Transgenic potato plants with altered expression levels of chloroplast NADP-malate dehydrogenase: interactions between photosynthetic electron transport and malate metabolism in leaves and in isolated intact chloroplasts

The contribution of the malate valve in the regulation of steady-state photosynthesis was studied in transgenic potato (Solanum tuberosum L. cv Désirée) plants with altered expression of plastidic NADP-dependent malate dehydrogenase (NADP-MDH; EC 1.1.1.82). Mutant plants were obtained after transformation with the homologous Nmdh gene in antisense orientation, or with the Nmdh gene from pea (Pisum sativum L.) in sense orientation. A total number of nine stable sense and antisense lines with 10% or 30%, and 400% of wild-type NADP-MDH capacity were selected. Intact chloroplasts were isolated from leaves of wild-type and mutant plants. In chloroplasts from sense transformants the increased enzyme amount was activated as in wild-type chloroplasts, but increased rates of oxaloacetate-dependent malate formation were only measured upon partial uncoupling. In contrast, chloroplasts from antisense transformants produced only little malate upon oxaloacetate addition. Measurements with intact leaves during steady-state photosynthesis yielded no differences in gas-exchange parameters and chlorophyll fluorescence. The leaf malate content was unchanged in NADP-MDH underexpressors, but twice as high in overexpressing plants. The altered NADP-MDH expression clearly influences the redox state of ferredoxin, especially in low light. Furthermore, the malate valve can successfully compete for electrons with cyclic electron flow, but the conditions under which this occurs are quite artificial. Received: 14 February 1998 / Accepted: 12 May 1998     

The chloroplastic 2-oxoglutarate/malate transporter has dual function as the malate valve and in carbon/nitrogen metabolism

Transport of dicarboxylates across the chloroplast envelope plays an important role in transferring carbon skeletons to the nitrogen assimilation pathway and exporting reducing equivalent to the cytosol to prevent photo-inhibition (the malate valve). It was previously shown that the Arabidopsis plastidic 2-oxoglutarate/malate transporter (AtpOMT1) and the general dicarboxylate transporter (AtpDCT1) play crucial roles at the interface between carbon and nitrogen metabolism. However, based on the in vitro transport properties of the recombinant transporters, it was hypothesized that AtpOMT1 might play a dual role, also functioning as an oxaloacetate/malate transporter, which is a crucial but currently unidentified component of the chloroplast malate valve. Here, we test this hypothesis using Arabidopsis T-DNA insertional mutants of AtpOMT1. Transport studies revealed a dramatically reduced rate of oxaloacetate uptake into chloroplasts isolated from the knockout plant. CO(2) -dependent O(2) evolution assays showed that cytosolic oxaloacetate is efficiently transported into chloroplasts mainly by AtpOMT1, and supported the absence of additional oxaloacetate transporters. These findings strongly indicate that the high-affinity oxaloacetate transporter in Arabidopsis chloroplasts is AtpOMT1. Further, the knockout plants showed enhanced photo-inhibition under high light due to greater accumulation of reducing equivalents in the stroma, indicating malfunction of the malate valve in the knockout plants. The knockout mutant showed a phenotype consistent with reductions in 2-oxoglutarate transport, glutamine synthetase/glutamate synthase activity, subsequent amino acid biosynthesis and photorespiration. Our results demonstrate that AtpOMT1 acts bi-functionally as an oxaloacetate/malate transporter in the malate valve and as a 2-oxoglutarate/malate transporter mediating carbon/nitrogen metabolism.     

Malate valves to balance cellular energy supply

In green parts of the plant, during illumination ATP and NAD(P)H act as energy sources that are generated mainly in photosynthesis and respiration, whereas in darkness, glycolysis, respiration and the oxidative pentose-phosphate pathway (OPP) generate the required energy forms. In non-green parts, sugar oxidation in glycolysis, respiration and OPP are the only means of producing energy. For energy-consuming reactions, the delivery of NADPH, NADH, reduced ferredoxin and ATP has to take place at the required rates and in the specific compartments, since the pool sizes of these energy carriers are rather limited and, in general, they are not directly transported across biomembranes. Indirect transport of reducing equivalents can be achieved by malateoxaloacetate shuttles, involving malate dehydrogenase (MDH) for the interconversion. Isoenzymes of MDH are present in each cellular compartment. Chloroplasts contain the redox-controlled NADP-MDH that is only active in the light. In addition, a plastid NAD-MDH that is permanently active and is present in all plastid types has been found. Export of excess NAD(P)H through the malate valves will allow for the continued production of ATP (1) in photosynthesis, and (2) in oxidative phosphorylation. In the latter case, the coupled production of NADH is catalysed by the bispecific NAD(P)-GAPDH (GapAB) in chloroplasts that is active with NAD even in darkness, or by the specific plastid NAD-GAPDH (GapCp) in non-green tissues. When plants are subjected to conditions such as high light, high CO(2), NH(4) (+) nutrition, cold stress, which require changed activities of the enzymes of the malate valves, changed expression levels of the MDH isoforms can be observed. In nodules, the induction of a nodule-specific plastid NAD-MDH indicates the changed requirements for energy supply during N(2) fixation. Furthermore, the induction of glucose 6-phosphate dehydrogenase isoforms by ammonium and of ferredoxin and ferredoxin-NADP reductase by nitrate has been described. All these findings are in line with the assumption that a changed redox state caused by metabolic variability leads to the induction of enzymes involved in redox poise.     

Up-regulation of mitochondrial alternative oxidase concomitant with chloroplast over-reduction by excess light

Alternative oxidase (AOX), the unique terminal oxidase in plant mitochondria, catalyzes the energy-wasteful cyanide (CN)-resistant respiration. Although it has been suggested that AOX might prevent chloroplast over-reduction through the efficient dissipation of excess reducing equivalents, direct evidence for this in the physiological context has been lacking. In this study, we examined the mitochondrial respiratory properties, especially AOX, connected to the accumulation of reducing equivalents in the chloroplasts and the activities of enzymes needed to transport the reducing equivalents. We used Arabidopsis thaliana mutants defective in cyclic electron flow around PSI, in which the reducing equivalents accumulate in the chloroplast stroma due to an unbalanced ATP/NADPH production ratio. These mutants showed higher activities of the enzymes needed to transport the reducing equivalents even in low-light growth conditions. The amounts of AOX protein and CN-resistant respiration in the mutants were also higher than those in the wild type. After high-light treatment, AOX, even in the wild type, was preferentially up-regulated concomitant with the accumulation of reducing equivalents in the chloroplasts and an increase in the activities of enzymes needed to transport reducing equivalents. These results indicate that AOX can dissipate the excess reducing equivalents, which are transported from the chloroplasts, and serve in efficient photosynthesis.     

Overexpression of plastidic maize NADP-malate dehydrogenase (<Emphasis Type="Italic">ZmNADP-MDH</Emphasis>) in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> confers tolerance to salt stress

The plastidic C4 Zea mays NADP-malate dehydrogenase (ZmNADP-MDH), responsible for catalysis of oxaloacetate to malate, was overexpressed in Arabidopsis thaliana to assess its impact on photosynthesis and tolerance to salinity stress. Different transgenic lines were produced having ~3–6-fold higher MDH protein abundance and NADP-MDH enzyme activity than vector control. The overexpressors had similar chlorophyll, carotenoid, and protein content as that of vector control. Their photosynthetic electron transport rates, carbon assimilation rate, and consequently fresh weight and dry weight were almost similar. However, these overexpressors were tolerant to salt stress (150 mM NaCl). In saline environment, the Fv/Fm ratio, yield of photosystem II, chlorophyll, and protein content were higher in ZmNADP-MDH overexpressor than vector control. Under identical conditions, the generation of reactive oxygen species (H₂O₂) and production of malondialdehyde, a membrane lipid peroxidation product, were lower in overexpressors. In stress environment, the structural distortion of granal organization and swelling of thylakoids were less pronounced in ZmNADP-MDH overexpressing plants as compared to the vector control. Chloroplastic NADP-MDH in consort with cytosolic and mitochondrial NAD-MDH plays an important role in exporting reducing power (NADPH) and exchange of metabolites between different cellular compartments that maintain the redox homeostasis of the cell via malate valve present in chloroplast envelope membrane. The tolerance of NADP-MDH overexpressors to salt stress could be due to operation of an efficient malate valve that plays a major role in maintaining the cellular redox environment.     

Control of reversible intracellular transfer of reducing potential.

Isolated rat liver mitochondria were incubated in the presence of a reconstituted malate-aspartate shuttle under carboxylating conditions in the presence of glutamate, octanoyl-carnitine and pyruvate, or a preset lactate/pyruvate ratio. The respiration and attendant energy state were varied with soluble F1-ATPase. Under these conditions reducing equivalents are exported due to pyruvate carboxylation. This was shown by lactate production from pyruvate and by a substantial increase in the lactate/pyruvate ratio. This led to a competition between malate export and energy-driven malate cycling via the malate-aspartate shuttle, resulting in a lowered redox segregation of the NAD systems between the mitochondrial and extramitochondrial spaces. If pyruvate carboxylation was blocked, this egress of reducing equivalents was also blocked, leading to an elevated value of redox segregation, delta G(redox) (in kJ) = -5.7 log(NAD+/NADHout)/(NAD+/NADHin) being then equal to approximately one-half of the membrane potential, in accordance with electrogenic glutamate/aspartate exchange. Reconstitution of malate-pyruvate cycling led to a further kinetic decrease in the original malate-aspartate shuttle-driven value of delta G(redox). Therefore, the value of segregation of reducing potential between mitochondria and cytosol caused by glutamate/aspartate exchange can be diminished kinetically by processes exporting reducing equivalents from mitochondria, such as pyruvate carboxylation and pyruvate cycling.     

Towards understanding the functional difference between the two PsbO isoforms in Arabidopsis thaliana—insights from phenotypic analyses of psbo knockout mutants

The extrinsic PsbO subunit of the water-oxidizing photosystem II (PSII) complex is represented by two isoforms in Arabidopsis thaliana, namely PsbO1 and PsbO2. Recent analyses of psbo1 and psbo2 knockout mutants have brought insights into their roles in photosynthesis and light stress. Here we analyzed the two psbo mutants in terms of PsbOs expression pattern, organization of PSII complexes and GTPase activity. Both PsbOs are present in wild-type plants, and their expression is mutually controlled in the mutants. Almost all PSII complexes are in the monomeric form not only in the psbo1 but also in the psbo2 mutant grown under high-light conditions. This results either from an enhanced susceptibility of PSII to photoinactivation or from malfunction of the repair cycle. Notably, the psbo1 mutant displays such problems even under growth-light conditions. These results together with the finding that PsbO2 has a threefold higher GTPase activity than PsbO1 have significance for the turnover of the PSII D1 subunit in Arabidopsis.     

Adaptation responses in C4 photosynthesis of maize under salinity

The effect of salinity on C(4) photosynthesis was examined in leaves of maize, a NADP-malic enzyme (NADP-ME) type C(4) species. Potted plants with the fourth leaf blade fully developed were treated with 3% NaCl solution for 5d. Under salt treatment, the activities of pyruvate orthophosphate dikinase (PPDK), phosphoenolpyruvate carboxylase (PEPCase), NADP-dependent malate dehydrogenase (NADP-MDH) and NAD-dependent malate dehydrogenase (NAD-MDH), which are derived mainly from mesophyll cells, increased, whereas those of NADP-ME and ribulose-1,5-bisphosphate carboxylase, which are derived mainly from bundle sheath cells (BSCs), decreased. Immunocytochemical studies by electron microscopy revealed that PPDK protein increased, while the content of ribulose-1,5-bisphosphate carboxylase/oxygenase protein decreased under salinity. In salt-treated plants, the photosynthetic metabolites malate, pyruvate and starch decreased by 40, 89 and 81%, respectively. Gas-exchange analysis revealed that the net photosynthetic rate, the transpiration rate, stomatal conductance (g(s)) and the intercellular CO(2) concentration decreased strongly in salt-treated plants. The carbon isotope ratio (δ(13)C) in these plants was significantly lower than that in control. These findings suggest that the decrease in photosynthetic metabolites under salinity was induced by a reduction in gas-exchange. Moreover, in addition to the decrease in g(s), the decrease in enzyme activities in BSCs was responsible for the decline of C(4) photosynthesis. The increase of PPDK, PEPCase, NADP-MDH, and NAD-MDH activities and the decrease of NADP-ME activity are interpreted as adaptation responses to salinity.     

The mitochondrial CMSII mutation of Nicotiana sylvestris impairs adjustment of photosynthetic carbon assimilation to higher growth irradiance

The CMSII mutant of Nicotiana sylvestris, which lacks a functional mitochondrial complex I, was used to investigate chloroplast-mitochondria interactions in light acclimation of photosynthetic carbon assimilation. CMSII and wild-type (WT) plants were grown at 80 micromol m(-2) s(-1) photosynthetic active radiation (PAR; 80) and 350 micromol m(-2) s(-1) PAR (350). Carbon assimilation at saturating PFD was markedly higher in WT 350 leaves as compared with WT 80 leaves, but was similar in CMS 80 and CMS 350 leaves, suggesting that the mutant is unable to adjust photosynthesis to higher growth irradiance. WT 350 leaves showed several general characteristic light acclimation responses [increases in leaf specific area (LSA), total chlorophyll content, and chlorophyll a/b ratio, and a higher light compensation point]. In contrast, a similar chlorophyll content and chlorophyll a/b ratio were measured for both CMS 80 and CMS 350 leaves, while LSA and the light compensation point acclimated as in the WT. The failure of CMSII to adjust photosynthesis to growth PFD did not result from lower quantum efficiency of PSII, lower whole-chain electron transport rates (ETRs), or lower ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) and sucrose phosphate synthase (SPS) capacities. Excess ETR not used for carbon assimilation was even higher in CMS 350 than in WT 350. Since photochemical fluorescence quenching and the initial activity of NADP malate dehydrogenase (NADP-MDH) were identical in WT 350 and CMS 350 leaves but the activation state of NADP-MDH was different, redox signals from primary ETR are not involved in the signal transduction of light acclimation, while a contribution of stromal redox state cannot be excluded. When mature plants were transferred between 350 and 80 conditions, the mutant showed acclimatory tendencies, although adjustments were not as rapid or as marked as in the WT, and the response of the initial activities of Rubisco and NADP-MDH was impaired or altered. Initial activities of Rubisco and SPS at limiting concentration were also affected in CMS 350 as compared with WT plants when compared at growth irradiance or after in situ activation at 1000 micromol m(-2) s(-1) PAR. The data demonstrate that chloroplast-mitochondria interactions are important in light acclimation, and modulation of the activation state of key photosynthetic enzymes could be an important mechanism in this cross-talk.     

Responses of the photosynthetic electron transport system to excess light energy caused by water deficit in wild watermelon

In plants, drought stress coupled with high levels of illumination causes not only dehydration of tissues, but also oxidative damage resulting from excess absorbed light energy. In this study, we analyzed the regulation of electron transport under drought/high-light stress conditions in wild watermelon, a xerophyte that shows strong resistance to this type of stress. Under drought/high-light conditions that completely suppressed CO(2) fixation, the linear electron flow was diminished between photosystem (PS) II and PS I, there was no photoinhibitory damage to PS II and PS I and no decrease in the abundance of the two PSs. Proteome analyses revealed changes in the abundance of protein spots representing the Rieske-type iron-sulfur protein (ISP) and I and K subunits of NAD(P)H dehydrogenase in response to drought stress. Two-dimensional electrophoresis and immunoblot analyses revealed new ISP protein spots with more acidic isoelectric points in plants under drought stress. Our findings suggest that the modified ISPs depress the linear electron transport activity under stress conditions to protect PS I from photoinhibition. The qualitative changes in photosynthetic proteins may switch the photosynthetic electron transport from normal photosynthesis mode to stress-tolerance mode.     

Redox regulation of chloroplast enzymes in Galdieria sulphuraria in view of eukaryotic evolution

Redox modulation is a general mechanism for enzyme regulation, particularly for the post-translational regulation of the Calvin cycle in chloroplasts of green plants. Although red algae and photosynthetic protists that harbor plastids of red algal origin contribute greatly to global carbon fixation, relatively little is known about post-translational regulation of chloroplast enzymes in this important group of photosynthetic eukaryotes. To address this question, we used biochemistry, phylogenetics and analysis of recently completed genome sequences. We studied the functionality of the chloroplast enzymes phosphoribulokinase (PRK, EC 2.7.1.19), NADP-dependent glyceraldehyde 3-phosphate dehydrogenase (NADP-GAPDH, GapA, EC 1.2.1.13), fructose 1,6-bisphosphatase (FBPase, EC 3.1.3.11) and glucose 6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49), as well as NADP-malate dehydrogenase (NADP-MDH, EC 1.1.1.37) in the unicellular red alga Galdieria sulphuraria (Galdieri) Merola. Despite high sequence similarity of G. sulphuraria proteins to those of other photosynthetic organisms, we found a number of distinct differences. Both PRK and GAPDH co-eluted with CP12 in a high molecular weight complex in the presence of oxidized glutathione, although Galdieria CP12 lacks the two cysteines essential for the formation of the N-terminal peptide loop present in higher plants. However, PRK inactivation upon complex formation turned out to be incomplete. G6PDH was redox modulated, but remained in its tetrameric form; FBPase was poorly redox regulated, despite conservation of the two redox-active cysteines. No indication for the presence of plastidic NADP-MDH (and other components of the malate valve) was found.     

Redox-shuttling between chloroplast and cytosol: integration of intra-chloroplast and extra-chloroplast metabolism

Reducing equivalents produced in the chloroplast are essential for many key cellular metabolic enzyme reactions. Two redox shuttle systems transfer reductant out of the chloroplast; these systems consist of metabolite transporters, coupled with stromal and cytosolic dehydrogenase isozymes. The transporters function in the redox shuttle and also operate as key enzymes in carbon/nitrogen metabolism. To maintain adequate levels of reductant and proper metabolic balance, the shuttle systems are finely controlled. Also, in the leaves of C(4) plants, cell-specific division of carbon and nitrogen assimilation includes cell-specific localization of the redox shuttle systems. The redox shuttle systems are tightly linked to cellular metabolic pathways and are essential for maintaining metabolic balance between energy and reducing equivalents.     

NADP-malate dehydrogenase from unicellular green alga Chlamydomonas reinhardtii. A first step toward redox regulation?

The determinants of the thioredoxin (TRX)-dependent redox regulation of the chloroplastic NADP-malate dehydrogenase (NADP-MDH) from the eukaryotic green alga Chlamydomonas reinhardtii have been investigated using site-directed mutagenesis. The results indicate that a single C-terminal disulfide is responsible for this regulation. The redox midpoint potential of this disulfide is less negative than that of the higher plant enzyme. The regulation is of an all-or-nothing type, lacking the fine-tuning provided by the second N-terminal disulfide found only in NADP-MDH from higher plants. The decreased stability of specific cysteine/alanine mutants is consistent with the presence of a structural disulfide formed by two cysteine residues that are not involved in regulation of activity. Measurements of the ability of C. reinhardtii thioredoxin f (TRX f) to activate wild-type and site-directed mutants of sorghum (Sorghum vulgare) NADP-MDH suggest that the algal TRX f has a redox midpoint potential that is less negative than most those of higher plant TRXs f. These results are discussed from an evolutionary point of view.