The role of mitochondrial respiration in salinity tolerance

Review

The role of mitochondrial respiration in salinity tolerance Richard P. Jacoby, Nicolas L. Taylor and A. Harvey Millar ARC Centre of Excellence in Plant Energy Biology and Centre for Comparative Analysis of Biomolecular Networks, M316, The University of Western Australia, Crawley, WA 6009, Australia

NaCl is the most abundant salt in salinity-affected land. The ability of plants to sift the water table, limit NaCl uptake, compartmentalise Na+/Cl– ions and prevent negative ionic and osmotic effects on cell function, are the foundations of salinity tolerance mechanisms. In this review, we show that although the quantitative response of respiratory rate to changes in salt concentration is complex, the properties of respiratory processes are crucial for tolerance during ion exclusion and tissue tolerance. We consider whole-plant gas exchange and carbon balance analysis alongside the salt responses of mitochondrial properties and genetic studies manipulating respiratory processes. We showcase the importance of efficient ATP generation, dampened reactive oxygen species and mitochondrial osmolytes for salinity tolerance in plants. Causes and consequences of salinity Salinity describes soils that contain high concentrations of water-soluble salts, mainly NaCl. Salinity is a major impairment to agricultural production owing to the toxic effects of these salts upon plant growth. Salinity can be caused by rising saline groundwater or the combination of poor drainage and poor-quality irrigation [1]. The hazardous effects of salinity are forecast to increase in the coming decades owing to increased reliance on marginal land, the continued use of poor-quality irrigation water in the developing world, as well as the recognition that the biological consequence of salinity often lags behind its hydrological causes by decades [2,3]. In a laboratory context, salinity is a well-studied environmental stress and an excellent example of abiotic stress signalling [4,5]. However, salt stress is still an enigma because of the overlapping impact of salt on plant function and development, as well as the complexity of avoidance versus tolerance mechanisms used by tolerant plant species (Box 1) [6]. Osmotic stress (see Glossary) occurs as salinity lowers the osmotic potential (p) of the soil; therefore, to maintain flux of water from root to shoot, plants must either assimilate toxic ions or synthesise high concentrations of osmolytes. In this respect, there is significant overlap in the nature of salt and drought stress on plants [7]. Ionic stress occurs when Na+ and Cl– ions enter the transpiration stream and ultimately accumulate in leaf tissue. High Na+ and Cl– concentrations are toxic to many cellular processes because they alter protein– protein interactions, disrupt electrochemical gradients Corresponding author: Millar, A.H. ([email protected]).

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across membranes, increase cellular reactive oxygen species (ROS) production and displace vital ionic cofactors of enzymes [6,8,9]. Understanding the respiratory contribution to salinity A broad understanding of how cellular respiration aids salinity tolerance can be gained by integrating data from molecular to whole-plant responses. Two important foundations underpin this approach. First, the detrimental effects of salinity stress at physiological and cellular levels and the mechanisms of salinity tolerance deployed by resistant species have been explored (reviewed in [6,8,10]). Second, the role of mitochondrial respiration in plants has been defined [11,12], and the responses of glycolysis, the tricarboxylic acid (TCA) cycle and the electron transport chain (ETC) to salinity stress have been documented using physiological, biochemical and ‘omics techniques [13–17]. By integrating the molecular changes Glossary ADP/O ratio: the amount of oxygen consumed by isolated mitochondria during ADP-stimulated respiration relative to the amount of ADP added. Alternative pathway: the plant-specific mitochondrial ETC terminating at alternative oxidase. Anaplerotic: describes metabolites that are produced by catabolism and consumed by anabolism, such that their provision for anabolism is dependent on continued catabolic flux. Carbon balance: a physiological framework where plant growth is the sum total of carbon fixed through photosynthesis minus carbon lost by respiration. C/N: the ratio of free organic acids to free amino acids. Cytochrome pathway: the classical mitochondrial ETC terminating at cytochrome oxidase. Growth respiration: the proportion of respiration that is allocated to producing ATP to fuel the growth of new tissue. Ion exclusion: the ability of roots to limit uptake of Na+ and Cl– ions. Ionic stress: cellular toxicity caused by high concentrations of Na+ and Cl– ions. Maintenance respiration: the proportion of respiration that is allocated to producing ATP to fuel the maintenance of existing tissue. Non-stomatal photosynthetic limitations: reductions in photosynthesis caused by biochemical limitations rather than by stomatal closure. Osmotic stress: growth impairment caused by low turgor. Osmotic tolerance: the ability to maintain transpiration and turgor under high salinity. Osmolytes: organic molecules that accumulate to high concentrations under salinity to enable water uptake and balance osmotic potential in the vacuole. Protein turnover: the rate at which old proteins are degraded and new proteins are synthesised. Respiratory homeostasis: the ability to maintain constant respiration rates under differing environmental conditions. Retrograde signalling: the process by which organellar signals regulate nuclear gene expression. Tissue tolerance: the ability of leaves to maintain cellular function despite high concentrations of Na+ and Cl– ions. Vacuolar compartmentation: the ability to sequester high concentrations of Na+ and Cl– ions in the vacuole to prevent cellular toxicity.

1360-1385/$ – see front matter . Crown Copyright ß 2011 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.tplants.2011.08.002 Trends in Plant Science, November 2011, Vol. 16, No. 11

Review

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that occur under salt stress with physiological studies of salinity tolerance, we aim to show how respiratory properties are strongly linked to salinity tolerance in a variety of contexts and in a range of plant tissues (Figure 1). In this review, we begin by considering the nature of salinity and its physicochemical impact on plants. We then consider the contribution of carbon balance analysis and the diversity of molecular responses of plant mitochondria to salt stress. Finally, we consider their integration to showcase the importance of respiratory properties in different plant tissues during salinity stress.

Box 1. Division of tolerance mechanisms in plants Salinity tolerance strategies for plants involve a trade-off between mitigation of osmotic stress and ionic stress. Arguably, the easiest way to avoid osmotic stress is to take up Na+ or Cl–, which imposes ionic stress, whereas the easiest way to avoid ionic stress is to exclude Na+ or Cl–, which exacerbates the osmotic stress. Strategies that alleviate the osmotic component of salinity stress are called osmotic tolerance, and typically involve production of osmolytes to promote water uptake into cells. Meanwhile, two separate strategies are used to cope with the ionic component of salt stress: ion exclusion and tissue tolerance. Ion exclusion is the ability to prevent Na + and Cl– from accumulating in leaves, whereas tissue tolerance is the ability to maintain cellular function despite high concentrations of these ions in leaves. Ion exclusion is the better characterised of these two mechanisms, and it involves direct exclusion of Na+ at the epidermis, as well as retrieval of Na+ ions from the xylem [6]. Molecular mechanisms of tissue tolerance are poorly characterised, but a major component probably involves the vacuolar sequestration of Na+ ions. There is direct evidence that Na+ and Cl– ions are compartmentalised in the vacuole [114,115], and notably the Na+ concentrations typically found in salt-exposed shoot tissue significantly inhibit the activity of many metabolic enzymes [116–118]. However, not all Na+ and Cl– ions are excluded from the cytosol [114], meaning that they inevitably interfere with metabolic processes. A significant demand is placed on cytosolic ATP provision to maintain these transport processes against concentration gradients [29]. This accounts for the elevated percentage of respiration dedicated to maintenance in plants under salt stress [36,37].

Carbon flux balance analysis

Tissue tolerance

Salinity and tolerance

Respiration and salinity

Ion exclusion

Root transport and energetics

Respiration rates, carbon balance and salinity tolerance Biomass accumulation in plants, which is often adversely affected by salinity, is largely determined by the balance between photosynthesis and respiration. There is often a strong negative correlation between growth rates and the amount of daily fixed carbon that is respired [18–20], although this phenomenon is dependent on growth conditions [21]. In data collected across a range of species, it is evident that 25–60% of assimilated CO2 is lost through respiration over the course of each day [18,22]. Respiratory gas exchange measurements and salinity The key reactions of O2 consumption or CO2 production from respiring plants can be measured through gas exchange from plant tissues, whole plants, field sites and

Energy efficiency ROS and ROS signalling

ETC inhibition

Mitochondrial metabolism

Mitochondrial function and salt response Energy for ion exclusion and vaculolar compartmentation

Metabolites and osmotic homeostatis

Organic acids ATP GABA Proline

NADH

Na + Plasma membrane Tonoplast membrane Na +

Root respiration ATP formation and Na + transport ATP

O2 CO2

ADP ATP ATP Mitochondrial membrane

O2

NADH ROS ATP Carbon-use efficiency and

ROS-induced stress and

p hotosynthesis support

signalling TRENDS in Plant Science

Figure 1. Salinity and respiratory function across scales. Salinity tolerance strategies can be divided into tissue tolerance and ion exclusion, both of which require the cooperation of respiratory processes. Ion exclusion is a major cellular ATP demand in roots, and tissue tolerance requires carbon flux balance as salts accumulate in shoot cells. The ionic and osmotic stress imposed during salt accumulation in cells affects respiratory processes and can be balanced by mitochondrial processes and osmolytes. Maintenance of ATP provision and oxidation of redox equivalents to support photosynthesis is a crucial role for mitochondria. Finally, increasing mitochondrial reactive oxygen species (ROS) production under saline conditions can not only alter mitochondrial function, but also acts as a signalling agent to coordinate a transcriptional response to salinity. Abbreviation: ETC, electron transport chain; GABA, gamma-aminobutyric acid.

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even ecosystems [18,23–25]. However, the respiratory rate responses to salinity in plant tissues are complex, with 37% of studies reporting increases, 34% reporting decreases and 29% reporting no consistent change in respiratory rate, based on our analysis of the current literature (Figure 2; Table S1 in the supplementary material online). This variability in respiratory responses was also noted in a review of respiration rates under drought stress [26].

Although the more general decrease in photosynthesis in the face of salinity can be largely attributed to lower CO2 availability through stomatal closure, there is no such limitation on cellular O2 concentrations under salt stress. This leaves the control of respiration rates to substrate supply and biochemical regulation, which probably vary significantly between species. In this respect, the respiratory response to salinity is more akin to the long-term

Tolerant plant Respiratory response

Physiological trait

Biochemical mechanism Provide ATP to fuel defence processes (ie vaculolar compartmentation, protein turnover)

Tissue tolerance Shoot respiration rate increases

Robust ROS defences that enable high respiratory flux despite high ion concentration

Osmotic tolerance Shoot respiration rate decreases Positive carbon balance

Provision of ATP and carbon skeletons for synthesis of compatible solutes Low proportion of fixed carbon allocated to respiration

Root respiration rate increases

Ion exclusion

Provide ATP to fuel ion exclusion

Root respiration rate decreases

Positive carbon balance, reliance on tissue tolerance

Decreased carbon allocation to roots

Sensitive plant Respiratory response

Physiological trait

Biochemical mechanism High proportion of fixed carbon expended by shoot respiration

Shoot respiration rate increases

Negative carbon balance

Shoot respiration rate decreases

Lack of tissue tolerance

Damaged respiratory components High flux unattainable owing to inadequate ROS defences

High proportion of fixed carbon expended by root respiration

Root respiration rate increases

Negative carbon balance

Root respiration rate decreases

Lack of ion exclusion

Damaged respiratory components Inadequate ATP provision does not meet demand of ion exclusion TRENDS in Plant Science

Figure 2. Complexity of respiratory responses to salinity tolerance and susceptibility. The link between salt tolerance and respiratory rate response to salinity is complex, with evidence supporting the value of higher, lower, or stable respiration rates in particular circumstances leading to susceptibility or tolerance. However, although respiratory rate per se might not be a key determinant of salinity tolerance, we argue that the biochemical mechanisms underpinning respiratory rates can aid physiological traits associated with salinity tolerance, so the respiratory component of salinity tolerance involves optimal deployment of these mechanisms. Abbreviation: ROS, reactive oxygen species.

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Review variation in non-stomatal photosynthetic reduction during salinity [27,28]. There is currently no clear expectation of the respiratory rate response to variance in whole-plant salinity tolerance from the literature. Through collation of literature, we have found that tolerant plants displayed an increased respiratory rate in 33% of reports and a decreased rate in 42% of reports, whereas sensitive plants displayed an increased respiratory rate in 58% of reports and a decreased rate in 17% of reports (Table S1 in the supplementary material online). Although these numbers might suggest a trend toward respiratory rate increases in sensitive plants, they also indicate that high respiration rates under salinity can be either beneficial or detrimental to growth rate, depending on organ and tolerance strategy (Figure 2). The benefit of a high respiration rate is that more ATP is produced, which provides vital energy for growth of new tissue and defence processes, such as osmotic adjustment, sodium exclusion or tissue tolerance [29]. However, the cost of high respiration rates is that carbon is expended on respiration instead of being allocated to synthesis of new tissue, therefore limiting growth capacity [18]. There is evidence that tolerant respiratory homeostasis in the shoot is linked to salt tolerance [30,31], although it would be desirable to confirm this link in a wider range of species. In roots, high respiration rates can enhance salinity tolerance by facilitating ion exclusion in rice [32], although certain halophytes show decreases in root respiration under salinity [33,34], perhaps because they are deploying their carbon reserves in the shoot to mediate tissue tolerance. As a result, it has been unclear which gross respiratory response to screen for in a bid to find tolerance in a particular tissue or species. Respiratory carbon balance analysis under salinity The prevalent framework for understanding plant biomass accumulation uses the concept of carbon balance, which positions growth as the sum of carbon fixed through photosynthesis minus the carbon consumed by respiration, and allocates net assimilated carbon to roles within plant tissue. This approach correlates well with growth rate under control conditions across a wide range of species [18]. Investigations of carbon balance under salinity show that salt stress can slightly slow respiration rate, but that the net carbon balance of the plant is still lower under salinity, owing to the dramatic reduction in photosynthesis caused by salt stress [35,36]. Thus, the contribution of respiratory changes under salt stress seems to be secondary compared with the photosynthetic contribution, because respiration rates change within a relatively narrow range compared with the dramatic decreases in photosynthesis [14]. In the context of control versus salinity treatments, this argument seems to hold true, given that growth rate will always be dramatically slower under salt stress and photosynthetic rate is probably the major determinant of this. However, respiratory changes might be crucial to determining the relatively small growth differences between plant varieties under salt stress. For instance, a sensitive wheat (Triticum aestivum) variety can exhibit a dramatically increased shoot respiration rate under salinity, whereas the tolerant variety maintains

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respiratory homeostasis under salinity [30], suggesting that the tolerant variety allocates less of its fixed carbon into respiration, and more into growth. Growth versus maintenance respiration changes under salinity Another framework for understanding growth is the division of total respiration rate into two components: growth respiration (which fuels the synthesis of new tissue), and maintenance respiration (which fuels the preservation of existing tissue). This explanation for reduced growth under salinity stress was explored during the 1980s [36,37]. Data showed that salinity usually increases maintenance respiration, leading to the theory that increased maintenance respiration is an adaptive mechanism present in tolerant species [36]. Maintenance respiration in salinity tolerance is linked to processes such as vacuolar compartmentation, synthesis of osmotica and protein turnover, which all consume significant amounts of cytosolic ATP [29]. Mitochondrial function and the response to salinity The primary function of mitochondria is ATP generation via oxidative phosphorylation. This provides researchers with a mechanistic platform to consider links between molecular properties, cellular function and whole-plant physiology in response to salinity in terms of energy provision. However, the electron chemistry of oxygen leading to ROS generation [38], the plant-specific aspects of respiration [11] and the anaplerotic role of plant mitochondrial metabolism [39] can widen this picture to provide a broader appreciation of the mitochondrial involvement in the salinity response. Analyses of mitochondria isolated from plants subjected to saline conditions show that respiratory rate is inhibited in 90% of reports (Table S2 in the supplementary material online). These reports show the capacity of respiratory enzymes and pathways in isolated organelles are decreased, but do not necessary mean that the activity of respiration in vivo will be altered in the same way. However, a variety of other mitochondrial activities appear to be stimulated by salinity treatment, giving clues to the impact of pathway capacity changes on organelle function in vivo. For instance, in studies on salinity that refer to changes in proteins involved in mitochondrial ROS defence and protein turnover, increased abundances under salinity were reported in 95% of cases; in studies referring to salinity responses of mitochondrial proteins involved in nitrogen and phosphate metabolism and photorespiration, increased abundances under salinity treatments were reported in 75% of cases (Table S3 in the supplementary material online). The combination of transcript and protein abundance studies shows the transcriptional response of mitochondrial functions that rapidly respond to saline conditions (Box 2; Table S4 in the supplementary material online). Collectively, these changes indicate that respiratory operations are subject to a combination of ionic, osmotic and oxidative stresses. Effect of salt on the ETC The ETC involves a series of protein complexes that operate to produce ATP by utilising an electrochemical gradient across the mitochondrial inner membrane. In plants, 617

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Box 2. Transcriptional and translation control in the mitochondrial salinity response. Determining the degree of transcriptional and translational control in stress response can be garnered by comparison of transcript profiling and proteome analysis. By overlaying data on salinity stress [119,120] with information on the localisation of Arabidopsis proteins [121], we observed that there was an unexpected preponderance of mitochondrial delocalised proteins in the list of salinity-responsive proteins [16]. Here, we have attempted to gauge the nature of this response in mitochondria versus the rest of the cell by assessing the correlation of transcriptional and protein changes in those published data. This shows that, although the list of all salinity-responsive proteins (Table S4 in the supplementary material online) had no clear relationship between transcript and protein abundance, mitochondria showed a

notably inverse relationship. This was because of the decrease in abundance of some mitochondrial proteins in the first 6 hours of salinity but an increase in abundance of their corresponding transcripts, whereas in other cases proteins increased in abundance in the absence of a transcriptional response (Table I) [119,120]. Proteins decreasing in abundance included cytochrome oxidase, heat shock proteins and GDH, whereas MnSOD and CI proteins increased in abundance (Table S4 in the supplementary material online). This suggests that plant cells exposed to salinity rapidly induce the transcription of nuclear-encoded genes for mitochondrial proteins in response to loss or damage of mitochondrial function to protect this organelle.

Table I. Nuclear-encoded mitochondrial transcripts and/or proteins changed in abundance after 6 hours of salinity Arabidopsis genome initiative locus At1g22450 At5g07440 At1g53240 At2g20360 At2g47510 At3g10920 At4g11600 At5g09590

Description Cytochrome c oxidase subunit 6b (COX 6b-1) Glutamate dehydrogenase 2 (GDH2) Malate dehydrogenase (MDH-2) NADH dehydrogenase 39-kDa subunit Fumarase (FUM1) Manganese superoxide dismutase (Mn-SOD) Glutathione peroxidase 6 (ATGPX6) Mitochondrial heat shock protein (mtHSP70-2)

the mitochondrial ETC is more complex and flexible than in mammals and yeast, owing to the presence of several alternative NAD(P)H dehydrogenases and a quinol oxidase, the alternative oxidase (AOX). It has been shown that activities of some ETC complexes are stimulated by MgCl2 and KCl salts in vitro [40], although their activities are inhibited at high NaCl concentrations owing to protein denaturation and complex disassembly [13]. Therefore, it has been speculated that salinity stress will alter the rate at which ETC complexes provide or accept electrons. As this effect will differ between ETC complexes, it will inevitably lead to high levels of reduction of certain sites of the ETC, which manifests in electron leakage to O2 and ROS generation. This provides a mechanistic explanation for the higher salt tolerance of Arabidopsis (Arabidopsis thaliana) overexpressing the gene encoding AOX, as increased AOX capacity prevents high levels of reduction of the ubiquinone pool [41]. There is evidence that the respiration rate of mitochondria isolated from salt-stressed seedlings of durum wheat (Triticum durum) or barley (Hordeum vulgare) can be significantly decreased [42–44]. In durum, the magnitude of this decrease was far greater when seedlings were under salinity stress compared with the mannitol stress of equivalent osmotic potential [42]. This suggests that the ionic component of salinity stress exerts a greater toxic effect on the mitochondrial electron transport chain than does the osmotic component. The exact site at which this inhibition occurs is of interest. It has been shown that salt stress can impair the activities of NADH dehydrogenase (Complex I: CI) and succinate dehydrogenase (Complex II: CII), with heat shock protein 22 (Hsp22) restoring CI activity and compatible solutes restoring CII activity [45]. Given that the bulk of mitochondrial ROS is generated via electron leakage from the ETC, it is interesting that these particular ETC complexes appear to be 618

Salinity response (transcript/protein) Down/down Up/down Down/up Down/up Down/down Down/up Up/down Up/down

sensitive to salt stress, because it implies that robust protection of these sites would alleviate ROS production under salinity stress. Plant mitochondria can be thought of as having two competing respiratory chains, the cytochrome pathway and the alternative pathway. Under salinity stress, it has been noted that the capacity of the cytochrome pathway can decrease, whereas the capacity of the alternative pathway usually increases, or at least stays constant [43,46,47]. Of course, some of this effect will be the result of altered protein abundance and biochemical regulation, but it is also evident that fundamental mechanical differences between the two chains can confer differing sensitivities to ionic stress. Key to this are the environments in which the two electron carriers are found; the ubiquinone pool operates inside the inner membrane and is shielded from ionic salts, whereas cytochrome c is more prone to ionic interference because it is located in the intermembrane space, electrostatically associated with the outside of the inner membrane and is not protected by the inner membrane barrier. Therefore, it can be postulated that the alternative pathway is a bypass during salinity when the cytochrome pathway is limited. This hypothesis is supported by the lower ADP/O ratios observed when electron transport capacity of isolated mitochondria was measured under high concentrations of NaCl and KCl [13,48], because low ADP/O ratios are indicative of high alternative pathway contribution to state 3 respiration rate. Mitochondrial ROS defence and salinity tolerance Increased ROS production is thought to be a common effect of all abiotic stresses [49], and many studies have identified higher ROS levels under salt stress [50–53]. ROS are particularly important in the mitochondrial context because mitochondria generate a significant portion of cellular ROS,

Review mainly owing to leakage from ETC [54], and the mitochondrial redox status can also orchestrate antioxidant capacity in other cellular compartments [55,56]. There is evidence that the ionic rather than osmotic component of salinity stress is responsible for mitochondrial ROS production, because the amount of oxidised reduction–oxidation-sensitive (ro)GFP detected was higher under salt stress compared with mannitol stress [53]. There are very strong links between mitochondrial antioxidant defences and whole-plant salinity tolerance. Most convincing are transgenic experiments showing that salt tolerance in Arabidopsis can be enhanced by overexpressing the genes encoding the mitochondrial antioxidant proteins AOX [41] and manganese superoxide dismutase (MnSOD) [57]. These reports provide causal evidence that mitochondrial composition and function is a major contributor to whole-plant salt tolerance in Arabidopsis. Furthermore, there is an abundance of correlative evidence showing that salt-tolerant genotypes exhibit more robust mitochondrial antioxidant defences than do salt-sensitive genotypes, and that salt stress can induce the expression of mitochondrial antioxidant defences (Box 2, Tables S3 and S4 in the supplementary material online). A study of two wheat varieties, one salt-tolerant and the other salt-sensitive, demonstrated that the salt-tolerant variety expressed high levels of a particular MnSOD isoform that was not detected in the sensitive variety, and had constitutively high AOX levels [58]. One explanation for the strong link between mitochondrial antioxidant defence and salinity tolerance is that robust mitochondrial ROS defences enable higher respiratory flux under salt stress. This argument is supported by evidence that ROS are known to damage certain mitochondrial proteins [59–61] and that salinity stress tolerance requires large amounts of ATP to fuel ion exclusion, osmotic adjustment and vacuolar compartmentation [29]. Decreased respiration rates under salt stress are usually measured in intact tissue and isolated mitochondria (Tables S1 and S2 in the supplementary material online), and it is possible that ROS damage contributes to this decrease. Therefore, robust antioxidant defences could prevent this decrease from occurring. Mitochondrial ROS signalling and salinity tolerance Another explanation for the strong link between salinity tolerance and mitochondrial ROS defences is that ROS signals emanating from the mitochondrion are key players in signalling processes that orchestrate whole-cell ROS capacity and determine survival or death signals under salinity stress. Studies of mitochondrial retrograde signalling have conclusively shown that other cellular compartments are affected by perturbations to mitochondrial proteins, such as CI [55], MnSOD [56] and Hsp22 [62]. Salt stress and osmotic stress elicit different transcriptional responses in a set of H2O2-responsive genes [15], suggesting that the ionic component of salt stress has a unique ROS signature. Undoubtedly, some of these salt-induced ROS signals will emanate from the mitochondrion and, although it is difficult to quantify the mitochondrial contribution to cellular H2O2 and O2– pools accurately, it has been proposed that proteolytic peptides derived from damaged mitochondrial proteins transmit specific retrograde

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signals from the mitochondrion to the nucleus [63]. Thus, the lists of mitochondrial proteins known to differ in abundance under salt stress (Tables S3 and S4 in the supplementary material online) provide a set of candidate proteins that might be cleaved to generate messenger peptides that then transmit salt-specific mitochondrial retrograde signals. Mitochondria and osmotic tolerance The synthesis of high concentrations of proline is an extremely well-defined response to salinity. In vitro work shows that proteins can maintain their native structures under high concentrations of proline [64], making it a classic osmoprotectant; it can also directly scavenge ROS [65], highlighting the broad-spectrum utility of high concentrations of proline. The catabolism of proline occurs in the mitochondrion via proline dehydrogenase (ProDH) and delta-1-pyrroline-5-carboxylate dehydrogenase (P5CDH), so the abundance or activity of these mitochondrial enzymes can regulate the abundance of proline and, hence, promote tolerance. Decreased ProDH-dependent proline catabolism in mitochondria occurs in durum shoots under salt stress [42], implying that downregulation of ProDH leads to more proline and, hence, tolerance. However, the story is complicated because other evidence shows that faster proline catabolism might also enable tolerance. ProDH can increase in expression under salt stress [66], and the overexpression of the gene encoding ProDH enhances tolerance to a combined salt and cold stress [67]. The fact that proline accumulates to such high levels and is catabolised in the mitochondrion has led many researchers to suspect that it serves as an alternative energy source during abiotic stress, and shoot tissues appear to accumulate proline, whereas developing root tissues catabolise proline at a high rate, suggesting that proline is an important source of energy for some tissues [68]. Therefore, uniformly decreasing the activity of ProDH seems an unwise way to promote salt tolerance, because it would deprive some tissues of an important energy source. An alternative view on proline proposes that it can act as electron shuttle to balance redox potentials between chloroplasts and mitochondria [26]. Proline is synthesised in the chloroplast or cytosol and catabolised in the mitochondrion, leading to glutamate synthesis, which can in turn be recycled back into proline synthesis in the chloroplast and/ or cytosol [69]. Therefore, it can be argued that proline metabolism is a means to achieving organellar redox balance; perhaps with the side-benefit that proline preserves native protein structure better than do photorespiratory intermediates. Furthermore, proline synthesis from glutamate consumes cytosolic NADPH, whereas its catabolism releases FADH2 and NADH in the mitochondrion; therefore, proline metabolism could be a vector to balance the abundance and subcellular location of these different electron carriers [26]. The role of gamma-aminobutyric acid (GABA) can be likened to the role of proline under salt stress, because GABA also accumulates [70,71], and is synthesised in the chloroplast or cytosol and is catabolised in the mitochondrion [72]; therefore, it can again be suggested that mitochondrial properties regulate the accumulation of this key 619

Review stress-related metabolite. Catabolism of GABA provides substrate to both the TCA cycle and the ETC, so it would be a useful metabolic substrate to provide energy and carbon skeletons under salt stress. It has been shown that mutants of the mitochondrial GABA-transaminase protein are sensitive to salt stress [73], showing that GABA metabolism is important in mediating salt tolerance. Several studies show a general rebalancing of carbon to nitrogen ratio (C/N) under salinity stress, with a lower abundance of organic acids and a higher abundance of free amino acids [71,74–76]. This C/N rebalancing can be driven by mitochondrial properties relating to photorespiration, 2-oxoglutarate (2-OG) and perhaps glutamate dehydrogenase (GDH). Under salinity, NH4+ concentrations have been reported to increase [77], probably as a result of higher rates of proteolysis and photorespiration [78,79]. NH4+ is toxic to plants unless swiftly detoxified. The main enzymes of nitrogen assimilation in the cytosol and chloroplast, such as nitrate reductase (NR), nitrite reductase (NiR), glutamine synthetase (GS) and glutamine-oxoglutarate aminotransferase (GOGAT), show decreased activities under salinity [80–82]; whereas mitochondrial GDH, which can assimilate NH4+ by the reductive amination of 2OG, has selectively increased activity in the aminating direction under salinity [51,80,81]. The in vivo magnitude of ammonium assimilation through GDH has been debated because isolated mitochondria assimilate ammonium at a very slow rate [83], and GDH also has a relatively low affinity for ammonium compared with GS [84]. However, regardless of whether the reassimilation of photorespiratory ammonium is catalysed by GS or GDH, both enzymes rely upon the provision of 2-OG from a partial TCA cycle, and flux through this pathway increases under photorespiratory conditions [85,86]. Therefore, mitochondria have an increasing role in NH4+ assimilation under salinity to drive cellular C/N rebalancing, which could enable osmotic adjustment for cells under stress. Mitochondrial respiratory properties and salt tolerance physiology Carbon balance and the links between rates of respiration and photosynthesis Respiration is required for optimal photosynthesis and the two processes are interdependent. This was shown by considering the impact of respiratory inhibitors on photosynthetic function [87] and reiterated by evidence that knockout of certain mitochondrial proteins can modulate photosynthesis [88,89]. Simultaneous measurements of photosynthesis and respiration demonstrate that mitochondrial CO2 evolution is inhibited in the light, whereas O2 uptake is unaffected or sometimes stimulated [90]. These shifts could be driven by metabolite shuttles involving oxaloacetate and/or malate or 3-phosphoglycerate and/ or dihydroxyacetone phosphate between chloroplasts and mitochondria; alternatively, cytosolic NAD(P)H derived from photosynthetic products can be oxidised by the mitochondrial external NAD(P)H dehydrogenases to fuel mitochondrial respiration and relieve photoinhibition in the plastid [91]. Hence, the non-stomatal inhibition of photosynthesis observed when plants are under salt stress could be partially induced by the loss of respiratory competency. 620

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Recent papers have dissected the role of respiration in supporting photosynthesis under drought or osmotic stress [92–94], pointing out the need for comparable studies in salt-treated plants. Interconnection of ROS defence, osmotic tolerance and ROS signalling Although the primary function of the TCA cycle is to provide reductant for the ETC, it also functions to provide carbon skeletons used for the biosynthesis of many molecules, such as amino acids and compatible solutes. TCA cycle enzymes are acutely inhibited by ROS species [59,95], suggesting that robust mitochondrial ROS defences are crucial in maintaining TCA cycle flux under salinity to provide the building blocks for osmolyte synthesis. Furthermore, although AOX is often considered an antioxidant pathway, it is also important in redirecting metabolism, because AOX activity can decouple TCA cycle flux from adenylate control, thus enabling TCA cycle flux despite low ADP/ATP ratios, which can serve to meet high cellular demand for carbon skeletons [96]. Therefore, AOX capacity can be a component of osmotic tolerance, as well as antioxidant defence. This is supported by evidence that young, rapidly expanding leaves express higher levels of the gene encoding AOX under osmotic stress [93]. It can be argued that mitochondrial ROS is a more logical signalling pathway than chloroplastic ROS because the initial perception of salt stress would occur in the root. Although ROS themselves are short lived, they do leave significant after-effects, such as oxidative protein modifications. It has been shown that mitochondria are the main cellular target of protein carbonylation following drought stress [97], and that mitochondrial redox poise takes longer than the plastids to return to baseline levels after H2O2 application [53], perhaps as a result of feed-forward loops, where ROS damage proteins in the ETC, thus creating more ROS. In this context, a suggested function of proteins such as uncoupling protein (UCP) and AOX is to bypass the conventional ETC to prevent overreduction and ROS generation, therefore ‘dampening’ mitochondrial ROS signals [98]. MnSOD could have a similar dampening effect on the superoxide signal from mitochondria. ATP synthesis, ATP efficiency and the energetic requirements of ion exclusion and tissue tolerance Ion exclusion is believed to involve the selective uptake of essential ions (e.g. K+, NO3–, SO42– and PO43–), and the selective exclusion or efflux of Na+ and Cl–. The precise bioenergetics of Na+ and Cl– transport in plants is not fully understood [9,99], but using assumptions regarding the energetic costs of active transport, two publications have calculated the energetic cost of Na+ and Cl– exclusion at 100 mM NaCl concentration, and both calculations show that exclusion processes have a considerable and constant ATP demand [9,100]. Given that respiration is the primary source of ATP in roots, it is evident that root respiration ‘fuels’ ion exclusion, as shown by the tight coupling of Na+ exclusion to root respiration rate in rice (Oryza sativa) cultivars [32]. The link between respiration and exclusion is further supported by studies showing that O2 deficiency in roots dramatically inhibits sodium exclusion owing to a

Review lack of cellular energy [101–104], whereas plants with aerenchyma can maintain respiration and sodium exclusion in O2-deficient root media [105]. Conclusions and future directions Through reviewing literature to link mitochondrial function with salinity stress, we have attempted to integrate mitochondrial molecular function with whole-plant respiratory physiology. We propose discrete roles of respiration in root ion exclusion tolerance, and in both ionic and osmotic leaf tissue tolerance. There are strong links between salinity and the mitochondrial production of ATP, osmolytes and ROS, shown through biochemical experiments, whole-plant measurements and transgenic studies. Although a range of experiments have now defined species and varieties that exhibit high tissue tolerance [106–108], the molecular mechanisms that underpin tissue tolerance are still poorly defined. Many authors agree that defining the molecular tissue tolerance mechanisms will become a priority over the coming years, using imaging technologies [109], quantitative trait loci (QTL) marker strategies [110] and high-throughput integration of ‘omic data in studies of different varieties of crop plants under salinity stress [58,71,111–113]. We contend that measurements of photosynthetic and respiratory rates in carbon balance calculations have an important place in furthering understanding of salinity tolerance and should help to explain complex molecular data sets and aid their integration in future screens for salinity tolerance. Acknowledgements This work was supported by the Australian Research Council (ARC) through the ARC Centre of Excellence for Plant Energy Biology (CE0561495). RPJ is supported by a Grains Research and Development Corporation (GRDC) PhD scholarship and AHM as an ARC Australian Professorial Fellow (DP0771156). We thank Ben Biddulph (Department of Food and Agriculture Western Australia, DAFWA), Tim Colmer (School of Plant Biology, UWA) and Rana Munns (CSIRO Plant Industry) for helpful discussions.

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