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Journal of Experimental Botany, Vol. 59, No. 7, pp. 1675–1684, 2008 doi:10.1093/jxb/ern002 Advance Access publication 5 February, 2008 SPECIAL ISSUE REVIEW PAPER The enigmatic contribution of mitochondrial function in photosynthesis Adriano Nunes-Nesi*, Ronan Sulpice, Yves Gibon and Alisdair R. Fernie Max-Planck-Institut für Molekulare Pflanzenphysiologie, Am Mühlenberg 1, D-14476 Potsdam-Golm, Germany Received 27 September 2007; Revised 8 November 2007; Accepted 13 November 2007 Abstract Introduction Considerable cumulative evidence has accrued suggesting a vital role for mitochondrial function in optimizing photosynthesis. Both pharmacological approaches using respiratory inhibitors and reverse genetic approaches have recently underscored the high degree of interconnection between photosynthesis and respiration—the major pathways of energy production which are largely confined to the plastid and mitochondria, respectively. Here recent studies into the nature of these interactions are reviewed, with particular focus on (i) the recently described link between the mitochondrial electron transport chain activity, ascorbate biosynthesis, and photosynthesis; and (ii) the contribution of mitochondrial metabolism to the photorespiratory process. Whilst there is increasing evidence of a role for ascorbate in coordinating the rates of respiration and photosynthesis, some data are presented here for plants grown under extreme environmental conditions that suggest that this relationship is not absolute. It thus seems likely that interactions between these compartments are perhaps more numerous and complicated than previously thought. This observation suggests that although the elucidation of the genetic bases of both photorespiration and the Wheeler–Smirnoff pathway of ascorbate biosynthesis has recently been completed, much further research is probably necessary in order to understand fully how energy metabolism is coordinated in the illuminated leaf. Mitochondrial oxidative metabolism has been postulated to be a prerequisite for the maintenance of high rates of photosynthesis (Padmasree et al., 2002). Depending on the developmental stage and/or the environment, reducing equivalents generated by the photochemical reactions accumulate in the chloroplast stroma, causing the overreduction of the photosynthetic electron chain and the generation of reactive oxygen species (ROS), leading to photoinhibition (Foyer and Noctor, 2000; Allen, 2002). It has been proposed that this excess of reducing equivalents generated in the chloroplasts can be dissipated by their export from the chloroplast to the mitochondria, via the malate valve (Krömer, 1995; Scheibe, 2004; Scheibe et al., 2005). Within the mitochondria, the reducing equivalents are subsequently oxidized by the respiratory electron transport chain (Noctor et al., 2007), thus allowing continued high rates of photosynthesis. That said, recent studies have revealed that mitochondria– chloroplast interactions are by no means limited to this example, with considerable evidence suggesting roles for the tricarboxylic acid (TCA) cycle, both cytochrome and alternative respiration, and the uncoupling protein in the mediation of this cross-talk. Since the roles of the mitochondrial electron transport chain have been extensively reviewed previously (Bartoli et al., 2005; NunesNesi et al., 2007a; Sweetlove et al., 2007), they will not be documented here, but rather attention will be focused on the mechanisms by which modulation of the activities of TCA cycle enzymes and uncoupling protein exert their effects. Key words: Ascorbate biosynthesis, mitochondrial function, photorespiration, photosynthesis. A role for the TCA cycle in photosynthesis The TCA cycle is a fundamental component of mitochondrial respiration, linking glycolysis and/or * To whom correspondence should be addressed. E-mail: [email protected] ª The Author [2008]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved. For Permissions, please e-mail: [email protected] 1676 Nunes-Nesi et al. extramitochondrial malate synthesis to the mitochondrial electron transport chain (Fernie et al., 2004; Millar et al., 2004). As part of an ongoing project, a wide range of transgenics and mutants with deficient expression of enzymes from the TCA cycle have been analysed. Interestingly, modifications in the photosynthetic rate were found in a subset of these genotypes. Thus, tomato plants with less fumarase activity showed a decrease in photosynthesis (Nunes-Nesi et al., 2007b). In contrast, tomato plants with reduced expression of aconitase or malate dehydrogenase showed an enhanced photosynthetic performance (Carrari et al., 2003; Nunes-Nesi et al., 2005). Finally, in contrast to these effects, tomato plants with less succinyl CoA ligase (Studart-Guimarães et al., 2007) or less mitochondrially localized isocitrate dehydrogenase (Lemaitre et al., 2007) have no change in photosynthesis. While the reasons why decreased succinyl CoA ligase and isocitrate dehydrogenase are not essential to photosynthesis are currently unclear, the reduced photosynthetic activity found in fumarase antisense transgenics could be shown to be related to the impairment of stomatal function (Nunes-Nesi et al., 2007b). However, this phenotype is very specific to manipulation of fumarase activity and lies outside the scope of this article. Furthermore, a comprehensive molecular and metabolic characterization of lines with altered aconitase and malate dehydrogenase (MDH) activities revealed a specific increase in ascorbate in leaves (Carrari et al., 2003; NunesNesi et al., 2005), coupled to a general up-regulation of levels of transcripts encoding genes associated with photosynthesis (Urbanczyk-Wochniak et al., 2006). These transgenesis experiments hint at an important role for ascorbate in the co-ordination of major pathways of energy metabolism in the leaf. Moreover, they are in keeping with the current thinking that redox signals emanating from the mitochondria are important in setting the cellular machinery to maintain the overall redox balance (Sweetlove et al., 2007). To summarize, recent studies have revealed that photosynthetic performance can be modulated by modifications in mitochondrial pathways, such as the electron transport chain (Bartoli et al., 2005) or TCA cycle (Carrari et al., 2003; Nunes-Nesi et al., 2005). These studies have suggested ascorbate as a linkage between mitochondrial and chloroplast metabolism. Recently, another mitochondrial protein, an uncoupling protein (UCP-1), has been found to be involved in a novel mechanism contributing to photosynthesis through efficient oxidation of glycine produced via photorespiration (Sweetlove et al., 2006). The aim of this article is to review the current understanding of the contribution of the mitochondrial metabolism to photosynthesis, with a focus on the mitochondrial components of ascorbate metabolism and photorespiration. In the next sections, the intimate relationship between ascorbate metabolism and general mitochondrial metabolism is described in order to provide a possible context for the results described above. Ascorbate metabolism in mitochondria Ascorbate is an abundant antioxidant present at millimolar concentrations in plant leaves and in storage organs, which contributes significantly to the cellular redox state (Noctor and Foyer, 1998; Smirnoff, 2000; Smirnoff et al., 2001; Ishikawa et al., 2006). Ascorbate has also been reported to be involved in a range of other cellular processes such as photoprotection (Kiddle et al., 2003), establishment of whole-plant morphology, root architecture, and development (Olmos et al., 2006; Horemans et al., 2003). Furthermore, its oxidized form, i.e. dehydroascorbate (DHA), has been found to be involved in the regulation of the cell cycle (Potters et al., 2000, 2004) and in the control of stomatal closure in tobacco leaves (Chen and Gallie, 2004). Recently, the last missing intermediate steps of the Smirnoff–Wheeler pathway of ascorbate synthesis (Fig. 1) have been elucidated (Laing et al., 2007; Linster et al., 2007). A link between mitochondria and ascorbate biosynthesis has been acknowledged for many years, with the enzymatic conversion of L-galactono-c-lactone to L-ascorbate, the final reaction of the Smirnoff–Wheeler pathway, being demonstrated in the 1950s (Mapson et al., 1953; Mapson and Breslow, 1958). However, direct evidence that L-galactono-1,4-lactone dehydrogenase (GalLDH) is a membrane-intrinsic protein linked to the inner membrane of mitochondria was provided only recently (Siendones et al., 1999; Bartoli et al., 2000). Thus, in intact potato mitochondria, cytochrome c was found to be the electron acceptor for the reaction catalysed by GalLDH (Bartoli et al., 2000). It was also demonstrated that the activity of cytochrome c oxidase can modulate ascorbate synthesis. More recently, studies performed on mitochondria isolated from Arabidopsis leaves suggested that ascorbate synthesis is coupled to the cytochrome c pathway (Millar et al., 2003). However, rotenone, which inhibits the transfer of electrons from the Fe–S centres of complex I to ubiquinone, was found to inhibit the rate of ascorbate synthesis to the same extent as KCN, which blocks electron transport at the level of the cytochrome oxidase (Millar et al., 2003). Interestingly, when electrons entered the electron transport chain via complex II (succinate dehydrogenase) and without complex I being engaged, the addition of rotenone had no effect on the rate of ascorbate synthesis (Millar et al., 2003). Similarly, when electrons were introduced at cytochrome c via the oxidation of L-galactono-c-lactone as the sole substrate, rotenone exerted no effect on the rate of ascorbate synthesis (Millar et al., 2003). These observations suggest that the electron flow through complex I affects the rate of ascorbate synthesis, and thus implies that GalLDH is Contribution of mitochondrial metabolism to photosynthesis 1677 physically associated with complex I in Arabidopsis. Reverse genetics approaches have also been undertaken with this enzyme. Thus, the reduction of its expression in BY2 tobacco cells resulted in an impairment of cell division and growth (Tabata et al., 2001). More recently, an RNA interference (RNAi) approach was used in tomato to reduce the expression of GalLDH, in order to evaluate the effects of the modification of ascorbate pools on plant and fruit development (Alhagdow et al., 2007). Up to 80% reduction of the activity of GalLDH led to a strong reduction of leaf size, as a result of reduced cell expansion. Intriguingly, this was accompanied by an altered mitochondrial respiration (Alhagdow et al., 2007). Ascorbate recycling in mitochondria Ascorbate becomes rapidly oxidized to DHA via reactions involving oxidative species, and must therefore be efficiently recycled to prevent the degradation of DHA Fig. 1. Simplified representation of the Smirnoff–Wheeler pathway. HXK, hexokinase; PGI, phosphoglucose isomerase; PMI, phosphomannose isomerase; PMM, phosphomannose mutase; GMPP, GDPmannose pyrophosphorylase; GME, GDP-mannose-3,5-epimerase; GGP, GDP-L-galactose phosphorylase; L-GalP, L-galactose-1-phosphatase; L-GalDH, L-galactose dehydrogenase; GalLDH, L-galactono-1,4-lactone dehydrogenase. via ring opening (Winkler, 1987). Ring opening can deplete ascorbate pools within minutes; however, it is believed to be a non-enzymatic process and the physiological function of its product is currently unknown. That said, in plants, ascorbate turnover is relatively fast, for example, 13% per hour in pea seedlings (Pallanca and Smirnoff, 2000) and 40% in a 22 h period in Arabidopsis leaves (Conklin et al., 1997). In both plants and animals, mitochondria are also responsible for the regeneration of ascorbate from its oxidized form (Jiménez et al., 1997; Li et al., 2002; Chew et al., 2003; Szarka et al., 2007). DHA can be reduced to ascorbate via two mechanisms involving small electron carriers such as the reduced form of glutathione, GSH (Jiménez et al., 1999; Chew et al., 2003), or lipoic acid (Xu and Wells, 1996), or by the respiratory electron transport chain (Szarka et al., 2007). In tobacco BY2 cells, the regeneration of ascorbate was found to be stimulated by succinate and abolished by malonate, a competitive inhibitor of the reaction catalysed by succinate dehydrogenase, complex II (Szarka et al., 2007). It has also been suggested that the reduction of DHA is coupled to complex II and may contribute to the redox homeostasis of the mitochondria, with the transport of DHA through the inner mitochondrial membrane potentially playing a crucial role in this process (Szarka et al., 2007). However, exact details regarding the mechanism of its transport are currently lacking, and further work is required to substantiate these claims. It has also recently been demonstrated in both tobacco and maize leaves that ascorbate levels can be increased by enhancing the recycling of ascorbate via the overexpression of dehydroascorbate reductase, DHAR (Chen et al., 2003). DHAR catalyses the reduction of DHA to ascorbate in a GSH-dependent reaction (Fig. 2). The implication of this enzyme in regulating the ascorbate redox state has also been associated with the regulation of the foliar ROS level and subsequent photosynthetic activity during leaf development and ageing (Chen and Fig. 2. Simplified representation of the ascorbate–glutathione cycle in mitochondria. GSSG, glutathione disulphide; GSH, reduced glutathione; AsA, ascorbate; MDHA, monodehydroascorbate, DHA, dehydroascorbate; MDHAR, MDHA reductase; APX, ascorbate peroxidase; DHAR, DHA reductase; GR, glutathione reductase. The dashed black arrow indicates the spontaneous and irreversible hydrolytic ring rupture of DHA. 1678 Nunes-Nesi et al. Gallie, 2006). In addition, the GSH-dependent DHAR together with other enzymes of the ascorbate–GSH cycle (Fig. 2) has been found in purified Arabidopsis (Chew et al., 2003) and pea (Jiménez et al., 1997) mitochondria. Furthermore, in vitro import experiments showed dual targeting of ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), and glutathione reductase (GR) proteins to mitochondria and chloroplasts (Chew et al., 2003). In the same study, APX was reported to be located on the outside of the inner membrane, while the three enzymes involved in the ascorbate–GSH cycle system, i.e. MDHAR, GSH-dependent DHAR, and GR, were largely found in the matrix. The authors proposed that the association of APX with the inner membrane allows the coupling with GalLDH, in order to produce MDHA and DHA, which are needed in the matrix. The location of MDHAR, DHAR, and GR largely in the matrix is then proposed to ensure the recycling of NADH and NADPH (Chew et al., 2003). Whilst this theory is highly attractive, further evidence is required to support it. Nevertheless, the mitochondrial location of the ascorbate– glutathione cycle suggests that an interaction of key importance is taking place between mitochondrial and extramitochondrial metabolism in illuminated leaves. Interaction of ascorbate metabolism, respiration, and photosynthesis There are now several lines of correlative evidence that link the rates of ascorbate biosynthesis and respiration. It has been observed that plants exhibiting a down-regulation of mitochondrially localized enzymes, and displaying up to 50% reduction in dark respiration, contained increased levels of total ascorbate (Carrari et al., 2003; Nunes-Nesi et al., 2005; Urbanczyk-Wochniak et al., 2006). Similar results were obtained in studies on tomato mitochondria, wherein intact mitochondria isolated from green tomato fruits of plants with reduced activity of mitochondrial MDH were assayed. These experiments revealed no difference in the rate of oxygen consumption in the presence of malate and pyruvate, but a significantly enhanced rate of oxygen consumption when L-galactono-lactone and NADH were provided as respiratory substrates (Nunes-Nesi et al., 2005), suggesting that GalLDH can effectively act as an alternative electron donor in cases where flux through the TCA cycle is impaired. Clear and direct evidence that light and respiration affect the capacity for ascorbate synthesis was provided recently (Bartoli et al., 2006). The authors also demonstrated that the relative capacities of the cytochrome and alternative oxidase (AOX) pathways, as well as the overall capacity for leaf respiration, also influence the extent of leaf ascorbate accumulation. It has been further postulated that ascorbate biosynthesis is influenced by AOX activity, suggesting that ascorbate production is intimately linked to the mitochondrial electron transport chain (Foyer and Noctor, 2005). In light of previous work it is additionally tempting to speculate that ascorbate is the key link between light quality, respiration, and photosynthesis. However, recent data, shown in Fig. 3, reveal that this link is not absolute. Growing plants deficient in the expression of mitochondrial MDH under short-day conditions resulted in a dwarf phenotype (potentially indicative of less efficient photosynthesis; Fig. 3A–C), although displaying elevated levels of ascorbate and alteration in the redox state of this pool (Fig. 3D–F). These data thus indicate that despite a clear role for ascorbate, under many environmental conditions, other currently undefined mechanisms can prevent the upregulation of photosynthesis if the prevailing conditions are not suitable to support it. The most likely explanation for this observation is that the TCA cycle is, at least to a limited extent, inhibited in the light (Hanning and Heldt, 1993; Tovar-Mendez et al., 2003; Tcherkez et al., 2005) whereas in the dark it becomes the only functional pathway of energy metabolism. It could perhaps, therefore, be anticipated that extension of the dark period would produce the observed phenotype. That said, the fact that under optimal conditions inhibition of TCA cycle enzymes can result in improved plant performance, presumably by an effect on ascorbate biosynthesis, remains of great interest. The exact mechanism by which this effect is mediated is currently unclear, since although photosynthetic gene expression is clearly affected in the transgenics, ascorbate is capable of exerting a multiplicity of other effects on photosynthesis (Smirnoff and Wheeler, 2000). Indeed, results of a previous study demonstrated that loading of leaves with ascorbate for a mere 2 h in the dark resulted in an increase in carbon assimilation, suggesting that the effect seen in the transformants may be mediated by the metabolite directly. Genetic screens are currently being set up in Arabidopsis in the hope of being able to dissect this process (and the factors that prevent it from functioning under short-day conditions). The role of UCP in photosynthesis As described above, the multiplicity of mechanisms linking mitochondrial and plastidial function serve to reinforce the high degree of metabolic co-ordination inherent in the plant cell. Another mechanism that highlights the essentiality of the mitochondria for plant photosynthetic metabolism has been recently proposed that extends the concept of metabolic co-ordination to the cytosol and peroxisome. Biochemical and physiological analyses of a T-DNA insertional mutant of Arabidopsis deficient in the expression of the uncoupling protein AtUCP-1 revealed a specific inhibition of photorespiration Contribution of mitochondrial metabolism to photosynthesis 1679 Fig. 3. Effect of down-regulation of antisense mitochondrial MDH tomato plants under short-day conditions. The left-hand panel shows the growth phenotype of transgenic plants under short-day conditions (6 h light). (A) Photograph showing representative plants at 3 weeks growth. (C) Rate of growth over the developmental period. (E) Biomass accumulation in various plant organs at 4 weeks growth. The right-hand panel shows the ascorbate content of fully expanded leaves from plants at 4 weeks growth. (B) Total ascorbate content; (D) reduced ascorbate content; (F) reduced ascorbate/DHA ratio. Samples were taken from mature source leaves in the middle of a light period. Total ascorbate and reduced ascorbate were measured as described by Kampfenkel et al. (1995). DHA was calculated as the difference between total ascorbate and reduced ascorbate. Values are presented as mean 6SE of determinations on six individual plants per line; an asterisk indicates values that were determined by the t-test to be significantly different (P <0.05) from the control plants. (Sweetlove et al., 2006). Uncoupling proteins are integral to the inner mitochondrial membrane and catalyse proton conductance across this membrane (Fig. 4), dissipating the mitochondrial proton gradient as heat (Krauss et al., 2005). It has been postulated that this is important when the demand for oxidation of NADH is high, and may facilitate high TCA cycle flux (Smith et al., 2004). Consistent with this suggestion, the ucp1 mutants displayed a reduced photosynthetic carbon assimilation rate that was linked to a reduced rate of photorespiratory glycine oxidation (Sweetlove et al., 2006). This finding thus strongly corroborates both pharmacological and genetic evidence that mitochondrial respiratory processes play an important role in the co-ordination of metabolism in the illuminated leaf and introduces the much studied photorespiratory pathway as another area worthy of discussion here. Mitochondrial reactions of photorespiration Environmental stresses can affect the efficiency of the Calvin cycle in consuming NAD(P)H, and result in changes in the redox state of the chloroplasts and in a rapid increase of free radicals and ROS, a response that has to be tightly regulated to allow optimal functionality of photosynthesis (Foyer and Noctor, 2000; Noctor et al., 2007). A number of metabolic responses may provide protection against this oxidative process, including the process of photorespiration that is believed to mitigate 1680 Nunes-Nesi et al. Fig. 4. Simplified representation of the photorespiratory pathway and the relationship between UCP and photosynthesis. RuBP, ribulose-1,5bisphosphate; PGP, phosphoglycolate phosphatase; GOX, glycolate oxidase; SGAT, serine/glyoxylate aminotransferase; GDC/SHMT, glycine decarboxylase/serine hydroxymethyltransferase; HPR; hydroxypyruvate reductase; GK, glycerate kinase; GS, glutamine synthetase; GOGAT, glutamate/oxoglutarate aminotransferase; Fdred, reduced ferredoxin; Fdox, oxidized ferredoxin; Glu, glutamate; Gln, glutamine; 2-OG, 2-oxoglutarate; OAA, oxaloacetate. Black arrows indicate chemical reactions, while grey dashed arrows show membrane transport. photooxidative damage by functioning as an electron sink to prevent the over-reduction of the photosynthetic electron transport chain and photoinhibition (Kozaki and Takeba, 1996; Wingler et al., 2000; Noctor et al., 2002). Photorespiration is effectively a salvage pathway that returns some of the carbon lost to phosphoglycolate (produced under atmospheric conditions by the oxygenase reaction of Rubisco) to the Calvin cycle. This pathway, which involves both light-dependent carbon dioxide evolution and oxygen uptake, occurs concurrently with photosynthesis in the green leaves of C3 plants. Photorespiration restricts photosynthesis under conditions that lead to a high oxygenation of Rubisco such as high light, low external CO2 concentrations, high temperatures, and stress conditions that lead to stomatal closure and reduction in intercellular CO2 concentrations (Wingler et al., 2000). The reactions of the photorespiratory pathway are spatially separated across three distinct subcellular compartments: the chloroplast, mitochondrion, and peroxisomes (Fig. 4). The photorespiratory pathway is, however, commonly regarded to be highly inefficient given the loss of CO2 inside the mitochondria. This loss of carbon has been estimated as between 17% and 25% (Sharkey, 1988; Cegelski and Schaefer, 2006) of that fixed during photosynthesis. However, from the pathway itself, it would be postulated that from the carbon used for oxygen fixation, two out of five atoms enter photorespiration, and one out of four of these carbon atoms is released. Irrespective of the exact amount of carbon atoms lost, it is clearly a ‘wasteful’ process and as such photorespiration has been the issue of many studies which aim to alleviate the negative effects of this pathway. A good example of this is the work performed by Kebeish et al. (2007) showing that photorespiratory losses in Arabidopsis thaliana can be reduced by introducing into chloroplasts a bacterial pathway for the catabolism of the photorespiratory substrate, glycolate. This manipulation resulted in stunning increases in biomass which are presumably at least in part due to the bypass of the mitochondrial reactions of photorespiration. The exact Contribution of mitochondrial metabolism to photosynthesis 1681 reason, from an evolutionary perspective, why the plant photorespiratory pathway was retained, is of great interest, and the fact that the pathway is spread across the cell is both important and of general interest with respect to interorganellar co-ordination of metabolism. Within the mitochondrial matrix, the glycine decarboxylase–serine hydroxymethyltransferase (GDC–SHMT) complex convert two molecules of glycine to one molecule of serine with the simultaneous evolution of carbon dioxide and ammonium, and the production of NADH (Douce et al., 2001). GDC is a mitochondrial multienzyme complex, which consists of four different component enzymes, the P-, H-, T-, and L-proteins, involved in two pathways, photorespiration and one-carbon metabolism, in all photosynthesizing organs and in all biosynthetically active tissues (Douce et al., 2001; Bauwe and Kolukisaoglu, 2003). Whether GDC is obligatory for processes other than photorespiration was recently studied by Engel et al. (2007) who isolated and characterized T-DNA insertion mutants of both Arabidopsis P-protein genes, which encode the glycine decarboxylating subunit (Bauwe and Kolukisaoglu, 2003). The authors concluded that the independent knockout of either of the two genes did not significantly alter growth. However, when both P-protein knockouts were combined, seedling development was arrested at the cotyledon stage even under non-photorespiratory conditions, suggesting that GDC activity is required for processes other than photorespiration (Engel et al., 2007). A second mitochondrial enzyme which cooperates with GDC in photorespiration is SHMT, which converts glycine to serine (Douce et al., 2001). SHMT mutants in Arabidopsis, previously named as smt mutants, later renamed as shm, and recently termed shm1-1 (Voll et al., 2006), were among the first photorespiratory mutants described (Somerville and Ogren, 1981). Early studies of the shm1-1 mutant revealed that it contained no mitochondrial SHMT activity and exhibited 40-fold increases in the foliar level of glycine with respect to the wild-type control (Somerville and Ogren, 1981). More recently, Moreno et al. (2005) showed that SHMT activity plays a role in controlling cell damage caused by abiotic stress, probably because photorespiration forms part of the dissipation mechanisms that minimize production of ROS in the chloroplast and then mitigate oxidative damage. A novel mitochondrially targeted protein, glycolate dehydrogenase (AtGDH), has been identified in Arabidopsis (Bari et al., 2004). This enzyme has no homology on the protein level and differs in important enzymatic properties from the peroxisomal isoform (Bari et al., 2004). AtGDH transcripts are present in all tissues, but Fig. 5. Schematic representation of the proposed shuttle systems for ammonium transport in the form of amino acids between mitochondria and plastids. Dashed red arrows indicate the citrulline–ornithine shuttle, and the black pathway indicates the glutamate–glutamine shuttle. CPS, carbamoylphosphate synthetase; Cit, citrulline; Orn, ornithine; CarbP, carbamoylphosphate; OCT, Orn carbamoyltransferase; CK, carbamate kinase. 1682 Nunes-Nesi et al. accumulate preferentially in illuminated leaves, with higher accumulation under photorespiratory conditions, implying its contribution to this process (Niessen et al., 2007). However, T-DNA insertion lines for AtGDH, showing drastic reduction in mitochondrial GDH activity and photorespiratory CO2 release from glycolate, interestingly display no clear phenotype under ambient growth conditions (Niessen et al., 2007), suggesting that mitochondrial glycolate oxidation might not be essential for photorespiration. Although the physiological role of mitochondrial glycolate oxidation remains unclear, it has been reasonably postulated that it may act in combination with peroxisomal glycolate metabolism to provide the plant with flexibility to adapt to environmental conditions (Niessen et al., 2007). A further complexity of photorespiration was brought to light by the recent demonstration by Taira et al. (2004) that Arabidopsis GLN2 encodes GS2 precursors that can be translocated to both leaf mitochondria and chloroplasts. This surprising finding could potentially change the previously held view that NH+4 ammonia is generated in the mitochondria during photorespiration and subsequently actively or passively transported to plastids to be assimilated into glutamate by the glutamine synthetase 2 (GS2) and Fd-dependent glutamate synthetase (FdGOGAT) system (Fig. 5). These authors postulated an alternative model for this process in which a citrulline– ornithine shuttle between chloroplasts and mitochondria is proposed. This model would transfer not only ammonia from the mitochondria to the chloroplast but also carbon dioxide. This finding is largely in keeping with the observation that transgenic potato plants expressing SHMT in the antisense orientation displayed a lower photosynthetic capacity and accumulate glycine in the light (Schjoerring et al., 2006). Surprisingly, the nocturnal serine synthesis and concomitant release of postphotorespiratory NH+4 were increased in the SHMT transgenic plants. Also increased were the GS2 and FdGOGAT protein and activity levels (Schjoerring et al., 2006). However, it should be borne in mind that subcellular compartmentalization was not considered in this study. Furthermore, such a function can only be provided at the cost of higher energy consumption (Linka and Weber, 2005). Additional models have recently been put forward in which a less costly alternative glutamate– glutamine shuttle between mitochondria and chloroplasts is proposed (Linka and Weber, 2005) (Fig. 5), and therefore it seems prudent that more in-depth experimentation is required to resolve this issue. Conclusions Although the final genes encoding components of the photorespiratory (Boldt et al., 2005) and Smirnoff– Wheeler (Dowdle et al., 2007; Laing et al., 2007; Linster et al., 2007) pathways have recently been cloned, our understanding of the interaction of these processes and those of photosynthesis and respiration remains far from complete. The combined works reviewed here provide a strong case for the essentiality of mitochondrial function in the maintenance of efficient photosynthesis and, furthermore, begin to define the mechanisms by which this is mediated. Many open questions remain regarding the finer details of these interactions, and, as illustrated in the cases that are presented here, little is known concerning the hierarchical control, if any, that ensures that metabolism and function are effectively co-ordinated in the illuminated leaf. 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