Plants in Extreme Environments

Provided for non-commercial research and educational use only. Not for reproduction, distribution or commercial use. This chapter was originally published in the book Advances in Botanical Research, Vol. 57, published by Elsevier, and the attached copy is provided by Elsevier for the author's benefit and for the benefit of the author's institution, for non-commercial research and educational use including without limitation use in instruction at your institution, sending it to specific colleagues who know you, and providing a copy to your institution’s administrator.

All other uses, reproduction and distribution, including without limitation commercial reprints, selling or licensing copies or access, or posting on open internet sites, your personal or institution’s website or repository, are prohibited. For exceptions, permission may be sought for such use through Elsevier's permissions site at: http://www.elsevier.com/locate/permissionusematerial From: László Szabados, Hajnalka Kovács, Aviah Zilberstein and Alain Bouchereau, Plants in Extreme Environments: Importance of Protective Compounds in Stress Tolerance. In Jean-Claude Kader and Michel Delseny, editors: Advances in Botanical Research, Vol. 57, Burlington: Academic Press, 2011, pp. 105-150. ISBN: 978-0-12-387692-8 © Copyright 2011 Elsevier Ltd. Academic Press.

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Plants in Extreme Environments: Importance of Protective Compounds in Stress Tolerance

´ SZLO ´ CS,* ´ SZABADOS,*,1 HAJNALKA KOVA LA { AVIAH ZILBERSTEIN AND ALAIN BOUCHEREAU{

*Institute of Plant Biology, Biological Research Center, Szeged, Hungary { Faculty of Life Sciences, Tel Aviv University, Ramat Aviv, Tel Aviv, Israel { Universite´ de Rennes 1, Campus de Beaulieu, Baˆtiment 14A, Rennes Cedex, France

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Global Metabolic Consequences of Osmotic Stress . . . . . . . . . . . . . . . . . . . . . . . . III. Osmoprotective Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Quaternary Amines: Glycine Betaine........................................ B. Sugars: Trehalose ............................................................... C. Polyalcohols: Mannitol, Pinitol, Inositol ................................... D. Amino Acids: Proline .......................................................... IV. Osmoprotective Compounds and Adaptation to Extreme Environments . . V. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Corresponding author: E-mail: [email protected]

Advances in Botanical Research, Vol. 57 Copyright 2011, Elsevier Ltd. All rights reserved.

0065-2296/11 $35.00 DOI: 10.1016/B978-0-12-387692-8.00004-7

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ABSTRACT Extreme environmental conditions such as drought, cold or high soil salinity impede plant growth and require specific adaptation capacity. In response to environmental stresses, a number of low-molecular-weight compounds can accumulate in plants: protective amino acids, sugar alcohols, sugars and betaine-type quaternary amines. The function of these compounds includes the stabilisation of cellular structures, photosynthetic complexes, specific enzymes and other macromolecules, the scavenging of reactive oxygen species or acting as metabolic signals in stress conditions. Although a correlation between the accumulation of certain osmoprotective compounds and stress tolerance certainly exists, a causal relationship between osmolyte accumulation and enhanced tolerance could not always be confirmed. Nevertheless, the importance of osmoprotective compounds for the adaptation to extreme environmental conditions is supported by numerous studies obtained with natural variants, mutants or transgenic plants with different capabilities to accumulate these metabolites. Combining genetic analysis with metabolic profiling approaches could considerably increase our understanding of plant stress responses and the importance of the protective metabolites in the adaptation to stress conditions.

I. INTRODUCTION Environmental conditions determine plant growth and development. Abiotic stresses such as drought, heat, cold and soil salinisation restrain the optimal growth of plants and require certain degree of adaptation to such extreme environments. Climate change needs more intense research on reprogramming of physiological, metabolic events during stress, regulation of growth and development under suboptimal conditions and adaptation of plants to extreme environments. Approximately 40% of the arable earth is arid, semi arid or affected by soil salinity, which reduces crop yield leading to increasing ecological, agronomical and economical impact. A correlation between increased frequency of extreme environmental events and global warming requires more efficient, environmentally compatible agricultural practices including the implementation of new crop cultivars with enhanced tolerance to environmental stresses (Ahuja et al., 2010; Boyer, 1982; Etterson and Shaw, 2001; Gregory et al., 2005; Kintisch, 2009). Water shortage, extreme temperatures or high salinity lead to a depletion of cellular water content, enhance the cellular osmotic potential generating osmotic stress in plants. Physiological responses to drought, cold and salt stress are similar and include reduced shoot growth and photosynthetic activity, accumulation of reactive oxygen species (ROS), alterations in ion transport and compartmentalisation, and changes in metabolite profiles (Lugan et al., 2009, 2010; Munns, 2002; Shulaev et al., 2008). One of the metabolic consequences of osmotic stress is the accumulation of low-molecular-weight organic compounds that do not interfere with normal metabolic reactions and

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are considered to have protective functions (Ashraf and Harris, 2004; Bartels and Sunkar, 2005; Bohnert et al., 1995; Hare et al., 1998; Hasegawa et al., 2000; Yancey et al., 1982). This chapter is intended to give an overview of osmoprotective compounds with an emphasis on recent advances describing their function and significance to adaptation in improving stress tolerance.

II. GLOBAL METABOLIC CONSEQUENCES OF OSMOTIC STRESS Metabolism is of essential functional importance to support growth and development by providing building blocks and energy for plant structures and reserves, to generate signalling compounds for coordination and defence or to produce protective substances to cope with adverse environmental conditions. At a systemic level, metabolism can be viewed as the most integrated (and possibly informative) action reflecting both genotypic and environment-dependent regulations and resulting in the phenotype. Control of energy inputs and outputs is mainly driven through metabolic processes to support optimal growth and reproduction, which is a major goal for all organisms (Baena-Gonzalez and Sheen, 2008; Stitt et al., 2010). Metabolic adjustment is a challenging task for plants, which have to resist fluctuating environmental conditions frequently perceived as stress factors. As a matter of fact, plant metabolic networks are under constraint and metabolic reconfigurations are observed under abiotic stress exposition (Bohnert and Sheveleva, 1998; Guy, 1990) Major trends frequently observed in many plant species exposed to high salt, low water availability, and high or low temperatures include the accumulation of primary metabolites like amino acids, non-structural sugars and organic acids (Ahuja et al., 2010; Sanchez et al., 2008). Multiple functions have been proposed for these stress-induced metabolic adjustments like osmotic potential regulation, thermo- and osmoprotection, chaperon-like roles of macromolecules, adjustments in the carbon/nitrogen balance, scavenging of ROS or reactive nitrogen species, redox buffering, pH adjustment, carbon and nitrogen reserves deposition and signal transduction (Bartels and Sunkar, 2005; Noctor, 2006). The impact of stress on metabolic network is often associated with concurrent depression of growth and development depending on the amplitude and the duration of the physico-chemical pressure (Chaves et al., 2003). Exploring correlations between biomass production and metabolite profiles in recombinant inbred line (RIL) populations of Arabidopsis, Meyer and collaborators proposed that growth may drive metabolism and not the opposite and biomass can be described as a function of metabolic composition. Significant correlation


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could be observed between biomass production and specific combination of metabolites (Meyer et al., 2007). Through the analysis of quantitative trait loci (QTL) with RIL and introgression line (IL) populations of Arabidopsis thaliana, it was demonstrated that there was a substantial and significant overlap of at least a subset of the biomass QTL, with metabolic QTL, suggesting a strong link between biomass and primary metabolism (Lisec et al., 2008). In contrast to secondary metabolism, it is proposed that perturbation of the primary metabolic network should have strong detrimental effects on plant performance. Metabolic adjustments in plants under osmotic stress are often observed through accumulation of specific metabolites. Such changes can be interpreted either as regulated adaptive or acclimation events or as biochemical consequences of growth impairment and trophic disruption. Plants that are tolerant to harsh environments (high salt, low water, high temperature) generally display low relative growth rate and often possess high solute concentrations (Alpert, 2006; Lugan et al., 2010). Although much evidence is now available about the adaptive or protective role of specific accumulation of compatible osmoprotectants, antioxidant and ROS scavenging compounds, a clear explanation of functional consequences of symptomatic global metabolic changes is still missing. Studies of metabolic adjustments in plants under stressful conditions have been encouraged in the past years, as technologies available for metabolomic approaches have been significantly improved (Fiehn et al., 2000; Ratcliffe and Shachar-Hill, 2005; Sumner et al., 2003; Weckwerth, 2003). Together with transcriptomic and proteomic approaches, metabolomic analysis gives a more comprehensive view of systems biology studies to investigate biological networks in order to understand plant responses to environmental cues and to develop tolerance improvement strategies. Metabolic profiling has been used recently for the description of plant adaptation to a wide range of biotic and abiotic stresses, creating the emerging field of environmental metabolomics (Bundy et al., 2009; Lugan et al., 2009; Schauer and Fernie, 2006; Shulaev et al., 2008). Complex changes in the metabolome during temperature stress have been characterised by studying the metabolic events associated with cold acclimation or acquired thermotolerance (Browse and Lange, 2004; Guy et al., 2008; Hannah et al., 2006; Kaplan et al., 2007; Korn et al., 2010; Shuman et al., 2006; Stitt and Hurry, 2002). Reconfiguration of central carbohydrate metabolism under stress appears to play a major role in plant response to temperature, where proposed triple function of sugars as nutrients, signalling compounds and putative ROS scavengers complicates the elucidation of the mechanisms involved (Guy et al., 2008; Rolland et al., 2006; Rosa et al., 2009; Stitt and Hurry, 2002). Moreover, the importance of DREB1/CBF


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transcription factors in the regulation of the low-temperature metabolome, associated with cold acclimation has been demonstrated in Arabidopsis (Cook et al., 2004; Maruyama et al., 2009). Changes in lipid molecular profiles during cold acclimation and freezing have also been described in Arabidopsis using ESI-MS/MS-based technologies (Wang et al., 2006; Welti et al., 2002). Few studies have used holistic metabolic fingerprinting or metabolic profiling in plants to highlight biomarkers and understand the molecular basis of tolerance to salt or drought stress (Sanchez et al., 2008). Valuable information has been obtained about global management of metabolic networks under osmotic stress (induced either by exposure to high salt concentrations or water deprivation) in crop species like grapevines (Cramer et al., 2007), tomato (Johnson et al., 2003; Semel et al., 2007), rice (Zuther et al., 2007), pear (Larher et al., 2009) or lupin (Pinheiro et al., 2004). Halophytic extremophiles and desiccation-tolerant species have also been the subject of metabolic profiling, searching for biochemical attributes of salt or dehydration tolerance (Gagneul et al., 2007; Ksouri et al., 2010; Lugan et al., 2010; Moore et al., 2009; Weber et al., 2007). Halophytes belonging to different species collected in the same inland salt marsh habitat have been compared and characterised in terms of nitrogenous compound production, thus highlighting some competitive regulations between betaines and proline accumulation (Tipirdamaz et al., 2006). The importance of antioxidant systems in desiccation tolerance has been supported by a study on Myrothamnus flabellifolia (Kranner et al., 2002). In addition to antioxidant metabolism, carbohydrate metabolism was shown to be reconfigured in resurrection plants undergoing dehydration (Moore et al., 2007; Whittaker et al., 2001). Due to the available genetic and genomic resources most of the recent molecular and integrated studies on metabolic adjustment under salt or drought stress have used Arabidopsis, a typical glycophyte species (Lugan et al., 2009; Tester and Bacic, 2005). Such analysis can combine transcriptomic, proteomic and metabolic profiling approaches, and could markedly increase our understanding of global plant stress response and adaptation to stress conditions such as drought (Mittler and Blumwald, 2010; Seki et al., 2007; Shulaev et al., 2008; Urano et al., 2010). Through multiparallel and iterative combinatorial experiments, the central role of abscisic acid in stressregulated metabolic pathways and redox control has been illustrated (Ghassemian et al., 2008; Kempa et al., 2008; Urano et al., 2010). Genetic variation between naturally occurring populations of Arabidopsis has also provided a valuable source of information to unravel metabolic traits and genetic architecture associated with the complex mechanisms of abiotic stress tolerance (Bouchabke et al., 2008; Brosche et al., 2010; Cross et al., 2006; Hannah et al., 2006; Katori et al., 2010; Wingler and


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Roitsch, 2008). To decipher the metabolic attributes and the genetic bases of extreme stress tolerance, Arabidopsis-relative model systems (ARMS) have recently been developed (Amtmann, 2009; Amtmann et al., 2005; Orsini et al., 2010). Recent comparative metabolic profiling experiences between Arabidopsis and Thellungiella salsuginea (halophila), a relative halophyte, contributed to the detection of relevant variation in metabolic composition and function in response to salt or drought treatments (Gong et al., 2005; Lugan et al., 2010; Pedras and Zheng, 2010). The metabolic composition of the salt tolerant T. salsuginea seems to be more compatible with dehydration, suggesting that this halophyte has more efficient osmoprotection than Arabidopsis (Lugan et al., 2010). It can be predicted that more and more studies will include metabolomics as a comprehensive addition to the systems biology approach to decipher plant stress response (Saito and Matsuda, 2010). The predicted changes in climatic conditions will likely create combinations of various abiotic stresses and make it necessary to adapt novel breeding or genetic engineering approaches to provide new crop varieties for modern agriculture (Mittler and Blumwald, 2010; Shulaev et al., 2008). Such practices will certainly encourage the development of metabolic phenotyping procedures and their adaptation in knowledge-based breeding programmes.

III. OSMOPROTECTIVE COMPOUNDS The most common osmoprotective compounds are amino acids, sugar alcohols, sugars and betaine-type quaternary amines (Table I; Chen and Murata, 2008; Hare and Cress, 1997; Hare et al., 1998; Hasegawa et al., 2000; Kerepesi et al., 1998; Parida and Das, 2005; Shulaev et al., 2008; Szabados and Savoure, 2010; Yancey et al., 1982). Composition and concentration of the solutes in stressed plants can vary considerably, depending on species and are influenced by the environmental conditions (Evers et al., 2010; Kumar, 2009; Lugan et al., 2010; Murakeozy et al., 2003; Sanchez et al., 2008). In contrast to inorganic ions, which can be harmful at high concentrations, osmolytes are considered compatible solutes, which can contribute to cell turgour, protect cellular structures, replace inorganic salts and alleviate ion toxicity. Compatible osmolytes were thought to mediate osmotic adjustment when water supply is limited, to replace water in biochemical reactions, stabilise the internal osmotic potential and to protect macromolecular structures. Therefore, osmotic adjustment has traditionally been accepted to be the primary function of osmolytes in plants (Crowe et al., 1992; Ford, 1984; Hasegawa et al., 2000; Ingram and Bartels, 1996; Parida and Das, 2005;


TABLE I Metabolism of the Most Common Osmoprotective Compounds in Plants

Osmolyte

Type of compound

Glycine betaine Quaternary ammonium compound

Proline

Amino acid

Trehalose

Sugar

Biosynthesis pathway Choline -(CMO)Betaine aldehyde -(BADH)Glycine betaine Glutamate -(P5CS)Pyrroline-5carboxylate -(P5CR)Proline

Enzymes in biosynthesis

Degradation pathway

Enzymes in catabolism

CMO: choline monooxygenase BADH: betaine aldehyde dehydrogenase P5CS: pyrroline5-carboxylate synthase P5CR: pyrroline5-carboxylate reductase

UDP TPS: trehalose phosphate glucose þ glucosesynthase 6-phosphate TPP: trehalose phosphate -(TPS)phosphatase Trehalose-6phosphate -(TPP)Trehalose

References Burnet et al. (1995), Hanson et al. (1994), Weigel et al. (1986)

Proline -(ProDH)Pyrroline-5-carboxylate -P5CDH)Glutamate

ProDH: proline dehydrogenase P5CDH: pyrroline5-carboxylate dehydrogenase

Trehalose -(TRE)2 Glucose

TRE: trehalase

Yoshiba et al. (1995), Hu et al. (1992), Szoke et al. (1992), Delauney and Verma (1993), Verbruggen et al. (1993), Strizhov et al. (1997), Kiyosue et al. (1996), Peng et al. (1996), Deuschle et al. (2004) Blazquez et al. (1998), Frison et al. (2007), Gussin et al. (1969) Lopez et al. (2008), Muller et al. (2001), Pramanik and Imai (2005), Vogel et al. (1998, 2001)

(continues)


Table I

Osmolyte

Type of compound

Mannitol

Polyalcohol

Myo-inositol

Polyalcohol

Pinitol

Polyalcohol

Biosynthesis pathway

(continued )

Enzymes in biosynthesis

Fructose-6P -(M6PI)Mannose-6P -(M6PR)Mannitol-1P -(M1PP)D-Mannitol D-Glucose-6P -MIPSMyo-inositol-1PMyo-inositol

M6PI: mannose-6P isomerase M6PR: mannose-6P reductase M1PP: mannose-1P phosphatase

Myo-inositol -(IMT1)D-ononitol Pinitol

IMT1: inositol-Omethyltransferase ononitol epimerase

MIPS: myo-inositol1-phosphate synthase

Degradation pathway Mannitol -(MTD)Mannose D-Mannose-6P -(M6PI)Fructose-6P

Enzymes in catabolism MTD: mannitol dehydrogenase M6PI: mannose-6P isomerase

References Loescher (1987, 1992), Rumpho et al. (1983)

Majumder et al. (1997), Johnson and Sussex (1995), Yoshida et al. (1999, 2002), Abreu and Aragao (2007), Wei et al. (2010a,b) Rammesmayer et al. (1995), Sengupta et al. (2008)


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Yancey et al., 1982). This model has been challenged by results obtained with transgenic plants and mutants, suggesting that osmolytes could have alternative protective functions. Concentrations of organic osmolytes were found to be lower than inorganic solutes in several halophytes, suggesting that these compounds might not be important for osmotic adjustment in these plants (Gagneul et al., 2007). For example, proline overproducing transgenic tobacco plants were shown to be more tolerant to salt and drought stress, without unequivocal data on osmotic adjustment (Blum et al., 1996; Kishor et al., 1995). Some osmoprotectants might have other protective functions, such as stabilisation of redox balance, maintenance of proper protein folding, mediating sugar or stress signals (Chen and Murata, 2008; Hare et al., 1998; Parida and Das, 2005; Rosgen, 2007; Szabados and Savoure, 2010). One important function of osmoprotective compounds is the stabilisation of proteins under conditions that can lead to protein denaturation. Equilibrium between native and unfolded forms of proteins is influenced by solvent composition. Unfavourable environmental conditions such as extreme temperatures, high salinity or dehydration can alter secondary and tertiary structure of proteins and modify the ratio of active and inactive proteins. Protein denaturation, formation of protein aggregates and accelerated protein degradation can be the cellular consequence of such stress conditions. However, the maintenance of native folding of proteins is essential for their function and can be facilitated by osmoprotective compounds (Bolen and Baskakov, 2001; Burg, 1995; Yancey et al., 1982). Protective osmolytes were shown to stabilise proteins and push the balance of the folding equilibrium towards native, actively folded proteins through raising the free energy of the unfolded proteins (Auton and Bolen, 2005; Street et al., 2006). The protein backbone but not the side chains were shown to be the preferred target for the stabilising function of osmolytes which explains their universal protecting effect (Auton and Bolen, 2004; Liu and Bolen, 1995). Protecting osmolytes improve thermodynamic stability of proteins via hydrogen bonding, which in contrast to hydrophobic interactions, does not affect other cellular functions during stress conditions (Holthauzen and Bolen, 2007; Kumar, 2009). Cyclic polyalcohols such as pinitol and myo-inositol were shown to protect plant and bacterial enzymes against thermally induced inactivation (Jaindl and Popp, 2006). By preserving the native conformation, proteins are protected from aggregation or degradation, which can therefore be considered as principal functions of osmoprotective compounds (Bolen, 2001; Kumar, 2009; Rosgen, 2007; Street et al., 2006). Different osmolytes most probably act independently and have no synergistic or competing effects in their interactions with proteins (Holthauzen and Bolen, 2007).


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Osmolytes can stabilise the structure of large multiprotein complexes as well. Glycine betaine (GB) was shown to support the oxygen-evolving activity of photosystem II (PSII) complex by preventing the dissociation of regulatory proteins from the core complex (Papageorgiou and Murata, 1995) and stabilise the efficiency of PSII photochemistry (Zhang et al., 2008). GB can ameliorate the damage of PSII and thereby stabilise photosynthesis under salt and cold stress (Ohnishi and Murata, 2006). Protection of cellular structures such as membranes and thylakoids against destabilisation during high temperatures or freezing was attributed to some of the protective compounds such as GB (Jolivet et al., 1982; Zhao et al., 1992). Some compounds can function as sinks of reducing power or carbon and nitrogen source after stress is relieved (Greenway and Munns, 1980; Hare et al., 1998). Accumulation of ROS is the result of various abiotic stresses including drought and high salinity. The protective function of osmolytes can include the suppression of radical oxygen production or scavenging of ROS. Accumulation of proline, GB and fructans in transgenic tobacco plants leads to reduced oxidative damage after freezing (Hong et al., 2000; Parvanova et al., 2004). Protection of the photosynthetic apparatus by GB during stress can reduce ROS accumulation and minimise lipid peroxidation during salt stress (Chen and Murata, 2008; Demiral and Turkan, 2004). Activation and protection of the ROS detoxification system is another key component of stress tolerance (Moradi and Ismail, 2007). Osmoprotective compounds can scavenge ROS directly, or contribute to the protection of the enzymes involved in the antioxidant system. Proline was suggested to be a singlet oxygen quencher during osmotic stress as it could reduce ROS damage such as lipid peroxidation in different plants (Hong et al., 2000; Matysik et al., 2002; Mehta and Gaur, 1999; Siripornadulsil et al., 2002; Smirnoff and Cumbes, 1989; Wang et al., 2009). GB increased catalase activity in cold-stressed tomato plants and thereby reduced ROS levels and improved chilling tolerance (Park et al., 2006). Stabilisation of the detoxifying enzymes such as ascorbate peroxidase and glutathione reductase was recently attributed to proline and can be important for the elimination of ROS during salt and drought stress (Sze´kely et al., 2008). Accumulation of ROS can be ameliorated by enhanced rate of proline biosynthesis during stress, which can help to maintain photosynthetic electron flow in the chloroplasts, stabilise redox balance and reduce photoinhibition (Hare and Cress, 1997; Szabados and Savoure, 2010). Osmolytes can control ROS-dependent damage through other, less-known pathways also. ROS was shown to promote Kþ efflux in root epidermal cells, which was significantly reduced by several osmolytes, including proline, GB, mannitol, myo-inositol and trehalose (Cuin and Shabala, 2007).


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Osmolytes can have regulatory functions and modulate metabolic processes and gene activity. Proline and GB were shown to induce the expression of defence genes such as catalase and peroxidase, which can suppress cell death during stress (Banu et al., 2009). GB induced a group of low-temperature-responsive genes, which could elicit cold acclimation and freezing tolerance (Allard et al., 1998). Transcript profiling showed that exogenous GB treatment enhances the expression of numerous Arabidopsis genes, encoding transcription factors, membrane transporters and ROS detoxifying enzymes (Einset et al., 2008). Expression of catalase and an NADPH-dependent ferric reductase (FRO2) was enhanced by GB treatment, which contributed to antioxidant activity and chilling tolerance (Einset et al., 2007; Park et al., 2006). Certain regulatory functions were attributed to proline as well. Transcript profiling revealed that part of the rehydration-inducible Arabidopsis genes can be activated by proline (Oono et al., 2003). bZIP-type transcription factors were suggested to recognise the conserved PRE element in the promoter region of these genes and to activate their transcription (Satoh et al., 2004; Weltmeier et al., 2006). A. QUATERNARY AMINES: GLYCINE BETAINE

The quaternary ammonium compound GB is a methylated derivative of glycine, which accumulates at high concentrations in many halophyte plants (Chen and Murata, 2008; Hanson et al., 1983). GB is synthetised from choline in two steps. Betaine aldehyde hydrate generated from choline by the action of choline monooxygenase (CMO) produces betaine aldehyde hydrate from choline, which is converted spontaneously to betaine aldehyde and subsequently oxidised to GB by NADþ-dependent betaine aldehyde dehydrogenase (BADH; Burnet et al., 1995; Fitzgerald et al., 2009; Fujiwara et al., 2008; Hanson et al., 1983). GB accumulation can be induced by different stress conditions such as osmotic stress (Hanson and Nelsen, 1978), salinity (Hanson et al., 1991), drought (Guo et al., 2009), heat (Jolivet et al., 1982) and cold stresses (Allard et al., 1998; DeRidder and Crafts-Brandner, 2008). The significance of GB accumulation for stress tolerance has been investigated in transgenic plants engineered to produce GB by overexpressing plant-derived CMO and BADH genes (Guo et al., 1997; Nakamura et al., 1997; Shirasawa et al., 2006). Alternatively, bacterial betaine aldehyde dehydrogenase (betB) or choline oxidase (COX or codA) genes were introduced and expressed in transgenic plants, leading to increased GB accumulation (Holmstrom et al., 1994; Mohanty et al., 2002; Park et al., 2007; Sakamoto et al., 1998; Su et al., 2006) .The beneficial effects of GB accumulation


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regarding salt and osmotic stress tolerance have been demonstrated in a number of engineered GB-accumulating plants, including tobacco (McNeil et al., 2001; Nuccio et al., 1998; Zhang et al., 2008), tomato (Park et al., 2004, 2007) and rice (Chen and Murata, 2002, 2008; Guo et al., 1997; Kathuri et al., 2009; Nakamura et al., 1997; Sakamoto et al., 1998; Shirasawa et al., 2006). GB accumulation in BADH-overexpressing transgenic tobacco led to enhanced salt or heat tolerance mainly by protecting photosynthetic activity through the maintenance of Rubisco activity and PSII activity (Yang et al., 2005, 2007, 2008). Targeted accumulation of GB in chloroplasts has been achieved by engineering a plastid-expressed CMO gene, leading to higher PSII activity during salt and drought stress (Zhang et al., 2008). Therefore protection of the PSII by GB-mediated osmoprotection can be a promising strategy to improve drought and salt tolerance in crops. Rice was engineered to produce GB by introducing the chloroplast-targeted choline oxidase (codA9) gene from Arthrobacter globiformis. GB synthesis enhanced the activity of PSII, led to better ROS detoxification and improved physiological and agronomic performance (Kathuria et al., 2009). Plants are usually very sensitive to environmental stress during reproduction. GB was shown to have a particularly important protective effect on reproductive organs, such as inflorescence apices and flowers during drought and cold stress (Chen and Murata, 2008; Sakamoto and Murata, 2000). Engineering of GB accumulation reduced chilling damage on tomato flowers, leading to a 10–30% increase in fruit production (Park et al., 2004). These data confirm that GB has osmoprotective qualities, which can therefore be explored to improve tolerance to salinity and probably to other abiotic stresses such as drought and cold. B. SUGARS: TREHALOSE

Trehalose is a nonreducing disaccharide of glucose, which was first described in desiccation-tolerant organisms capable of surviving dehydration, including resurrection plants (Drennan et al., 1993; Fernandez et al., 2010; Liu et al., 2008; Moore et al., 2009). Later, trehalose accumulation was detected in numerous other plants under different stress conditions such as drought, cold, high salinity (Iordachescu and Imai, 2008; Kaplan et al., 2004; Kosmas et al., 2006; Lopez et al., 2008; Pramanik and Imai, 2005) and during various plant–microbial interactions (Brodmann et al., 2002; Dominguez-Ferreras et al., 2009; Farıás-Rodrı´guez et al., 1998; Lopez et al., 2008). Trehalose biosynthesis is a two-step pathway. First, trehalose-6-phosphate is produced from UDP glucose and glucose-6-phosphate by trehalose phosphate synthase, which is converted to trehalose by the enzyme trehalose phosphate


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phosphatase (Vogel et al., 1998, 2001). Trehalose is catabolised by trehalase, which converts it to glucose (Brodmann et al., 2002; Goddijn et al., 1997). The importance of trehalose in modulating plant stress responses was demonstrated by engineering the trehalose biosynthetic pathway in transgenic plants. Stress-dependent trehalose accumulation was engineered in transgenic rice by the regulated overexpression of the fused bacterial trehalose biosynthetic genes otsA and otsB. Enhanced trehalose levels resulted in improved salt, drought and cold tolerance, and lower photo-oxidative damage, which was suggested to modulate carbohydrate metabolism (Garg et al., 2002). Several other transgenic plants were produced that accumulated trehalose at high levels, and subsequently improved tolerance to drought, freezing or high salinity (Jang et al., 2003; Romero et al., 1997; Stiller et al., 2008; Yeo et al., 2000). However, constitutive overexpression of the trehalose biosynthetic genes led to pleiotropic growth alterations, dwarfism and abnormal root structure (Garg et al., 2002; Romero et al., 1997; Schluepmann et al., 2004; Yeo et al., 2000). Using stress-induced promoters such as the drought-induced Arabidopsis AtRAB18 or potato StDS2 promoters, the environmentally controlled upregulation of trehalose biosynthesis was important to generate transgenic plants with enhanced stress tolerance, but without morphological abnormalities (Karim et al., 2007; Stiller et al., 2008). The function of trehalose in stress responses is controversial. Trehalose was shown to have the ability to stabilise membranes and protect proteins in desiccated tissues and was suggested to function as chemical chaperon (Crowe, 2007; Crowe et al., 1984). However, trehalose levels in the engineered plants usually remained well below 1 mg/g fresh weight, suggesting that trehalose does not act as a compatible solute, but has an alternative function (Garg et al., 2002). The fact that trehalose-accumulating transgenic plants exhibit abnormal morphology can also indicate that this compound is not a neutral osmolyte. Recently, trehalose-6-phosphate was proposed to function as a metabolic signal involved in the control of the SnRK1 activity, which is a central regulator of sugar and energy homeostasis (Paul et al., 2010; Zhang et al., 2009). Therefore, the signalling function of trehalose and trehalose-6P could be more important than the previously suggested chaperone or osmolyte function, although in some tissues such a protective role cannot be excluded (Fernandez et al., 2010). C. POLYALCOHOLS: MANNITOL, PINITOL, INOSITOL

Accumulation of polyalcohols, such as mannitol and pinitol, has been detected in several water-stressed plants and are considered as important compatible solutes, which serve as ROS scavengers and molecular


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chaperones (Bohnert et al., 1995; Ford, 1984; Sengupta et al., 2008). Mannitol is the most common sugar alcohol and is an important photosynthetic product in a number of plant species (Loescher et al., 1992; Rumpho et al., 1983). In plants, mannitol is synthetised from fructose-6P by subsequent action of mannose-6P isomerase (phosphomannose isomerase), mannose6P reductase (M6PR) and mannose-1P phosphatase (Loescher et al., 1992; Rumpho et al., 1983). Catabolism of mannitol is controlled by mannitol dehydrogenase, which produces mannose. Phosphorylation of mannose results in mannose-6P, which is converted to fructose-6P by mannose-6P isomerase (Loescher, 1987). One of the first demonstrations of the osmoprotective function of an osmolyte was the overexpression of a bacterial mannitol 1-phosphate dehydrogenase (mtlD) in transgenic tobacco plants, which led to increased mannitol accumulation in conjunction with enhanced salt tolerance (Tarczynski et al., 1993). In agreement with these results, engineering of mannitol biosynthesis by overexpression of mtlD led to enhanced mannitol accumulation and improved salt tolerance in various transgenic plants including wheat (Abebe et al., 2003), poplar (Populus tomentosa; Hu et al., 2005) and loblolly pine (Pinus radiata; Tang et al., 2005). Overexpression of celery M6PR is an alternative way to enhance mannitol biosynthesis and was shown to be an efficient way to improve salt tolerance of Arabidopsis (Zhifang and Loescher, 2003). As an alternative use, M6PR was employed as a selectable marker for plant transformation, using mannose tolerance as selection criteria (Song et al., 2010). Myo-inositol is an essential polyalcohol in plants and all eukaryotes. Biosynthesis starts from D-glucose-6P, which is converted to myo-inositol1P by myo-inositol-1P synthase (MIPS; Johnson and Sussex, 1995; Majumder et al., 1997). Myo-inositol is produced from myo-inositol-1P by dephosphorylation and is used for the subsequent biosynthesis of all inositolcontaining compounds, including phospholipids. MIPS genes were shown to be salt-induced, leading to accumulation of myo-inositol in the halophyte ice plant, but not in the glycophyte Arabidopsis (Ishitani et al., 1996). MIPS genes can be regulated by several environmental stress factors such as drought, heat and cold stress, high light and were shown to be controlled by ABA signals (Abreu and Aragao, 2007; Wei et al., 2010a,b; Yoshida et al., 1999, 2002). Myo-inositol serves not only as osmoprotectant compound, but also functions as signal that controls metabolic responses to stress, such as sodium uptake in saline environments (Nelson et al., 1999). Phosphorylated derivatives of myo-inositol are important signalling compounds, which are involved in numerous regulatory pathway and control diverse aspects of plant development, responses to biotic and abiotic stresses.


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Other polyalcohols with osmoprotective features such as D-ononitol and are synthetised from myo-inositol through the extension of the metabolic pathway. Pinitol is a methylated inositol, which is synthetised from myo-inositol by inositol-o-methyltransferase (IMT1) and ononitol epimerase (OEP1) (Bohnert et al., 1995; Rammesmayer et al., 1995; Sengupta et al., 2008). Pinitol accumulation is a characteristic feature of a number of halophytic plants in saline environment and occurs in several glycophytic plants grown under osmotic stress conditions (Ford, 1984; Fougere et al., 1991; Murakeozy et al., 2003; N’Guyen and Lamant, 1988; Sengupta et al., 2008). Salt-induced pinitol hyperaccumulation was found in Porteresia coarctata, a halophytic wild relative of rice, which is missing in domesticated rice. The inositol methyl transferase 1 (PcIMT1) gene is strongly upregulated by salt in wild rice, suggesting that this gene controls the stress-induced pinitol accumulation in this halophytic plant which is an essential metabolic response for salt tolerance (Sengupta et al., 2008). Engineering of complex metabolic pathways by altered expression of single genes has limitations. Simultaneously regulated expression of several genes encoding key enzymes could have more profound effects on the metabolic pools. Biosynthesis of myo-inositol was enhanced by introduction and overexpression of the MIPS gene from P. coarctata in tobacco leading to inositol accumulation and enhanced salt tolerance (Majee et al., 2004). Even higher levels of salt tolerance were reported by the introgression and simultaneous overexpression of the MIPS coding gene from P. coarctata and the IMT1 coding gene from M. crystallinum in transgenic tobacco. By comparison, double transgenics accumulated more inositol and pinitol than plants transformed by single genes, which conferred improved growth, higher photosynthetic activity and lower oxidative damage during salt stress (Patra et al., 2010). D-pinitol

D. AMINO ACIDS: PROLINE

Proline is an essential amino acid, a common denominator of many stress responses being accumulated during diverse abiotic and biotic stresses such as high salinity (Ben Hassine et al., 2008; Voetberg and Stewart, 1984; Yoshiba et al., 1995), drought (Ben Hassine et al., 2008; Choudhary et al., 2005; Huang and Cavalieri, 1979; Rhodes et al., 1986), UV irradiation (Saradhi et al., 1995), heavy metals (Mehta and Gaur, 1999; Schat et al., 1997; Singh et al., 2010) and oxidative stress (Yang et al., 2009). Moreover, proline was reported to accumulate in plants infected by avirulent bacteria (Fabro et al., 2004) or Agrobacterium (Haudecoeur et al., 2009). In plants, proline is synthesised from glutamate in the cytosol and likely also in the chloroplast by the sequential action of delta-1-pyrroline-5-carboxylate


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Synthesis

synthetase (P5CS) and P5C reductase (P5CR). P5CS produces glutamate semialdehyde, which is unstable and is immediately converted to pyrroline-5carboxylate (P5C). P5CR reduces P5C to proline, a reaction that takes place in the cytosol and according to biochemical data also in the chloroplast (Fig. 1; Delauney and Verma, 1993; Hu et al., 1992; Rayapati et al., 1989; Strizhov et al., 1997; Szabados and Savoure, 2010; Szoke et al., 1992; Verbruggen et al., 1993; Yoshiba et al., 1995). Proline synthesis is considered as an evolutionary conserved process based on the similarity that exists among the prokaryotic and eukaryotic pathways and the high homology of the involved enzymes. The human and plant P5CSs are bifunctional enzymes representing evolved fusion products of the domains responsible for the catalytic activity in the prokaryotic proB and proA genes, encoding glutamate kinase (GK) and g-glutamyl phosphate reductase (GPR), respectively (Csonka, 1989; Hu et al., 1992; Perez-Arellano et al., 2010). These genes are arranged in a single operon in bacteria and their paralogs in lower eukaryotes, such as yeast, still form two separate enzymes (Takagi, 2008). Despite sharing functional homology, the mammalian and plant P5CSs are localised to different cellular compartments. The human P5CS isoforms are functioning in the mitochondria, utilising innermitochondrial glutamate and energy sources (Perez-Arellano et al., 2010),

Chloroplast

Cytosol

Mitochondrion

Glutamate

Glutamate

Arginine

NADPH+ + H+ NADP+ ATP ADP

NADPH+ + H+

P5CS1

P5CS1 & 2

Arginase

NADP+

GSA P5C P5CR

GSA P5C

Ornithine P5CR

NADP+

Proline

Proline

NADPH+ + H+

OAT Accumulation during stress in cytosol, vacuole, and PRPs Transport to the cytosol

Transport into mitochondria

Mitochondrion

Degradation

ProDH 1 & 2

Proline

P5C FAD+

FADH2

P5CDH NAD+/NADP+

eElectron transport chain

NADH+ + H+/ NADPH+ + H+

Glutamate

Fig. 1. Scheme of proline metabolism in plants. Proline synthesis occurs in the cytosol and likely also in the chloroplast. Proline degradation is carried out in the mitochondrion. The dashed arrows indicate the proline cycle. See text for details.


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whereas the two plant isoforms, P5CS1 and P5CS2, are functioning in the cytosol, with a likely abiotic-stress-induced shift of P5CS1 to the chloroplasts (Sze´kely et al., 2008). In yeast, both enzymes (GK and GPR) function in the cytosol (Takagi, 2008). Another source for P5C is the mitochondrial degradation pathway of arginine, whose first step is catalysed by arginase that forms ornithine and urea. In the second step, ornithine amino transferase (OAT) produces P5C by ornithine deamination in the mitochondria (Brownfield et al., 2008; Roosens et al., 1998; Xue et al., 2009). The importance of this pathway in proline biosynthesis has recently been questioned as proline levels were not altered in the oat mutant (Funck et al., 2008). Although P5C transporters have not been identified in plant mitochondrial or chloroplast membranes, biochemical evidence support the movement of P5C from the mitochondria to the cytosol in human and plant cells which enables the reduction of mitochondrial-produced P5C to proline in the cytosol (Miller et al., 2009; Yoon et al., 2004). Proline catabolism occurs in the inner-mitochondrial membrane of all eukaryotes. Proline degradation provides electrons and glutamate for mitochondrial usage. Proline dehydrogenase (ProDH), a FAD-enzyme localised to the inner-mitochondrial membrane, catalyses the first oxidising step of proline to P5C and meanwhile delivers electrons to the mitochondrial electron transport chain (Kiyosue et al., 1996; Peng et al., 1996; Rayapati and Stewart, 1991). P5C is further oxidised to glutamate or transported back to the cytosol for proline re-synthesis by the proline cycle (Fig. 1; Deuschle et al., 2004; Miller et al., 2009). Proline cycle exists in yeast, mammals and plants and contains the cytosolic P5CR and mitochondrial ProDH (Miller et al., 2009; Phang et al., 2010; Takagi, 2008). In human cells, proline cycle acts as a suppressor of carcinogenesis. It oxidises proline and provides an excess of electrons producing ROS that are responsible for initiating programmed cell death that prevents tumour development (Phang et al., 2010). P5CDH is the second enzyme in proline degradation, catalysing the oxidation of P5C to glutamate in the mitochondria and is responsible for maintaining proline-P5C homeostasis (Deuschle et al., 2001, 2004; Forlani et al., 1997). When P5CDH activity is impaired, hyperactivity of proline-P5Cproline cycle provides excess electrons to the mitochondrial electron chain and generates ROS by using O2 as the electron acceptor (Miller et al., 2009). ROS signals can be produced during the first hour of dehydration in Arabidopsis leaves, when a transient increase in ProDH transcription occurs (Kiyosue et al., 1996; Peng et al., 1996). In some plant species, derivatisation of proline occurs following its stress-induced accumulation. In the salt tolerant salt-cider (Tamarix spp.), part of the stress-accumulated proline is


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modified to N-methylproline analogues including N-methyl-L-proline, trans4-hydroxy-N-methyl-L-proline and trans-3-hydroxy-N-methyl-L-proline whose function is still unknown (Jones et al., 2006). Proline accumulation during stress has multiple protective functions. For a long time, proline was considered a neutral osmolyte that protects cellular structures and stabilises enzymes (Delauney and Verma, 1993; Kavi Kishor et al., 2005; Mishra and Dubey, 2006; Sharma and Dubey, 2005; Sharma et al., 1998). Besides osmoprotection, proline was shown to have antioxidant activity, activate detoxification pathways, contribute to cellular homeostasis by protecting the redox balance, function as protein precursor, energy source for the stress-recovery process and even as a signalling molecule (Hoque et al., 2008; Islam et al., 2009; Khedr et al., 2003; Matysik et al., 2002; Szabados and Savoure, 2010; Sze´kely et al., 2008). In plants, maintenance of PSII and PSI activity as well as electron flux through the photosynthetic electron transport chain is very important in stress conditions. Inhibition of Calvin cycle and pentose phosphate pathway can channel NADPH, ATP and glutamate for proline synthesis in the chloroplasts. Thus, proline synthesis in the chloroplast may allow an efficient oxidation of photosynthetically produced NADPH providing the required NADPþ for electron acceptor avoiding the use of O2 that leads to ROS generation (Hare and Cress, 1997; Szabados and Savoure, 2010). Numerous reports describe the importance of proline accumulation in salt and drought tolerance, although a clear relationship between proline accumulation and tolerance could not always be confirmed (Kavi Kishor et al., 2005; Lehmann et al., 2010; Szabados and Savoure, 2010; Verbruggen and Hermans, 2008). Convincing evidence for the protective function of proline was provided by studies of mutants and transgenic plants with proline deficiency or proline hyperaccumulation. Arabidopsis p5cs1 insertion mutants had only 10% proline of the wild type and were hypersensitive to salt stress, produced more ROS and lipid peroxidation products, which confirmed the importance of proline in stress tolerance, in particular, in ROS scavenging (Sze´kely et al., 2008). However, enhanced proline accumulation and improved salt tolerance was achieved by increasing the biosynthetic pathway through constitutive overexpression of the Vigna P5CS cDNA in transgenic tobacco (Kishor et al., 1995) and Clamydomonas (Siripornadulsil et al., 2002). Overexpression of feedback-insensitive P5CS in tobacco or rice could enhance proline levels even more than the wild-type enzyme, showing that post-translational regulation needs to be considered for metabolic engineering (Hong et al., 2000; Kumar et al., 2010). Alternatively the feedback-insensitive proAB gene from Bacillus subtilis was expressed in Arabidopsis, which produced more free proline and improved


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osmotolerance (Chen et al., 2007a,b). While overexpression of Arabidopsis P5CR in transgenic soybean improved drought and heat tolerance, antisense expression of P5CR leads to stress sensitivity (De Ronde et al., 2004).These reports confirm that proline accumulation in dehydrated plants is not only a consequence of stress, but also a part of the metabolic defence system against abiotic stress. 1. Regulation of proline metabolism While proline biosynthesis is augmented and proline catabolism is repressed during stress, the opposite happens during recovery, when synthesis declines rapidly and catabolism is activated. Accumulation of cellular free proline levels has been attributed to increased transcription of P5CS and silencing of ProDH, while reciprocal transcriptional regulation suppress P5CS transcrip´ braha´m et al., 2003; Kiyosue et al., tion and activates ProDH after stress (A 1996; Peng et al., 1996; Ribarits et al., 2007; Strizhov et al., 1997; Verbruggen et al., 1996; Yoshiba et al., 1995). Whereas the stress-induced drastic reduction in ProDH transcription is a general phenomenon in plants, some species lack the concomitant increase in P5CS transcription (Ginzberg et al., 1998; Miller et al., 2005). Stress-dependent P5CS activation and ProDH repression is controlled by both ABA-dependent and independent signalling pathways, ´ braha´m et al., 2003; modulated by light and brassinosteroid signals (A Sharma and Verslues, 2010; Strizhov et al., 1997; Sze´kely et al., 2008; Verslues et al., 2007). During incompatible plant–pathogen interactions P5CS activation is controlled by SA-dependent ROS signals (Fabro et al., 2004). In the ornithine pathway, OAT transcription is upregulated by salt stress in young Arabidopsis seedlings but not in mature plants, suggesting the involvement of developmental and temporal regulation in OAT expression (Roosens et al., 1998). Regulation of key enzymes involved in proline metabolism seems to vary according to environmental conditions and also during plant development (Lehmann et al., 2010; Szabados and Savoure, 2010). Calmodulin and MYB2 are likely involved in P5CS activation under salt stress, as interaction of MYB2 and the salt-induced CaM isoform (GmCaM4) was shown to upregulate P5CS1 transcription and leads to proline accumulation (Yoo et al., 2005). In addition to the MYB2, bZIP transcription factors (bZIP-TF) might also be involved in regulating transcription of the genes encoding enzymes of proline metabolism. Promoter analyses by several servers that identify promoter motifs (e.g., Agris, http://arabidopsis.med.ohio-state.edu) indicate that the promoters of all Arabidopsis genes encoding enzymes involved in proline metabolisms possess bZIP-TF recognition motifs: six sites in P5CS1 (At2g39800), 11 sites in P5CS2 (At3g55610), 14 in P5CR (At5g14800), four in ProDH1 (At3g30775), six in ProDH2 (At5g38710), two


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in P5CDH (At5g62530) and five in OAT (At5g46180). As yet, only the molecular basis of induced ProDH transcription by the drought and salinity stressrecovery process has been unravelled, indicating the importance of bZIP transcription factors. bZIP-TFs from the S and C groups AtbZIP53 and AtbZIP10 (Jakoby et al., 2002) form homo- or heterodimers and promote the recruitment of the transcription complex to the ProDH promoter region. Interaction of these transcription factors with the PRE motif ACTCAT of the ProDH promoter strongly enhances its transcription (Satoh et al., 2002, 2004; Weltmeier et al., 2006). Interactions of bZIP11/ATB2 and other heterodimerforming partners from groups S and C with this PRE motif is also responsible for upregulating ProDH transcription in response to sugar depletion, an effect that also blocks the post-transcriptional inhibition of bZIP11 by sucrose (Hanson et al., 2008). The possible involvement of bZIP-TFs in the regulation of the other genes controlling proline metabolism still has to be confirmed. Upon recovery from stress, a reduction in P5CS and enhancement of ProDH transcription occur, leading to intensive proline degradation that provides energy and glutamate for the surviving cells (Kiyosue et al., 1996; Peng et al., 1996; Verbruggen and Hermans, 2008). In addition to changes in enzyme levels, mostly dictated by regulation at the transcriptional level, proline allosterically inhibits the enzymatic activity of bacterial GK/GPR and the plant P5CS, the initial enzyme in the synthesis pathway (Csonka et al., 1988; Fujita et al., 2003; Hu et al., 1992). The accumulation of proline during various stresses without exerting a feed-back regulation on P5CS activity is not well understood. Although expression of ProDH genes is induced by exogenous proline, ProDH transcription is silenced during stress, despite the high levels of accumulated free proline in the cells (Kiyosue et al., 1996; Miller et al., 2005). Hence, it is likely that a certain compartmentalisation of the stress-accumulated proline exists to avoid interaction with P5CS and cancel the silencing of ProDH transcription. Further studies are required to clearly understand the signalling pathways and define all the regulatory proteins involved. 2. Proline rich proteins and stress response Proline rich proteins (PRP) are plant-specific proteins that are mainly localised to the cell wall, playing different roles in cell wall maintenance and linkage to the plasma membrane. Their synthesis utilises a large portion of the free proline pool and normally occurs in most of the plant cells during development. These proteins are characterised by reiterated proline-rich sequence motifs. Many of the proline residues in these proteins are hydroxylated and thereafter glycosylated through their processing in the ER and Golgi network before being secreted to the outer side of the plasma membrane or linked to the membrane by a GPI-anchore or hydrophobic


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transmembrane domain/s. The hydroxyproline-rich glycoproteins comprise a huge super family in Arabidopsis and other plants, which is divided into three subfamilies; arabinogalactans, extensins and PRP (Showalter et al., 2010). Most of these proteins are involved in cell wall assembly in all plant organs and along the developmental processes or are more specific to certain organs (Fowler et al., 1999). Oxidative cross-linking of tyrosine residues within the subfamily of extensin molecules strengthens the cell wall and increases resistance to invading pathogens. Whereas many hydroxyproline-rich proteins are involved in plant response to wounding and pathogen attacks, only a limited number of these PRP are involved in abiotic stress responses (Deepak et al., 2010). Most of them belong to the HyPRP family. HyPRPs, which are only found in plants, contain a proline-rich N-terminal repetitive domain suggested to protrude from the plasma membrane towards the cell wall and a hydrophobic C-terminal transmembrane domain with a certain conserved arrangement of eight cysteine residues (Jose-Estanyol et al., 2004). MsPRP2 is an HyPRP that is induced by water stress in alfalfa (Medicago sativa) cells under saline conditions (Deutch and Winicov, 1995), whereas another alfalfa HyPRP, MsACIC, reveals enhanced expression in cold-tolerant plants (Castonguay et al., 1994). The Brassica napus HyPRP, BNPRP, is also highly expressed at low temperatures, whereas low level of the BNPRP transcript is also present under normal growth conditions (Goodwin et al., 1996). As yet, the functional role of these proteins and their contribution to stress tolerance are not clear. It is likely that the Arabidopsis cell wall linker protein (CWLP) that shows 85% amino acid sequence homology to BNPRP (Goo et al., 1999), and is induced by cold treatment in Arabidopsis, is involved in the adhesion of the cell wall to the plasma membrane (Ziberstein’s group preliminary results). Since cold-induced synthesis of these proteins is linked to proline accumulation, availability of free proline might support the synthesis of PRP.

IV. OSMOPROTECTIVE COMPOUNDS AND ADAPTATION TO EXTREME ENVIRONMENTS Numerous earlier papers and reviews have discussed the importance of osmoprotective compounds in stress tolerance and the value they might have in the adaptation process to extreme environmental conditions (Hare et al., 1998; Rontein et al., 2002; Table II). While a number of reports described the coincidence of osmolyte accumulation with salt or drought tolerance, the adaptive value of osmoprotective compounds for stress tolerance was unequivocally demonstrated in a few cases only.

Author's personal copy TABLE II Plant Species or Genotypes in Which Osmolyte Accumulation is Important for Tolerance to Extreme Environmental Conditions Species

Compound

Environment

Atriplex halimus L (xero-halophyte)

Glycine betaine, sugars, proline

Arid environment, saline soil

Camphorosma annua (halophyte)

Glycine betaine, pinitol

salty–sodic soil

Cicer arietinum (chickpea genotypes with contrasting copper tolerance) Hordeum vulgare L (salt tolerant/ sensitive barley)

Proline

Copper toxicity

Proline, glycine betaine, sugars, hexose phosphates

Saline soil

Lepidium crassifolium (halophyte)

Proline, sugars

salty–sodic soil

Limonium spp. (halophyte, Plumbaginaceae spp.) Limonium gmelini (halophyte)

b-Alanine betaine, choline-Osulphate b-Alanine betaine, choline-Osulphate, pinitol Sugars, chiro-inositol, proline Proline, sugars

Hypoxic saline, sulphate-rich saline soils salty–sodic soil

Limonium latifolium (halophyte) Medicago truncatula, M. laciniata, (contrasting landraces in drought tolerance) Mesembryanthemum crystallinum (ice plant, halophyte) Oryza sativa (drought tolerant/ sensitive rice varieties) Oryza sativa (drought tolerant/ sensitive indica rice variety)

Polyols (myo-inositol, D-ononitol, D-pinitol) Proline Reducing sugars, proline, polyols

Reference Shen et al. (2002), Wang and Showalter (2004), Martinez et al. (2004) Murakeozy et al. (2003) Singh et al. (2010)

Saline soil Drought, arid environment

Chen et al. (2007b), Widodo et al. (2009) Murakeozy et al. (2003), this study Hanson et al. (1991, 1994) Murakeozy et al. (2003) Gagneul et al. (2007) Yousfi et al. (2010)

Saline soil

Nelson et al. (1999)

Drought, osmotic stress

Choudhary et al. (2005) Zuther et al. (2007), Roychoudhury et al. (2008)

Saline soil


Proline

Tolerance to Hg2þ-induced oxidative stress Drought, arid environment

Glycine betaine

Saline soil

Plumbaginaceae spp. (halophytes) Porteresia coarctata (wild rice, halophyte) Solanum tuberosum L. (drought tolerant/sensitive potato cultivars) Sorghum bicolor (sorghum: salt tolerant/sensitive variety) Thellungiella salsugiana (halophila) (halophyte)

Proline betaine Pinitol

Arid environment Saline soil

Hanson et al. (1991, 1994) Hanson et al. (1994) Sengupta et al. (2008)

Proline, inositol, galactose, galactinol Sugars, proline

Drought treatment

Evers et al. (2010)

Drought, arid environment

Proline, sugars (sucrose, fructose, glucose, raffinose, melibiose), sugar alcohols (inositol, galactinol)

Saline soil

Triticum aestivum (drought tolerant/ sensitive wheat genotypes) Triticum durum Desf. (drought tolerant/sensitive variety) Various contrasting legume species in drought tolerance

Sugars, fructan

Drought, arid environment

Premachandra et al. (1995) Lugan et al. (2010), Gong et al. (2005), Inan et al. (2004), Arbona et al. (2010), Taji et al. (2004) Kerepesi et al. (1998)

Sugars, proline

Arid environment, osmotic stress Drought, water stress

Oryza sativa (proline accumulating mutant) Phaseolus vulgaris (contrasting landraces in drought tolerance) Plumbaginaceae spp. (halophytes)

Proline

Pinitol

Such examples are listed where experimental evidence suggests the value of osmolyte accumulation for stress tolerance.

Wang et al. (2009) Tari et al. (2008)

Bajji et al. (2001) Ford (1984)


L. SZABADOS ET AL.

Osmoprotective compounds can accumulate to very high concentrations in extremophile plants in saline or arid environments, suggesting that these metabolites contribute considerably to the adaptation of these plants to the harsh environment. Some species produce high levels of sugars or polyols, others preferentially accumulate nitrogenous compounds. Halophytes usually accumulate one dominant compatible solute, which can be proline, GB, sorbitol, b-alanine betaine, choline-O-sulphate or sugar (Arbona et al., 2010; Hanson et al., 1991, 1994; Inan et al., 2004; Lugan et al., 2010; Tipirdamaz et al., 2006). Halophytic Limonium species were shown to accumulate high levels of quaternary ammonium compounds such as choline-O-sulphate, GB and b-alanine betaine, suggesting that these compounds are important for the salt tolerance of these species (Hanson et al., 1991). Sucrose accumulated in Juncus maritima, Phragmites communis and Scirpus maritimus, while maltose and rhamnose were abundant in Atriplex hastata and Plantago maritima, respectively. Polyols were present in Aster tripolium, Juncus maritimus, P. maritima and P. communis (Briens and Larher, 1982). Distribution of osmoprotective ammonium compounds among different species of the Plumbaginaceae family suggested that particular compounds have selective advantage in different environments. GB was dominant in species adapted to dry environments, choline-O-sulphate was advantageous in sulphatecontaining soils, b-alanine betaine is apparently more typical in species growing on hypoxic saline soils, and proline–betaine was detected in plants adapted to arid environments (Hanson et al., 1994). Composition and concentration of compatible osmolytes had species-specificity and showed seasonal fluctuation in three halophyte species in salty–sodic grasslands in Hungary. GB and pinitol was characteristic of Camphorosma annua, b-alanine betaine and pinitol accumulated in Limonium gmelini, while proline was most characteristic of Lepidium crassifolium. The highest osmolyte concentrations were measured in spring, characterised by low temperatures, hypoxic conditions and high salt concentrations in the habitats of the tested species (Murakeozy et al., 2003). Among the extremophile Chenopodiaceae species, Artiplex halimus is a known xero-halophyte plant, characterised by GB and proline accumulation in stressful environments (Shen et al., 2002; Wang and Showalter, 2004). Accumulation of GB and sugars were suggested to be responsible for osmotic adjustment during osmotic stress in this species (Martinez et al., 2004). In a more recent study, P. maritima was found to accumulate sorbitol, proline and GB, while species belonging to the Chenopodiaceae family are typical accumulators of GB in saline environments (Tipirdamaz et al., 2006). In other species, the importance of osmoprotecting compounds in stress tolerance has been studied by comparing genotypes with contrasting


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drought or salt tolerance and osmolyte accumulation. The importance of osmoprotectants for salt and drought stress tolerance of cereals has long been discussed (Garcia et al., 1997). Proline and sugar levels were higher in drought and salt tolerant than sensitive rice varieties, suggesting that these protective compounds can contribute to stress tolerance of rice (Choudhary et al., 2005; Roychoudhury et al., 2008). In durum wheat, main osmolytes that correlated with drought tolerance were sugars, followed by proline and quarternary ammonium compounds (Bajji et al., 2001). A positive correlation between drought tolerance of wheat and glucose, fructose, sucrose and fructan contents was observed, suggesting that soluble carbohydrates have adaptive value for wheat (Kerepesi et al., 1998). However, accumulation of proline and GB did not correlate with salt tolerance in contrasting barley genotypes, while hexoses and TCA intermediates did, suggesting that carbohydrates contribute to salt tolerance of barley (Chen et al., 2007a,b; Widodo et al., 2009). Transcript profiling of drought tolerant and sensitive barley varieties revealed that tolerance correlated with the drought-dependent activation of genes, which are involved in the synthesis, and transport of GB, but not proline (Guo et al., 2009). GB is therefore important for drought but not for salt tolerance in barley, hexoses are significant for salt tolerance, while proline accumulation is rather a symptom of salt susceptibility. In sorghum, drastic increase of both proline and GB levels were recorded upon water deficit (Wood et al., 1996). Accumulation of solutes was higher in a drought-tolerant sorghum line than in a drought sensitive one, with the notable exception of proline, which does not seem to contribute to drought tolerance in this plant (Premachandra et al., 1995). In saline environments, pinitol accumulated in the halophytic wild rice, P. coarctata Roxb., but not in the cultivated rice. Inositol methyl transferase (IMT1) mediates pinitol synthesis in P. coarctata, while this gene is missing in the rice genome (Sengupta et al., 2008). Moreover, a salt tolerant form of L-MIPS was identified in P. coarctata, containing a 37 amino acid stretch, which conferred superior thermodynamic stability and elevated activity to the MIPS enzyme leading to enhanced myo-inositol synthesis in saline environments (Ghosh Dastidar et al., 2006). The capacity to synthesise and accumulate myo-inositol and pinitol can therefore be an important adaptive feature in P. coarctata and probably a number of other extremophile plants (Sengupta and Majumder, 2009). In legumes, drought or salt tolerance correlated with osmolyte accumulation in some but not all species. Drought-induced proline accumulation was not different in contrasting bean landraces or in Medicago truncatula and M. laciniata ecotypes characterised by contrasting drought tolerance. Therefore, proline does not seem to contribute significantly to osmotic stress tolerance in these legumes (Tari et al., 2008; Yousfi et al., 2010). In several


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tropical legumes, water stress induced the accumulation of sugar alcohols and proline, while betaine contents were not changed. Tolerance to low water potential correlated with pinitol accumulation, suggesting that this sugar alcohol contributes to drought tolerance in these species (Ford, 1984). In contrasting, chickpea (Cicer arietinum) genotypes the capacity to accumulate proline correlated with copper tolerance, suggesting that in some species proline can contribute to heavy metal tolerance (Singh et al., 2010). Metabolite analysis of potato cultivars with different drought tolerance showed that enhanced galactose, inositol and proline content in drought-stressed plants correlate with higher degree of tolerance (Evers et al., 2010). The above listed examples confirm the importance of osmoprotectants for stress adaptation not only in extremophiles but in other species as well, including in crops. The elucidation of the importance of osmolyte accumulation in extremophiles became recently possible through comparative analysis of closely related halophyte and glycophyte species, especially those which are related to the model plant Arabidopsis (Orsini et al., 2010).The halophyte T. salsuginea (halophila) and L. crassifolium are close relatives of A. thaliana (a glycophyte), and can grow under extreme saline conditions. Proline contents in T. salsuginea and L. crassifolium are higher than those in Arabidopsis, even in optimal growth conditions, and proline accumulates to higher levels when salt stress is imposed (Fig. 2). Metabolite profiling revealed that in saline environments, proline is the dominant osmoprotective compound in T. salsuginea, followed by sugars, while betaines and polyols do not accumulate in this species (Arbona et al., 2010; Inan et al., 2004; Lugan et al., 2010). Sucrose, galactose and melibiose are among the carbohydrates that also accumulate at high levels in T. salsuginea (Lugan et al., 2010). Transcript analysis showed that the P5CS gene, which controls the glutamate-derived proline biosynthetic pathway, has higher constitutive expression level in T. salsuginea than in Arabidopsis, and is induced more rapidly under stress (Inan et al., 2004; Taji et al., 2004). High proline contents in T. salsuginea could also be the consequence of lower transcription of the ProDH gene, which controls proline catabolism (Kant et al., 2006). Such comparative analysis suggests that proline levels are key for the salt tolerance of T. salsuginea, which is controlled by the activities of its key biosynthetic and catabolic genes.

V. CONCLUSIONS The specific physiological responses elicited by various stresses on higher plants are relatively well described. The accumulation of osmoprotective compounds represents a specific metabolic response that is important to


PLANTS IN EXTREME ENVIRONMENTS

A

Arabidopsis

Thellungiella

Lepidium

Salt stress

Control B

Proline (mM/gFW)

400

300 Control

200

Salt 100

0 Arabidopsis

Thellungiella

Lepidium

Plants

Fig. 2. Comparison of salt tolerance and proline accumulation of Arabidopsis thaliana and two close relatives: Thellungiella salsuginea and Lepidium crassifolium. (A) Plants were grown in soil for 4 weeks and then watered twice a week with water or 0.5 M NaCl for 2 weeks. (B) Relative proline accumulation in the three brassicaceae species after 1 week of salt stress. Note that both halophytes contained more free proline in the absence of salt stress and accumulated more proline than Arabidopsis under saline conditions.

withstand harmful conditions. Although osmoprotective compounds belong to various categories of bioactive chemicals, they have similar cellular functions such as the stabilisation of cellular structures, protein complexes or specific enzymes or the control of redox balance and ROS production. Sugars or proline could function as metabolic signals and therefore have broader influence on physiological responses and metabolic adjustment to stress conditions.


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Despite of the enormous amount of information accumulated in the past decades, the exact function of low-molecular-weight protective compounds in the adaptation to extreme environmental conditions is still not completely understood. Combining genomic, proteomic and metabolic profiling approaches could increase our understanding of plant stress responses on a global scale and of the metabolic bases of adaptation to drought, salinity or extreme temperatures. Engineering of model and crop plans via genetic transformation is a promising tool to study the significance of osmoprotective compounds in stress responses and to improve the performance of crop plants under suboptimal conditions. Enhanced accumulation of a metabolite can be achieved via activation of the biosynthetic pathway or inhibition of the catabolic pathway. Alternatively, novel pathways can be established in plants, by introducing genes from other species. Various examples have been published for engineering the synthesis and accumulation of osmoprotective compounds. Although osmoprotectant levels in such transgenic plants are often low, and increase in stress tolerance is small, manipulation of the metabolism of osmoprotective compounds have already produced promising results (Chen and Murata, 2008; Rontein et al., 2002; Szabados and Savoure, 2010; Verbruggen and Hermans, 2008). Adaptation to extreme environmental conditions is a complex trait, controlled by numerous genes. Engineering several traits by simultaneous manipulation of two or more genes is a real alternative to handle complex traits such as stress tolerance. Significant enhancement of drought tolerance have been achieved by pyramiding of transgenes in maize, by combining codA from E. coli and TsVP (V-H(þ)-PPase) from Thellungiella. Expression of the two transgenes led to higher GB accumulation and enhanced H(þ)-PPase activity compared with the parental lines, resulting in lower cell damage and higher yields under drought conditions (Wei et al., 2010a,b). Simultaneous engineering of osmolyte accumulation and various other traits by transgenic technology therefore is a feasible strategy to improve abiotic stress tolerance in crops.

ACKNOWLEDGEMENTS Authors are indebted to Ilse Kranner, Csaba Papdi and Laura Zsigmond for proofreading and correcting the chapter. Research and this publication were supported by OTKA grant K-68226, Cross-Border Cooperation Programme HURO/0801/167 and COST Action FA0901.


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