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Journal of Experimental Botany, Vol. 55, No. 394, pp. 49±57, January 2004 DOI: 10.1093/jxb/erh021 Advanced Access publication 17 November, 2003 FOCUS PAPER Use of mitochondrial electron transport mutants to evaluate the effects of redox state on photosynthesis, stress tolerance and the integration of carbon/nitrogen metabolism Graham Noctor1,*, Christelle Dutilleul2, Rosine De Paepe2 and Christine H. Foyer3 1 Laboratoire Signalisation Redox, Institut de Biotechnologie des Plantes, BaÃtiment 630, Universite Paris XI, F-91405 Orsay cedex, France 2 Laboratoire Mitochondries et MeÂtabolisme, Institut de Biotechnologie des Plantes, BaÃtiment 630, Universite Paris XI, F-91405 Orsay cedex, France 3 Crop Performance and Improvement, Rothamsted Research, Harpenden, Hertfordshire AL5 2JQ, UK Received 18 June 2003; Accepted 3 October 2003 Abstract Primary leaf metabolism requires the co-ordinated production and use of carbon skeletons and redox equivalents in several subcellular compartments. The role of the mitochondria in leaf metabolism has long been recognized, but it is only recently that molecular tools and mutants have become available to evaluate cause-and-effect relationships. In particular, analysis of the CMSII mutant of Nicotiana sylvestris, which lacks functional complex I, has provided information on the role of mitochondrial electron transport in leaf function. The essential feature of CMSII is the absence of a major NADH sink, i.e. complex I. This necessitates re-adjustment of whole-cell redox homeostasis, gene expression, and also in¯uences metabolic pathways that use pyridine nucleotides. In air, CMSII is not able to use its photosynthetic capacity as well as the wild type. The mutant shows up-regulation of the leaf antioxidant system, lower leaf contents of reactive oxygen species, and enhanced stress resistance. Lastly, the loss of a major mitochondrial dehydrogenase has important repercussions for the integration of primary carbon and nitrogen metabolism, causing distinct changes in leaf organic acid pro®les, and also affecting downstream processes such as the biosynthesis of the spectrum of leaf amino acids. Key words: C/N metabolism, complex I, mitochondrial electron transport mutants, photosynthesis, redox state, stress tolerance. Introduction There is growing interest in the role of redox state in the orchestration of plant cell metabolism and in the determination of cell fate. Even in the soluble phase of the cell, `redox state' can mean many things, because, under most conditions, the major soluble redox-active couples (e.g. pyridine nucleotides, thioredoxins, glutathione, ascorbate) are unlikely to be in equilibrium with each other (Noctor et al., 2000). Thus, consideration of the in¯uence of redox state depends on the redox couple under discussion. For dehydrogenase activities, the major immediate in¯uence will be the reduction state of NAD or NADP. For the regulation of protein function by thiol/disuphide exchange, the redox states of thioredoxins and/or glutathione are likely to be most important. Other important redox-active compounds that interact with these components are reactive oxygen species (ROS), and their rates of production and destruction. Growing evidence demonstrates that all the above elements are not only key to the control of metabolism but are also sensed to initiate changes in gene expression. The primary aim of this review is to discuss * To whom correspondence should be addressed. Fax: +33 1 69 15 34 25. E-mail: [email protected] Abbreviations: AOX, alternative oxidase; APX, ascorbate peroxidase; Ci, leaf intercellular CO2 concentration; CMS, cytoplasmic male sterile; GDC, glycine decarboxylase; NDin, mitochondrial internal alternative NADH dehydrogenase; NR, nitrate reductase; 2-OG, 2-oxoglutarate; ROS, reactive oxygen species; TCA, tricarboxylic acid; WT, wild type. Journal of Experimental Botany, Vol. 55, No. 394, ã Society for Experimental Biology 2004; all rights reserved 50 Noctor et al. recent evidence derived from studies of the Nicotiana sylvestris mutant, CMSII, relating to the role of mitochondrial redox state in leaf metabolism and defence. Leaf mitochondria play key roles in photosynthesis, e.g. glycine decarboxylation in C3 plants and malate decarboxylation in certain C4 plants. Moreover, leaf mitochondrial function will depend on whether the cells are photosynthetic or non-photosynthetic. Despite extensive work on puri®ed leaf mitochondria and mitochondrial components (Douce and Neuburger, 1989; Douce et al., 2001; Mùller and Rasmusson, 1998; Mùller, 2001), much remains to be elucidated concerning the role of the mitochondrial electron transport chain in the integration of leaf metabolism (for reviews, see KroÈmer, 1995; Hoefnagel et al., 1998; Foyer and Noctor, 2000). The key questions addressed below are: How important is mitochondrial electron transport in the C3 photosynthetic process? What is the signi®cance of changes in mitochondrial redox state for leaf redox signalling and stress tolerance? Do changes in mitochondrial redox state impact on the co-ordination of carbon and nitrogen assimilation in leaves? An incisive approach to answering these questions is the analysis of plants in which key components of the mitochondrial electron transport chain have been manipulated genetically. Isolated in Orsay, the Nicotiana sylvestris mutant, CMSII, is one of the few such stable mutants characterized to date. Phenotypic and developmental characteristics of CMSII Numerous studies on the consequences of genetic modi®cation of enzyme expression have emphasized the impressive ¯exibility of leaf metabolism. It might therefore be predicted that leaves possess considerable redundancy in redox exchange between subcellular compartments, and that the impact of non-lethal mutations would be absorbed by metabolic adjustments, preventing marked perturbations or phenotypic effects. The essential feature of CMSII is a deletion mutation in the mitochondrial gene, nad7, leading to the absence of functional complex I (Gutierres et al., 1997). Thus, the mutant has lost the function of a major NADH sink, resulting in partial male sterility, i.e. decreased production of pollen. Although the mutation also causes slower shoot growth during the vegetative phase, the ®nal biomass attained by the mutant is not signi®cantly different from the wild type (WT). It is therefore possible to analyse the two genotypes at comparable stages of shoot development, and thus to obtain information on the importance of mitochondrial electron transport status in leaf physiology and metabolism. A comparable stage of development is achieved by sowing CMSII seeds 3 weeks earlier than the WT. Figure 1 compares the morphology of CMSII with the WT at about 10 d prior to the stage at which sampling is performed. Fig. 1. Photograph of Nicotiana sylvestris WT (right) and the CMSII mutant, de®cient in mitochondrial complex I (left). Six plants of each genotype are shown. WT and CMSII plants were germinated 9 and 12 weeks, respectively, prior to the time at which the photograph was taken. The data discussed in this review were obtained by analysis of plants at a developmental stage corresponding to 10 d later than that shown in the photograph, when both genotypes are comparable in shoot size and leaf number. Respiratory activities of the CMSII mutant Despite the absence of complex I function, CMSII does not show decreased rates of leaf respiration in the dark, suggesting that TCA cycle activity is maintained in the mutant by complex II and alternative dehydrogenases that bypass complex I (Sabar et al., 2000; Dutilleul et al., 2003a). In mitochondria isolated from CMSII leaves, O2 consumption is insensitive to rotenone and ADP/O values are signi®cantly lower than in WT mitochondria (Sabar et al., 2000). Flux to O2 in mutant mitochondria is probably facilitated by the increased reduction state of mitochondrial NAD(P) pools, which allows the internal alternative dehydrogenases to be engaged (Mùller, 2001). At ®rst sight, therefore, the response of respiration to the loss of complex I seems to point to a further example of enzyme redundancy in plants. However, one of the principal themes of this article is to show that, even if respiration is not decreased when complex I is nonfunctional, the absence of this NADH sink has important consequences for leaf function, and necessitates numerous adjustments in leaf metabolism and gene expression that uncover the potentially critical in¯uence of mitochondrial redox status. A key observation in isolated CMSII mitochondria is that respiratory capacity relative to the WT is substratedependent (Sabar et al., 2000). Gly metabolism is inhibited about 70% relative to the WT, whereas respiration of malate, in the presence of pyruvate, is unchanged (Sabar et al., 2000). Subsequent work showed that, in the dark, Redox interactions in mitochondrial complex I mutants CMSII leaf discs converted Gly to Ser slower than WT leaf discs (Dutilleul et al., 2003a). These observations cannot be explained by a possible absence of catalytically active Gly decarboxylase (GDC). First, the mutant shows a pronounced burst of CO2 evolution during the ®rst minute following a light±dark transition and, second, Gly/Ser ratios in the light are not increased relative to the WT (Dutilleul et al., 2003a). Thus, the internal alternative NADH dehydrogenase (NDin) appears to be able to function with malate dehydrogenase and/or malic enzyme but much less ef®ciently with GDC, probably re¯ecting the con¯icting kinetic properties of NDin and GDC: the KmNADH of NDin is about 5-fold higher than the KiNADH of GDC (Douce et al., 2001; Bykova and Mùller, 2001). Hence, in the absence of complex I and signi®cant extramitochondrial reductant sinks (i.e. in isolated mitochondria or in leaves in the dark), the accumulation of NADH to a concentration required to engage NDin substantially is incompatible with optimal function of GDC. Intercompartmental redox shuttles during photosynthesis The role of mitochondrial matrix enzymes in C3 photosynthesis is well established (Douce and Neuburger, 1989), and work with Arabidopsis and barley `photorespiratory' mutants has demonstrated that GDC and serine hydroxymethyl transferase are indispensable for photorespiratory carbon and nitrogen cycling (Somerville and Ogren, 1981; Blackwell et al., 1990). The fate of the matrix NADH produced by GDC is less clear (Leegood et al., 1995; Hoefnagel et al., 1998; Foyer and Noctor, 2000; GardestroÈm et al. 2002). Studies in vitro led to the conclusion that the reducing equivalents are shared between the mitochondrial electron transport chain and exchange with the peroxisome via malate/oxaloacetate shuttles (Hanning and Heldt, 1993). If this is so in vivo, the mitochondrial electron transport chain must deal with a considerable ¯ux of electrons from GDC and a large fraction of the reductant necessary for peroxisomal glycerate synthesis must be produced elsewhere, presumably the chloroplast, via the malate valve (Hanning and Heldt, 1993; Hoefnagel et al., 1998; Scheibe, 2003). When one considers that the malate valve may also be coupled to the mitochondrial electron transport chain via cytosolic and mitochondrial redox shuttles, the picture that emerges is one of chloroplast and mitochondrial reductant export co-existing with peroxisomal and mitochondrial reductant import. Furthermore, in the cytosol, enzymes such as glyceraldehyde-3-phosphate dehydrogenase may also make a contribution to NAD(P)H production, whereas nitrate reductase (NR) acts as an electron sink. According to this view, the roles of the chloroplast and peroxisome in redox exchange in illuminated leaves are relatively clear. Stated simply, the former is a reductant exporter, the latter 51 a reductant importer. Mitochondrial function in the light is a more complex question. Because the mitochondrial NADH/NAD ratio is maintained about 100-fold higher than the cytosolic ratio, KroÈmer and Heldt (1991) concluded that malate/oxaloacetate exchange must operate in the direction of net reductant export from the mitochondria, and that mitochondrial oxidation of NAD(P)H produced in the cytosol probably occurs through external dehydrogenases (reviewed by Mùller, 2001). In the leaves of most C3 species, photosynthesis (and, in many conditions, the accompanying process of photorespiration) can be very much faster than dark respiration. The light-dependent photorespiratory pathway complicates the measurement of respiration in the light, though most data indicate that non-photorespiratory CO2 evolution is signi®cantly inhibited in the light relative to the dark, whereas inhibition of mitochondrial O2 consumption is less apparent (Avelange et al., 1991; PaÈrnik and Keerburg, 1995; reviewed by KroÈmer, 1995; Hoefnagel et al., 1998). Thus, under many conditions, mitochondrial electron transport activity in the light is probably maintained, in part, by processes other than `dark' respiration. What is the predicted effect of photorespiratory Gly oxidation on total mitochondrial electron transport activity? Given that ¯uxes of carbon into glycollate approach the rate of net photosynthesis under physiological conditions, it might be considered that when C3 photosynthesis is rapid, signi®cant partitioning of reductant from GDC activity to the mitochondrial electron transport chain would entail much higher respiratory electron transfer rates than those operating in darkened leaves. Measurements of respiratory O2 consumption during active photorespiration are technically dif®cult, notably because ribulose-1,5-bisphosphate oxygenation and associated glycollate oxidation account for most of the observed gross O2 uptake (Rey and Peltier, 1989). Nevertheless, it can be inferred that even during high rates of photosynthesis in air, a substantial fraction of Gly oxidation could be coupled to the mitochondrial electron transport chain without a huge increase in overall electron transfer rates relative to those that are commonly observed in darkened leaves (Table 1). This is because (1) only half the Gly produced must be oxidized, (2) at least part of the reducing equivalents is exported from the mitochondrion, and (3) tricarboxylic acid (TCA) cycle activity in the light is probably signi®cantly less than in the dark (PaÈrnik and Keerburg, 1995). Nevertheless, the values given in Table 1 suggest that signi®cant coupling of Gly decarboxylation to the mitochondrial chain would cause some increase in overall electron transport rates. An attractively simple mechanism, involving inhibition of pyruvate dehydrogenase by photorespiration-stimulated protein phosphorylation (Gemel and Randall, 1992), has been identi®ed that could explain how, in the light, mitochondria in photosynthetic cells substitute GDC activity for TCA cycle 52 Noctor et al. Table 1. Predicted effect of photorespiration on mitochondrial electron transport rates in the photosynthetic cells of C3 leaves The data compare typical rates of leaf O2 consumption in the dark with maximum and likely rates of mitochondrial O2 consumption at moderately fast rates of photosynthesis. It is assumed that the ratio of ribulose-1,5-bisphosphate (RuBP) carboxylation:oxygenation is 2.5, which is a likely value for non-stressed C3 leaves (Keys, 1999); and that `dark' respiration is inhibited 50% by light (see text for references). Maximum [G] and likely [I] rates of Gly-linked O2 consumption in the light assume that Gly oxidation is coupled either 100% [G] or 40±60% [I] to the mitochondrial electron transport chain. The basic equation for calculating Gly-linked O2 consumption by the electron transport chain can be represented by 2 Gly+0.5 O2 ® Ser+CO2+NH3. For purposes of simplicity, O2 consumption that could be linked to export of reductant from the chloroplast is not considered. For further discussion, see text. Parameter Derivation Value (mmol m±2 s±1) [A] `Dark' respiration (in dark) [B] `Dark' respiration (in light) [C] RuBP carboxylation [D] RuBP oxygenation [E] Glycine oxidation [F] Net CO2 uptake [G] Maximum Gly-linked O2 consumption [H] Maximum total mitochondrial O2 consumption in light [I] Likely Gly-linked O2 consumption in light [J] Likely total O2 consumption in light Typical leaf respiration rate 0.5 [A] Moderately fast C3 photosynthesis [C] / 2.5 0.5 [D] [C] ± ( [B] + [E] ) 0.5 [E] [B] + [G] 0.4 [G] to 0.6 [G] [B] + [I] 1 0.5 25 10 5 19.5 2.5 3 1 to 1.5 1.5 to 2 turnover. A principal mitochondrial function in the light could be to generate precursors, notably 2-oxoglutarate (2OG) for net ammonia assimilation (i.e. incorporation of ammonia other than reassimilation of, for example, photorespiratory ammonia). This function, which requires only partial TCA cycle activity, is discussed further below. Complex I is required for optimal CO2 ®xation under physiological conditions Much of the data available on the role of mitochondrial electron transport in photosynthesis has been generated by inhibitor treatment of leaf pieces or protoplasts (KroÈmer et al., 1988, 1993; Igamberdiev et al., 1997, 1998; Padmasree and Raghavendra, 1999). This and other work has pointed to several mechanisms by which mitochondrial electron transport and associated oxidative phosphorylation could be important in optimizing photosynthesis. First, mitochondrial ATP production could be crucial to support UDP-glucose formation for sucrose synthesis in the cytosol. Second, as discussed above, mitochondrial electron transport is probably responsible for the regeneration of part of the NAD used by GDC. Third, the mitochondria might act as the ultimate acceptors for reductant generated in the chloroplast and, therefore, prevent over-reduction of this organelle. In the mutant, CMSII, non-function of complex I does not cause decreased photosynthetic capacity. At saturating CO2 concentrations, where photorespiration is negligible, CMSII displays photosynthetic rates at least equal to the WT (Dutilleul et al., 2003a). Despite the clear restriction over Gly oxidation in CMSII mitochondria in the dark, in illuminated leaves the Gly/Ser ratio is not increased relative to the WT (Dutilleul et al., 2003a). Thus, though inhibited in the dark, Gly metabolism in CMSII is supported in the light by components other than complex I, presumably extra-mitochondrial reductant sinks. As discussed above, NDin is unlikely to be able to substitute completely for complex I, though a contribution from NDin to photorespiratory Gly metabolism cannot be excluded, given that (1) Gly oxidation is only 70% inhibited in isolated CMSII mitochondria (Sabar et al., 2000) and (2) NDin shows some evidence of lightinduction in potato leaves (Svensson and Rasmusson, 2001). Although light restores Gly metabolism in the mutant, this comes at a price for the overall photosynthetic process, suggesting that, in the WT, complex I plays a signi®cant role in regenerating NAD for GDC activity. Carbon ®xation is consistently decreased in the mutant by 20± 30% in air (Sabar et al., 2000; Dutilleul et al., 2003a). Analysis of Ci curves and O2 sensitivity demonstrates that photosynthesis is less inhibited in CMSII, relative to the WT, under conditions where CO2 ®xation (and, therefore, sucrose synthesis) is enhanced. This suggests that the primary cause of decreased photosynthesis in CMSII in air is unlikely to be insuf®cient mitochondrial ATP production for sucrose synthesis (Dutilleul et al., 2003a). Even more striking than the decrease in steady-state photosynthesis is the effect on the photosynthetic induction period, which is greatly prolonged relative to the WT (Dutilleul et al., 2003a). From these data the following conclusions can be inferred. First, mitochondrial complex I is largely dispensable for photosynthesis when photorespiration is inactive, probably because, in this condition, C3 photosynthesis is limited to chloroplastic events, accompanied by sucrose synthesis in the cytosol. Second, in air, active complex I plays an important role in the inter-compartmental redox cycling involved in C3 photosynthesis (Fig. 2A). Third, when extra-mitochondrial sinks are Redox interactions in mitochondrial complex I mutants available in the light, Gly metabolism can continue in the absence of complex I, but greater demand for other reductant sinks impacts on redox cycling in the rest of the cell, decreasing the ef®ciency of the photosynthetic process (Fig. 2B). This last conclusion is supported by increased activation state of the chloroplast NADP-malate dehydrogenase in CMSII, suggesting that the reduction state of the stroma is increased relative to the WT (Dutilleul et al., 2003a). One interpretation of the inability of the mutant to deploy its photosynthetic capacity as ef®ciently as the WT is that the photosynthetic cell has a relatively narrow window of redox ¯exibility and that the mitochondrial electron transport chain plays an important part in setting the width of this window. Other components can compensate for the absence of complex I in maintaining Gly oxidation, but this lowers the rate of photosynthesis that can be achieved. Mitochondria and leaf redox homeostasis: in¯uence on diurnal regulation and the determination of stress resistance The redox changes in CMSII are accompanied by induction or diurnal adjustment of antioxidant systems located within and outside the mitochondria (Dutilleul et al., 2003b). Although modulation of antioxidant expression is usually taken as an indicator of oxidative stress, modi®ed expression in CMSII occurs despite the absence of any evidence of increased oxidative load or overall leaf ¯ux to reactive oxygen species (ROS). In fact, the mutant shows decreased leaf H2O2 contents, both in the light, where mitochondrial H2O2 formation is probably a minor component of total leaf production, and in the dark, where the mitochondria are probably a major source of H2O2 (Dutilleul et al., 2003b). Associated with lower leaf H2O2 is signi®cantly enhanced resistance to both biotic and abiotic stress, linked at least in part to a higher capacity of antioxidative enzymes (Dutilleul et al., 2003b). The increased availability of reductant, as discussed above, might also play a signi®cant role in a more general upregulation of defence metabolism that underpins observations that CMSII has a higher constitutive resistance than the WT to a broad spectrum of environmental constraints and stresses (authors' unpublished results). As for several other C3 species, there are three genes encoding catalase in N. sylvestris, and the transcripts of these genes all vary throughout the day/night cycle. Whereas CAT1 is most strongly expressed in the dark, CAT2 and CAT3 transcripts peak in the light (Dutilleul et al., 2003b). These diurnal changes are enhanced in CMSII, as is the rhythm for cytosolic ascorbate peroxidase (cAPX) transcripts, which are highest towards the end of the photoperiod (Dutilleul et al., 2003b). In contrast to these transcripts, for which WT expression and/or light/ dark rhythms are reinforced in CMSII, the diurnal pattern 53 of alternative oxidase (AOX) expression is completely inverted in the mutant. At each point measured throughout the light/dark cycle, AOX transcripts are more abundant in CMSII than in the WT. However, AOX transcripts peak in the light in the WT but are more abundant in the dark in CMSII, so that the relative increase in AOX transcripts (CMSII/WT) is about 2-fold in the light but 10-fold or more in the dark (Dutilleul et al., 2003b). The expression of AOX has been shown to be inversely correlated with mitochondrial ROS availability in tobacco (Maxwell et al., 1999), and AOX capacity can play a role in determining cell fate in oxidative conditions (Robson and Vanlerberghe, 2002; Vanlerberghe et al., 2002). While the factors controlling AOX expression remain fully to be elucidated, it is possible that mitochondrial redox state, manifested either as the relative reduction of NAD(P) or of key electron transport components such as ubiquinone, is one of the principal determinants of AOX transcript abundance. Although the intermediaries that link changes in mitochondrial redox state to nuclear gene expression remain to be identi®ed, the initial sensing could involve components such as mitochondrial thioredoxins (Laloi et al., 2001). Engagement (i.e. in vivo activity) of AOX protein is dependent on reductive activation, linked to the reduction status of mitochondrial pyridine nucleotide pools, probably either through the mitochondrial glutathione system or, more likely perhaps, NADPH-dependent reduction of mitochondrial isoforms of thioredoxin (Mùller and Rasmusson, 1998; Vanlerberghe and Ordog, 2002). Effects on nuclear transcripts that result from mitochondrial redox sensing could be multifactorial in nature, with thioredoxin(s) perceiving the NADPH reduction state and other components such as ROS relaying information on the status of the electron transport chain itself. Mitochondria-linked redox modulation impacts on the co-ordination of carbon and nitrogen assimilation Co-ordination of carbon and nitrogen metabolism in leaves involves intricate cross-talk at the level of gene expression and enzyme activity (Stitt et al., 2002; Foyer et al., 2003). Complementarity and competition also occurs in the use of metabolites and energy, and the interlocking of C and N assimilation entails communication between several compartments of the leaf cell, including the chloroplast, the cytosol, and the mitochondrion (Noctor and Foyer, 1998). Comparison of foliar metabolite pro®les in CMSII and the WT indicates that complex I dysfunction impacts strongly on the leaf C/N interaction (authors' unpublished results). Thus, even though respiration rates in the dark are not decreased in the mutant, the relative amounts of metabolites involved in the TCA cycle are modi®ed. Most strikingly, 2-OG, the carbon skeleton for the glutamine 54 Noctor et al. Redox interactions in mitochondrial complex I mutants synthetase/glutamate synthase (GS-GOGAT) pathway (reviewed by Lea and Mi¯in, 2003), is decreased in the mutant. By contrast, malate and citrate are generally increased in CMSII, so that the citrate/2-OG ratio is typically increased by a factor of 10±20 relative to the WT. These changes are accompanied by increased ammonia and Gln/Glu, together with sharp increases in amino acids with low C/N (Asn, Arg). Increases in citrate/2-OG, and in Gln/Glu, contrast with the absence of changes in Gly/Ser in illuminated leaves of the mutant, and suggest that changes in mitochondrial redox state impact more strongly on the mitochondria± chloroplast interactions required for ammonia assimilation than on GDC activity. Consistently enhanced citrate/2-OG ratios indicate a restriction over the mitochondrial conversion of citrate to 2-OG, either through modulation of aconitase or isocitrate dehydrogenase, or both. As the latter enzyme is NAD-dependent, the simplest explanation of increased citrate/2-OG in CMSII is that it is a response to increased reduction of the matrix NAD pool. Thus, the adjustment in CMSII of the relative pool sizes of TCA cycle intermediates leads to a perturbation of the GSGOGAT pathway, as shown by an increase in Gln/Glu and in ammonia. These effects can be predicted to occur when 2-OG supply is low relative to that of ammonia (Novitskaya et al., 2002). This observation is interesting given the uncertainty over which isocitrate dehydrogenases (mitochondrial or cytosolic) are most important in anaplerotic production of 2-OG (reviewed by Hodges et al., 2003). Theoretical arguments have been advanced in favour of a predominant role for the cytosolic NADPdependent form (Chen and Gadal, 1990) and indeed transcripts encoding this enzyme are induced by nitrate (Scheible et al., 1997). In CMSII, however, the abundance of this protein is similar to the WT, whereas the mitochondrial NAD-dependent enzyme is signi®cantly enhanced (authors' unpublished results). The induction of the mitochondrial NAD-dependent form indicates that the de®cit in 2-OG acts, directly or indirectly, as a signal that speci®cally up-regulates this enzyme. The accompanying up-regulation of phosphenolpyruvate carboxylase (authors' unpublished results) is further evidence that homeostatic activation of anaplerosis occurs in the mutant in response to 2-OG de®cits, and the consequent perturb- 55 ation of the GS-GOGAT pathway, that result from the absence of mitochondrial complex I activity. Complex I de®ciency also causes changes in C/N interactions downstream of the GS-GOGAT pathway. Co-ordination of the synthesis of minor amino acids is a key feature of N metabolism in fungi. This phenomenon, known as the general control of amino acid synthesis, is largely unexplored in plants (Foyer et al., 2003). Recent data suggest the operation of some form of inter-pathway cross-talk in wheat, potato and barley leaves (Noctor et al., 2002). The contents of several minor amino acids also appear to be co-ordinated in N. sylvestris. In the WT, amino acids with relatively high C/N are most closely coordinated whereas in the mutant, the modulation of the C/ N interaction causes minor amino acid pools to become dominated by amino acids rich in N (Arg, Lys). Hence, the perturbation of the GS-GOGAT pathway, involving decreased 2-OG, increased Gln/Glu, and increased ammonia, feeds downstream to the synthesis of minor amino acids (authors' unpublished results). This causes a weakening in the co-ordination of minor amino acids, favouring N-rich amino acids at the expense of C-rich amino acids. Concluding remarks The data discussed above demonstrate that mitochondrial redox state is in¯uential in co-ordinating major leaf metabolic functions. Redox changes of mitochondrial origin modulate metabolism and nuclear gene expression. Key questions concern how such redox changes are sensed. First, by analogy to the chloroplast plastoquinone pool, the redox state of an electron transfer component such as ubiquinone could be important. Second, redox effects on leaf metabolism and function could be mediated by changes in pyridine nucleotide pools, which would directly in¯uence dehydrogenase activities and feed downstream to in¯uence processes located in other subcellular compartments, such as CO2 ®xation and ammonia assimilation. Third, effects could occur through modi®ed protein function and/or gene expression mediated by protein thiol/disulphide exchange linked to thioredoxins and/or protein glutathionylation. Thiol-modulation of proteins is a well characterized mode of post-translational regulation, particularly in chloroplast metabolism, where thiol/dis- Fig. 2. Simpli®ed cartoon of the importance of the mitochondrial electron transport chain in redox shuttles during C3 photosynthesis. (A) WT leaves with optimized photosynthetic activity. (B) One possible explanation of why non-functional complex I renders the Nicotiana sylvestris mutant, CMSII, less ef®cient for CO2 assimilation under physiological (photorespiratory) conditions (Dutilleul et al., 2003a). It should be noted that the red bars are meant to indicate restrictions over ¯ux, and not absolute blocks, notably because of the presence of other components that can at least partly replace complex I function (see text for further discussion). In (B), the proposed sequence of cause±effect is indicated by numbers. Brie¯y, in spite of the presence of alternative dehydrogenases, the absence of functional complex I (1) limits NADH oxidation by the mitochondrial electron transport chain (2), which puts extra pressure on reductant export, notably to the peroxisome (3). This encroaches on a minor, but signi®cant, electron sink for chloroplast reducing equivalents (4), leading to perturbation of redox poise and decreased photosynthesis (5). 56 Noctor et al. ulphide exchange is linked to the ¯ux of electrons through the electron transport chain. In the mitochondria, thioredoxin is reduced by NADPH, which is generated by enzymes such as NADP-dependent isocitrate dehydrogenase. Finally, modi®ed mitochondrial ROS production might be one factor responsible for the observed changes in nuclear gene expression that result from the absence of a major NADH sink in complex I-de®cient plants. References Avelange MH, Thiery JM, Sarrey F, Gans P, ReÂbeille F. 1991. Mass-spectrometric determination of O2 and CO2 gas-exchange in illuminated higher plant cells. Evidence for light-inhibition of substrate decarboxylations. Planta 183, 150±157. Blackwell RD, Murray AJS, Lea PJ. 1990. Photorespiratory mutants of the mitochondrial conversion of glycine to serine. 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