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Annals of Botany 103: 581 –597, 2009 doi:10.1093/aob/mcn094, available online at www.aob.oxfordjournals.org

REVIEW

The crucial role of plant mitochondria in orchestrating drought tolerance Owen K. Atkin1 and David Macherel2,* 1

Functional Ecology Group, Research School of Biological Sciences, Australian National University, Canberra, ACT 0200, Australia and 2IFR 149 QUASAV, Unite´ Mixte de Recherche 1191 Physiologie Mole´culaire des Semences, Universite´ d’Angers/ Institut National d’Horticulture/Institut National de la Recherche Agronomique, ARES, 49045 Angers Cedex 01, France Received: 17 December 2007 Returned for revision: 21 April 2008 Accepted: 9 May 2008 Published electronically: 13 June 2008

† Background Around the world, the frequency and intensity of droughts is increasing as a result of global climate change, with important consequences for the growth and survival of agricultural and native plant species. Understanding how plants respond to water stress is thus crucial for predicting the impacts of climate change on the crop productivity and ecosystem functioning. In contrast to the large number of studies assessing drought impacts on photosynthesis, relatively little attention has been devoted to understanding how mitochondrial respiratory metabolism is altered under water stress conditions. † Scope This review provides an overview of the impacts of water stress on mitochondrial respiration (R), combining studies at the whole-plant, individual organ, cellular and organelle levels. To establish whether there are clear patterns in the response of in vivo R to water stress, a wide range of root, leaf and whole-plant studies are reviewed. It is shown that water stress almost always inhibits R in actively growing roots and whole plants. However, in fully expanded, mature leaves the response is more variable, with water stress reducing R in near two-thirds of reported studies, with most of the remainder showing no change. Only a few studies reported increases in leaf R under severe water stress conditions. The mechanisms responsible for these variable responses are discussed. Importantly, the fact is highlighted that irrespective of whether drought increases or decreases respiration, overall the changes in R are minor compared with the large decreases in photosynthetic carbon gain in response to drought. Based on recent work highlighting the link between chloroplast and mitochondrial functions in leaves, we propose a model by which mitochondrial R enables survival and rapid recovery of productivity under water stress conditions. Finally, the effects of water stress on mitochondrial function, protein abundance and overall metabolism are reviewed. Key words: Respiration, photosynthesis, water stress, drought, desiccation tolerance, mitochondria, chloroplast, metabolic shuttle, alternative pathway, organelle cross-talk, oxidative stress, photorespiration.

IN T RO DU C T IO N One hundred years ago, while homesick in England, Dorothea Mackellar published an iconic poem (later known as ‘My Country’) that highlighted the dominance of drought in her homeland (Australia). Over the subsequent century, the frequency and intensity of such droughts has increased, not only in her homeland (Wang and Hendon, 2007), but also in many other regions around the globe, with the latest IPCC report predicting further increases as a result of ongoing global climate change (www.ipcc.ch/SPM6avr07.pdf ). Because water is crucial for plant growth (Boyer, 1982), such increases in the prevalence of drought will have important impacts on the productivity of agricultural (Passioura, 2007) and natural ecosystems (Ciais et al., 2005), distribution of plant species (Engelbrecht et al., 2007) and the concentration of CO2 in the atmosphere (Buermann et al., 2007). An example of the importance of drought is shown by the finding that the decrease in the European-wide plant productivity in 2003 was not primarily caused by high temperatures but rather by drought (Reichstein et al., 2007). Decreases in carbon gain by photosynthesis are a major factor contributing to the decrease in productivity of plants * For correspondence. E-mail [email protected]

experiencing drought, with the inhibitory effect of drought on photosynthesis often being rapid (Fig. 1A; Hsiao, 1973; Lawlor and Cornic, 2002; Flexas et al., 2006). For example, cessation of watering for 3–5 d results in an 80–100 % decline in lightsaturated net photosynthesis in controlled environment-grown soybean leaves (Ribas-Carbo et al., 2005). Similarly, withholding water for 17 d leads to 80–95 % reduction in photosynthetic carbon gain in a wide range of plant functional types, with the inhibition of photosynthesis being greater in herbaceous species (i.e. with high ratios of leaf area to leaf dry mass) than their evergreen shrub counterparts whose leaves are often thicker and more dense (Galmes et al., 2007). Reductions in stomatal and mesophyll conductance (Boyer, 1982; Flexas et al., 2006), combined with rapid changes in cellular metabolism (Lawlor and Fock, 1978, Lawlor and Cornic, 2002, Zrenner and Stitt, 1991), contribute to decreased photosynthesis and overall productivity under water-stressed conditions. A further factor determining the impact of water stress on plant productivity is the effect it has on mitochondrial respiration (R; see Table 1 for list of abbreviations) in roots, stems and leaves, and the extent to which the effect of water stress on R differs from that of photosynthesis. Although specific rates of R is typically an order of magnitude lower than that of net photosynthesis (Lambers et al., 1998), collectively R

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Atkin & Macherel — Plant mitochondria and drought tolerance TA B L E 1. Abbreviations used in the text

25 Photosynthesis (arbitrary units)

A AOX COX R leaf Rdark Mal/OAA PDC Rlight ROS RWC UCP

20 15 10 5 0

Rewatered

3·0 B

R (arbitrary units)

2·5 2·0 1·5 1·0 Increased R No change Decreased R

0·5

Rewatered

0·0 3·0

C

R/P ratio

2·5 2·0 1·5 1·0 0·5 0·0 Rewatered

0

10 20 30 Days after withdrawal of watering

40

F I G . 1. Diagrammatic representation of the potential impacts of water stress on leaf gas exchange. Shown are (A) rates of CO2 uptake by photosynthesis and (B) respiratory CO2 release by leaf respiration (R) in darkness, and (C) the ratio of respiration to photosynthesis (R/P). The effects of rewatering are shown after an initial 20 d of withholding water. In (B) and (C), three scenarios are shown: one where imposition of water stress has no effect on leaf R, and scenarios where R is increased or decreased in response to reduced water availability. Irrespective of how R responds to water stress, R/P increases in response to increasing water stress, and decreases in response to rewatering.

by roots and shoots play a major role in determining the carbon balance and productivity of plants. Of the CO2 fixed each day by net photosynthesis in well-watered plants, 30– 70 % is released back into the atmosphere by plant R (Poorter et al., 1990; Atkin et al., 1996; Loveys et al., 2002), with 50– 70 % of whole-plant R taking place in leaves (Atkin et al., 2007). The percentage of daily fixed carbon that is respired is likely

Alternative oxidase Cytochrome c oxidase Respiration Non-photorespiratory mitochondrial R in darkness Malate/oxaloacetate Pyruvate decarboxylase Non-photorespiratory mitochondrial R in the light Reactive oxygen species Relative water content Uncoupling protein

to be even higher in water-stressed plants due to the fact that drought has typically a greater proportional inhibitory effect on photosynthesis than on plant R (Flexas et al., 2005, 2006), and because in some cases, leaf R actually increases in response to drought (see next section; Hsiao 1973; Flexas et al., 2005). Viewed from this perspective, maintenance of R in water-stressed plants has a negative effect on the carbon balance of plants due to the continued (or even increased) loss of CO2 into the atmosphere. The maintenance of R can also serve several positive roles in helping plants grow and survive, both in well-watered and water-stress conditions. For example, irrespective of the availability of water, chloroplasts and leaf mitochondria are highly inter-dependent (Kro¨mer, 1995), reflecting the considerable energetic and metabolic cross-talk between the two organelles and the cytosol (reviewed by Hoefnagel et al., 1998; Raghavendra and Padmasree, 2003). A clear example of the interdependence of the organelles within the photosynthetic cell was reported by Kro¨mer and Heldt (1991), who showed that gentle rupturing of protoplasts immediately relieved the inhibition of photosynthesis brought about by the inhibition of mitochondrial oxidative phosphorylation by oligomycin. Numerous other examples of inter-dependence are available; for example, the mitochondrial uncoupling protein (UCP) has recently been ascribed a major role with respect to the maintenance of the mitochondrial redox poise to facilitate carbon assimilation (Sweetlove et al., 2006). Moreover, mitochondria can dissipate excess redox equivalents from the chloroplast in the light (Noguchi and Yoshida, 2008). At the genomic level, a complex dialogue is required between the two organelle genomes and the nucleus to orchestrate their biogenesis. An elaborate network of anterograde and retrograde signalling between the organelles and nucleus is emerging, with redox status and reactive oxygen species (ROS) figuring as important actors (Leister, 2005; Rhoads and Subbaiah, 2007). The pivotal signalling role of mitochondria is nicely illustrated by a mutant of N. sylvestris impaired in Complex I, which revealed the influence of mitochondria in controlling the expression of anti-oxidative genes in other cell compartments (Dutilleul et al., 2003) and in adjusting carbon assimilation regime to high light (Priault et al., 2006). Such studies show that mitochondria and chloroplasts are intimately connected by metabolic and signalling networks, and that, in intact leaves, photosynthesis depends to a large extent on mitochondrial functions. This reliance on mitochondria has the potential to be further enhanced under water-stress conditions, as discussed in later sections of this review. Plant mitochondria display a remarkable flexibility in electron transfer and energy dissipation which is of prime

Atkin & Macherel — Plant mitochondria and drought tolerance importance in the fluctuating environment of plants, and certainly in the case of water stress. The non-proton pumping alternative dehydrogenases (Rasmusson et al., 2004) and the alternative oxidase (AOX) (Vanlerberge and McIntosh, 1997) provide electron transfer by-passes while proton gradient can be dissipated by UCP (Sluse et al., 2006) or by the combined action of the Kþ/Hþ antiporter and potassium Kþ channel (Pastore et al., 1999). These systems allow mitochondria to reoxidize substrates independently of adenylate control, and prevent accumulation of ROS that could result from overreduction of electron transfer chain (Fig. 2). In this review, an overview is provided of the impacts of water stress on mitochondrial R, combining studies at the whole plant, individual organ, and cellular and organelle levels. To establish whether there are clear patterns in the response of in vivo respiration to water stress, we review a wide range of root, leaf and whole-plant studies and show that while water stress invariably inhibits R in actively growing tissues, increases in R can also occur in mature, fully expanded leaves that are no longer growing. The mechanisms responsible for this variable response are discussed. Importantly, we highlight the fact that irrespective of whether drought increases or decreases respiration, overall the changes in R are minor compared with the large decreases in photosynthetic carbon gain in response to drought. Based on recent work highlighting the link between chloroplast and mitochondrial functions, we propose a model by which mitochondrial R enables survival and rapid recovery of productivity under water-stress conditions. Finally, the effects of water stress on mitochondria, gene expression, proteins and metabolism are reviewed.

WAT E R S T R E S S A N D I N V I VO G A S E XC H A NG E Impacts of water stress on respiration rates exhibited by intact tissues

To what extent does the effect of water stress on R differ between individual organs and whole plants? In roots, exposure to water stress eventually results in root respiratory CO2 release declining during the development of water stress (e.g. in Citrus volkameriana, root respiration is 30– 50 %

lower in water-stressed roots than roots that are well-watered; Bryla et al., 1997, 2001; Table 2); only when root R is measured as O2 uptake is water stress associated with a transient increase in respiratory flux (Greenway, 1970; Collier and Cummins, 1993). Similarly, all of the whole-plant R studies shown in Table 2 found substantial decreases in CO2 efflux under reduced water supply, particularly under conditions of severe water stress (De Vries et al., 1979). However, compared with measurements done using roots and whole plants, the impact of water stress on leaf metabolism is more varied — although leaf R decrease under water stress in two-thirds of the measurements shown in Table 2, approximately one-third found that leaf R is unaffected by water stress. Moreover, there are an additional three cases where, rather than decreasing, leaf R actually increases under severe water stress (Zagdanska, 1995; Ghashghaie et al., 2001; Bartoli et al., 2005). Additionally, in four cases listed in Table 2, water stress resulted in a bi-phasic response (i.e. both an inhibition and stimulation leaf respiration, depending on the duration of exposure and/or the severity of water stress; Pallas et al., 1967; Collier and Cummins 1993, 1996; Flexas et al., 2005); in such bi-phasic cases, the stimulatory effect of water stress on leaf R appears to occur when the relative water content (RWC) of leaves falls below 50– 55 %, with falls in RWC from 100 % to 70 % being associated with a decline in leaf R (Lawlor and Cornic, 2002; Flexas et al., 2005). Leaves also exhibit less consistent responses (compared with roots and whole plants) to rewatering; for example, whereas rewatering resulted in root and whole-plant R increasing in all cases shown in Table 2, leaf R increased in only five of the 12 cases shown (in six cases, rewatering had no effect on leaf R). Thus, while changes in water supply have a predictable, consistent effect on root and whole-plant R, the response of leaf R to water stress is more varied depending on the duration and severity of drought. Mechanisms responsible for variability in respiratory responses to water stress

As a complex process at the interface between cellular carbon supply and energy demand, respiration is regulated at ATP synthesis

D µH+ NAD(P)H

Dissipation

H+

H+ NDex

IMS

583

H+

H+

H+

K+

Cyt c Electron transfer

CI

UQ/UQH2 CII

Matrix

Succ

CIII

CIV

UCP KHap

Kch

AOX NDin

O2

O2

NADH F I G . 2. Energy dissipating systems in plant mitochondria membrane. The scheme shows the pathway of electrons from the substrates to oxygen via the different complexes of the mitochondrial respiratory chain (bold arrows) and the coupled export of protons (dotted arrows). Plant alternative dehydrogenases (NDin, NDex) and alternative oxidase (AOX) are indicated in blue. The major sites of ROS formation, namely superoxide, are indicated by stars. The systems capable of dissipating the electrochemical proton gradient which slows down electron transfer and favours ROS formation are the uncoupling protein (UCP), and the combination of the Kþ/Hþ antiporter (KHap) with the potassium channel (Kch).

Species/spatial scale/organ Whole plants Lolium perenne (intact tissues) Rosmarinus officinalis (intact tissues) Sorghum bicolour (intact tissues)

Zea mays (intact tissues) Leaves Arnica alpina (intact tissues)

Short-term osmotic treatment

Impact of water stress

No effect at low-moderate water stress; decrease under severe water stress Decreased

Withholding of water for 10 d; 5 d rewatering/ recovery Withholding of water for 15 d; 4 d of rewatering/ Decreased recovery Germinating seedlings exposed to two concentrations Decreased of osmotica and/or saline solutions for short periods Short-term osmotic treatment No effect at low-moderate water stress; decrease at under severe water stress Short-term osmotic treatment No effect at low-moderate water stress; decrease at under severe water stress

Changes during recovery

References

Vries et al., 1979 Increased

Nogues et al., 2001

Sharp increase upon rewatering

McCree et al., 1984; Richardson and McCree, 1985 Flagella et al., 2006 Vries et al., 1979 Vries et al., 1979

Withholding of water for 11 d followed by rewatering; 5 d of osmotic treatment (two concentrations) Withholding of water for 17 d

Leaf respiration transiently increased, followed by decline as level of water stress increased No change

Increased

Galmes et al., 2007

Withholding of water for 17 d

Decreased

Fully recovered

Galmes et al., 2007

Sustained low watering regime Withholding of water for 17 d Withholding of water for 17 d Withholding of water for extended period Sustained low watering regime

Decreased No change Decreased Decreased Decreased Decreased Decreased

Boyer, 1970 Ribas-Carbo et al., 2005

Glycine max (intact tissues/isolated mitochondria) Gossypium hirsutum (intact tissues)

Withholding water for several days Watered to 100 %, 50 % (mild stress) and 0 % (severe stress) of the water lost each day for 5 d Shoots treated with increasing osmoticum prior to mitochondrial isolation Withholding water for 11 d

Decreased

Flowers and Hanson, 1969 Pallas et al., 1967

Helianthus annuus (intact tissues) Helianthus annuus (intact tissues)

Withholding water for 4 d Withholding water

Initially decreased with increasing severity, after which it increased at most severe water stress Decreased No change

Helianthus annuus (intact tissues) Helianthus annuus (intact tissues)

Withholding water for several days Withholding water for 20 d

Hypericum balearicum (intact tissues) Lavandula stoechas (intact tissues) Lavatera maritime (intact tissues) Limonium magallufianum (intact tissues) Limonium gibertii (intact tissues) Lycopersicon esculentum (intact tissues) Lysimachia minoricensis (intact tissues)

Withholding of water for 17 d

Beta maritima subsp. Marcosii (intact tissues) Beta maritima subsp. Maritima (intact tissues) Capsicum annuum (intact tissues) Cistus albidus (intact tissues) Diplotaxis ibicensis (intact tissues) Eucalyptus spp. (nine genotypes) Festuca arundinaceae (intact tissues) Glycine max (intact tissues) Glycine max (intact tissues)

Collier and Cummins, 1992, 1993

No change Partial recovery

Decreased Increased in response to more severe loss in leaf water content No change Decreased

Gonza´lez-Meler et al., 1997 Galmes et al., 2007 Galmes et al., 2007 Callister and Adams, 2006 Huang and Fu, 2000

Boyer, 1971 Lawlor and Fock, 1975 (as cited in Lawlor and Fock, 1977) Boyer, 1970 Ghashghaie et al., 2001 Galmes et al., 2007

Withholding of water 10 d, followed by 5 d recovery Decreased Withholding of water for 17 d No change Withholding of water for 17 d Decreased

Increased Increased No recovery

Nogues et al., 2001 Galmes et al., 2007 Galmes et al., 2007

Withholding of water for 17 d Withholding of water for 8 d

No change Decreased

No change

Withholding of water for 15 d

Decreased at moderate stress, followed by an increase at more severe stress

Galmes et al., 2007 Haupt-Herting et al., 2001; Haupt-Herting and Fock, 2002 Flexas et al., 2005

Atkin & Macherel — Plant mitochondria and drought tolerance

Triticum durum (isolated mitochondria) Triticum aestivum (intact tissues)

Treatment/duration

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TA B L E 2. Summary of the impacts of water stress and recovery from water stress on rates of mitochondrial respiration for a variety of plant tissues and spatial scales

Lysimachia minoricensis (intact tissues) Matteuccia struthiopteris (intact tissues) Mentha aquatica (intact tissues) Nicotiana sylvestris (intact tissues) Nicotiana tabacum (intact tissues)

Triticum aestivum (intact tissues/ isolated mitochondria) Triticum aestivum Zea mays Zea mays Roots Acer saccharum (intact tissues)

Decreased

No recovery

3 –4 weeks of osmotic treatment

Decreased in young fronds, but no change in mature tissues Withholding of water for 15 d No change Withholding water for 20 d Decreased at moderate water stress Watered to 100 %, 40 % (mild stress) and 15 % No change in dark R; decrease in R in the (severe stress) of field capacity for sustained period light under severe drought Sustained low watering regime Decreased Withholding of water for 15 d Decreased at moderate to severe stress Withholding of water for 17 d Decreased No change Withholding of water for 15 d No change Withholding of water for 17 d No change No change Sustained low watering regime Decreased Withholding water for several days on two soil Decreased when stress developed at substrates differing in water-holding capacity moderate speed; Transient increase, then decline upon sharp stress Withholding water to 14 % of field water capacity for Increased several days Withholding water for extended period Increased Withholding water for several days Decreased Shoots treated with increasing concentrations of Decreased osmoticum prior to mitochondrial isolation Increased

Citrus volkameriana (intact tissues)

Seasonal variation in soil moisture moisture over 3 Decreased years Withholding water for 11 d followed by rewatering; 5 Root respiration transiently increased, d of osmotic treatment followed by decline as level of water stress increased Withholding water for 3– 4 months Decreased

Ferocactus acanthodes (intact tissues)

Withholding water for several days followed by 3 d of rewatering/recovery

Decreased, irrespective of root type

Increased in established roots, but not in ‘rain’ roots

Festuca arundinaceae (intact tissues) Glycine max (intact tissues)

Sustained low watering regime

Decreased

Arnica alpine (intact tissues)

Opuntia ficus-indica (intact tissues) Pinus radiate (intact tissues) Poa pratensis (intact tissues) Triticum aestivum (intact tissues) Triticum aestivum (intact tissues) Zea mays (intact tissues/isolated mitochondria) Zea mays (intact tissues/isolated mitochondria) Zea mays (intact tissues) Flower apices Triticum aestivum (intact tissues)

Hydroponically grown roots removed from liquid for Decreased 5 h each day during daytime period for 19 d, kept in water saturated air Withholding water for several days, followed by 3 d Decreased, irrespective of root type of rewatering/recovery Decreased Decreased Decreased Decreased

Roots treated with osmoticum for short periods

Decreased

Roots treated with osmoticum for 24 h

Decreased

Withholding water for 14 d and treatment of apices with osmoticum for short periods

Decreased

Prange et al., 1983 Flexas et al., 2005 Ghashghaie et al., 2001 Galmes et al., 2006 Gonza´lez-Meler et al., 1997 Flexas et al., 2005 Galmes et al., 2007 Flexas et al. 2005 Galmes et al., 2007 Huang and Fu, 2000 Collier and Cummins, 1996 Bartoli et al., 2005 Zagdanska, 1995 Boyer, 1970 Bell et al., 1971 Burton et al., 1998 Collier and Cummins, 1992, 1993 Bryla et al., 1997, 2001; Espeleta et al., 1998 Palta and Nobel, 1989a, 1989b Huang and Fu, 2000

Increased, full recovery of root O2 uptake

Thomas and Andre, 1982

Increased in established roots, but not in ‘rain’ roots Increased

Palta and Nobel, 1989a, b Carlyle and Than, 1988 Huang and Fu, 2000 Nicolas et al., 1985 Liu and Li, 2005 Greenway, 1970; Greenway and Leahy, 1970; Greenway and West, 1973 Nir et al., 1970

O2 uptake transiently increased, thereafter decreased; CO2 efflux decreased with time

Increased, full recovery of root O2 uptake

Kolarovic et al., 2006 Pheloung and Barlow, 1981

585

Seasonal variation in soil moisture over 262 d Sustained low watering regime Withholding water for 14 d Watered to 50 and 30 % of field water capacity for sustained period Roots treated with osmoticum prior to and after isolation of mitochondria

Galmes et al., 2007

Atkin & Macherel — Plant mitochondria and drought tolerance

Phaseolus vulgaris (intact tissues) Phlomis italica (intact tissues) Phlomis italica (intact tissues) Pistacia lentiscus (intact tissues) Pistacia lentiscus (intact tissues) Poa pratensis (intact tissues) Saxifraga cernua (intact tissues)

Withholding of water for 17 d

586

Atkin & Macherel — Plant mitochondria and drought tolerance No change in electron partitioning with the mitochondrial ETC No change in respiration

Change in partitioning between the COX and AOX pathways, resulting in changes in the efficiency of ATP synthesis

Constant rate of ATP synthesis

Regulatory changes in enzyme activity Alterations in protein abundance

Acclimated to sustained water stress Increase in flux, resulting in increased ATP synthesis and/or increased heat release

Regulatory increase in enzyme activity Increase in mitochondrial protein concentration

Change in the rate of respiration Regulatory decrease in enzyme activity Decrease in flux Decrease in mitochondrial protein concentration F I G . 3. Schematic presentation of the types of respiratory responses to drought. Changes in protein abundance are depicted by coloured borders around the relevant boxes, with regulatory changes in enzyme activity (e.g. in response to changes in substrate supply, adenylate control and/or concentrations of allosteric activators such as pyruvate) being shown as shaded boxes.

several levels ranging from metabolic control to gene expression (Fig. 3). An almost online rheostat is provided by proton gradient modulation and electron partitioning between cytochrome c oxidase (COX) and AOX, while the overall process is regulated by metabolites and protein expression level. The diversity of respiratory responses under water stress and upon acclimation may in fact result from various combinations between theses regulatory processes (Fig. 3). In this schematic view, one can ask the question: why is it that the response of root R to water stress generally is more consistent than that of R of mature leaves? To answer this question, it is necessary to consider what factors regulate respiratory flux in the two organs, and how such factors might be impacted on by changes in water supply. Water-stress induced changes in respiratory flux could reflect changes in the maximum activity of respiratory enzymes (as determined by the abundance of active protein in glycolysis, the TCA cycle and the mitochondrial electron transport chain), changes in substrate supply (e.g. due to reduced rates of carbon fixation and subsequent sugar production) and/or demand for ATP (associated with growth, cellular maintenance and ion transport). At moderate temperatures, R of well-watered plants is rarely limited by enzymatic capacity (Atkin et al., 2005). Although little is known about whether enzymatic capacity per se limits flux in water-stressed plants, Herppich and Peckmann (2000) found that the in vitro activity of several key respiratory enzymes was unaffected by mild to moderate water stress in two CAM species. Moreover, the speed of water stress-induced changes in respiratory flux suggests that

changes in enzymatic capacity alone are unlikely to be responsible for the changes in R, or why leaves and roots sometimes differ in response to drought. Rather, changes in substrate supply and/or the extent to which ATP turnover regulates respiratory flux are likely to play an important role, particularly when considering changes in flux over the short- to mediumterm in response to mild to moderate water stress. To what extent might substrate availability limit R in water-stressed plants? Water stress-induced reductions in photosynthesis (Lawlor and Cornic, 2002), shifts in carbon allocation away from R to the formation of compounds involved in osmotic adjustment (e.g. Nicolas et al., 1985; Dekankova et al., 2004), and water stress-induced reductions in sugar concentrations (Lawlor and Fock, 1977) could potentially result in R becoming substrate limited. Moreover, the fact that the ‘sink-strength’ of roots is often poor, with assimilates exported to roots representing those left over by other sinks (Brouwer, 1963; Lambers and Atkin, 1995; Farrar and Jones, 2000), suggests that the potential for substrate limitations may be greater for roots than leaves under water-stress conditions, depending on the extent to which drought increases carbon allocation to roots (e.g. Huang and Fu, 2000). However, in many studies, the decline in photosynthesis is not matched by concomitant decreases in R (Table 2), and soluble sugar concentrations were found to decrease (Lawlor and Fock, 1977), increase (Dekankova et al., 2004) or remain stable (Ghashghaie et al., 2001) during onset of drought. Thus, while we lack a full understanding of the extent to which substrates limit R under water-stress conditions, the available data suggest that declines in substrate availability

Atkin & Macherel — Plant mitochondria and drought tolerance are unlikely to be the only factor responsible for droughtinduced declines in leaf R, at least in the short to medium term. A further factor that is likely to regulate changes in respiratory flux under water stress conditions are drought-induced changes in the demand for ATP. In fully expanded, mature leaves (i.e. that are not growing and thus have no growth R requirement), ATP is required for sucrose synthesis, phloem loading and maintenance processes (e.g. protein turnover and maintenance of ion gradients; Bouma et al., 1994; Hoefnagel et al., 1998). In well-watered plants, nocturnal phloem loading consumes, on average, 29 % of the energy produced by dark R in starch-storing species (Bouma et al., 1995), while protein turnover accounts for an a further 20 % in mature leaves (Bouma et al., 1994). How drought impacts on such processes in mature leaves is not clear. On one hand, we might expect a decline in energy demand if rates of sucrose synthesis and/or phloem loading are reduced [NB Even though sucrose concentrations can, in some cases, remain constant in drought-treated leaves (Ghashghaie et al., 2001), decreases in sucrose synthesis/phloem loading rates could occur, so long as reduced rates of supply are matched to reduced consumption/export of sucrose.] Certainly, energy costs associated with sucrose synthesis/phloem loading are likely to fall in leaves where sucrose concentrations and rates of CO2 assimilation decline in response to water stress (e.g. Lawlor and Fock, 1977). Reduced energy demand by sucrose synthesis and/or phloem loading would increase the likelihood that R becomes adenylate restricted (in response to reduced turnover of ATP to ADP) and may explain why R rates of mature leaves often decrease in drought-treated plants (Table 2), particularly as part of the bi-phasic response to declining RWC (Lawlor and Cornic, 2002; Flexas et al., 2005). As part of the bi-phasic response to declining RWC, drought could also result in an increase in energy demand if the demand for maintenance respiratory energy is increased and/or there is a need to compensate for reduced rates of chloroplast ATP synthesis (Tezara et al., 2008). Droughtinduced reductions in photosynthesis increase the likelihood that cellular redox equivalents accumulate to excessive levels in illuminated leaves (Lawlor and Khanna-Choppra, 1984), which in conjunction with photorespiration, can contribute to the production of ROS (Noctor et al., 2002) that can impact on mitochondria (Bartoli et al., 2004). Increased ROS production and associated protein turnover can increase the demand for mitochondrial ATP (Hoefnagel et al., 1998). Increased R may also be required to oxidize excess cellular redox equivalents (e.g. via engaging non-phosphorylating pathways; Fig. 2, 3). Thus, severe levels of water stress could result in an increased demand for respiratory energy to deal with increased maintenance costs and/or a requirement for oxidation of excess cellular redox equivalents. [NB With the exception of Pallas et al. (1967) and Ghashghaie et al. (2001), all of the studies reporting increases in leaf R measured leaf R as O2 uptake; if widespread, this might point to water stress having a stimulatory effect on rates of leaf mitochondrial electron transport that is not coupled to increases in leaf TCA cycle flux (e.g. via increases in use of the cytosolic NADH by the electron transport chain), with the increase in electron transport being used either to produce additional ATP or to oxidize excess cellular redox equivalents.]

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In roots, 25– 45 % of respiratory energy is used for biosynthesis, with a further 50– 70 % being used for ion uptake (Poorter et al., 1991). Only 5 – 10 % of respiratory energy is used to support cellular maintenance processes (Penning de Vries, 1975), and thus any drought-mediated changes in cellular maintenance processes are unlikely to have a large quantitative effect on total R rates (Burton et al., 1998). On the other hand, drought is likely to reduce the rate of ion uptake and associated energy demand, which may explain why rates of root R are almost always lower under water stress conditions (Table 2). Drought-mediated decreases in root growth rate, where they occur (e.g. Van den Boogaard et al., 1996) would also result in a concomitant decline in demand for respiratory ATP. The inhibitory effect of water stress on growth R energy requirements could also explain why the apparent discrepancy between consistent reductions in whole-plant R versus the inconsistent responses in mature leaves (Table 2). Unlike in mature leaves, developing tissues in the shoots and roots contribute to respiratory CO2 release in whole plants. Given that water stress will invariably reduce the demand for growth R in above- and below-ground organs (De Vries et al., 1979), one would anticipate a lower demand for respiratory energy at the whole-plant level under water stress conditions. In contrast, drought-mediated decreases in growth rate would be of less consequence for measurements of R in fully expanded, mature leaves (depending on the extent to which water stress impacted on metabolic rates in tissues that were no longer expanding). As stated above, leaf R is unaffected by water stress in nearly one-third of the cases listed in Table 2. Does this mean that drought had no effect on mitochondrial metabolism in such cases? Not necessarily. For example, in a study of soybean leaf R, Ribas-Carbo et al., (2005) found that, although O2 uptake in darkness was not affected by water stress, the partitioning between the two terminal oxidases (i.e. COX and AOX) was (Fig. 2); severe water stress resulted in a shift away from COX to the AOX pathway, with the latter increasing from near 10 % of total flux under well-watered conditions to near 40 % under severe water stress (Ribas-Carbo et al., 2005). This increase in AOX activity was not associated with an increase in AOX protein abundance, suggesting that the increase reflected changes in the regulation of flux via the terminal oxidases (e.g. increased adenylate restriction of COX, and/or increased reduction of the ubiquinone pool resulting from greater electron flow through Complex I and/ or the non-phoshorylating dehydrogenases; Fig. 2). Further work is needed to assess whether there are consistent patterns in the effects of water stress on relative AOX and COX engagement of roots and leaves, irrespective of whether overall flux has increased, decreased or not changed. Balance between carbon gain and carbon loss: impacts of water stress

The overall effect of water stress on plant carbon balances depends on the extent to which photosynthesis versus R are affected by changes in water supply. As outlined above, leaf photosynthesis and R (of whole-plants and roots) invariably decrease following development of water stress; in many cases, R of mature leaves also decreases. Importantly,

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however, the proportional change in photosynthesis (both during the development of water stress and following rewatering) is almost always greater than the change in R (Lawlor, 1976; Lawlor and Fock, 1977, 1978; Nogues et al., 2001; Galmes et al., 2006, 2007). Because of this, cessation of watering typically results in the ratio of R to photosynthesis (R/P) being greater in water-stressed plants than their well-watered counterparts (e.g. Galmes et al., 2007), irrespective of whether R increased, decreased or remained unchanged in water-stressed tissues (Fig. 1B; but see McCree et al., 1984). Although the extent to which R/P increases under water stress depends on whether leaf R decreases, increases or remains unaltered (Fig. 1C), overall the most important factor determining how negative a plant’s carbon balance becomes under water stress is the absolute and proportional change in photosynthesis (Flexas et al., 2006; Galmes et al., 2007). Even in the few cases where R/P remains constant under water stress (e.g. due to proportional declines in R being matched by proportional declines in photosynthesis, such as that for Nicotiana sylvestris in Ghashghaie et al., 2001), drought is likely to result in reduced growth. This is because for a equal proportional declines in photosynthesis and R (e.g. 75 %), the absolute decrease in photosynthesis (e.g. from 20 to 5 mmol CO2 m22 s21) will invariably be greater than the absolute decline in R (e.g. 2 – 0.5 mmol CO2 m22 s21), resulting in overall net carbon gain being reduced under water stress conditions. In reality, both the absolute and proportional declines in photosynthesis are likely to be greater than the declines in R, further exacerbating the negative effects of water stress on productivity. If one assumes that drought-induced decreases in growth (and thus demand for growth R) are responsible for much of the decline in R rates exhibited by roots and whole-plants, one might expect that water stress would affect R less in inherently slow-growing species, whose growth rate is less inhibited by drought, than in comparable faster-growing species. Indeed, Flexas et al. (2005) provide several examples of where water stress has less effect on R of several slow-growing species. Interestingly, Galmes et al. (2007) also found that water stress has less effect on the R of mature, fully expanded leaves (i.e. not growing) of slow-growing evergreen shrubs than in faster-growing, herbaceous species; given that growth R does not contribute to gas exchange in fully developed leaves, the differences in impact of water stress on R in leaves of contrasting species differing in leaf traits must reflect inherent differences in the underlying rates and/or susceptibility of maintenance R to water stress in those species. Does water stress impact equally on leaf respiration taking place in light as in darkness?

In contrast to the large number of studies investigating the effects of water stress on leaf R measured in darkness (Rdark), relatively little is known about how decreases in water availability impact on non-photorespiratory, mitochondrial R in the light (Rlight). Galmes et al. (2006) found that Rlight (measured as CO2 release using the method of Laisk, 1977) was substantially reduced in tobacco leaves experiencing increasing levels of water stress (Table 2). Similarly, by

quantifying rates of 12CO2 release into an atmosphere containing 3000 mL L21 13CO2, Haupt-Herting et al. (2001) and Haupt-Herting and Foch (2002) found that Rlight was markedly lower in water-stressed wild-type and hp-1 (high-pigment) mutant tomato, compared with their well-watered counterparts. In the same experiment, Rdark also decreased in response to increasing water stress; however, water stress resulted in no change in photorespiratory CO2 release in hp-1 leaves. Water stress has also been shown to reduce rates of Rlight in the intertidal macroalga Ulva lactuca (Zou et al., 2007), particularly at high measuring temperatures. Thus, not withstanding the lack of studies assessing the effects of water stress on Rlight, the available studies suggest that water stress is likely to inhibit rates of Rlight in leaves where Rdark is inhibited. In most studies carried out using well-watered plants, rates of Rlight are lower than Rdark (Brooks and Farquhar, 1985; Pa¨rnik and Keerberg, 1995; Villar et al., 1995; Atkin et al., 1998a, 1998b, 2000b, 2006; Hoefnagel et al., 1998; Wang et al., 2001; Zaragoza-Castells et al., 2007) even when re-fixation of respiratory CO2 is taken into account (Pa¨rnik and Keerberg, 1995). Available evidence suggests that light inhibition of leaf R is the result of light-mediated reductions in pyruvate decarboxylase (PDC) activity (Randall et al., 1990) and transition to the partial TCA cycle in the light (Igamberdiev et al., 2001; Tcherkez et al., 2005), with changes in cellular energy status and NH3 production associated with photorespiration being responsible for both factors (Hurry et al., 2005). The inhibition of PDC activity by light is due to reversible phosphorylation, with inactivation of PDC being prevented by inhibitors of photorespiration (Budde and Randall, 1987, 1990; Gemel and Randall, 1992). This link between light inhibition of leaf R and photorespiratory metabolism may explain why the degree of light inhibition is often (but not always) greatest at high temperatures (Atkin et al., 2000a, 2006; Zaragoza-Castells et al., 2007; Zou et al., 2007) and why reductions in photorespiration (brought about by reduced O2 concentrations) result in an increase in Rlight (Atkin et al., 1998a). Given this, and because water stress often coincides with increased daytime temperatures, conditions favouring photorespiration, one might expect that the degree of light inhibition of leaf R (measured as non-photorespiratory CO2 release) to increase during drought. Further experimental data on rates of Rlight and Rdark in well-watered and water-stressed leaves in genotypes differing in rates of photorespiration (e.g. using photorespiratory mutants) are needed to test this hypothesis. When comparing effects of leaf R measured in the light and in darkness, it is important to distinguish between R measured as non-photorespiratory CO2 release and as mitochondrial O2 uptake. As discussed above, it is likely that the former will be lower in the light than in darkness, and that the degree of inhibition will increase with increasing water stress. In contrast, rates of mitochondrial O2 uptake have the potential to be higher in the light than in darkness (Hoefnagel et al., 1998; Hurry et al., 2005), reflecting mitochondrial oxidation of excess cellular redox equivalents whenever the redox poise of the chloroplast is excessive (e.g. in leaves exposed to bright light and with limited demand for NADPH). Given that water stress can limit the demand for NADPH by the Calvin cycle, probably as a consequence of ATP reduction

Atkin & Macherel — Plant mitochondria and drought tolerance (Tezara et al., 1999, 2008), we would predict that mitochondrial O2 uptake in the light would be greater in water-stressed leaves than their well-watered counterparts. Unfortunately, no data are yet available to test this hypothesis. There is also a lack of information regarding the interactive effects of water stress and other abiotic factors (e.g. short and long-term changes in temperature, variations in nutrient supply and/or changes in atmospheric CO2 concentration). Understanding how such factors impact on the water-stress response of plant R is essential if we are to predict better how future climates will effect plant growth and performance. P IVOTAL ROL E S OF M I TO CHO ND RI A I N P H OTO S Y N T H E T I C CE L L S Mitochondrion – chloroplast co-operation

In leaves, the nature and intensity of the metabolic interconnections between plastids and mitochondria is largely dependent on irradiance, since light energy drives chloroplast NADPH and ATP production, which in turn are the driving force for carbon uptake, nitrogen assimilation and photorespiration. In the dark or in heterotrophic tissues, the relationship between the plastids and mitochondria are different since mitochondria become the main energy providers (Hoefnagel et al., 1998). Nevertheless, irrespective of the tissue type, mitochondria always maintain their basic function of being the powerhouse of the cell, with the capacity for mitochondrial energy production being ubiquitous among contrasting tissue types and developmental stages. This is reflected at the biogenesis level since genes encoding Complex I subunits show much fewer diurnal variations in their expression than genes encoding alternative respiratory pathway components (Svensson and Rasmusson, 2001) or photorespiratory enzymes (McClung et al., 2000). The major metabolic connections between the two organelles in a non-water-stressed C3-type photosynthetic cell in the light have been tentatively represented in Fig. 4. In an optimal situation (open stomata, no heat stress), most atmospheric CO2 entering leaf tissue is converted into triose phosphates which are exported to the cytosol. The two major fates of the triose phosphates are: (1) to enter glycolysis and mitochondrial R to provide carbon skeletons for biosyntheses and ATP; and (2) to be converted into sucrose in the cytosol for storage and export. Some of the photosynthetic reducing power and ATP may also be used for nitrogen and sulphur assimilation. In this most favourable scenario, mitochondria essentially function as providers of the carbon skeletons necessary for nitrogen assimilation, and ATP for sucrose synthesis and cell metabolism (Fig. 4). Mitochondria support chloroplasts during water stress

What happens to the mitochondrion – chloroplast interplay in periods of water stress is a crucial question that was recently addressed in a comprehensive review about the effect of water stress on photosynthesis and R (Flexas et al., 2006). Here, we consider a schematic view in which mitochondria play a major role to support chloroplast functions, and ultimately plant survival, under conditions of sustained or severe water-stress

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conditions (Fig. 4). When plants experience drought, their primary response is the closure of stomata, which inevitably leads to a decrease of CO2 assimilation and the engagement of the photorespiratory pathway to recycle phosphoglycolate (Lorimer and Andrews, 1981). In the process, mitochondria have to cope with a high rate of glycine oxidation that requires a continuous re-oxidation of matrix NADH (Fig. 4). Experiments with inhibitors (Igamberdiev et al., 1997) indicates that photorespiratory mitochondrial NADH can be re-oxidized without energy conservation using the internal type II NADH dehydrogenase (whose expression is lightdependent; Michalecka et al., 2003) and AOX. In addition, NADH re-oxidation can be coupled to downstream reduction in the photorespiratory pathway through the malate/oxaloacetate shuttle coupled to hydroxypyruvate reduction in the peroxisome (Raghavendra et al., 1998). Since photosynthesis (and net CO2 assimilation) is strongly reduced under water stress (Fig. 1A), the efflux of chloroplastic triose phosphate, glycolysis and sucrose synthesis are much reduced, and glycine oxidation becomes prevalent in mitochondria of illuminated leaves. Under water-stress conditions, there should also be less need for mitochondrial export of carbon skeletons (e.g. citrate), because de novo nitrogen assimilation, especially at the level of nitrate reductase, is reduced under drought conditions (Foyer et al., 1998; Fresneau et al., 2007). However, it is crucial that the photorespiratory ammonia released in mitochondria be recycled; this requires the involvement of the glutamine synthetase – glutamate oxoglurate aminotransferase (GS-GOGAT) system in chloroplasts (Somerville and Ogren, 1980). Since glutamine synthetase is also localized in leaf mitochondria, it has been proposed that photorespiratory ammonia does not diffuse but is rather re-assimilated by the mitochondrial glutamine synthetase and directed to the plastid either by a glutamine/glutamate shuttle, or by a more complex ornithine/citrulline shuttle, that allows simultaneous export of photorespiratory CO2 released in mitochondria (Taira et al., 2004). Collectively, such observations demonstrate that here is a dramatic reorientation of leaf mitochondrial metabolism in the light under water-stress conditions in order to cope with the photorespiratory flux and consequences as well as with the arrest of net CO2 fixation. Under water-stress conditions, leaf mitochondria also need to contribute to the maintenance of cell energy balance and redox status. Reducing equivalents produced in the chloroplast (as a result of light absorption) can be exported from chloroplasts to mitochondria by the photorespiratory cycle and malate/oxaloacetate shuttle (Fig. 4), and subsequently oxidized via the mitochondrial electron transport chain (either resulting in phosphorylation of ADP to ATP or releasing energy as heat). However, the system is dependent on the functioning of the Calvin cycle, which in turn requires a higher ratio of ATP/NADPH than is provided by non-cyclic photophosphorylation. Because of this, part of the reducing power produced in the chloroplast under water-stressed conditions must be re-oxidized in other processes in the plastid (e.g. antioxidant systems) or in other compartments (see Kro¨mer, 1995). In sunflower, water stress has been shown to inhibit photosynthesis through impairment of chloroplast ATP synthase, preventing proper operation of Calvin cycle (Tezara et al., 1999). Interestingly, even under severe water stress, leaf ATP

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Atkin & Macherel — Plant mitochondria and drought tolerance Atm CO2

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F I G . 4. Metabolic implication of leaf mitochondria during water stress. The scheme figures the main metabolic fluxes occurring in a typical C3 leaf cell in optimal well-watered conditions, and under water stress. The thickness of the lines corresponds to the estimated importance of the fluxes. Diffusion is indicated with dotted lines, and metabolic shuttles with dashed blue lines. Restriction of carbon dioxide diffusion by closure of stomata and specific inhibition of plastid ATPase under water deficit are indicated by arrows in red circles. TN, Plastidial ATP/ADP antiporter; Mal//OAA, malate oxaloacetate shuttle; DHAP//PGA, dihydroxyacetone phosphate 3-phosphoglycerate shuttle; Glu//Pro, glutamate/proline shuttle; Gln//Glu, glutamine/glutamate shuttle; Orn//Citrn, ornithine/citrulline shuttle; PGA, 3-phosphoglycerate; TP, triose phosphate; Pyr, pyruvate; Ac-CoA, acetyl-coenezyme A; BBC, Benson– Bassham– Calvin cycle; TCA, tricarboxylic acid cycle; RubP, ribulose 1,5 bis-phosphate; Rubisco, ribulose 1,5 bis-phosphate carboxylase oxygenase; pdc, pyruvate decarboxylase complex; gdc, glycine decarboxylase complex; GS-GOGAT, glutamine synthetase– glutamate oxoglutarate aminotransferase; Cyt pathway, phosphorylating electron transfer pathway; Alt pathway, non-phosphorylating electron transfer pathway.

levels remained at around one-third of the well-watered control. This, together with the fact that R is generally less affected than P by water stress (see Fig. 1 and associated text), suggests that leaf mitochondria might play an important role in supplying chloroplasts with ATP under drought conditions (Keck and Boyer, 1974). The import of mitochondrial ATP into plastids can be mediated by an ATP/ADP nucleotide transporter (Heber and Heldt, 1981) which was recently shown to play a role in the nocturnal import of ATP into plastids (Reinhold et al., 2007). Under water stress, the respective

nucleotide transporters would allow a remote regeneration of chloroplastic ATP that originated in leaf mitochondria, using NADPH produced in illuminated chloroplasts (Fig. 4). Interestingly, the two arabidopsis genes encoding the chloroplast nucleotide transporter (At1g80300 and At1g15500) are both transcriptionally up-regulated by a transient drought stress or a prolonged osmotic stress (http://bar.utoronto.ca/; Toufighi et al., 2005; Winter et al., 2007), which seems to support the hypothesis that mitochondrial ATP can be imported into chloroplasts under water stress and thereby

Atkin & Macherel — Plant mitochondria and drought tolerance partially compensate for the impairment of chloroplast photophosphorylation under water stress. Such cooperation would be crucial to allow photorespiratory flux, including the regeneration of RuBP and the assimilation of ammonia by plastidial glutamine synthetase, to be maintained. A further problem facing chloroplasts under water-stress conditions is the potential for an imbalance between light energy absorption and dissipation. Photosynthetic cells are constitutively endowed with an array of safety valves to dissipate excess light energy and redox equivalents, including constitutive and non-photochemical quenching, chlororespiration, water – water cycle, photorespiration and redox equivalent poising (Niyogi, 2000). Importantly, however, mitochondria are also heavily involved in preventing over-reduction in the chloroplast through photorespiration, and by offering various ways to re-oxidize redox equivalents exported from the plastid. Both the malate/oxaloacetate (Mal/OAA) and the dihydroacetone phosphate/3-phosphoglycerate (DHAP/PGA) shuttles export excess NAD(P)H from plastid to the cytosol (Heineke et al., 1991; Niyogi, 2000). Evidence of this comes from the observation that an arabidopsis mutant defective in cyclic electron flow around photosystem I, and hence accumulating excess NADPH, exhibits an increased capacity in the Mal/OAA shuttle and mitochondrial AOX, suggesting a long-distance recycling of redox equivalents via malate from the plastid to mitochondria in low- and high-light conditions (Yoshida et al., 2007). While external NADH was elegantly shown to be preferentially re-oxidized by a reconstituted mitochondrial Mal-OAA shuttle (Pastore et al., 2003), under photorespiratory conditions the shuttle should rather export reducing power for peroxisomal hydroxypyruvate reduction (Fig. 4). Excess reducing equivalents in the cytosol might then be directly re-oxidized by the external mitochondrial NAD(P)H dehydrogenases, with electrons then passing either via the complexes (Complexes III and IV) associated with proton translocation (necessary for ATP synthesis) or via the non-phosphorylating AOX (Fig. 2). Dissipation of energy through non-phosphorylating electron transfer pathways [e.g. via the external NAD(P)H dehydrogenase and AOX] allows mitochondria to adjust energy production versus substrate oxidation, as well as to prevent ubiquinone over-reduction and subsequent oxidative stress (Møller, 2001). This is well illustrated by the up-regulation of the AOX pathway in response to water deficit in wheat leaves, and the fact that AOX inhibition affects photosynthetic electron transfer (Bartoli et al., 2005). Centrality of mitochondria in regulating proline concentrations

Mitochondria have an important role regarding proline homeostasis during water stress. In many plants, proline (which is a compatible osmolyte) accumulates in response to water stress and rapidly disappears upon recovery, due to variations in its cytosolic synthesis and mitochondrial degradation rates (Kiyosue et al., 1996); mitochondria thus play an important role during recovery of water stress when accumulated proline is rapidly degraded (Mani et al., 2002). Moreover, since proline synthesis from glutamate requires two molecules of cytosolic NADPH, and its reverse oxidative degradation in mitochondria generates FADH2 and NADH, it has been

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suggested that glutamate– proline cycling between cytosol and mitochondria could have a major role in redox homeostasis and metabolism (Hare and Cress, 1997). This is supported by the recent identification in durum wheat mitochondria of a proline/glutamate antiporter (Di Martino et al., 2006). The increase of the proline pool observed during water stress could support a proline/glutamine shuttle re-oxidizing cytosolic NADPH exported from the chloroplast or generated by the oxidative pentose phosphate pathway independently of the mitochondrial Mal-OAA valve (Fig. 4). When taken together, the available literature thus suggests that leaf mitochondria act as a versatile safety engine able to cope with considerable variations in chloroplast metabolism under water stress. The combination of metabolic shuttles, together with the exquisite balancing of the energy-conserving and energy-dissipating electron transfer pathways, is likely to allow cells to maintain energy and redox homeostasis under water stress, and chloroplast functionality for rapid resumption of growth upon recovery. I M PAC T S OF WAT E R ST RE S S O N P L AN T MI TO CH ON DR IA Plants subjected to abiotic stress inevitably face a disruption of cellular homeostasis with inevitable consequences for the functioning of mitochondria, including their ability to adjust cellular energy status to cope with adverse conditions and during recovery. Because of the crucial role of mitochondria in eukaryotic cells, one might expect that cells with compromised mitochondria should not be able to survive stress. This is illustrated by the decisive role of mitochondria during cellular life switches in animal cells (Kakkar and Singh, 2007) and by the primordial role of mitochondria in the autophagy process of cellular components in carbondeprived heterotrophic plant cells (Aubert et al., 1996). Here, we consider whether mitochondrial function is adversely affected by changes associated with drought conditions. As discussed in a previous section, R is generally affected by water stress to a lesser extent than photosynthesis, which fits well with the essential role of mitochondria. Nevertheless, water stress should have a significant impact on mitochondrial properties since mitochondria isolated from drought-stressed corn were shown in the 1970s to display altered membrane permeability and decreased rates of R without loss of coupling (Bell et al., 1971; Miller et al., 1971). More recently, mitochondria isolated from potato cells acclimated to water stress were found to have an increased capacity of several transporters (dicarboxylate, nucleotide, K channel, UCP) which could possibly be related to oxidative stress and energy management (Fratianni et al., 2001). Moreover, mitochondrial protein import has been shown to be stimulated by drought, which suggests a higher capacity for biogenesis and repair (Taylor et al., 2003). Increased capacity for repair is likely to be necessary, as oxidative damage of mitochondrial proteins increases in response to drought stress (Taylor et al., 2002, 2005; Bartoli et al., 2004). It should be stressed that different responses of mitochondria to water deficit may arise according to the tissues or organs. For instance, ionic permeability and phospholipid composition were clearly different between root and shoot

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mitochondria isolated from osmotically challenged wheat seedlings (Klein et al., 1986). Mitochondrial shape and structure in water stressed plants

An important issue related to water stress is how changes in osmotic potential impact on mitochondrial ultrastructure, morphology and movement. Mitochondrial swelling in vitro has been largely used for demonstrating ion and metabolite transport (Phillips and Williams, 1973), and, in animals, variations of matrix volume induces major functional and pathological events (Kaasik et al., 2007). Although plant mitochondria are highly dynamic organelles exhibiting movement, fusion and fission within live cells (Logan, 2006), little is known about the occurrence and functional consequences of variations in their matrix volume. In mammals, potassium cycling through the Kþ/Hþ antiporter and an ATP-sensitive gated potassium channel is a determinant of mitochondrial volume regulation (Garlid and Paucek, 2003). In plants, mitochondrial potassium channels exhibiting high conductivity were recently discovered, and have been suggested to play a major role in energy dissipation (Fig. 2) and prevention of oxidative stress (Pastore et al., 1999; Ruy et al., 2004). Such a system, which shows potential gating of the potassium channels by different compounds (nucleotides, NADH, metals), might also have an important role in the adjustment of mitochondrial matrix volume to the fluctuating water status of plants. Although the fate of mitochondrial morphology in vivo during water deficit is unknown, it has been known for many years that osmotic stress applied to isolated organelles depressed their substrate oxidation rates and phosphorylation capacity (Flowers and Hanson, 1969; Sells and Koeppe, 1981). In euglena, fully reversible alterations of the mitochondrial network were recorded upon shrinkage and rehydration of osmotically shocked but viable cells (Morris et al., 1985), and it is likely that similar events occurs in higher plant cells during dehydration and rehydration. Mitochondrial involvement in drought and desiccation tolerance

As far as water stress is concerned, it is interesting to look at models that show adaptation to water deficit, such as drought-resistant species and desiccation-tolerant organisms like resurrection plants and orthodox seeds. Durum wheat is a drought-tolerant cereal for which mitochondrial biology in relation to water stress has been extensively studied (Pastore et al., 2007). In addition to having an active AOX, durum wheat mitochondria exhibit a highly active UCP and an ATP-sensitive potassium channel, both of which are activated by ROS, suggesting the energy-dissipating systems may decrease ROS production in isolated mitochondria (Pastore et al., 2007). In addition, Pastore et al. (2007) found that moderate osmotic or salt stress applied to seedlings resulted in the activation of UCP and the potassium channel, suggesting an mitochondrial acclimation to water deficit (Pastore et al., 2007). The particularly high activity of the Mal/OAA shuttle in durum wheat mitochondria (Pastore et al., 2003) led to the suggestion that the Mal/OAA shuttle was the main route for re-oxidation of cytosolic NADH accumulating in stress

conditions (Pastore et al., 2007). While understanding the complexity of redox homeostasis in the illuminated stressed leaves of durum wheat as well as in other plants remains a challenge (see earlier text), the durum wheat model provides a sharp demonstration that mitochondria are deeply involved in the stress response, and that their energy-dissipating systems are crucial (Pastore et al., 2007). Resurrection plants are remarkable in that they can survive desiccation at the vegetative stage, which is common in lichens and mosses but sporadic among angiosperm taxa. Resurrection plants can lose up to 95 % of their cellular water content for prolonged periods, and regain rapidly their physiological activity upon re-hydration. Interestingly, while photosynthesis rapidly declines when water content falls, respiratory metabolism is well preserved until very low water content, suggesting the maintenance of energy supply is crucial for preservation and resumption of activity (Tuba et al., 1997, 1998; Farrant, 2000). To our knowledge, nothing is known about the features of mitochondria in resurrection plants, which have to manage not only with the consequences of moderate water deficit like in a typical leaf, but also with those related to full desiccation. Whatever the mechanisms that underpin resurrection capabilities, the efforts invested by plants in preserving R under various stress situations highlight the prominent role of mitochondria in cell life and fate. Seeds as a model for studying the role of mitochondria in desiccation tolerance

Seeds, which rely on the resumption of mitochondrial R to power germination, are an interesting model to investigate the properties of mitochondria with respect to desiccation tolerance (Benamar et al., 2003; Macherel et al., 2007). Most higher-plant seeds are desiccation tolerant, a property that permits the life cycle to be suspended for long periods of time, thus allowing the seedling to settle in favourable conditions. The so-called orthodox seeds (desiccation-sensitive seeds are called recalcitrant) lose all their free cellular water during the last stage of seed maturation, to reach a low water content of 5 – 15 % (fresh weight basis) depending on the species. Maize and pea mitochondria isolated from imbibing seeds were found to oxidize principally succinate and external NADH oxidation at high rates, while TCA cycle activities developed later during germination (Logan et al., 2001; Benamar et al., 2003). Interestingly, rice mitochondria were shown to be highly competent for protein import in early stages of imbibing, with external NADH being the major energy provider (Howell et al., 2006). Following desiccation, mitochondria with intact membranes would then rely on the simplest machinery to generate ATP during early stages of imbibing for resumption of metabolism and repair process. Resumption of R in dry tissues is very rapid in seeds (Benamar et al., 2003) as well as in resurrection plants (Tuba et al., 1998), which indicates that mitochondria are preserved throughout desiccation, and suggests the existence of protective mechanisms. Indeed, mitochondria from pea seed were shown to accumulate high concentrations of stress proteins such as HSP22 and LEAM, a late embryogenesis abundant protein protein (Bardel et al., 2002; Grelet et al., 2005).

Atkin & Macherel — Plant mitochondria and drought tolerance HSP22 belongs to the small HSP family, which contains prominent stress proteins in plants, with several cytosolic members expressed in seeds under a developmental programme, suggesting a role in desiccation tolerance (Wehmeyer et al., 1996; Wehmeyer and Vierling, 2000). However, the molecular function of HSP22 has not been elucidated so far. Late embryogenesis abundant proteins are hydrophilic proteins with repeated motifs that accumulate to high concentrations in seeds and other anhydrobiotic organisms, and which are expected also to be major contributors to desiccation tolerance. Recently, the mitochondrial protein LEAM was shown to be a natively unfolded protein soluble in the hydrated matrix space, but was able to fold into helices upon drying to immerse laterally into the inner membrane, and thus to provide protection in the dry state (Tolleter et al., 2007). In addition to the presence of stress proteins, mitochondria from pea seeds were shown to have a lower phosphatidylethanolamine : phosphatidylcholine ratio and less polyunsaturated fatty acids than their counterparts from epicotyls, a desiccation sensitive tissue (Stupnikova et al., 2006). Such a composition was proposed to be advantageous in the context of dehydration because phosphatidylethanolamine could destabilize membranes at low hydration, and polyunsaturated fatty acids would be targeted by oxidative stress which is a component of seed physiology (Stupnikova et al., 2006). Seed mitochondria are thus gifted with specific protective mechanisms (e.g. stress proteins, membrane adaptation) that contribute to their preservation through dry periods, and thus to desiccation tolerance of the cells. They are also surprisingly tolerant of extreme physiological temperatures, as shown by the fact that exogenous NADH can drive oxidative phosphorylation at temperatures as low as – 3.5 8C or þ40 8C in pea leaf mitochondria (Stupnikova et al., 2006). The ability of mitochondria from seeds, and possibly from some resurrection plants or other anhydrobiotes, to cope with severe drying indicates that mitochondria play a vital role in desiccation tolerance. Whether some of the specific features of seed mitochondria can play a role in a water-stress situation in vegetative tissues remains to be established. Although a faint expression for the seed mitochondrial LEAM protein could be induced in pea leaves by very severe water stress (Grelet et al., 2005), the abundance of the protein was too low to afford protection, and the response was attributed to a background activity of abscisic acid-regulated gene expression. It is reasonable to hypothesize that during periods of severe water stress, when mitochondrial operation becomes a life and death question for cells, the organelles may use seed-type strategies such as preferential use of exogenous NADH and succinate for ATP regeneration. In summary, although mitochondria require specific protective mechanisms to face the ultimate stress imposed by physiological desiccation as in seeds, they appear less sensitive than chloroplasts to the consequences of water deficit. Both the relative robustness of respiration in water-stressed tissues, and the rapid resumption of respiration in imbibing seeds suggest that mitochondrial ATP synthesis is not easily compromised by water stress. This contrasts with the situation in plastids where ATP synthase is a primary target of dehydration (Tezara et al., 1999). The reasons underlying the differential sensitivity of the organellar ATP synthases to dehydration are unknown,

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although the two enzymes are very similar in structure and function. They display however differences in subunit composition and architecture, the mitochondrial enzyme being more complex (Hong and Pedersen, 2003), and function in markedly different membrane and aqueous environments that may have a decisive influence on their functional state upon dehydration. CO NC LUS IO NS A ND F UTU RE PROSP E CTS This review has highlighted the fact that when plants are exposed to water stress, mitochondria display an array of metabolic adaptations involving metabolic shuttles and taking advantage of their intrinsic ability to dissipate energy. Under severe water stress, the preservation of mitochondrial function should be vital, and in some anhydrobiotic organisms such as resurrection plants and orthodox seeds, R appears to be extremely tolerant to water deficit. Accordingly, seed mitochondria are endowed with protective mechanisms such as energydissipating valves, stress proteins and phospholipid adaptations that contribute to stress tolerance. Although there is compelling evidence for a crucial role of mitochondria in water stress because of their pivotal role in energy, redox and metabolic fluxes, we have a limited understanding of the mechanisms involved and their role in determining cell responses to water stress. More generally, there is little knowledge about the changes in mitochondrial biology that accompany acclimation to sustained water stress, and almost none about the consequences of co-occurring environmental changes in temperature and light (shade or sunny). There is now little doubt that the Earth and humanity are facing rapid climate change that will result in frequent periods of drought, with dramatic consequences for natural and agricultural ecosystems. Respiration being central to survival and productivity, the aim would be to sharpen our vision of its contribution to the response to water deficit, and what could be the bottlenecks limiting adaptation to drought. It is thus essential to rapidly gain more insights into the role of mitochondria and respiration in orchestrating drought tolerance, both at the molecular and the whole-plant level. Here, we would like to highlight some research directions that deserve priority. Model plants offer the opportunity to address the impact of environmental stress at the physiological and functional genomic levels in a detailed and integrated manner. However, it will be of great importance to establish physiological models that account for multiple changes in the abiotic environment that accompany drought (rather than relying on the classical single parameter experiments). The respective contributions/interactions of water and co-occurring stresses at the level of gas exchange in intact tissues and isolated mitochondria will have to be quantified; the underlying metabolic changes will also need to be elucidated. It will also be important to examine the evolution of genome expression associated with acclimation to drought conditions. As the response is unlikely to be ontogenetically uniform, it is essential to gain information about the situation at different developmental stages. Establishing the extent to which respiratory acclimation to drought differs among different organs and tissues at different times of the day will also be important because of the considerable inter-organ variations in the physiology, and the

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likely diurnal differences leaves resulting from the intimate connection with photosynthesis. Most approaches have concerned the impact of water stress on R and mitochondria in leaves, and we know little about the impact on the respiratory flux in other tissues. Efforts need to be made to test the hypotheses involving energy-dissipating systems and the various metabolic shuttles to cope with drought-associated metabolism and oxidative stress management. Our core proposal that maintenance of mitochondrial ATP synthesis during water stress is essential for preserving plastid functions under water stress, and the subsequent recovery of net carbon gain following rewatering, also needs to be tested. The power of reverse genetic approaches, and screening for respiratory mutants in connection with drought tolerance, should shed light on the links between R and water stress. More effort also should be directed toward the exploration of the proteome and the lipidome of mitochondria in response to water deficit, not only in drought-sensitive plants but also in drought- and desiccation-tolerant plants, as well as in seeds which cope with loss of all free water while maintaining the integrity of mitochondria. Collectively, such approaches should provide a better understanding of the morphological, biochemical and functional responses of mitochondria to onset and recovery from water deficit, and, in so doing, provide important insights into how plants cope with one of the most common stresses experienced in nature. Given the globally widespread occurrence of water stress and the importance of mitochondria in all tissue types, and in helping maintain the functionality of chloroplasts in photosynthetic organs, understanding how mitochondrial metabolism is affected by water stress will be crucial for the development of more water stress-tolerant genotypes of crop species. Establishing a process-based understanding of how water stress impacts on respiratory metabolism in plants is also essential for understanding/predicting ecosystem responses to current and future climates. L I T E R AT U R E CI T E D Atkin OK, Botman B, Lambers H. 1996. The causes of inherently slow growth in alpine plants: an analysis based on the underlying carbon economies of alpine and lowland Poa species. Functional Ecology 10: 698–707. Atkin OK, Bruhn D, Hurry VM, Tjoelker MG. 2005. The hot and the cold: unraveling the variable response of plant respiration to temperature. Functional Plant Biology 32: 87– 105. Atkin OK, Evans JR, Ball MC, Lambers H, Pons TL. 2000a. Leaf respiration of snow gum in the light and dark: interactions between temperature and irradiance. Plant Physiology 122: 915– 923. Atkin OK, Evans JR, Ball MC, Siebke K, Pons TL, Lambers H. 1998a. Light inhibition of leaf respiration: the role of irradiance and temperature. In: Moller IM, Gardestrom P, Gliminius K, Glaser E eds. Plant mitochondria: from gene to function. Leiden: Bluckhuys Publishers, 25–32. Atkin OK, Evans JR, Siebke K. 1998b. Relationship between the inhibition of leaf respiration by light and enhancement of leaf dark respiration following light treatment. Australian Journal of Plant Physiology 25: 437–443. Atkin OK, Millar AH, Gardestro¨m P, Day DA. 2000b. Photosynthesis, carbohydrate metabolism and respiration in leaves of higher plants. In: Leegood RC, Sharkey TD, von Caemmerer S eds. Photosynthesis, physiology and metabolism. Dordrecht, The Netherlands: Kluwer Academic Publishers, 153–175. Atkin OK, Scheurwater I, Pons TL. 2006. High thermal acclimation potential of both photosynthesis and respiration in two lowland Plantago

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