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Journal of Experimental Botany, Vol. 49, No. 329, pp. 1895–1908, December 1998 REVIEW ARTICLE A re-evaluation of the ATP :NADPH budget during C 3 photosynthesis: a contribution from nitrate assimilation and its associated respiratory activity? Graham Noctor1 and Christine H. Foyer Department of Biochemistry and Physiology, IACR-Rothamsted, Harpenden, Herts AL5 2JQ, UK Received 11 July 1998; Accepted 4 August 1998 Abstract The purpose of this review in reanalysing the ATP5reductant balance in illuminated leaf cells is to stress that photosynthesis in vivo does not involve CO 2 fixation alone, but embraces other processes, chief among which is N assimilation. Prior to the demonstration of CO fixation and photophosphorylation by isol2 ated chloroplasts, the mitochondria were thought likely to provide all the ATP required for CO fixation 2 (discussed in Arnon et al., 1954). During the 1960s, the development of techniques for the isolation of chloroplasts able to fix CO at rates approaching those 2 of the parent tissue induced a paradigm shift, leading to the establishment of a dominant (if not unanimous) view that chloroplasts in vivo must by themselves meet all their ATP requirements. More recent studies, however, indicate that the reality lies somewhere between these two extremes. The present work places emphasis on the integrated nature of photosynthesis and proposes that much of the respiratory ATP necessary for whole cell photosynthesis may be generated during the production of C skeletons for N assimilation. Rather than considering dissipative electron transport pathways as necessary to uncouple respiratory precursor synthesis from ATP production, the present analysis emphasizes the metabolic value of ATP produced during N-linked respiration, with cellular ATP supply being tailored to ATP demand. Key words: Photosynthesis, ATP :NADPH ratio, CO fixa2 tion, nitrogen assimilation, photorespiration, respiration. The ATP: reductant requirements of photosynthetic CO fixation 2 The reductive assimilation of inorganic compounds into the cellular matter of plants is mediated by low potential electrons (principally reduced ferredoxin, NADPH, NADH ) and nucleoside triphosphates (mainly ATP). These two types of molecule are required in specific reaction sequences at fixed stoichiometries and their intracellular pools turn over rapidly (typically, in less than 1 s). Their relative rates of production must, therefore, be closely co-ordinated with their relative rates of consumption. Nowhere has this question engendered more discussion than in the area of photosynthesis where, since the first demonstration of photophosphorylation and CO fixation by isolated chloroplasts (Arnon et al., 2 1954), much attention has focused on the chloroplastic processes responsible for producing ATP and NADPH at the stoichiometry required for CO fixation via the 2 RPP pathway (1.5 at the steady-state; Edwards and Walker, 1983; Horton, 1985). This ratio is increased by photorespiratory flux and by the consumption of ATP in the conversion of sugar phosphates to sucrose or starch. It is still uncertain whether the ATP requirements of C assimilation can be met solely by linear electron flow to NADP (for a review, see Horton, 1985). Consequently, 1 To whom correspondence should be addressed. Fax: +44 1582 760981. E-mail: [email protected]. Abbreviations: Ala, alanine; asp, aspartate; 1,3-bPGA, 1,3-bisphosphoglycerate; C:O, ratio of carboxylation:oxygenation of RuBP; FBP, fructose 1,6bisphosphate; Fd, ferredoxin; GAP, glyceraldehyde 3-phosphate; GAPDH, GAP dehydrogenase; gln, glutamine; glu, glutamate; gly, glycine; GOGAT, glutamine:2-oxoglutarate amidotransferase; GS, glutamine synthetase; HPR, hydroxypyruvate reductase; ICDH, isocitrate dehydrogenase; MDH, malate dehydrogenase; ME, malic enzyme; NiR, nitrite reductase; NR, nitrate reductase; OAA, oxaloacetate; 2-OG, 2-oxoglutarate; PDH, pyruvate dehydrogenase; PEP, phospoenolpyruvate; PGA, 3-phosphoglycerate; Pi, phosphate; PK, pyruvate kinase; PSI, photosystem I; PSII, photosystem II; QR, quantum requirement; Rubisco, RuBP carboxylase/oxygenase; Ru5P, ribulose 5-phosphate; RuBP, ribulose 1,5-bisphosphate; RPP, reductive pentose phosphate; ser, serine; TCA, tricarboxylic acid; TP, triose phosphate. © Oxford University Press 1998 1896 Noctor and Foyer alternative pathways of chloroplastic electron flow, enabling ATP production without NADP reduction, have frequently been considered necessary. Of these, the most often considered are cyclic electron flow around PSI (Arnon et al., 1954; Arnon and Chain, 1975; Heber et al., 1978; Furbank and Horton, 1987) and O reduction on 2 the reducing side of PSI ( Egneus et al., 1975; Marsho et al., 1979; Behrens et al., 1982; Furbank and Badger, 1983; Robinson, 1988). Because of the difficulty of directly measuring the former and the problems of distinguishing the latter from other routes of O uptake in the leaf, most 2 studies addressing the importance of these mechanisms have been carried out with in vitro systems. Such studies have demonstrated the potential of these mechanisms in supporting light-dependent ATP synthesis in the chloroplast (Arnon et al., 1954; Egneus et al., 1975; Heber et al., 1978; Furbank and Horton, 1987; Robinson, 1988), though the actual extent to which they contribute to ATP synthesis in the intact leaf remains unresolved. Investigation of relationships between the photochemical yields of the two photosystems and the quantum yield of CO fixation have generally failed to provide strong 2 evidence for high rates of either cyclic electron flow or photosynthetic O reduction in vivo (Genty et al., 1989; 2 Harbinson and Foyer, 1991). The ATP :reductant requirements of photosynthetic N assimilation In the intact plant, photosynthesis cannot be considered merely as the assimilation of carbon. The second largest sink for photosynthetic energy in many higher plants is nitrate. Since the assimilation of nitrate in many species occurs predominantly in the leaves (Smirnoff and Stewart, 1985), this process will often be ongoing simultaneously with CO fixation in photosynthetic cells. Elemental ana2 lysis suggests that net rates of N assimilation are around 6–13% of net C assimilation ( Edwards and Walker, 1983; Raven, 1988; Cram, 1990; de la Torre et al., 1991). Measured rates show that the capacity for N assimilation is typically 5–10% of that for CO assimilation 2 ( Robinson, 1988). In terms of the required reductant and ATP, incorporation of nitrate into glu, via NR, NiR, GS, and GOGAT, can be represented simply by the following equations: NO−+NADHNO−+NAD (1) 3 2 NO−+6Fd NH++6Fd (2) 2 red 4 ox Glu+NH++ATPGln+ADP+Pi (3) 4 Gln+2-OG+2Fd 2Glu+2Fd (4) red ox In photosynthetic cells, the reaction represented by equation 1 takes place in the cytosol (Smirnoff and Stewart, 1985) but may use reducing power generated in the chloroplast (Gray and Cresswell, 1984). The reactions depicted by equations 2–4 occur in the chloroplast stroma, using photosynthetically reduced Fd (Miflin, 1974; Lea and Miflin, 1974; Keys et al., 1978). The overall ATP and reductant balance of equations 1–4 can be represented as follows: NO−+2-OG+10e−+ATPglu+ADP+Pi (5) 3 Thus, NO− assimilation into glu requires 2.5 times as 3 many electrons as the reduction of CO to the redox level 2 of TP but only 0.33 times as much ATP. The assimilation of N is tightly co-ordinated with C assimilation through a complex array of regulatory processes involving transcriptional and post-transcriptional controls (Foyer et al., 1995). For example, NO− reduction in leaves is activated 3 by de-phosphorylation of NR, which prevents binding of the 14-3-3 protein inhibitor (Nussaume et al., 1995; Bachmann et al., 1996). If plants are deprived of CO , 2 then N assimilation ceases in both shoots and roots ( Kaiser and Forster, 1989; Pace et al., 1990). Foliar nitrate assimilation is markedly stimulated by light (Reed et al., 1983) and can lead to pronounced increases in light-dependent O evolution (Bloom et al., 2 1989; de la Torre et al., 1991). The associated photosynthetic electron transport activity is expected to produce ATP in amounts well in excess of that required for the reaction catalysed by GS. Thus, as discussed by other authors ( Edwards and Walker, 1983; Horton, 1985), the amount of ATP which must be generated during the production of NADPH for CO fixation is less if nitrate 2 assimilation is also occurring. The above equations describe simply the reductive assimilation of NO− into the a-amino group of glu. In 3 order to evaluate the full impact of N assimilation on cellular ATP status, however, the production of 2-OG has to be considered. For nitrate assimilation to continue in the light, 2-OG must be continuously generated, ultimately from photosynthate. This will necessitate at least partial operation of the glycolytic pathway and the TCA cycle. It is clear that inorganic N sources stimulate respiration, increasing the incorporation of newly-fixed photosynthate into TCA cycle intermediates and amino acids while diverting carbon from sucrose and starch (Graham and Chapman, 1979; Larsen et al., 1981; Foyer et al., 1994; Scheible et al., 1997; Turpin et al., 1997). Partial operation of glycolytic and TCA cycle activities to provide carbon skeletons for N assimilation is therefore another potential source of ATP, in addition to that generated in the chloroplast during photosynthetic nitrate reduction. Dark respiration in the light? The extent to which respiratory substrate decarboxylations and O uptake occur in illuminated photo2 ATP5NADPH budget during C photosynthesis 1897 3 synthetic cells is still a matter of debate. Using mass spectrometry to distinguish actual rates of CO uptake 2 and CO release, Avelange et al. (1991) concluded that 2 respiratory decarboxylations were partially inhibited by light. In contrast, other gas-exchange studies suggested that the rate of respiratory CO release in the light is 2 equal to, if not higher than, the rate in the dark (AzcónBieto and Osmond, 1983). It seems clear from metabolite measurements that considerable flux though glycolysis and the TCA cycle continues in the light (reviewed by Graham and Chapman, 1979; Krömer, 1995). Indeed, some oxidative C metabolism must occur, in order to generate C skeletons for biosynthetic purposes. Significant amounts of C precursors will be required for processes such as the shikimate pathway (Hermann, 1995) and tetrapyrrole synthesis (Castelfranco and Beale, 1983). Most attention, however, has focused on the role of respiratory metabolism in meeting the C requirements of primary N assimilation in the illuminated leaf (Graham and Chapman, 1979; Larsen et al., 1981; Foyer et al., 1994; Scheible et al., 1997; Turpin et al., 1997). In addition to supplying C skeletons, oxidative C flux in leaves may also be necessary to provide organic acids important in acid-base regulation during the assimilation of nitrate in the shoot (Raven, 1988). If it is now accepted that some respiratory C flow must occur in the light, in order to sustain biosynthetic pathways, the role of oxidative phosphorylation in the light is less clear. Data from mass spectrometric analysis suggest that respiratory O uptake is not markedly influenced 2 by light (Gerbaud and André, 1980; Avelange et al., 1991). However, the mitochondrial inner membrane contains several sites for reductant entry and O reduction, 2 and the ATP yielded during NAD(P)H oxidation can vary considerably (Azcón-Bieto et al., 1983; Douce and Neuburger, 1989; Lambers, 1997). Nevertheless, three studies point to continuing mitochondrial ATP production during photosynthesis. Firstly, it is unlikely that the cytosolic ATP5ADP ratio in illuminated leaves reaches the high values required to inhibit oxidative phosphorylation in the mitochondrion (Stitt et al., 1982; Gardeström, 1987). Secondly, respiration can support autotrophic growth in Chlamydomonas mutants lacking the thylakoid ATP synthase (Lemaire et al., 1988). Thirdly, perhaps most importantly, selective inhibition of mitochondrial ATP production leads to lower rates of photosynthetic O evolution in leaf protoplasts ( Krömer et al., 1988, 2 1993) and in leaves ( Krömer and Heldt, 1991). This observation has been rationalized in terms of a requirement for mitochondrial ATP production to support sucrose synthesis in the cytosol; according to this interpretation, inhibition of mitochondrial ATP synthesis restricts sucrose synthesis, leading to feedback inhibition of photosynthesis ( Krömer et al., 1988, 1993). An integrated approach to cellular ATP5reductant balance during C photosynthesis 3 Although several authors have considered the influence of the reductive processes of nitrate and nitrite assimilation on the ATP balance during photosynthesis ( Edwards and Walker, 1983; Horton, 1985), we are not aware of any discussion of the impact of the associated, necessary, oxidative C flow. In this study an integrated analysis of ATP supply and demand in a leaf cell carrying out photosynthesis has been undertaken. Photosynthesis will be considered as the two major reductive assimilations of inorganic elements (C and N ), together with the synthesis of carbohydrate and amino acids, photorespiration and ‘dark’ respiration necessary for ongoing N assimilation in the steady-state. Together, these processes can be considered as the principal energy-requiring and energyyielding metabolic sequences in the illuminated photosynthetic cell. The calculated data illustrate the potential importance of N-linked respiratory ATP production in sustaining high rates of CO fixation. Central to this 2 approach is the recognition that N assimilation in the illuminated leaf involves two interdependent but distinct processes: (1) oxidative formation of oxoacids from triose phosphate, and (2) reductive incorporation of nitrate into oxoacids to form amino acids. Underlying assumptions Analyses of this type must make certain fundamental assumptions, notably concerning the ratio of ATP5NADPH production by the chloroplast electron transport chain and coupling factor, which is still a matter of debate. Here, the value of 1.33 has been chosen as the starting point for these calculations. This value is derived assuming that 2 H+ are translocated per electron transported and that 3 H+ (Hangarter and Good, 1982; Strotmann and Lohse, 1988) are required per ATP synthesized by the chloroplast coupling factor (for further discussion of these questions, see Edwards and Walker, 1983; Horton, 1985; Ort and Oxborough, 1992; Cramer et al., 1996). Figure 1 illustrates the metabolic processes considered in the analysis. Carbon assimilation, photorespiration and NH+ incorporation are assumed to occur 4 via accepted pathways ( Edwards and Walker, 1983; Keys et al., 1978; Lea and Miflin, 1974; Leegood et al., 1995). For N assimilation and its associated respiratory activity, several metabolic alternatives must be considered ( Krömer, 1995). These are indicated on Fig. 1 by numbers, and are described in the legend. It is likely that these alternative pathways are simultaneously active, to some degree, under in vivo conditions. However, because of the difficulty of predicting their relative importance, various exclusive combinations of these alternatives have been considered (see Table 2). It should be noted that only the minimum respiratory activity required for N 1898 Noctor and Foyer Fig. 1. Diagram of energy production and utilization during the synthesis of carbohydrate and major foliar amino acids in the leaf cell of a C 3 plant. Reaction sequences are not depicted stoichiometrically and not all substrates, co-factors and exchange-transport metabolites are shown. The processes illustrated are photosynthetic and mitochondrial electron transport, the RPP pathway, the photorespiratory C cycle, starch synthesis, 2 sucrose synthesis, and N assimilation and its associated respiratory C flow. Reaction sequences are shown in detail only for N assimilation. Net products are denoted by capital letters in boxes. Solid arrows show chemical conversions; dashed arrows indicate translocation between compartments. For reasons of simplicity, re-incorporation of photorespiratory NH , which is assumed to be complete and to occur via the 3 chloroplastic GS/GOGAT system, is not shown (this process will involve utilization of photosynthetic electrons and ATP and is included in the calculations of Tables 1 and 2). Unless stated otherwise, the calculations of Tables 1 and 2 assume that hydroxypyruvate reduction is NADHdependent and is perfectly coupled to gly oxidation in the mitochondrion via a malate/OAA shuttle (Hanning and Heldt, 1993; Leegood et al., 1995). In calculating gas exchange parameters ( Table 1) and cellular ATP balance ( Table 2), it is assumed that the production of oxoacids from TP occurs by the shortest metabolic route possible, as shown (i.e. the calculations of Tables 1 and 2 consider the minimum oxidation of TP necessary to support given rates of net amino acid synthesis). Transaminations of pyruvate to ala and OAA to asp are shown to be cytosolic for reasons of diagrammatic simplicity: these reactions can also occur in the mitochondrion and chloroplast (Ireland, 1997). The data of Tables 1 and 2 are calculated according to different combinations of alternative assumptions about the processes involved in N assimilation. These are marked by numbers on the figure and are as follows. (1) Reductant for NR activity is provided by glycolytic NADH (1a) formed during conversion of TP to oxoacids or by export of malate (1b) from the chloroplast (Smirnoff and Stewart, 1985; Scheibe, 1987). (2) TP is oxidized to PGA in the cytosol by NAD-GAPDH/PGA kinase ( left) or by NADP-GAPDH (right; Kelly and Gibbs, 1973). (3) Pyruvate is produced from PEP via PK (3a) or via PEPC, NAD-MDH and NAD-ME (3b). (4) Citrate is converted to 2-OG via mitochondrial aconitase and NAD-ICDH (4a) or via cytosolic aconitase and NADP-ICDH (4b; Chen and Gadal, 1990). For pyruvate production via NAD-ME, it is assumed that the high-affinity mitochondrial OAA transporter ( Ebbighausen et al., 1985) competes effectively with cytosolic MDH so that OAA is transported into the mitochondrion before being reduced to malate. Since the two-step conversion of OAA to pyruvate produces and consumes matrix NADH in equal amounts, no ATP gain will result from import of reducing equivalents into the mitochondria when considering oxidation of cytosolic NAD(P)H by mitochondrial external dehydrogenases (see Table 2). Abbreviations as in the list, except: NQR, NADH-Q oxidoreductase (complex I ); ext, externally-oriented NAD(P)H dehydrogenases; bc , cytochrome bc (complex III ), aa , cytochrome c oxidase (complex IV ); FNR, ferredoxin-NADP oxidoreductase; 1 1 3 CF , thylakoid ATP synthase; F , mitochondrial ATP synthase. 1 1 assimilation is considered (partial operation of glycolysis and the TCA cycle; Fig. 1). Regardless of the route of pyruvate production, mitochondrial PDH activity would be required to produce acetyl CoA. Although this activity is lower in the light than the dark (Budde and Randall, 1990; Gemel and Randall, 1992), it is noteworthy that only half of the C flowing through PEP is converted to acetyl units and CO during anapleurotic respiratory 2 activity (Fig. 1). Through the chloroplastic GS/GOGAT cycle, glu is an ATP5NADPH budget during C photosynthesis 1899 3 value for C plants under typical atmospheric conditions 3 (Sharkey, 1988; De Veau and Burris, 1989). Values for two rates of N assimilation are shown that give a ratio of net C5N assimilation of 19–20.1 (N=5) and 9–10.1 (N=10). These values may be considered conservative estimates of N assimilation rates in mature and young leaves, respectively (de la Torre et al., 1991). The %C incorporated into amino acid pools will vary as a function of net N assimilation rates and the nature of the amino acid products ( Table 1). Although the amount of C incorporated into amino acids may seem substantial, it should be noted that these values are predicted by likely in vivo molar ratios of C5N assimilation, at least for the initial incorporation of N into major foliar amino acid pools. Early labelling experiments investigating the proportion of fixed C incorporated into total free amino acids (including ser and gly) gave typical values of 25–40% after 15–30 min light (Smith et al., 1961; Ongun and Stocking, 1965). The calculations presented here assume that, under steady-state conditions, the synthesis of gly and ser from phosphoglycollate will be matched by their conversion to sugar phosphates (Fig. 1; Berry et al., 1978). Photorespiration increases the required ATP5reductant ratio in two ways. Firstly, because of lower rates of net CO fixation per RuBP metabolized, C flow through the 2 early product of N assimilation (Lea and Miflin, 1974). In the steady-state, transamination reactions will regenerate 2-OG and consume other oxoacids, and amino acids will be used in further syntheses (for example, proteins, nucleotides, haem, and chlorophyll ). The complexity of these reactions forbids a comprehensive assessment of their influence. Nevertheless, an attempt can be made to approximate the likely net oxoacid requirements of N assimilation by considering production of those amino acids from which the majority of others are synthesized. Hence, in addition to the synthesis of glu alone, net production of asp, ala and gln has been considered ( Fig. 1). These compounds, along with photorespiratory ser and gly, typically represent the major amino acid pools in illuminated leaves of C plants (Larsen et al., 3 1981; Noctor et al., 1997). This is reflected in the relative activities of foliar aminotransferases, of which the highest (along with GOGAT and the photorespiratory aminotransferases) are glu:pyruvate, glu:OAA, and asp:pyruvate ( Ireland, 1997). The influence of N assimilation on O and CO exchange 2 2 Table 1 shows the influence of N assimilation and its associated respiration on gas exchange parameters calculated for given rates of CO fixation (set at 125) and 2 photorespiration (set at 50). A C5O ratio of 2.5 is a likely Table 1. Effect of N assimilation into different amino acid products on gas exchange parameters Rate of N assimilation Amino acid products 0 5 10 — Glu Glu Asp Ala Gln Asp Ala Glu Glu Asp Ala Gln Asp Ala 100 — 0 100 0 — 95 19 26 102.5 9.8 1.25 99.5 19.9 21 107 5.1 2.3 100.5 20.1 15.4 108.5 3.2 3.4 90 9 56 105 19 1.25 99 9.9 41 114 9.6 2.3 101 10.1 31 117 6 3.4 Parameter Net CO fixation 2 C5N ratio (net assimilation) % Net C fixed into amino acids Net O evolution 2 Respiratory O uptake (% net O evolution) 2 2 O evolved5O consumed in N assimilation 2 2 Values are in arbitrary units, calculated for absolute rates of RuBP carboxylation and oxygenation of 125 and 50 (C5O=2.5: Sharkey, 1988; De Veau and Burris, 1989). For operation of the photorespiratory pathway, the calculations assume: (1) all ammonia released from gly is reincorporated via chloroplastic GS/GOGAT activities; (2) catalase activity results in stoichiometric re-evolution from H O of 50% of the O consumed by 2 2 2 glycollate oxidase activity; (3) gly oxidation is coupled to hydroxypyruvate reduction via redox shuttles, so that the rate of gly-linked respiratory O uptake is 0; (4) 75% of the C converted to phosphoglycollate is recycled to PGA, and 25% is lost in gly decarboxylation. Thus, for an 2 oxygenation rate of 50, the rate of photorespiratory CO release is 25. Net photorespiratory O consumption is also 25 (50 O consumed at 2 2 2 Rubisco+50 O consumed at glycollate oxidase—25 O released by catalase—50 O evolved at PSII (12.5 linked to GOGAT activity, 37.5 linked 2 2 2 to reduction of 75 1,3-bPGA)). In the absence of N assimilation, therefore, net CO assimilation and net O evolution are both 100 ( left-hand 2 2 column). It is assumed that all net C fixed that is not used for amino acid synthesis is converted to sucrose or starch. N assimilation affects gas exchange in two ways: (1) O evolution at PSII, linked to the reductive incorporation of NO− into C-amino groups; (2) respiratory CO evolution 2 3 2 and O consumption (Fig. 1). Values on the lowest line represent the rate of photosynthetic O evolution associated with the first process divided 2 2 by the rate of respiratory O consumption associated with the second. The overall C balance for production of the different amino acids can be 2 represented simply as follows: 2TP2-OG+CO (glu and gln); TP+CO OAA (asp); TPpyruvate (ala). For production of glu+asp+ala, and 2 2 gln+asp+ala, relative rates of synthesis are set at 45353 (glu5asp5ala) and 25353 (gln5asp5ala). For the values shown, it is assumed that photosynthetic electron transport supplies the reductant for conversion of NO− to NO− in the cytosol. The possibility that this conversion utilizes 3 2 NADH generated during respiratory C metabolism is considered in Table 2; coupling of nitrate reduction to respiratory NADH would lead to a lower rate of respiratory O consumption, but would not change the calculated rates of respiratory CO release, net CO fixation or net O 2 2 2 2 evolution. The above values for gas exchange parameters will be the same for all other alternative pathways and conditions of oxoacid production considered in Fig. 1 and Table 2. 1900 Noctor and Foyer regenerative phase of the RPP pathway must be increased relative to PGA reduction. Thus, for an equivalent rate of TP production, the ATP5NADPH ratio required in the RPP pathway increases from 1.5 (no photorespiration) to approximately 1.54 (C5O=2.5). Secondly, operation of the photorespiratory pathway introduces an extra ATP sink at the level of glycerate kinase (Leegood et al., 1995). This increase in ATP5reductant requirement is slightly offset by photorespiratory amino acid metabolism and the necessary re-incorporation of NH ( Keys et al., 1978). 3 Ammonia re-incorporation sustains ATP production during photosynthetic electron transport to 2-OG (GOGAT reaction; 1 NADPH equivalent=1.33 ATP produced per NH re-incorporated ) and utilizes ATP 3 during synthesis of gln (GS reaction; 1 ATP required per NH re-incorporated ). The net ATP balance will not be 3 affected by possible transamination of glyoxylate by amino donors other than ser and glu (Betsche, 1983; Ta et al., 1985). Assuming that gly oxidation and hydroxypyruvate reduction are redox-linked (discussed below), then the ATP52e− requirement of steady-state incorporation of CO into TP at C5O=2.5 is about 1.57. 2 Where synthesis of several amino acids is considered, relative rates are set at 45353 (for glu+asp+ala) and 25353 (for gln+asp+ala), ratios which approximate to the relative pool sizes of these amino acids in leaves from C plants (see, for example, Noctor et al., 1997). As 3 Table 1 shows, net synthesis of glu alone decreases net CO fixation by 5%, due to evolution of CO during 2 2 2-OG production. This effect is less marked when synthesis of other amino acids are considered. In fact, net synthesis of gln, asp and ala leads to a small increase in net CO fixation, since anapleurotic CO fixation at 2 2 PEP carboxylase exceeds respiratory decarboxylations ( Table 1). Due to its high reductant requirements, N assimilation markedly increases O evolution, but this 2 effect is offset by respiratory O uptake, so that net O 2 2 evolution is increased only moderately ( Table 1). The ratio between N-linked O evolution and O consumption 2 2 is lowest for synthesis of glu exclusively ( Table 1, lowest line), where N assimilation leads to little increase in net O evolution. When other amino acids are being produced 2 as well as, or instead of glu, the required C flow produces less NAD(P)H (Fig. 1), potential mitochondrial O 2 uptake is lower and so N assimilation has a bigger effect on net O evolution ( Table 1). It should be noted that 2 the amount of N-associated O evolution at PSII is 2 independent of the nature of the amino acid end-products, apart from a slightly lower electron requirement for net synthesis of gln. The present analysis does not consider formation of organic acids for acid-base regulation ( Raven, 1988), but is restricted to the production of C skeletons necessary for ongoing N assimilation. The assumed rates of oxoacid production required during N assimilation are therefore conservative estimates. In photoautotrophic cells at saturating CO , mass 2 spectrometric analysis showed that gross CO fixation 2 was only 75% of gross O evolution (Avelange et al., 2 1991), indicating reduction of electron acceptors other than CO . Other acceptors could include nitrate (equa2 tions 1, 2, 4). Indeed, foliar N assimilation in the light leads to a drop in the assimilatory quotient (net CO 2 fixed5net O evolved; Bloom et al., 1989) and stimulates 2 O evolution (de la Torre et al., 1991). Significantly, 2 however, the observed stimulation of O evolution was 2 less than that predicted from N assimilation rates (de la Torre et al., 1991). As the values of Table 1 suggest, this may be explained by concurrent stimulation of photosynthetic O evolution and respiratory O uptake, with 2 2 the latter partially offsetting the former. Cellular ATP balance during photosynthesis Table 2 shows a range of values calculated for the cellular ATP balance when N assimilation and carbohydrate synthesis are occurring under photorespiratory conditions in the steady-state. Under the assumptions made for ATP yields of photosynthetic NADP reduction (ATP5NADPH=1.33), the RPP pathway operating in isolation (all net CO fixed converted to TP) would show 2 an ATP deficit of 11.1%. In the leaf cell at ambient CO 2 and O concentrations, photorespiration and carbohyd2 rate synthesis will increase the demand for ATP so that, acccording to our model calculations, the cellular ATP deficit will be 16.4% and 17.6% for sucrose and starch synthesis, respectively. As Table 2 shows, even low rates of N assimilation must remove or at least mitigate this deficit, due both to the high electron requirement of reductive N assimilation and to the associated necessary respiratory C flux (Fig. 1). Important factors affecting the contribution of N assimilation are the absolute rate, the nature of the amino acids formed, and the source of NADH for cytosolic NR activity ( Table 2). The relative contributions of the reductive and oxidative components of N assimilation are dependent on these and other assumptions concerning the route of oxoacid synthesis. For example, for synthesis of glu, asp and ala at net N= 5 and assuming that photosynthetic reductant drives nitrate reduction, 33.3 ATP can be formed during production of the necessary 50 low-potential electrons by the photosynthetic electron transport chain. Under these conditions, a further 43.5 ATP can be generated if the required 2-OG, OAA and pyruvate (2.051.551.5) are formed via NAD-GAPDH/PGK kinase and PK, assuming internal oxidation of NADH. In this case, 21% of fixed C enters amino acid pools (Table 1) and the %ATP balance is −3.2 or −4.3 (remaining C converted to sucrose or starch, respectively; Table 2). The total amount of ATP produced as a result of N assimilation (76.8) is then 16.5% of that produced by photosynthetic electron Table 2. The contribution of N assimilation to cellular ATP balance (% deficit or excess) The calculations use the model pathways and rates given in Fig. 1 and Table 1. Absolute rates of carboxylation and oxgenation of RuBP are set at 125 and 50 (Table 1). For each set of products at a given rate of N assimilation, the two lines show ATP balance calculated assuming that net C assimilated (Table 1) is converted to the amino acids shown+sucrose (upper line) or starch ( lower line). In the absence of N assimilation, the %ATP balance is calculated as −16.4 (all assimilated C converted to sucrose) and −17.6 (all assimilated C converted to starch). Assumptions and conditions additional to those detailed in Fig. 1 and Table 1 are described in the footnote. Source of reductant for NRa Oxidation of TP to PGA Mitochondrial oxidation of cytosolic NADHb Route of pyruvate production Amino acid products Glu NAD-GAPDH/PGA kinase NADP-GAPDH Internal Internal External External NAD-GAPDH/PGA kinase NADP-GAPDH Internal Internal External ME PK External PK ME PK ME PK ME PK ME PK ME PK ME PK ME 5 +2.5 +1.5 +21.3 +20.6 +1.6 +0.6 +19.5 +18.8 −0.1 −1.2 +16.0 +15.3 −1.0 −2.1 +14.2 +13.5 +0.7 −0.3 +17.7 +17.0 −0.1 −1.2 +16.0 +15.3 −1.9 −2.9 +12.4 +11.7 −2.8 −3.8 +10.7 +9.9 −1.3 −2.4 +13.6 +12.9 −2.2 −3.2 +11.8 +11.2 −3.1 −4.1 +10.1 +9.4 −4.0 −5.0 +8.3 +7.6 −3.1 −4.1 +10.1 +9.4 −4.0 −5.0 +8.3 +7.6 −5.8 −6.8 +4.7 +4.1 −6.7 −7.7 +3.0 +2.3 5 −3.2 −4.3 +9.8 +8.9 −3.8 −5.0 +8.6 +7.7 −4.8 −5.9 +6.6 +5.7 −5.4 −6.5 +5.4 +4.5 −4.5 −5.6 +7.3 +6.4 −5.1 −6.2 +6.1 +5.2 −6.1 −7.2 +4.2 +3.3 −6.7 −7.8 +2.9 +2.0 −7.1 −8.1 +2.2 +1.3 −7.7 −8.8 +0.9 +0.1 −7.8 −8.9 +0.7 −0.1 −8.4 −9.5 −0.5 −1.4 −6.0 −7.1 +4.3 +3.2 −6.6 −7.7 +3.0 +2.2 −7.6 −8.7 +1.1 +0.2 −8.2 −9.3 −0.1 −1.0 10 5 −6.2 −7.4 +3.9 +2.8 −6.6 −7.8 +3.0 +1.9 −7.3 −8.4 +1.7 +1.1 −7.7 −8.9 +0.9 −0.2 −7.1 −8.3 +2.1 +1.0 −7.5 −8.7 +1.2 +0.2 −8.2 −9.3 0 −1.0 −8.6 −9.8 −0.9 −1.9 −10.0 −11.2 −3.8 −4.8 −10.5 −11.6 −4.7 −5.7 −10.2 −11.4 −4.2 −5.1 −10.7 −11.8 −5.0 −6.0 −7.8 −9.0 +0.5 −0.5 −8.3 −9.5 −0.3 −1.4 −8.9 −10.1 −1.6 −2.6 −9.4 −10.5 −2.6 −3.5 10 Assumptions: reduction of NADP by the photosynthetic electron transport chain is accompanied by the translocation of 4H+ into the intrathylakoid space (for a review, see Horton, 1985). The H+/ATP requirement of the chloroplast coupling factor is 3 (Hangarter and Good, 1982; Strotmann and Lohse, 1988). The cytosolic NADH/NAD and NADPH/NADP redox couples are not metabolically linked. Synthesis of the necessary oxoacids for the amino acids shown occurs by the shortest routes possible (Fig. 1). Starch synthesis from TP requires I ATP/hexose unit (ADP-glucose pyrophosphorylase). Sucrose synthesis from TP requires 1 ATP/sucrose ( UDP-glucose pyrophosphorylase, nucleoside diphosphate kinase). The other ATP sinks are: chloroplastic PGA (PGA kinase), Ru5P (Ru5P kinase), glycerate (glycerate kinase), glutamate (GS, in photorespiration and net N assimilation). Electron acceptors which sustain chloroplastic ATP production are: 1,3-bPGA (GAPDH ), 2-OG (GOGAT, in photorespiration and net N assimilation), nitrate (NR, via OAA reduction catalysed by chloroplastic NADP-MDH; see below), nitrite (NiR). aPhotosynthetic reductant for nitrate reduction in the cytosol ( left half of table) is provided by a malate/OAA shuttle and so involves no net exchange of ATP between the cytosol and the chloroplast (Scheibe, 1987). Respiratory carbon flow can provide NADH for NR (right half of table) from NAD-GAPDH, PDH complex or mitochondrial NAD-ICDH activities (it is assumed that cytosolic NADH from GAPDH is used preferentially). In cases where these activities do not provide sufficient NADH within the present model, the shortfall is assumed to be made good by export of photosynthetic reductant. bInternal oxidation of NADH by the mitochondrial electron transport chain has an ADP/O value of 3 whereas this value is 2 for oxidation of cytosolic NAD(P)H by the externallyoriented dehydrogenases of the inner membrane (for a review of ATP yields during operation of glycolysis and the TCA cycle, see Lambers, 1997). In calculating the ATP balance for ‘external’ oxidation of NAD(P)H, 2-OG is assumed to be produced by cytosolic ICDH (Chen and Gadal, 1990): the only dehydrogenase producing matrix NADH is then the PDH complex. Values for ‘internal’ oxidation of NADH assume full stoichoimetric ‘conversion’ of cytosolic NAD(P)H to matrix NADH by any or all of the following processes: (1) oxidation of isocitrate inside the mitochondrion rather than in the cytosol; (2) coupling of NAD-GAPD/NADP-GAPDH and NADH-HPR/NADPH-HPR activities (Givan and Kleczkowski, 1992) via redox shuttles, liberating an equivalent amount of NADH from gly oxidation for oxidative phosphorylation; (3) import of cytosolic NADH into the mitochondrion as malate in exchange for OAA or aspartate (reversal of the direction of net exchange during coupling of gly oxidation and HPR activity or PDH/NAD-ICDH and NR activities). ATP5NADPH budget during C photosynthesis 1901 3 Gln Asp Ala Glycolytic/TCA cycle dehydrogenases Rate of N assimilation 10 Glu Asp Ala Photosynthetic electron transport chain 1902 Noctor and Foyer transport associated with C assimilation and photorespiration (466.7), and glycolytic activity and respiration provide 8% of the total cellular ATP. If the oxoacids are produced via NADP-GAPDH and ME, and the cytosolic NAD(P)H formed is externally oxidized by the mitochondria, the ATP generated as a result of oxoacid formation is only 24 (4.4% of the cell’s ATP) and the calculated deficit increases to 6.7% or 7.8% (remaining C converted to sucrose or starch, respectively; Table 2). When the rate of N assimilation is set at 10 (C5N= approximately 10; Table 1), the maximum calculated ATP deficit is 6% ( Table 2; net synthesis of gln, asp and ala, reduction of nitrate to nitrite by respiratory NADH, formation of pyruvate via NAD-ME, external oxidation of cytosolic NAD(P)H ). Other conditions yield a large ATP surplus (up to 21.3%; net synthesis of glu only, photosynthetic reduction of nitrate to nitrite, conversion of TP to PGA via NAD-GAPDH/PGA kinase, production of pyruvate via PK, internal oxidation of NADH by the mitochondria). Experimental data suggest that, under certain conditions, nitrate reduction may utilize respiratory NADH, as shown by nitrate-linked inhibition of respiratory O 2 uptake (Gray and Cresswell, 1984, and references therein). The extent of this phenomenon in the illuminated leaf cell is not clear. Coupling of reductant for NR to respiratory C flow has a marked effect on N-linked ATP production (cf. left and right halves of Table 2), since it decreases both the amount of ATP that can be formed during photosynthetic nitrate reduction and the amount obtainable by oxidation of respiratory NADH. This effect is analogous to that resulting from the assumption that the oxidation of photorespiratory gly is fully coupled to hydroxypyruvate reduction (Hanning and Heldt, 1993; Leegood et al., 1995; Raghavendra et al., 1998). The literature contains conflicting evidence concerning the fate of mitochondrial NADH during gly oxidation. Some studies have led to the conclusion that the principal route of NAD regeneration for ongoing gly oxidation is likely to be via the mitochondrial electron transport chain (Dry et al., 1983; Gardeström and Wigge, 1988; Gemel and Randall, 1992). Other authors have suggested that some or most of the NADH produced during gly oxidation is shuttled out of the mitochondria as malate (Journet et al., 1981; Hanning and Heldt, 1993; Raghavendra et al., 1998). Despite the theoretically higher ATP5reductant requirements of the photorespiratory cycle, compared to the RPP pathway, cytosolic ATP/ADP ratios were higher in barley protoplasts in photorespiratory conditions than at high CO , suggesting that gly oxidation may be at 2 least partly linked to mitochondrial ATP synthesis (Gardeström, 1987; Gardeström and Wigge, 1988). If complete coupling of these reactions was assumed, then within the confines of the model (oxygenation rate at Rubisco=50, gly oxidation=25), gly oxidation could potentially produce as much as 75 ATP in the mitochondria. This would necessitate an alternative source of reductant for conversion of hydroxypyruvate to glycerate. If this alternative source were photosynthetic electron transport, then a further 33.3 ATP could be produced in driving the reduction of hydroxypyruvate. Hence, complete uncoupling of gly oxidation and hydroxypyruvate reduction would imply a huge photorespiratory production of ATP, particularly in the mitochondria. In terms of the total cellular ATP balance, this would produce an ATP surplus of 3% (sucrose synthesis) or 1.5% (starch synthesis), even in the absence of N assimilation. This condition is probably unlikely in vivo (Hanning and Heldt, 1993; Raghavendra et al., 1998). Nevertheless, it should be noted that the model calculations of Table 2 do not assume kinetically heterogenous pools of NADH within the mitochondrion (Dry et al., 1983; Dry and Wiskich, 1985) and that the coupling of gly oxidation and hydroxypyruvate reduction is a formal assumption that merely serves to simplify the calculations. Under certain combinations of the alternative metabolic routes considered, it is assumed that cytosolic NAD(P)H produced during respiratory C flow will be coupled to hydroxypyruvate reduction and that an equivalent amount of NADH from gly oxidation will be used for ATP production (Table 2, footnote). The possibility that hydroxypyruvate serves as an acceptor for photosynthetic electrons has not been included in the present analysis. Under some conditions considered in Table 2, the amount of NADH produced in conversion of TP to oxoacids would not be sufficient to meet the demands of nitrate reduction (for example, operation of NADPGAPDH, assuming that foliar NADPH-dependent NR activity is negligible) and, in these cases, it has been assumed that the shortfall would be met by photosynthetic electron transport. Respiration additional to that linked to N assimilation may also occur in the light and abundant NADH for nitrate reduction and/or ATP production might be generated by complete turnover of the TCA cycle. However, the rate of full cycle turnover in the light continues to fuel debate. While experiments with labelled succinate suggest that complete turnover of the TCA cycle does occur (McCashin et al., 1988), work with isolated leaf mitochondria metabolizing malate or pyruvate showed that most of the product was exported as citrate and 2-OG (Hanning and Heldt, 1993). It may be noted that the present calculations predict that, under conditions where the ATP balance approaches 0 ( Table 2), respiratory O uptake will be 5–10% of net O 2 2 evolution ( Table 1). Alternative respiratory chain pathways In calculating the data of Table 2, mitochondrial NAD(P)H oxidation by internal rotenone-insensitive sites or the alternative terminal oxidase have not been considered (Douce and Neuburger, 1989). In algae, Weger et al. (1988) have shown that high rates of NH+ assimilation 4 are associated with a marked increase in cyanide-sensitive O uptake. Rather than considering dissipative electron 2 transport pathways as necessary to uncouple respiratory precursor synthesis from ATP production, the present analysis emphasizes the metabolic value of ATP produced during N-linked respiration. It is apparent from Table 2 that potential ATP yields derived from oxoacid production vary considerably, and so the efficiency with which respiratory C flow is linked to ATP generation may be plastic, with cellular ATP supply being tailored to demand through, for example, regulation of dehydrogenases by NADH/NAD ratios and adenylate control of PK. ATP import into the chloroplast in the light? The need for mitochondrial ATP production in illuminated protoplasts has been rationalized in terms of ATP supply to sustain sucrose synthesis in the cytosol ( Krömer et al., 1988, 1993). However, calculations in this review suggest that oxoacid generation for N assimilation could produce significantly more ATP than that required for the synthesis of sucrose. It should be noted that, although inhibition of mitochondrial ATP synthesis in illuminated protoplasts most markedly depressed the cytosolic and mitochondrial ATP5ADP ratio, a significant effect on the stromal ratio was also observed ( Krömer and Heldt, 1991). In addition, although the effects of inhibition of the mitochondrial ATP synthase have been discussed predominantly in terms of the restriction of sucrose synthesis, several observations made with oligomycintreated barley leaves may be partly explicable in terms of inhibition of RuBP regeneration in the RPP pathway due to decreased ATP supply (eg, lowered RuBP but increased FBP and TP; Krömer and Heldt, 1991). Apart from sucrose synthesis, the ATP sinks considered in the present study are all chloroplastic (Fig. 1). The mitochondrial electron transport chain could increase chloroplastic ATP5reductant yields by oxidizing reducing equivalents exported from the chloroplast (Scheibe, 1987; Krömer et al., 1988). Since this effect would entail the mitochondria oxidizing photosynthetically produced reductant, in addition to reductant produced during respiratory C flow, even higher respiration rates than those calculated in Table 1 would result. A direct contribution of mitochondrial ATP synthesis to chloroplast metabolism would require a means of net ATP import (Lemaire et al., 1988). Although this could theoretically be provided by TP import, the net flux of TP in the light is thought to be from chloroplast to cytosol (Stitt, 1997). Moreover, in the light, the chloroplastic PGA kinase and GAPDH must catalyse net conversion of PGA to TP in the RPP pathway (Fig. 1). ATP5NADPH budget during C photosynthesis 1903 3 While these considerations do not rule out an influence of TP import in supplying ATP to the chloroplast, it must be recognized that chloroplastic conversion of TP to PGA would also produce NADPH. Although some import of TP into the chloroplast may occur, therefore, the 151 production of ATP and NADPH during its oxidation to PGA would exacerbate and not mitigate any shortfall in the required ATP5NADPH ratio. The chloroplast envelope adenylate carrier (Heldt, 1969) is another putative route through which ATP could be supplied from the cytosol to the chloroplast, in this case independently of concomitant transfer of reductant. The physiological function of this transporter is still a matter of conjecture, but it has generally been considered unlikely to play a significant role in photosynthetically competent leaf cells in the light (Stitt, 1997). One reason for this is that, in isolated chloroplasts from C plants, 3 maximum rates of the chloroplast ATP translocator are about 10-fold lower than those of other transporters, such as the Pi translocator (Flügge and Heldt, 1991). It is therefore worth considering the predictions of the present model concerning the relative rates of TP export from, and ATP import into, the chloroplast. Under conditions where the cellular ATP balance approaches 0 ( Table 2), extrachloroplastic ATP production has a value of 35–60 and net TP export from the chloroplast to cytosol is 33.3 (assuming all fixed C goes to amino acids and sucrose). Subtracting the amount of ATP required for sucrose synthesis means that respiratory ATP available for transport to the chloroplast would be 29–54 (about 5–10% of total ATP production by the cell ). Under these conditions, only 10–17% of the ATP produced extrachloroplastically can be consumed in sucrose synthesis and the potential net rate of chloroplastic ATP import would be similar to the net rate of TP export. At high rates of photosynthesis, therefore, where net fluxes are likely to be greatest, it would seem improbable that the chloroplast ATP translocator could keep pace with mitochondrial and cytosolic ATP production, within the confines of the present assumptions concerning the relative rates of net CO fixation and N-linked respiration. 2 Nevertheless, two points ought perhaps to be borne in mind. Firstly, unlike the ATP transporter, the Pi translocator is not directional, so that the net flux of TP may be considerably lower than maximum measured activities (Stitt, 1997). Secondly, highest activities of the chloroplast ATP translocator are observed in young leaves (Robinson and Wiskich, 1977), which have relatively low C5N ratios (de la Torre et al., 1991). A flexible view of cellular ATP production It must be stressed that the model calculations presented are not intended to provide an exact description of cellular ATP5reductant balance under all conditions. Clearly, the 1904 Noctor and Foyer required balance will depend on species and developmental stage, as well as on environmental factors such as light intensity, temperature and water status. The influence of N assimilation will vary according to the nature of the external N source and the proportion assimilated in the leaf. The sensitivity of photosynthetic CO fixation to inhibi2 tion of mitochondrial ATP synthesis has been shown to be dependent on ambient conditions ( Krömer et al., 1993). Other pathways which could potentially function to adjust the ATP5reductant ratio (for example, cyclic electron transport, O reduction at PSI ) also occur at 2 variable rates under different conditions, for example, different light intensities. In spinach chloroplasts fixing CO , the proportion of electrons allotted to O was 2 2 greater at low light (Heber et al., 1978). Other data suggest that O reduction (Marsho et al., 1979) and cyclic 2 electron transport (Heber et al., 1978; Furbank and Horton, 1987) both occur at maximal rates when light availability is high relative to the capacity of metabolism to use the products of photochemistry. One such condition, entailing an unusually high demand for ATP, is during autocatalytic generation of RPP pathway intermediates following a dark–light transition (Marsho et al., 1979; Furbank and Horton, 1987). The calculations presented in Table 2 assume that transport of one electron by the photosynthetic electron transport chain results in the transfer of 2 H+ across the thylakoid membrane. Higher values have sometimes been observed in isolated thylakoids and are usually explained in terms of translocation of an extra H+ during oxidation of plastoquinol at the cytochrome b f complex (Q-cycle; 6 for a review, see Cramer et al., 1996). It remains unclear whether this phenomenon occurs during intersystem electron transfer under all, or even most, conditions in vivo. Operation of a Q-cycle may be sensitive to the intrathylakoid pH and, therefore, more likely under conditions of low membrane energization, for example, low light ( Horton, 1985; Cramer et al., 1996). On the basis of data obtained in isolated chloroplasts, it has been suggested that concurrent operation of NADP reduction and cyclic electron transport will provide more than enough ATP for CO fixation (Arnon and Chain, 2 1975). Maximum rates of cyclic electron transport require appropriate redox poising of the electron transport chain (Arnon and Chain, 1975; Heber et al., 1978). Both cyclic electron transport and photosynthetic O reduction are 2 inhibited by electron acceptors such as NADP, OAA, nitrate or nitrite (Heber et al., 1978; Behrens et al., 1982; Furbank and Badger, 1983; Robinson, 1988). During reductive N assimilation, therefore, OAA and nitrite are likely to compete effectively with other pathways of electron transport. Equally, a contribution of respiratory ATP production to chloroplast metabolism could also diminish electron flow from Fd to O or the cyclic 2 pathway, by enabling ongoing oxidation of NADPH in the RPP pathway. According to this view, the extent to which electrons engage in alternative pathways would be flexible and self-regulating, as previously suggested (Heber et al., 1978). These calculations of the potential influence of N assimilation suggest that this process should be taken into account in considering the ATP balance during C photo3 synthesis, given that, in many photosynthetic cells, C and N assimilation will be occurring simultaneously. It should be noted, however, that the range of values shown in Table 2 emphasize the variable efficiency of ATP production during N assimilation. As in other areas of metabolism, plant cells are likely to show considerable flexibility in the mechanisms deployed to meet their ATP requirements. Chlororespiration (Garab et al., 1989; Bennoun, 1994), operation of the mitochondrial alternative oxidase (Azcón-Bieto et al., 1983; Douce and Neuburger, 1989), and possible increases in proton ‘leakiness’ through CF 0 CF under conditions of high membrane energization 1 (Ort and Oxborough, 1992), are other factors likely to be sensitive to ATP5reductant status, and therefore able to contribute in a flexible manner to cellular ATP requirements. The C5O ratio, and the extent to which gly oxidation and hydroxypyruvate reduction are coupled during photorespiration (discussed above), are other factors likely to affect, and perhaps be affected by, cytosolic and stromal ATP5reductant status. Alternative photosynthetic electron transport pathways may be particularly important in C photosynthesis, where CO fixation 4 2 requires high ATP5NADPH ratios ( Edwards and Walker, 1983). If nitrate reduction does make a significant contribution to the ATP requirements of CO fixation, then 2 this ATP contribution would obviously have to be met by other processes in plants that assimilate nitrate predominantly in the root. Likewise, in cases where N sources more reduced than nitrate are assimilated (Raven et al., 1992), other processes would have to compensate for a lower ATP yield associated with lower rates of nitrateand/or nitrite-linked photosynthetic electron transport. The quantum requirements of ATP production associated with N assimilation One line of evidence against high rates of photosynthetic electron flow through pathways other than non-cyclic electron transport to NADP is the relatively good correspondence found between the measured and expected quantum requirement (QR) of photosynthesis. The predicted minimum value for the accepted pathways of electron transport and CO fixation is 8 photons per CO 2 2 fixed (or per O evolved ). On averaging the QR for O 2 2 evolution of 37 different C species photosynthesizing 3 under non-photorespiratory conditions, Björkman and Demmig (1987) arrived at a value of 9.43. Under the ATP5NADPH budget during C photosynthesis 1905 3 production associated with the reductive component of N assimilation would be insufficient to meet the requirements of photosynthesis. As has been previously noted, the use of photosynthetically-generated reductant in the mitochondria is a more quantum-efficient way of producing ATP than via the reduction of alternative electron acceptors in the chloroplast (Raven, 1976; Krömer et al., 1988). This is the case whether the reductant is exported from the chloroplast in the form of malate (Scheibe, 1987) or as TP, from which reductant is generated during respiratory oxidation as in Fig. 1. Within the assumptions of the present calculations, photosynthetic reduction of NADP, followed by shuttling of reducing equivalents from the chloroplast to the mitochondrial matrix, could yield a total of 4.33 ATP per 4 photons or per 2e−. Accordingly, if both reductive and oxidative components of N assimilation are taken into account, this results in a slightly lower QR for photosynthesis (15.8) than the minimum calculated assuming that cyclic electron flow or O reduction 2 contributes the required ATP (16.5–17.0; Table 3). It may be noted that the two conditions for N assimilation which result in a cellular ATP balance approaching 0 were chosen since they involve disparate assumptions concerning the rate and routes of N assimilation, and the products formed. Other combinations of assumptions would not markedly change this value of 15.8. Equally, it is noteworthy that none of the calculated minimum QR values under photorespiratory conditions ( Table 3) are higher than values that have been determined experimentally for C plants in air (16.7–18.9; calculated from 3 present assumption concerning the amount of ATP formed during NADP reduction (ratio of 1.33), a QR of 8 is not possible ( Table 3). Eight photons can only produce 2.67 ATP in reducing 2 NADP via non-cyclic electron transport. If the ATP deficit is made good by cyclic electron transport or O reduction, only 1 extra 2 photon will be required and the minimum QR is 9 ( Table 3). If reductive N assimilation makes good the deficit, the minimum QR is approximately 9.2. The slight increase is due to the relatively small, but nevertheless significant ATP demands of gln synthesis. Although the ATP required for sucrose or starch synthesis further increases the calculated minimum QR, the latter never exceeds 10 in the absence of photorespiration ( Table 3). In contrast, under photorespiratory conditions, the QR increases drastically, reflecting the potential of photorespiration as a dissipative protective pathway ( Heber and Krause, 1980). Because the ATP52e− requirements of steady-state CO fixation are increased by photo2 respiration (from 1.5 to about 1.57 for TP production at C5O=2.5), the proportion of electrons flowing through the cyclic pathway or to O would have to increase from 2 0.11 to about 0.16 (QR=16.5; Table 3). If the ATP shortfall is removed purely by reductive N assimilation, then because of the extra ATP required by the reaction catalysed by GS, the minimum QR for CO fixation to 2 TP under photorespiratory conditions is slightly higher (about 16.9: Table 3). Fulfilment of the ATP requirements of CO fixation purely by reductive N assimilation would, 2 however, entail improbably low C5N ratios (6.8; Table 3) and so, within the constraints of the present model, ATP Table 3. Minimum quantum requirements of photosynthesis (quanta per net CO fixed) in the presence or absence of photorespiration 2 and N assimilation ATP balance (%) Additional route of ATP production Net CO 2 fixation C5O=2.5 95 TP Sucrose Starch TP Sucrose Starch Sucrose+glu 9.0 9.3 9.5 9.2 (17.2) 9.5 (13.6) 9.8 (11.3) — 16.5 16.8 17.0 16.9 17.2 17.5 15.8 99 Starch+glu+asp+ala — 15.8 (9.9) Cyclic electron transport or photosynthetic O reduction 2 100 0 Photosynthetic assimilation of nitrate into glu 100 −0.1b Assimilation of nitrate into amino acids and associated generation of oxoacids Assimilation of nitrate into amino acids and associated generation of oxoacids Minimum quantum requirement No photorespiration 0 −0.1a Products (6.8) (6.2) (5.7) (19) Figures in parentheses are net C5N ratios. Calculations assume that 1.33 ATP are generated during non-cyclic reduction of NADP, requiring absorption of 4 photons. All photosynthetic pathways of electron transport are assumed to produce 0.33 ATP/photon absorbed. Other assumptions as detailed in Fig. 1 and Tables 1 and 2. The conditions for the values shown in the lower half of the table are taken from Table 2 and are as follows: aTable 2, row 1, column 6: net rate of N assimilation=5; all N assimilated goes to glu; nitrate is reduced to nitrite using reductant exported from the chloroplast; TP is oxidized to PGA in the cytosol via NADP-GAPDH; cytosolic NAD(P)H is ‘converted’ into matrix NADH and oxidized by the mitochondrial complex I; pyruvate is produced via NAD-ME. This condition means that, of the 95C fixed, 25 are used for glu synthesis while 70 are converted to sucrose. bTable 2, row 8, column 11; net rate of N assimilation=10; N assimilated goes to glu (4N ), asp (3N ) and ala (3N ); nitrate is reduced to nitrite using reductant produced during TP oxidation; TP is oxidized to PGA in the cytosol via NAD-GAPDH and PGA kinase; cytosolic NAD(P)H is oxidized by the external mitochondrial dehydrogenases; pyruvate is generated via PK. In this condition, of the 99C fixed, 20 are used for glu synthesis, 12 for asp synthesis, 9 for ala synthesis, while 58 are incorporated into starch. 1906 Noctor and Foyer quantum yield values given in Björkman and Demmig, 1987). Conclusions A re-evaluation of the ATP5reductant requirements of photosynthesis has been presented, in which the photosynthetic process is defined as CO fixation, photorespiration, 2 carbohydrate synthesis, N assimilation, and net synthesis of major foliar amino acids. The calculated data suggest that ATP production during N assimilation may play an important role in complementing the relatively high ATP5reductant requirements of C assimilation. Respiratory C flow necessary for ongoing N assimilation may contribute significant amounts of ATP in the light, both through substrate-level and oxidative phosphorylation. Although alternative electron transport pathways almost certainly can occur in vivo, these are perhaps best viewed as facultative rather than obligatory events. Central to any calculation of photosynthetic ATP5NADPH balance is the assumed ratio of their production. This review has used a ratio of 1.33, derived from H+5e− and H+5ATP ratios of 2 and 3, respectively. It is possible, by assuming obligatory operation of a Q-cycle during plastoquinol oxidation at the cytochrome b f complex (H+52e−=6) and an H+5ATP requirement 6 of 4 (Rumberg et al., 1990), to arrive at an ATP5NADPH ratio of 1.5, which corresponds exactly to the ATP5NADPH requirements of CO fixation in the steady2 state (assuming no photorespiration and no starch or sucrose synthesis). It is hard to see why such a good correspondence should be expected to exist under all conditions, given the plethora of other ATP-consuming and ATP-generating reactions which occur in the illuminated leaf alongside CO fixation. Chief among these 2 reactions will be N assimilation. Other ATP-generating and ATP-consuming processes, which were not considered in the present analysis, include sulphate assimilation, active transport, synthesis of macromolecules, and the oxidative pentose phosphate pathway. Perfect correspondence of the RPP pathway’s ATP and NADPH requirements to ATP and NADPH production at the thylakoid would imply that other ATP-consuming reactions should be exactly matched to cellular ATP generation not linked to photosynthetic NADP reduction. In contrast to this view, which sees the RPP pathway as divorced from other cellular processes that produce and consume ATP, our aim has been to emphasize the integrated nature of photosynthetic metabolism. Limits over the extent of integration of photosynthetic metabolism will be set by intracellular compartmentation, but this is rarely absolute, since transporters enable flux between compartments. At the present moment, it remains unclear whether the net flux of ATP into the chloroplast, either directly or indirectly, would be sufficient to allow cytosolic and mitochondrial ATP synthesis to contribute significantly to chloroplast metabolism in the light. 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