, 46 ProDH, 45 Proline dehydrogenase (ProDH), 44–46, 53 Proline metabolism, 47–49 duplications and functional diversification, 49 embryo development, 61–62 flowering, 58–60 plant development, 56–57 pollen fertility, 60–61 regulatory mechanisms, 49 root growth, 58 seed germination, 57–58 transcription factors (TFs), 49 under stress, 62–63 Proline oxidase, 45 Proline-responsive element (PRE), 245 Proline transport intercellular transport, 48–49 intracellular transport, 47 and metabolism, 47–48 Proline transporter (ProT), 48, 61, 211 Putrescine (Put), 14, 25–26 Pyrroline-2-carboxylate reductase (P2CR), 42, 43 Pyrroline-5-carboxylate (P5C), 43–46, 48, 49, 55, 201 Pyrroline-5-carboxylate dehydrogenase (P5CDH), 245 Pyrroline-5-carboxylate reductase (P5CR), 44–47, 52, 61, 62 Pyrroline-5-carboxylate synthetase (P5CS), 201 Q Quantitative trait loci (QTLs), 327 Quaternary ammonium compounds (QACs), 2 R Reactive oxygen species (ROS), 4, 154, 257, 258, 262, 270, 272, 275 under cold stress, 24 regulation, 4 T6P/trehalose role, 188 Red beet, 159 Redox state catabolism and generation under stress, 87, 88 P5C-proline cycle, 86, 87 proline-glutamate interconversions, 85, 86 Regulatory proteins, 2 Relative water content (RWC), 294, 299, 300 Reserve carbohydrates, 319 RNA interference (RNAi), 2, 295

Index ROS scavenging abiotic and biotic stresses, 81 cultured tobacco BY2 cells, 82 GABA precursor, 83 hydroxyproline, 83 MDA formation, 81 mitochondrial electron leakage, 82 non-enzymatic antioxidants, 82 physiological conditions, 83 putative antioxidant properties, 82 TEMP oxidation, 82 RT-qPCR (real-time reverse transcription-­ polymerase chain reaction) technique, 7 S S-adenosylmethionine decarboxylase (SAMDC), 278, 290, 292, 301, 306, 307 S-adenosylmethionine (SAM), 276, 278, 309 Salicylic acid (SA), 146, 147, 276 Salinity, 258, 259, 262, 272, 273, 293, 295, 298, 302–304, 309 Salinity stress, 111 Salt stress, 3, 9–10, 14–16 brassinosteroids, 277 GABA, 280 harmful effect, 270 legume-rhizobia symbiosis, 272, 279 in legumes, 276 polyamines, 274, 275 root nodules of Medicago truncatula, 272 Salt-stressed bread wheat, 159 Serine hydroxymethyltransferase (SHMT), 215 Signaling process, 1 Soil water content (SWC), 300 Spd by spermidine synthase (SPDS), 273, 290, 292, 295, 301, 304 Spermidine (Spd), 14, 24–26 Spermidine/spermine N-1 acetyl transferase (SSATs), 291 Spermidine synthase (SPDS), 278 Spermine (Spm), 24–26 Spinacia BADH protein, 167 Spm synthase (SPMS), 273, 274, 278, 290, 307 Stress stimulus, 1 Stress-tolerant transgenic plants, 228, 230–231 Sucrose non-fermenting-related kinase-1 (SnRK1), 184 Sugar alcohols, 3, 16–17 Sugar beet, 210, 212 Sugars, 11 Superoxide dismutase (SOD), 259, 304

Index T Temperature stress, 258 Terminal drought stress, 324, 327, 328 Thiobarbituric acid reactive substances (TBARS), 304 Tolerance Al stress, 304 biotic and abiotic stress, 291 dehydration, 302 drought, 301, 304 osmotic and ionic, 302 and polyamines, 305–307 Put and Spd levels, 295 salinity, 303 salt and cadmium stress, 305 Spd-induced NO, 292 Spm and Spd levels, 300 stress, 293, 309 thermotolerance, 294 WT and GhPAO3 transgenic line, 305 TPS-TPP pathway, 176 Transcription, 232, 233 ABA-regulated stress-related genes, 235 Transcription factors (TFs), 2 bZIP, 245, 250 CREs site, 244 MYB102 and WRKY, 251 MYB2, 244 proline-, glycinebetaine- and trehalose-­ induced, 243 and stress-responsive genes, 242 Transcriptomic study description, 4 genomic-scale approach, 5 global expression of genes, 5 molecular targets, 5 osmoprotectants and potential related pathways, 5, 6 statistical analysis (p-values), 5 transcriptomic libraries, 4 Transfer DNA (T-DNA), 57 Transgenic plants, 20, 24, 26, 27, 293, 295, 300–304, 306–308 abiotic stress-tolerant, 234 drought-tolerant, 227 Gaff’s hypothesis, 231 leaves of, 225 and non-transgenic plants, 231 Trehalase (TRE), 3, 15, 16, 27, 180–181 abiotic stress (see Abiotic stress) anomers, 175 applications, 191–193 Attre1-1 and Attre1-2 mutants, 251 biosynthesis, 201, 234, 235, 252 biosynthesis pathways, 176

341 compatible solutes, 249 description, 175 as elicitor of plant defense responses, 189–190 endogenous, 208 environmental stresses, 249 exogenous treatments, 252 genome sequencing, 177 glucose monomers, 250 inter-organ transport, 216 metabolism, 249 in mycorrhizal fungi-plant relationships, 189 origin, 175 osmoprotectants, 225 pathway, 225 in plant growth and development, 181–185 R2R3-type MYB family, 251 SnRK1-upregulated genes, 250 stomata, 231, 232 as stress protectant, 181 stress-related genes, 249 TPP proteins, 180 TPS proteins, 177–180 TRE proteins, 180–181 trehalase (TRE), 177 trehalose-6-phosphate phosphatase, 249 uptake in plants, 206, 208 Trehalose synthase (TreS), 176, 227, 228, 234 pathway, 176 Trehalose-6-phosphate (T6P) in abiotic stress tolerance drought stress, 186–187 hypoxia, 188 oxidative stress, 188 salt stress, 185–186 temperature stress, 187–188 applications, 191–193 in biotic stress tolerance as elicitor of plant defense response, 189–190 pathogen attack, 190–191 and plant-microorganism symbiosis, 189 description, 182 in embryo development, 182 flowering time and inflorescence architecture, 184 on glucose, 178 on meristem development, 184 normal growth, 182 on plant development, 182 plant growth, 183 SnRK1 by T6P, 184 sucrose level, 183 sugar levels, 184 Trehalose-6-phosphate phosphatase (TPP), 176, 180, 202, 225–227, 230, 231, 234

342 Trehalose-6-phosphate synthase (TPS), 176–180, 201, 225, 226, 229, 230, 233, 249–251 TPS-TPP pathway, 176 TreZ-TreY pathway, 176 U UDP-glucose (UDPG), 225 Uridine diphosphate glucose (UDP-Glc), 249 Usually multiple amino acids move in and out transporters (UMAMITs), 49

Index W Water-deficit stress, 12–13 Water-soluble carbohydrates (WSCs), 325, 326 grain growth and filling, 319 identification, QTLs, 327 role, plants metabolism, 320 sucrose and starch, 320 variation, 326 in vegetative stems, 322 water-deficit conditions, 324 wheat and barley, 319