ROS homeostasis in halophytes in the context of salinity stress tolerance.

Journal of Experimental Botany Advance Access published December 24, 2013 Journal of Experimental Botany doi:10.1093/jxb/ert430

Review paper

ROS homeostasis in halophytes in the context of salinity stress tolerance Jayakumar Bose1, Ana Rodrigo-Moreno2 and Sergey Shabala1,* 1

School of Agricultural Science and Tasmanian Institute for Agriculture, University of Tasmania, Private Bag 54, Hobart, Tas 7001, Australia 2 LINV, University of Firenze, Viale delle idee, 30, 50019 Sesto Fiorentino, Italy

Received 16 September 2013; Revised 31 October 2013; Accepted 12 November 2013

Abstract Halophytes are defined as plants that are adapted to live in soils containing high concentrations of salt and benefiting from it, and thus represent an ideal model to understand complex physiological and genetic mechanisms of salinity stress tolerance. It is also known that oxidative stress signalling and reactive oxygen species (ROS) detoxification are both essential components of salinity stress tolerance mechanisms. This paper comprehensively reviews the differences in ROS homeostasis between halophytes and glycophytes in an attempt to answer the questions of whether stress-induced ROS production is similar between halophytes and glycophytes; is the superior salinity tolerance in halophytes attributed to higher antioxidant activity; and is there something special about the specific ‘pool’ of enzymatic and non-enzymatic antioxidants in halophytes. We argue that truly salt-tolerant species possessing efficient mechanisms for Na+ exclusion from the cytosol may not require a high level of antioxidant activity, as they simply do not allow excessive ROS production in the first instance. We also suggest that H2O2 ‘signatures’ may operate in plant signalling networks, in addition to well-known cytosolic calcium ‘signatures’. According to the suggested concept, the intrinsically higher superoxide dismutase (SOD) levels in halophytes are required for rapid induction of the H2O2 ‘signature’, and to trigger a cascade of adaptive responses (both genetic and physiological), while the role of other enzymatic antioxidants may be in decreasing the basal levels of H2O2, once the signalling has been processed. Finally, we emphasize the importance of non-enzymatic antioxidants as the only effective means to prevent detrimental effects of hydroxyl radicals on cellular structures. Key words: Antioxidant, hydrogen peroxide, hydroxyl radical, ionic homeostasis, oxidative stress, plasma membrane, potassium, programmed cell death, ROS scavenging, sodium.

Introduction Oxidative stress is defined as the toxic effect of chemically reactive oxygen species (ROS) on plant structures. Detrimental effects of ROS are a result of their ability to cause lipid peroxidation in cellular membranes, DNA damage, protein denaturation, carbohydrate oxidation, pigment breakdown,

and an impairment of enzymatic activity (Scandalios, 1993; Noctor and Foyer, 1998). ROS production is known to be increased dramatically under stress conditions. While traditionally a rapid rise in ROS (known as an ‘oxidative burst’ phenomenon) was associated with plant responses to pathogens

Abbreviations: AOX, alternative oxidase; APX, ascorbate peroxidase; BADH, betaine aldehyde dehydrogenase; CAM, Crassulacean acid metabolism; CAT, catalase; CAX, Ca2+/H+ exchanger; DPPH, 1,1-diphenyl-2-picrylhydrazyl; GR, glutathione reductase; GPX, glutathione peroxidase; GST, glutathione S-transferase; MDAR, monodehydroascorbate reductase; MIPS, l-myo-ionsitol 1-phosphate synthase; NADPH, nicotinamide adenine dinucleotide phosphate, PCD, programmed cell death; P5CS, pyrroline-5-carboxylate synthetase; PDH, proline dehydrogenase; PLC, phospholipase C; PLD, phospholipase D; POX, peroxidase; Prx, peroxiredoxin; PTOX, plastid terminal oxidase; ROS, reactive oxygen species; SOD, superoxide dismutase; SQDG, sulphoqupnovosyldiacylglycerol. © The Author 2013. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: [email protected]

Downloaded from http://jxb.oxfordjournals.org/ at Belgorod State University on December 28, 2013

* To whom correspondence should be addressed. E-mail: [email protected]

Page 2 of 17 | Bose et al. to provide the answers to the following questions. (i) Is stressinduced ROS production similar between halophytes and glycophytes? (ii) Is the superior salinity tolerance in halophytes attributed to higher antioxidant activity? (iii) Is there something special about the specific ‘pool’ of enzymatic and nonenzymatic antioxidants in halophytes? (iv) Could targeting the antioxidant activity improve salinity stress tolerance in glycophyte species?

Major sources of ROS production in plants ROS are formed in all aerobic organisms as a result of a multistep reduction of oxygen (O2). The first step during oxygen reduction is the formation of superoxide (O2·–) that is quickly converted to hydrogen peroxide (H2O2) by the action of superoxide dismutases (SODs). In the presence of transition metals, this species can form the highly toxic hydroxyl radicals (OH·) (for a review, see Rodrigo-Moreno et al., 2013b). As cells do not have mechanisms to detoxify OH· enzymatically, plants can minimize the OH·-induced damage to cell membranes by preventing its formation, or by detoxifying it by non-enzymatic antioxidants. Another non-radical ROS that can be produced in plant cells is singlet oxygen (1O2). This ROS has a really short half-life as it is quenched by water (Asada, 2006). H2O2 is the most stable of the ROS. While the half-lives of 1O2, O2·–, and OH· are $6 billion losses in crop and livestock production (Shabala, 2012). The worldwide cost of drought events to agriculture is at least an order of magnitude higher. As the extent of the global land salinization and occurrence of the drought events are expected only to increase, due to the global climate change scenario, breeding crops for salinity and drought stress tolerance is absolutely essential for future food security. Halophytes are defined as plants that are adapted to live in soil containing a high concentration of salt and benefiting from it, and thus represent an ideal model to understand complex physiological and genetic mechanisms of salinity stress tolerance. Moreover, reduced water availability due to a high salt level is one of the detrimental components of salt stress, implying strong correlation between drought and salt stress tolerance. In recent years, several comprehensive reviews have been published dealing with various aspects of halophyte physiology and their use for the purpose of saline agriculture (Flowers and Colmer, 2008; Riadh et al., 2010; Ruan et al., 2010; Shabala and Mackay, 2011; Ben Hamed et al., 2013) illustrating the renewed interest of researchers in this topic. However, with a few possible exceptions (e.g. Ozgur et al., 2013), none of them has elucidated in detail the difference by which halophytes and glycophytes respond to oxidative stress. Filling this gap was one of the aims of this work. The causal link between ROS production and stress tolerance is not as straightforward as one may expect. Explicit evidence was presented suggesting that ROS integrate signalling pathways involved in plant growth, development, gravitropism, hormonal action, and many other physiological phenomena (reviewed in Mittler, 2002; Apel and Hirt, 2004; Mittler et al., 2004; Foyer and Noctor, 2005; Miller et al., 2008). In many of these cases, production of ROS is genetically programmed, and ROS are used as second messengers (Foyer and Noctor, 2005). Thus, it appears that ROS have pleiotropic effects in plants as they do in animals (Storey, 1996). When ROS are produced in a controlled manner within specific compartments, they have key roles in plant growth and development. When ROS are produced in excess, the resultant uncontrolled oxidation leads to cellular damage and eventual cell death. To prevent damage, yet allow beneficial functions of ROS to continue, the antioxidant defences must keep active oxygen under control (Noctor and Foyer, 1998). The main aim of this work was to fill the above gap in our knowledge and, by comparing halophytes with glycophytes,

ROS homeostasis in halophytes | Page 3 of 17

and disproportionation of O2·– (Baker and Graham, 2002; del Río et al., 2002; Foyer and Noctor, 2003). They can also generate nitric oxide (Corpas et al., 2001). There are also two sites for O2·– production: the organelle matrix and the peroxisomal membrane (Corpas et al., 2001). The ROS generation by peroxisomes play an important role during senescence (del Río et al., 2006) and these organelles are also involved in heavy metal toxicity (Romero-Puertas et al.,1999, 2002). The levels of mitochondrial ROS in leaves are considerably lower than in peroxisomes or chloroplasts, especially under light conditions, and are estimated to be 30–100 times lower than in chloroplasts (Foyer and Noctor, 2003). However, in non-green tissues, mitochondria would be the main source of

ROS. Indeed, in roots, mitochondria are considered as a major source of ROS production (Rhoads et al., 2006). The two main sites of mitochondrial ROS production are complexes I and III in the mitochondrial electron transport chain, where O2·– is generated and rapidly catalysed into H2O2 (Raha and Robinson, 2000; Møller, 2001; Sweetlove and Foyer, 2004). ROS are also produced at the apoplastic space by NADPH oxidases, cell wall-associated peroxidases that generate O2·– by oxidizing NADPH and transferring the electron to oxygen (O2) (Sagi and Fluhr, 2006). This apoplastic ROS generation has important roles in the hypersensitive response under pathogen attack and also in developmental and growth processes, as well as in controlling programmed cell death


Fig. 1. Major sites of ROS production in plant cells and means by which their levels are controlled.

Page 4 of 17 | Bose et al. (PCD) (Torres et al., 2002; Foreman et al., 2003; Gechev and Hille, 2005; Gapper and Dolan, 2006; Sagi and Fluhr, 2006). Also there is a contribution to apoplastic ROS generation by amine and germin-like oxidases, and by pH-dependent oxalate peroxidases (Bolwell and Wojtaszek, 1997; Hu et al., 2003; Walters, 2003).

ROS production under stress conditions: halophytes versus glycophytes Stress-induced ROS production associated with photosynthesis

Stress-induced ROS production associated with respiration As mentioned above, mitochondrial respiration is another major source of salt-induced ROS production. Over-reduction of the ubiquinone pool during salt stress allows the electrons to leak from complexes I and III of the mitochondrial electron transport chain to molecular oxygen, resulting in O2·– production (Noctor et al., 2007; Miller et al., 2010). Thus, prevention of over-reduction of ubiquinones is an essential step to decrease salt-induced ROS production in mitochondria. To achieve this objective, plants use the alternative oxidase (AOX) to remove electrons from the ubiquinone pool


Plants usually respond to salinity stress by decreasing stomatal conductance to minimize the water loss, which often limits the CO2 availability for carbon fixation by the Calvin cycle, resulting in light absorption exceeding the demand for photosynthesis (Jithesh et al., 2006b; Ozgur et al., 2013). Together with toxic effects of Na+ and Cl– accumulated in the cytosol, this excess excitation light affects the rate of electron transport through the photosystems by depleting the electron acceptors (QA in PSII and NADP in PSI), and also increases ROS (mainly O2·– and 1O2) production through the reduction of O2 (Vass et al., 1992; Allakhverdiev et al., 2002). The limited CO2 supply in C3 plants also increases the rate of photorespiration, resulting in additional ROS production (Foyer and Noctor, 2003). Thus, in order to decrease salt-induced ROS production in leaves, plants have to (i) employ mechanisms that can aid in maintaining net photosynthesis under a limited CO2 supply and/or (ii) use alternative electron sinks that can prevent ROS formation from O2. Halophytes appear to have a much greater ability to maintain net photosynthesis and protect and stabilize both photosystems under saline stress conditions, as compared with glycophytes. A comparison between rice (Oryza sativa L.) and its halophytic relative Porteresia coarctate has revealed that the latter was more efficient in the active protection of the photosynthetic machinery by increasing the abundance of (i) 33 kDa Mn-stabilizing proteins of the oxygen-evolving complex in PSII; (ii) a chlorophyll a/b protein (CP47) involved in stabilization of the reaction centre protein D1 of PSII; (iii) a PSI subunit IV protein essential for crosslinking ferredoxin-NADP+ oxidoreductase; (iv) RubisCo large subunit; and (v) RubisCo activase, when exposed to 400 mM NaCl stress (Sengupta and Majumder, 2009, 2010). Similarly, proteomic comparison studies between common wheat (Triticum aestivum) and an introgression hybrid between wheat and halophytic wheat grass (T. aestivum/Thinopyrum ponticum) reported that, under 200 mM NaCl treatment, the abundance of a CP24 protein precursor involved in stabilization of PSII was higher in hybrid plants compared with wheat parents (Wang et al., 2008; Peng et al., 2009). Also, an increase in the sulpholipid content as well as modification of the fatty acid profile of sulphoquinovosyldiacylglycerol (SQDG), known to protect PSII, was observed during the salt treatment in the halophytes Aster tripolium and Sesuvium portulacastrum but not in the glycophyte Arabidopsis thaliana (Ramani et al., 2004).

Another important aspect of halophytes regarding salinity is related to the switch between different modes of carbon assimilation. It was reported that some halophytes are able to change the mode of carbon assimilation from C3 (e.g. Portulacaria afra or Mesembryanthemum crystallinum) or C4 (e.g. Portulaca oleracea) to Crassulacean acid metabolism (CAM) during salt stress (Kennedy, 1977; Ting and Hanscom, 1977; Cushman et al., 1990). This switch may be advantageous in decreasing salt-induced ROS production because CAM plants are well equipped to handle salt-induced low water availability by opening stomata during the night-time while maintaining net photosynthesis during the daytime. Indeed, H2O2 production decreased following the expression of CAM in the wild-type plants of M. crystallinum whereas H2O2 production remained high in a CAM-deficient mutant, under 400 mM salt stress (Sunagawa et al., 2010). In addition, other halophytes such as Atriplex lentiformis showed a shift from the C3 to the C4 mode of carbon fixation in response to salinity stress (Meinzer and Zhu, 1999). This shift is also believed to be a part of the adaptation mechanism to decrease ROS damage because few studies have demonstrated that C3 plants are more prone to oxidative damage through photorespiration than their C4 counterparts during salt and drought stresses (Stepien and Klobus, 2005; Uzilday et al., 2012). Taken together, it appears that avoiding the C3 mode of carbon fixation is the strategy utilized by halophytes that enables a decrease in ROS production while maintaining photosynthesis during stresses. A comparison of photosynthetic response between the halophyte Thellungiella salsuginea and the glycophyte Arabidopsis during salt stress revealed that electron transport through PSII is inhibited in Arabidopsis whereas it is increased in Thellungiella. Moreover, a plastid terminal oxidase (PTOX) protein capable of diverting up to 30% of total PSII electron flow via plastoquinone to O2, producing water instead of O2·–, is substantially up-regulated in Thellungiella, under both control and salt stress conditions (Stepien and Johnson, 2009). Such up-regulation was not observed in Arabidopsis, suggesting that alterative electron sinks have the potential to decrease salt-induced ROS production in halophytes. However, transgenic tobacco (Nicotiana tabacum) plants overexpressing PTOX showed increased O2·– and OH· production (Heyno et al., 2009), questioning the role of PTOX in counteracting the salt stress-induced ROS production.


Enzymatic control of ROS levels in halophytes Production of ROS is an unavoidable event for all organisms exposed to oxygen. Plants have evolved complex antioxidant defence mechanisms that consist of enzymatic and nonenzymatic pathways to eliminate excessive ROS accumulation while maintaining an optimum level of ROS for signalling. The threshold ROS concentrations required to inflict oxidative damage seem to be different between glycophytes and halophytes. For example, in a range of halophytes, the first significant increase in lipid peroxidation [content of thiobarbituric acidreactive substances (TBARS)] in shoots occurred only when the salt concentration in the soil solution exceeded 150 mM (Ozgur et al., 2013). This concentration is lethal for the vast majority of glycophytes. A high antioxidant capacity of halophytes compared with glycophytes has been suggested as one of the important reasons for the superior ability of the former to tolerate high levels of salinity stress (Jithesh et al., 2006b; Flowers and Colmer, 2008; Kosová et al., 2013; Ozgur et al., 2013). However, only a few studies have made a direct comparison between glycophytes and halophytes to elucidate the difference in regulation of the antioxidant machinery. The available information from those studies is reviewed in the following sections.

Superoxide dismutase (SOD; EC 1.15.1.1) SODs dismutate O2·– into H2O2 and have been considered to act as the ‘first line of defence’ against oxidative stress in plants (Alscher et al., 2002). While in glycophytes the number of reports suggesting the existence of a positive correlation between SOD activity and salinity stress tolerance is counterbalanced by about the same number of reports showing no, or negative, correlation between these two traits (see Maksimovic et al., 2013, and references therein), it appears that halophytes have an exceptional ability to utilize SOD to protect themselves from extreme environmental changes (Jithesh et al., 2006b; Ozgur et al., 2013; Table 1).

Based on the metal cofactors, SODs are classified into three groups: (i) Fe SOD localized predominantly in the chloroplast; (ii) Mn SOD localized in the mitochondria and peroxisomes; and (iii) Cu/Zn SOD localized in the cytosol and chloroplast. Such a diverse distribution is suggestive of the global role of SODs in scavenging O2·– in plants (Alscher et al., 2002). As a general rule, up-regulation of Fe SOD and Mn SOD correlated well with the salt tolerance of several halophytes (Jithesh et al., 2006b), while changes in Cu/Zn SOD activity showed no clear trends in halophytes. However, overexpression of cytosolic halophytic Cu/Zn SOD of Avicennia marina in glycophyte indica rice improved oxidative and salt tolerance, implying that Cu/Zn SOD also plays a significant role in stress tolerance (Prashanth et al., 2008). Also, overexpression of Fe SOD in tobacco and of Mn SOD in rice conferred tolerance against salt and oxidative stresses (Van Camp et al., 1996; Tanaka et al., 1999). In plants, each SOD can exist as a different isoform and shows differential activity during stress adaptation in plants (Alscher et al., 2002; Jithesh et al., 2006b). Interestingly, some halophytes were able to synthesize new isoforms of SOD during salt stress, with their appearance being dependent on the duration of salt stress as well as the plant developmental stage (Ozgur et al., 2013). As H2O2 is the end-product of SOD activity, comparing the H2O2 accumulation pattern between halophytes and glycophytes during the initial hours of stress may yield a useful clue to ROS signalling and damage. Indeed, such a comparison was made between Cakile maritime (a halophyte) and A. thaliana (Ellouzi et al., 2011). In C. maritime, the H2O2 concentration reached a maximum within 4 h of salt stress and rapidly declined afterwards. In contrast, the H2O2 concentration continued to rise in A. thaliana during the entire observation period (72 h after the onset of salt treatment). This difference clearly suggests that halophytes are quick to send stress signals through H2O2 and have an efficient antioxidant mechanism to scavenge H2O2 upon completion of signalling.

Catalase (CAT; EC 1.11.1.6) The main role of CAT is in catalysing the decomposition of H2O2 into water and oxygen (Willekens et al., 1997). The turnover rate of CAT is very high; every second, one unit of CAT protein complex can decompose millions of molecules of H2O2 (Deisseroth and Dounce, 1970). Like SODs, CATs also have different isoforms localized mainly in peroxisomes, but a mitochondrial localization ahs also been noted in some species (Jithesh et al., 2006b). In a range of halophytes, increasing the salt concentration has yielded all the possible scenarios (increase, decrease, and no change) in CAT expression/activity (Jithesh et al., 2006b). Despite this, only a few studies made a direct comparison of CAT expression/ activity between halophytes and glycophytes (Table 1). In all the cases, CAT expression/activity is found to be higher in halophytes than in glycophytes, reaffirming the importance of CATs in scavenging H2O2 during stresses.


and transfer them to oxygen to form H2O (Millar et al., 2011). The halophytic Populus euphratica has a substantial (3.1- and 1.8-fold) increase in AOX expression following 200 mM salt shock and gradual salt adoption, respectively (Ottow et al., 2005). Moreover, increased AOX activity correlated with decreased ROS production after completion of salt-induced conversion of C3 to CAM in the common ice plant, M. crystallinum (Sunagawa et al., 2010), and facultative CAM plants such as Kalanchoe daigremontian had a constitutive cyanideresistant AOX (Robinson et al., 1992). Constitutively overexpressing AOX (Ataox1a) in Arabidopsis decreased ROS formation and improved growth by 30–40% during salt stress (Smith et al., 2009). Thus it appears that AOX plays a key role in decreasing ROS production during salinity stress in both glycophytes and halophytes. However, information regarding potential difference in protein structure or regulatory pathways of AOX between glycophytes and halophytes is not currently available in the literature.

Page 6 of 17 | Bose et al. Table 1. Comparison of antioxidant enzymes between halophytes and glycophytes Halophyte

Glycophyte

Observed effect

References

Rhizophora stylosa and Rhizophora mangle Thellungiella salsuginea Hordeum marinum Plantago maritime Cakile maritime Lycopersicon pennellii

Pisum sativum

A 10- to 40-fold higher SOD activity in the halophyte

Cheeseman et al. (1997)

Arabidopsis thaliana Hordeum vulgare Plantago media Arabidopis thaliana Lycopersicon esculentuma

Taji et al. (2004) Seckin et al. (2010) Sekmen et al. (2007) Ellouzi et al. (2011) Mittova et al. (2000, 2003); Shalata et al. (2001)

Hordeum marinum

Hordeum vulgare

Thellungiella salsuginea Plantago maritime

Arabidopis thaliana Plantago media

Cakile maritime Lycopersicon pennellii

Arabidopis thaliana Lycopersicon esculentuma

Rhizophora stylosa and Rhizophora mangle Plantago maritima

Pisum sativum

Constitutively higher expression of FeSOD in the halophyte Increased SOD activity in the halophyte Increased SOD activity in the halophyte Increased SOD activity in the halophyte Constitutively higher SOD activity in the halophyte Increased SOD activities in chloroplast, mitochondria, and peroxisomes of the halophyte Constitutively higher expression and increase in CAT activity in the halophyte Abundance of caltalase 3 isoform in the halophyte No change in CAT activity at 100 mM NaCl but increased at 200 mM NaCl in the halophyte Increased CAT activity in the halophyte Constitutively higher peroxisome CAT activity and further increased during salt stress in the halophyte Approximately 40% higher APX activity in the halophyte

Sekmen et al. (2007)

Lycopersicon pennellii

Lycopersicon esculentuma

Thellungiella salsuginea Hordeum marinum

Arabidopis thaliana Hordeum vulgare

Cakile maritima Plantago maritima

Arabidopis thaliana Plantago media

Hordeum marinum Thellungiella salsuginea Rhizophora stylosa and Rhizophora mangle Lycopersicon pennellii

Hordeum vulgare Plantago major Pisum sativum

Rhizophora stylosa and Rhizophora mangle Hordeum marinum

Pisum sativum

No change in APX activity in the glycophyte but increased at 200 mM NaCl in the halophyte Constitutively higher APX activity and further increased during salt stress in chloroplast, peroxisome, and mitochondrial fraction of the halophyte Abundance of three APX isoforms in the halophyte A new isoenzyme APX5 identified in the halophyte Intensities of some isoenzymes (APX1, 2, 6, 7) increased in the halophyte Increased POX activity in the halophyte Increased POX activity and large number of POX isozymes in the halophyte Increased POX activity in the halophyte Constitutively higher POX activity in the halophyte MDAR activities were similar between the halophyte and glycophyte MDAR activity increased in the halophyte but decreased in the glycophyte GR activities were lower in the halophyte

Plantago maritime

Plantago media




Lycopersicon esculentum (Solanum lycopersicum)


Hordeum vulgare

A new isoenzyme GR5 identified in the halophyte Activities of isoenzymes GR1, 3, 6, 7 increased in halophyte Increased GR activity in the halophyte but decreased in the glycophyte Constitutively higher GR activity in mitochondrial and peroxisome fractions of the halophyte but decreased during stress in both the glycophyte and halophyte Higher GPX activity in the mitochondrial fraction of the halophyte

Wang et al. (2004) Sekmen et al. (2007) Ellouzi et al. (2011) Mittova et al. (2000, 2003); Shalata et al. (2001) Cheeseman et al. (1997)

Mittova et al. (2000, 2003); Shalata et al. (2001) Wang et al. (2004) Seckin et al. (2010)

Ellouzi et al. (2011) Sekmen et al. (2007) Seckin et al. (2010) Radyukina et al. (2007) Cheeseman et al. (1997) Mittova et al. (2000, 2003); Shalata et al. (2001) Cheeseman et al. (1997) Seckin et al. (2010) Sekmen et al. (2007) Mittova et al. (2003)

Mittova et al. (2003)

Now renamed Solanum lycopersicum.

Ascorbate peroxidase (APX; EC 1.11.1.11) Like CATs, the APXs also scavenge H2O2. The APX isozymes use ascorbate as a reductant and are located in stroma of chloroplasts, mitochondria, cytosol, and the membrane of peroxisomes and chloroplasts (Shigeoka et al., 2002). As a general rule, up-regulation of APX expression/activity increases the stress tolerance of both glycophytes and halophytes (Shigeoka

et al., 2002; Jithesh et al., 2006b). Comparison of the APX activity/expression between halophytes and glycophytes also reaffirms this notion (Table 1).

Peroxidase (POX; EC 1.11.1.7) POXs are a family of isoenzymes capable of scavenging H2O2 primarily in the apoplastic space (Fagerstedt et al., 2010).


a

Plantago media

Seckin et al. (2010)

ROS homeostasis in halophytes | Page 7 of 17 However, more often, excretion of POXs can produce ROS that have been shown to play an active extracellular signal transduction role for stomatal closer and cell elongation (Kawano, 2003). Considering the enhanced POX activity in halophytes (Jithesh et al., 2006b) in comparison with glycophytes (Table 1), it will be interesting to evaluate what role (signalling or scavenging) the POXs are actually playing in halophytes.

Redox regulatory enzymes

Non-enzymatic control of ROS homeostasis From the above, it is evident that the majority of research has primarily focused on enzymatic scavenging systems of ROS. However, several highly toxic ROS such as 1O2 and OH· cannot be scavenged by enzymatic means. Thus plants depend solely on non-enzymatic means to scavenge those ROS. The major non-enzymatic players implicated in ROS tolerance of halophytes are summarized here.

Ascorbate and glutathione Ascorbate and glutathione are the most common non-enzymatic antioxidants in plants. The mechanism of ROS detoxification in halophytes through the ascorbate–glutathione cycle is extensively reviewed in Ozgur et al. (2013). It was shown that the halophyte Lycopersicon pennellii increased the contents of reduced ascorbate and glutathione and their redox states compared with its glycophyte relative L. esculentum (now renamed Solanum lycopersicum; Shalata et al., 2001), implying better ascorbate–glutathione cycle operation in the halophyte. However, to the best of our knowledge, the above study is the only direct comparison of this sort, and more studies are needed to generalize this conclusion to all halophytes.

Glycine-betaine Glycine-betaine is classified as a ‘compatible solute’ which is accumulated predominantly in chloroplasts and is traditionally associated with osmotic adjustment (Ashraf and Foolad, 2007). However, considerable evidence exists in the

Proline Accumulation of proline during environmental stresses is a common phenomenon. Apart from its long known compatible osmolyte function, proline has been shown to perform multiple antioxidant functions such as (i) decreasing/quenching 1O2, H2O2, and OH·; (ii) stabilizing ROS-scavenging enzymes; (ii) maintaining a low NADPH to NADP+ ratio thereby decreasing 1O2 generation from PSI; (iv) stabilizing mitochondrial respiration by protecting complex II of the mitochondrial electron transport chain; (v) decreasing the destructive effects of 1O2 and OH· on PSII of isolated thylakoid membranes; and (vi) preventing PCD during stress signalling, adaptation, and recovery (Pardha Saradhi and Mohanty, 1997; Szabados and Savouré, 2010). Exogenous


Redox regulatory enzymes such as monodehydroascorbate reductase (MDAR; EC 1.6.5.4), glutathione reductase (GR; EC 1.8.1.7), glutathione peroxidase (GPX; EC 1.11.1.9), glutathione S-transferases (GST; EC 2.5.1.18), thiol peroxidase type II peroxiredoxin (Prx; EC 1.11.1.15), and APX (see above) also perform antioxidant functions in glycophytes and halophytes (Mittler, 2002; Ozgur et al., 2013). Some of these enzymes showed higher activity in halophytes than in glycophytes (Table 1). Moreover, a few studies demonstrated that overexpression of MDAR from either Arabidopsis or A. marina can improve tolerance of multiple stresses in transgenic tobacco (Eltayeb et al., 2007; Kavitha et al., 2010).

literature that glycine-betaine can also play a key role in ROS homeostasis. Indeed, glycine-betaine stabilized the structure and functioning of the oxygen-evolving complex of PSII by preventing high salt (Na+ and Cl–)-induced dissociation of the regulatory extrinsic proteins (the 18, 23, and 33 kDa proteins) (Papageorgiou and Murata, 1995). Moreover many studies confirmed that relatively low concentrations of glycine-betaine (90 μmol dry weight) is well documented (Rhodes and Hanson, 1993; Flowers and Colmer, 2008; Fitzgerald et al., 2009). Transgenic crops overexpressing halophyte betaine aldehyde dehydrogenases (BADH; a glycine-betaine synthesizing enzyme) were shown to possess enhanced salt and drought tolerance (Fitzgerald et al., 2009, and references therein). Similarly, amphiploids generated from wide crossing of wheat with high glycinebetaine-accumulating halophytic relatives showed increased glycine-betaine in the expanding leaves and consequently improved salt tolerance (Flowers and Colmer, 2008). Interestingly, overexpression of a plastid BADH in salt-sensitive carrot has resulted in a >50-fold higher glycine-betaine production than in the wild type and showed remarkable salt tolerance (up to 400 mM NaCl), similar to halophytes (Kumar et al., 2004). The above findings could be attributed to both the osmotic role of glycine-betaine and its ROS scavenging ability. However, overexpression of BADH in tobacco, Arabidopsis, and rice (all natural non-accumulators of glycine-betaine) increased salinity and ozone stress tolerance in transgenic plants without reaching glycine-betaine levels sufficient to contribute significantly to the osmotic adjustment process (Fitzgerald et al., 2009). Therefore, the primary role of glycine-betaine in ROS detoxification is more plausible. The above notion, however, should be evaluated by studying the ROS accumulation pattern in overexpressor and non-expressor lines during salt stress.

Page 8 of 17 | Bose et al.

Polyamines Polyamines (e.g. putrescine, spermidine, and spermine) are organic polycations known to be involved in plant growth and development, PCD, signalling, gene expression, and adaptation to environmental stresses (Kuznetsov and Shevyakova, 2007; Takahashi and Kakehi, 2010). An increase in polyamines accumulation is a common phenomenon during a variety of stresses and usually correlates with plant stress tolerance (Kuznetsov and Shevyakova, 2007; Takahashi and Kakehi, 2010). The polyamines can affect ROS homeostasis in two

ways (Takahashi and Kakehi, 2010). First, polyamine accumulation can decrease ROS production by scavenging free radicals and/or activating antioxidant enzymes. Moreover, conjugated polyamines showed more antioxidant ability than free polyamines (Kuznetsov and Shevyakova, 2007). Secondly, polyamines can also increase ROS production through catabolism in the apoplast (Mohapatra et al., 2009). Recently, apoplastic polyamines have been shown to potentiate hydroxyl radical-induced K+ efflux (Zepeda-Jazo et al., 2011), and the extent of this potentiation correlated with salinity stress tolerance among barley genotypes (Velarde-Buendia et al 2012). Although the role of polyamines has been examined in many species, comparative studies between glycophytes and halophytes are rare in the literature. It was reported that the free polyamine concentrations in leaves of a glycophyte (Vigna radiata) are several fold higher than in halophytes (Pulicaria undulate, Mesembryanthemum forskahlei, and Atriplex halimus) (Friedman et al., 1989). In another study, halophytes (M. crystallinum and T. salsuginea) were able to increase conjugated polyamines at a greater rate than glycophytes (Plantago major and Geum urbanum) during UV-B exposure (Mapelli et al., 2008). From these observations, it appears that halophytes were able to maintain a low level of free polyamines and accumulate a high level of conjugated polyamines during stresses. Interestingly, salt stress inhibited the activity of polyamine-synthesizing enzymes (l-arginine decarboxylase and l-ornithine decarboxylase) in the polyamine-hyperaccumulating glycophyte Vigna radiata but not in the halophyte P. undulate (Friedman et al., 1989), suggesting that polyamine biosynthesis is still essential for halophyte adaptive responses. Also, T. salsuginea increased its free polyamine concentration for up to 2 d upon salinity exposure, followed by its decrease, whereas a glycophyte species, P. major, showed a continuous decline in free polyamine concentrations for the entire duration of the stress (Radyukina et al., 2007). This suggests that polyamine signalling may be the essential component of superior salinity tolerance in halophytes.

Tocopherols Tocopherols (α-, β-,γ-, and γ-forms) are lipid-soluble molecules belonging to the group of vitamin E compounds known as active antioxidants which detoxify 1O2 and lipid peroxyl radicals (Munne-Bosch and Alegre, 2002; Falk and MunnéBosch, 2010). The tocopherols show a superior ability to detoxify lipid peroxyl radicals, thereby preventing lipid peroxidation during environmental stresses (Munne-Bosch and Alegre, 2002; Szarka et al., 2012). The antioxidant ability of tocopherols against Fe2+-ascorbate-induced lipid peroxidation declined in the order of γ > β ≈ γ > δ, with each single molecule of tocopherols protecting up to 220, 120, 100, and 30 molecules of polyunsaturated fatty acids, respectively, before being consumed (Fukuzawa et al., 1982). Among the isoforms, α-tocopherol is the predominant form in plant green tissues. This isoform is synthesized in a plastid envelope and is stored in plastoglobuli of the chloroplast stroma and in thylakoid membranes, suggesting that α-tocopherol is pivotal to decreased ROS production


application of proline also decreased hydroxyl radicalinduced K+ efflux from Arabidopsis roots (Cuin and Shabala, 2007). Proline synthesis (in the presence of light) as well as its catabolism in the mitochondria (in darkness) results in distinct diurnal fluctuation of the proline level in plant tissues (Sanada et al., 1995; Szabados and Savouré, 2010). Though free proline accumulation is beneficial to decrease ROS stress, its catabolism in the mitochondria can increase ROS production (Miller et al., 2009). A comparative study of the diurnal fluctuation in proline levels between halophyte species of M. crystallinum and two glycophytes (Hordeum vulgare and T. aestivum) showed that these diurnal fluctuations were much wider in glycophyte species grown under saline stress conditions (Sanada et al., 1995). This finding led the authors to suggest that the ability of halophytes to control proline catabolism may be a key to salt tolerance. This notion is further supported by the fact that a proline-oxidizing enzyme, proline dehydrogenase (PDH), was suppressed in proline hyperaccumulating halophytic T. salsuginea in comparison with the glycophyte A. thaliana (Taji et al., 2004; Kant et al., 2006). The beneficial effect of proline hyperaccumulation on salt tolerance has been demonstrated in a range of halophyte species (Radyukina et al., 2007; Szabados and Savouré, 2010). Thellungiella salsuginea and Lepidium crassifolium, two halophytic wild relatives of Arabidopsis, accumulated more proline under control and salt-stressed conditions (Murakeözy et al., 2003; Taji et al., 2004; Ghars et al., 2008). Proline hyperaccumulation in T. salsuginea was shown to be a result of enhanced proline synthesis via pyrroline-5-carboxylate synthetase (P5CS) and reduced proline catabolism by PDH (Taji et al., 2004; Kant et al., 2006). Interestingly, in Arabidopsis, phospholipase D (PLD) functions as a negative regulator of proline accumulation under control conditions (Thiery et al., 2004) whereas phospholipase C (PLC) acts as a positive regulator of proline accumulation during salt stress (Parre et al., 2007). However, this regulation is opposite in Thellungiella, where PLD functions as a positive regulator and PLC acts as a negative regulator (Ghars et al., 2012). This opposite regulatory function exerted by T. salsuginea on proline accumulation suggests that halophytes have a ‘stress-anticipatory preparedness’ strategy (Taji et al., 2004). Overall, it appears that overexpression of P5CS and suppression of PDH is the best option the plants have to increase free proline to mitigate environmental stresses.


Polyols Polyols such as sorbitol, mannitol, myo-inositol, ononitol, or pinitol are polyhydric alcohols that accumulate in large quantities during environmental stresses (Williamson et al., 2002). Apart from being implicated in osmotic adjustment, these molecules are also found to be efficient in scavenging hydroxyl radicals. In vitro experiments have suggested the following order: pinitol ≈ ononitol > myo-inositol > mannitol ≈ sorbitol (Smirnoff and Cumbes, 1989; Orthen et al., 1994). Subsequent experiments showed that a pinitol extract from the halophytic common ice plant (M. crystallinum) showed 2-fold higher scavenging of the stable free radical DPPH (1,1-diphenyl-2-picrylhydrazl) than glycophytic lettuce (Lactuca sativa) (Agarie et al., 2009). In addition to direct scavenging, polyols can be involved in signalling and protection of cellular structures by interacting with membranes, proteins, and enzymes (Valluru and Van den Ende, 2011). Among the polyols, pinitol was found to be the major compatible solute in the majority of halophytic mangrove plants, with its concentration on a per plant water basis ranging between 40 mM and 230 mM (Popp et al., 1985; Sun et al., 1999; Sengupta et al., 2008). These concentrations were clearly higher than in glycophyte species reported to accumulate pinitol (e.g. Pisum sativum, Glycine max, Vigna spp; Ford, 1982; Streeter et al., 2001). Pinitol is synthesized from glucose-6 phosphate through the action of l-myo-inositol

1-phosphate synthase (MIPS; gene INO1or INPS1) and inositol O-methyl transferase (gene IMT1) enzymes (Sengupta and Majumder, 2010). Although induction of INO1 and IMT1 homologues has been observed in a halophytic wild relative of rice (P. coarctata) and common ice plant (M. crystallinum) (Agarie et al., 2009; Sengupta and Majumder, 2010), such induction was absent in glycophytes such as Arabidopsis or tobacco (Ishitani et al., 1996; Sheveleva et al., 1997), suggesting that pinitol synthesis is the main determinant of salt tolerance between these two groups of plants. The purified PcINO1 enzyme showed unhindered activity up to 500 mM NaCl treatment in vitro, whereas the cultivated rice (O. sativa) homologue OsINO1 showed a progressive inhibition with increased salinities even at NaCl 600 compounds identified) are involved in quenching of 1O2 and peroxyl radicals that are generated during excess excitation of chlorophyll (Neubauer and Yamamoto, 1992; Demmig-Adams and Adams, 1996). Application of norflurazon, an inhibitor of carotenoid biosynthesis, increased photo-oxidative damage, mainly through the production of 1O2 (Knox and Dodge, 1985). Thus, maintaining a high carotenoid concentration may protect plants from 1O2 damage during environmental stresses. Compared with wheat, the halophytic monocot grass Chloris virgata had a constitutively high concentration of carotenoids under control conditions, and showed a much smaller decrease in the carotenoid concentration following exposure to increased salt concentrations (Yang et al., 2009). Similar results were reported from a comparative study of Arabidopsis and its halophytic relative Thellungiella. Thellungiella leaves showed higher expression of a PTOX protein (which acts as a cofactor in carotenoid biosynthesis) under both control and saltstressed conditions (Carol and Kuntz, 2001; Stepien and Johnson, 2009). It appears that glycophytes do not increase their carotenoid content during salt stress (see Koyro, 2006), while numerous papers report accumulation of carotenoids in halophytes (e.g Aghaleh et al., 2009; Youssef, 2009). This may

be indicative of the important role of carotenoids in detoxification of 1O2 in halophytes.

Polyphenols Polyphenols are a group of water-soluble antioxidants with a superior ability to scavenge O2·–, OH·, H2O2, and 1O2 compared with lipid-soluble carotenoids (Sakihama et al., 2002; Frary et al., 2010). In plants, >8000 phenolic compounds have been identified. Of these, flavonoids are the largest and beststudied group (Sakihama et al., 2002). There are many reports of polyphenol accumulation and enhanced antioxidant ability in halophytes (Basak et al., 1996; Ksouri et al., 2012b; Ozgur et al., 2013). The total polyphenol content (measured in gallic acid equivalents g–1 dry weight) was twice as high in halophyte species such as Tamarix gallica, Limoniastrum monopetalum, Limoniastrum guyonianum, Suaeda fruticosa, and Mesembryanthemum edule compared with glycopytes (Mentha pulegium and Nigella sativa) (Ksouri et al., 2012a, b). Moreover, shoot extracts from halophytes showed superior Fe-reducing power and DPPH radical-scavenging ability compared with glycophytes (Ksouri et al., 2012a, b), implying that polyphenols are also a key player to decrease ROS damage in halophytes. Plant phenolics are readily oxidized by the copper-containing polyphenol oxidase enzyme into highly reactive quinones, normally under circumstances of tissue damage (Vaughn and Duke, 1984; Mayer, 2006). These quinones alkylate proteins, leading to the commonly observed brown pigments in damaged plant tissues (Mayer, 2006). Interestingly, polyphenol oxidase activity was highest in salt-sensitive wheat and barley cultivars, intermediate in salt-tolerant genotypes, and least in halophytes (Desmostachya bipinnata, Panicum antidotale, Diplachna fusca, and Sporobolus marginatus) (Sharma et al., 1983), suggesting that salt stress induces tissue damage in glycophytes but not in halophytes.

Sulphated polysaccharides Sulphated polysaccharides such as glucans, galactans, and arabinogalactans are a group of water-soluble sugars mainly present in marine algae (Arad and Levy-Ontman, 2010). The extracts from marine algae show excellent ability to scavenge many ROS including hydroxyl radicals, oxygen radicals, H2O2, and DPPH radicals in vitro (Hu et al., 2001; Rupérez et al., 2002; Barahona et al., 2011). However, the in vivo role of these sulphated polysaccharides in ROS homeostasis has not been deciphered in plants. A recent survey found that accumulation of sulphated polysaccharides was observed only in halophytic plants (Ruppia maritima, Halophila decipiens, Halodule wrightii, Avicennia schaueriana, Rhizophora mangle, and Acrostichum aureum) but not in glycophytes (Zea mays, O. sativa, and Phaseolus vulgaris), suggesting that halophytes conserved this ability during plant evolution from marine algae (Aquino et al., 2011). Moreover, an increase in sulphated polysaccharide concentration was observed only when salt was present in the growth media (Aquino et al., 2011). Elucidating the role and synthetic pathways of


use ferritin, an iron-binding protein, to decrease the availability of free iron. A single molecule of ferritin can store up to 4500 iron atoms in its cavity in a soluble and bioavailable form (Theil, 1987; Briat et al., 1999). Involvement of ferritin in ROS homeostasis was confirmed when application of antioxidants (glutathione or N-acetylcysteine) completely abolished the iron-induced increase in abundance of ferritin mRNA (ZmFer1) in de-rooted maize plantlets and application of H2O2 promoted accumulation of this transcript (Lobréaux et al., 1995; Savino et al., 1997). Such induction of ferritin by H2O2 may play an important role during H2O2mediated signal propagation in response to environmental stresses. Many glycophytes use ferritin to decrease free iron availability during environmental stresses (Jithesh et al., 2006b). However, only a few studies examined the role of ferritin in halophytes. A histological study in halophytic M. crystallinum found that salt stress increased the abundance of ferritin deposits in all the cellular organelles where H2O2 is known to accumulate, including the chloroplast (Paramonova et al., 2004). Similarly, a halophytic green microalga (Dunaliella salina), found in particular in sea salt fields, accumulated triplicated transferrin-like protein (Liska et al., 2004). Moreover, the transcripts of the ferritin gene (Fer1) in the leaves of a mangrove A. marina increased transiently (within 12 h) but declined afterwards (Jithesh et al., 2006a). From the above evidence, it is plausible to suggest that halophytes use ferritin to prevent hydroxyl radicle formation in the early phase of H2O2-mediated signalling. Comparative studies on the fate of free iron during oxidative stress between glycophytes and halophytes may shed light on the difference in antioxidant capacity between these two groups of plants.

ROS homeostasis in halophytes | Page 11 of 17 sulphated polysaccharides during salt stress in halophytes may pave the way to improving salt tolerance in glycophytes.

Lessons for breeders and a way forward

Acknowledgements This work was supported by the Australian Research Council and Grain Research and Development Corporation grants to SS.

References Agarie S, Kawaguchi A, Kodera A, Sunagawa H, Kojima H, Nose A, Nakahara T. 2009. Potential of the common ice plant, Mesembryanthemum crystallinum as a new high-functional food as evaluated by polyol accumulation. Plant Production Science 12, 37–46. Aghaleh M, Niknam V, Ebrahimzadeh H, Razavi K. 2009. Salt stress effects on growth, pigments, proteins and lipid peroxidation in Salicornia persica and S. europaea. Biologia Plantarum 53, 243–248.


The idea of improving abiotic (and, specifically, salinity) stress tolerance by increasing antioxidant activity has long been, and still remains, attractive to plant breeders. A simple search on the Web of Science for three keywords ‘antioxidant’, ‘breeding or improvement’, and ‘plant’ reveals 791 entries. Unfortunately, while half of these reports claim a positive association between antioxidant production in plant tissues and plant salinity tolerance, the other half showed no, or even negative, correlation between these two traits. Several factors may contribute to this controversy. First, truly salt-tolerant species possessing efficient mechanisms for Na+ exclusion from the cytosol may not require a high level of antioxidant activity, as they simply do not allow excessive ROS production in the first instance. Such exclusion is most probably achieved by the orchestrated action of several complementary mechanisms including SOS1-mediated Na+ exclusion from the cell (Shi et al., 2000), vacuolar Na+ sequestration mediated by NHX tonoplast Na+/H+ exchangers (Blumwald, 2000), and efficient control of tonoplast fast (FV) and slow (SV) channels to prevent Na+ back-leak into the cytosol (Bonales et al., 2013a, b). Therefore, most probably, increased antioxidant activity should be treated as a damage control mechanism rather than a trait directly conferring salinity stress tolerance. In the light of the above, why then do halophytes often possess intrinsically higher levels of enzymatic antioxidants and especially SOD (Table 1)? The plausible answer may be found in the fact that the major role of SOD is in a rapid conversion of O2·– to H2O2. As the latter plays an important role as a second messenger triggering cascades of adaptive responses (at both the genetic and physiological levels, as discussed above), it may be envisaged that the rapid conversion of O2·– to H2O2 may be essential for early defence signalling in halophytes. In other words, it is not a detoxifying role of SOD but the fact that halophyte species are capable of rapidly inducing H2O2 levels, having enough SOD ‘in stock’, which gives them a certain adaptive advantage over glycophytes. While it still remains to be answered how the stress-induced increase in H2O2 production is ultimately converted into plant adaptive responses (Miller et al., 2010), the high tissue specificity and time dependence of H2O2 production allow us to propose that H2O2 ‘signatures’ may operate in plant signalling networks, in addition to well-known cytosolic calcium ‘signatures’ (Dodd et al., 2010). The specific details of such H2O2 ‘signatures’ and decoding mechanisms involved is the subject of a separate discussion. Building upon suggested qualitative similarities between Ca2+ and H2O2 stress-induced ‘signatures’, the role of other enzymatic antioxidants may be attributed to the need to decrease the basal levels of H2O2, once the signalling has been processed. In this context, the role of APX and CAT in the shaping of H2O2 signatures may be very similar to those that Ca2+ efflux systems (such as Ca2+-ATPases and CAX Ca2+/H+

exchangers) play in restoring the basal cytosolic Ca2+ levels (Bose et al., 2011). More work is needed to put a solid experimental base under this hypothesis. In the light of the above, it appears to be counterproductive to target overexpressing major antioxidant enzymes in crops, as, for example, constitutively high levels of APX and CAT may interfere with H2O2 signalling and diminish or completely abolish the beneficial role of H2O2 signalling. Also, given the number of enzymatic antioxidants and their isoforms, manipulating crop antioxidant activity by means of marker-assistant selection (MAS) is hardly practically feasible. This notion is supported by the recent report of Frary et al. (2010) who identified 125 quantitative trait loci for antioxidant content in experiments with an introgression population of tomatoes. For obvious reasons, this number is far too big to be handled practically in breeding programmes. While neither O2·– nor H2O2 per se appears to be completely harmful to plants, formation of hydroxyl radicals as a result of interaction between H2O2 and transition metals (Rodrigo-Moreno et al., 2013a, b) is a major concern. Not only is OH· highly reactive and causes significant damage to cell structures (as discussed above), it is also known to activate directly a range of Na+-, K+-, and Ca2+-permeable cation channels (Demidchik et al., 2002, 2003, 2010; Zepeda-Jazo et al., 2011) altering the cytosolic K+/Na+ ratio and triggering PCD (Huh et al., 2002; Shabala et al., 2007; Shabala, 2009; Demidchik et al., 2010). Thus, keeping OH· levels under control appears to be absolutely essential for plant survival under salt stress conditions. At the same time, none of the existing antioxidant enzymes has the capacity to scavenge OH·, and the only way to achieve this goal is via control of non-enzymatic antioxidants. Thus, according to our view, the breakthrough in breeding plants for increased salt stress tolerance may be achieved via two concurrent avenues: (i) understanding mechanisms responsible for generation and deciphering of H2O2 ‘signatures’; and (ii) revealing the role and interaction with signal transduction pathways responsible for stressinduced induction of non-enzymatic antioxidants involved in OH· scavenging and protecting the key cellular structures against its toxic action.

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Proteomics, metabolomics, and ionomics perspectives of salinity tolerance in halophytes.

Salinity Tolerance Mechanism of Economic Halophytes From Physiological to Molecular Hierarchy for Improving Food Quality.

Physiological and molecular mechanisms mediating xylem Na+ loading in barley in the context of salinity stress tolerance.

Genomics Approaches For Improving Salinity Stress Tolerance in Crop Plants.

Native-Invasive Plants vs. Halophytes in Mediterranean Salt Marshes: Stress Tolerance Mechanisms in Two Related Species.

Role of glycine in improving the ionic and ROS homeostasis during NaCl stress in wheat.

Constitutive expression of a salinity-induced wheat WRKY transcription factor enhances salinity and ionic stress tolerance in transgenic Arabidopsis thaliana.

Mitochondrial ROS signaling in organismal homeostasis.

A novel Azotobacter vinellandii (SRIAz3) functions in salinity stress tolerance in rice.

Evaluating contribution of ionic, osmotic and oxidative stress components towards salinity tolerance in barley.

Proteome Analysis for Understanding Abiotic Stress (Salinity and Drought) Tolerance in Date Palm (Phoenix dactylifera L.).

Linking salinity stress tolerance with tissue-specific Na(+) sequestration in wheat roots.

Potassium Retention under Salt Stress Is Associated with Natural Variation in Salinity Tolerance among Arabidopsis Accessions.

Nox-mediated ROS production under salinity stress.

Salinity tolerance in plants. Quantitative approach to ion transport starting from halophytes and stepping to genetic and protein engineering for manipulating ion fluxes.

Regulation of Na+ and K+ homeostasis in plants: towards improved salt stress tolerance in crop plants.

Tuning of Redox Regulatory Mechanisms, Reactive Oxygen Species and Redox Homeostasis under Salinity Stress.

Soybean Salt Tolerance 1 (GmST1) Reduces ROS Production, Enhances ABA Sensitivity, and Abiotic Stress Tolerance in Arabidopsis thaliana.

Guest editor's introduction: Energy homeostasis in context.

Macrophage iron homeostasis and polarization in the context of cancer.

The role of ethylene and ROS in salinity, heavy metal, and flooding responses in rice.

Response of PiCypA tobacco T2 transgenic matured plant to potential tolerance to salinity stress.

Effect of tempol on redox homeostasis and stress tolerance in mimetically aged Drosophila.

ROS-mediated Different Homeostasis of Murine Corneal Epithelial Progenitor Cell Line under Oxidative Stress.