Integration of photosynthetic carbon and nitrogen metabolism in higher plants.

Concomitant assimilation of C and N in illuminated leaves requires the regulated partitioning of reductant and photosynthate to sustain the demands of amino acid and carbohydrate biosynthesis. The short-term responses of photosynthesis and photosynthate partitioning to N enrichment in wheat (Triticum aestivum, L.) and maize (Zea mays L.) leaves were studied in order to understand the regulatory strategy employed in higher plants. Transgenic tobacco plants (Tobacco plumbaginifolia) over-expressing NR or with poor NR expression were used to compare plants differing in their capacities for NO3 (-) assimilation. Similar regulatory responses to NO3 (-) were observed in leaves having C4- and C3-type photosynthesis. It was shown that the extra- C needed in the short-term to sustain amino acid synthesis was not provided by an increase in photosynthetic CO2 fixation but rather by a rapid shift in the partitioning of photosynthetic C to amino acid at the expense of sucrose biosynthesis. The modulation of three enzymes was shown to be important in this C and N interaction, namely PEPCase (EC 4.1.1.31), SPS (EC 2.4.1.14) and NADH/NR (EC 1.6.6.1). The first two enzymes were shown to share the common feature of regulatory post-transcriptional NO3 (-)-dependent phosphorylation of their proteins on a seryl-residue. While PEPCase is activated, SPS activity is decreased. In contrast the NR phosphorylation state is unchanged and all N-dependent control of NR activity is regulated at the protein level. A number of arguments support the hypothesis that Gln, the primary product of NO3 (-) assimilation, is the metabolite effector for short-term modulation of PEPCase, and SPS in response to N enrichment. Since a major effect of NO3 (-) on the PEPCase-protein kinase activity in concentrated wheat leaf extracts was demonstrated, the hypothesis is put forward that protein phosphorylation is the primary event allowing the short-term adaptation of leaf C metabolism to changes in N supply.

Nitrate activation of cytosolic protein kinases diverts photosynthetic carbon from sucrose to amino Acid biosynthesis: basis for a new concept.

The regulation of carbon partitioning between carbohydrates (principally sucrose) and amino acids has been only poorly characterized in higher plants. The hypothesis that the pathway of sucrose and amino acid biosynthesis compete for carbon skeletons and energy is widely accepted. In this review, we suggest a mechanism involving the regulation of cytosolic protein kinases whereby the flow of carbon is regulated at the level of partitioning between the pathways of carbohydrate and nitrogen metabolism via the covalent modulation of component enzymes. The addition of nitrate to wheat seedlings (Triticum aestivum) grown in the absence of exogenous nitrogen has a dramatic, if transient, impact on sucrose formation and on the activities of sucrose phosphate synthase (which is inactivated) and phosphoenolpyruvate carboxylase (which is activated). The activities of these two enzymes are modulated by protein phosphorylation in response to the addition of nitrate, but they respond in an inverse fashion. Sucrose phosphate synthase in inactivated and phosphoenolpyruvate carboxylase is activated. Nitrate functions as a signal metabolite activating the cytosolic protein kinase, thereby modulating the activities of at least two of the key enzymes in assimilate partitioning and redirecting the flow of carbon away from sucrose biosynthesis toward amino acid synthesis.

NO(3) Enhances the Kinase Activity for Phosphorylation of Phosphoenolpyruvate Carboxylase and Sucrose Phosphate Synthase Proteins in Wheat Leaves: Evidence from the Effects of Mannose and Okadaic Acid.

The aim of this work was to determine which of the two reactions (i.e. phosphorylation or dephosphorylation) involved in the establishment of the phosphorylated status of the wheat leaf phosphoenolpyruvate carboxylase and sucrose phosphate synthase protein responds in vivo to NO(3) (-) uptake and assimilation. Detached mature leaves of wheat (Triticum aestivum L. cv Fidel) were fed with N-free (low-NO(3) (-) leaves) or 40 mm NO(3) (-) solution (high-NO(3) (-) leaves). The specific inhibition of the enzyme-protein kinase or phosphatase activities was obtained in vivo by addition of mannose or okadaic acid, respectively, in the uptake solution. Mannose at 50 mm, by blocking the kinase reaction, inhibited the processes of NO(3) (-)-dependent phosphoenolpyruvate carboxylase activation and sucrose phosphate synthase deactivation. Following the addition of mannose, the deactivation of phosphoenolpyruvate carboxylase and the activation of sucrose phosphate synthase, both due to the enzyme-protein dephosphorylation, were at the same rate in low-NO(3) (-) and high-NO(3) (-) leaves, indicating that NO(3) (-) had no effect per se on the enzyme-protein phosphatase activity. Upon treatment with okadaic acid, the higher increase of phosphoenolpyruvate carboxylase and decrease of sucrose phosphate synthase activities observed in high NO(3) (-) compared with low NO(3) (-) leaves showed evidence that NO(3) (-) enhanced the protein kinase activity. These results support the concept that NO(3) (-), or a product of its metabolism, favors the activation of phosphoenolpyruvate carboxylase and deactivation of sucrose phosphate synthase in wheat leaves by promoting the light activation of the enzyme-protein kinase(s) without affecting the phosphatase(s).

Effect of Light and NO(3) on Wheat Leaf Phosphoenolpyruvate Carboxylase Activity: Evidence for Covalent Modulation of the C(3) Enzyme.

Phosphoenolpyruvate carboxylase (PEPcase) activity was studied in excised leaves of wheat (Triticum aestivum L.) in the dark and in the light, in presence of either N-free (low-NO(3) (-) leaves) or 40 millimolar KNO(3) (high-NO(3) (-) leaves) nutrient solutions. PEPcase activity increased to 2.7-fold higher than that measured in dark-adapted tissue (control) during the first 60 minutes and continued to increase more slowly to 3.8-fold that of the control. This level was reached after 200 minutes exposure of the leaves to light and high NO(3) (-). In contrast, the lower rate of increase recorded for low-NO(3) (-) leaves ceased after 60 minutes of exposure to light at 2.3-fold the control level. The short-term NO(3) (-) effect increased linearly with the level of NO(3) (-) uptake. In immunoprecipitation experiments, the antibody concentration for PEPcase precipitation increased with the protein extracts from the different treatments in the order: control, illuminated low-NO(3) (-) leaves, illuminated high-NO(3) (-) leaves. This order also applied with regard to a decreasing sensitivity to malate and an increasing stimulation by okadaic acid (an inhibitor of P-protein phosphatases). Following these studies, (32)P labeling experiments were carried out in vivo. These showed that the light-induced change in the properties of the PEPcase was due to an alteration in the phosphorylation state of the protein and that this effect was enhanced in high-NO(3) (-) conditions. Based on the responses of PEPcase and sucrose phosphate synthase in wheat leaves to light and NO(3) (-), an interpretation of the role of NO(3) (-) as either an inhibitor of P-protein phosphatase(s) or activator of protein kinase(s) is inferred. In the presence of NO(3) (-), the phosphorylation state of both PEPcase and sucrose phosphate synthase is increased. This causes activation of the former enzyme and inhibition of the latter. We suggest that NO(3) (-) modulates the relative protein kinase/protein phosphatase ratio to favor increased phosphorylation of both enzymes in order to redirect carbon flow away from sucrose synthesis and toward amino acid synthesis.

Short-term effects of nitrate on sucrose synthesis in wheat leaves.

Experiments were carried out with fully expanded leaves from three-week-old seedlings of wheat (Triticum aestivum L.) raised without NO 3 (sup-) . Nitrate was supplied to the leaves through the transpiration stream in the light. Uptake of NO 3 (sup-) was linear with NO 3 (sup-) concentrations from 0 to 80 mM in the solution. Net sucrose synthesis showed inverse relationships versus nitrate uptake, assimilation, and accumulation, with correlation coefficients close to 1. By contrast, no alteration in sucrose synthesis was observed when KCl was substituted for KNO3 in the uptake solution. Sucrose synthesis was not affected by nitrate in seedlings treated with tungstate which absorbed but did not reduce NO 3 (sup-) . After 10 h, the final amount of sucrose in the tissues was only slightly decreased in the presence of NO 3 (sup-) , indicating that the effect of NO 3 (sup-) did not result from an altered sucrose-storage capacity. Comparison of the carbon skeleton and energy reductant necessary for NO 3 (sup-) and CO2 assimilation is consistent with the hypothesis that the processes of NO 3 (sup-) assimilation and sucrose synthesis compete for photosynthetic energy and carbon.

Nitrate reductases in hexaploid and tetraploid wheats and Aegilops.

Nitrate reductase activity (NR activity), protein content (NR protein) and polypeptides were compared in shoots of Triticum aestivum ssp. vulgare (L.) cv Fidel (bread wheat, AABBDD genome), Triticum dicoccum cv Vernal (AABB genome), Aegilops squarrosa var. strangulata (DD genome) and the amphiploid 365 (AABBDD genome), produced by crossing T. dicoccum cv Vernal and Ae. squarrosa var. strangulata. Constitutive NR protein and activity were found in shoots of all seedlings grown without nitrate, with the highest activity in the bread wheat. The inducible NR protein and activity developed upon the addition of nitrate. A 116-K polypeptide was identified as the main component of the NR from the bread wheat, while a faint, sometimes discernable 94-K band appeared on Western blots. Only one NR polypeptide could be identified in Ae. squarrosa -the 94 K. An intermediary situation was observed with the tetraploid T. dicoccum and the amphiploid: The 94-K polypeptide was the only one separated from NR of seedlings grown in the absence of nitrate. The 116-K polypeptide appeared after the addition of nitrate. The intensity of its band on the gel increased with the duration of the nitrate treatment. When comparing Ae. squarrosa and T. dicoccum, the constitutive isozyme (94-K polypeptide) was found in the D as well as in the AB genomes, while the inducible NR (116-K polypeptide) was absent from the D genome. Addition of the D genome into the AB genome slightly reinforced the expression of the inducible form (AB genome expression) in the amphiploid wheat. We postulate that the inducible form of NR in the bread wheat resulted from an evolutionary selection pressure favoured by cultivation.

Chromatographic and immunological evidence that chloroplastic and cytosolic pea (Pisum sativum L.) NADP-isocitrate dehydrogenases are distinct isoenzymes.

Two NADP-isocitrate dehydrogenase isoenzymes designated as NADP-IDH1 and NADP-IDH2 (EC 1.1.1.42) were identified in pea (Pisum sativum) leaf extracts by diethylaminoethylcellulose chromatography. The predominant form was found to be NADP-IDH1 while NADP-IDH2 represented only about 4% of the total leaf enzyme activity. These enzymes share few common epitopes as NADP-IDH2 was poorly recognized by the specific polyclonal antibodies raised against NADP-IDH1, and as a consequence NADP-IDH2 does not result from a post-translational modification of NADP-IDH1. Subcellular fractionation and isolation of chloroplasts through a Percoll gradient, followed by the identification of the associated enzymes, showed that NADP-IDH1 is restricted to the cytosol and NADP-IDH2 to the chloroplasts. Compared with the cytosolic isoenzyme, NADP-IDH2 was more thermolabile and exhibited a lower optimum pH. The data reported in this paper constitute the first report that the chloroplastic NADP-IDH and the cytosolic NADP-IDH are two distinct isoenzymes. The possible functions of the two isoenzymes are discussed.

Corn phosphoglycolate phosphatase: Modulation of activity by pyridine nucleotides and adenylate energy charge.

The activity of corn phosphoglycolate phosphatase (EC 3.1.3.18), a bundle sheath chloroplastic enzyme, is modulated, in vitro, both by NADP(H) and adenylate energy charge. The Vmax of the enzyme is increased by NADP (25%) and NADPH (16%) whatever the pH used, 7.0 or 7.9 respective pH of the stroma in the dark and in the light. At both pH, the adenylate energy charge alone has a positive effect with two peaks of activation, characteristics for this enzyme, at 0.2 and a maximum at 0.8 accentuated under nonsaturating concentration of phosphoglycolate. At low energy charge, NADP(H) increased the activation with an additive effect most particularly observed at pH 7.9 under saturating phosphoglycolate concentration; at high energy charge, NADP(H) had a positive or negative effect on the activation, depending on the pH value and the concentrations of substrate and NADP(H).The ferredoxin-thioredoxin system does not regulate the activity since i) DTT addition do not have any effect, ii) the light-reconstituted system containing ferredoxin, ferredoxin-thioredoxin reductase, thioredoxins and thylakoids is not effective either. However, light-dark experiments indicate that phosphophycolate phosphatase can be subjected to a fine tuning of its activity.All these data suggest that light cannot induce a modification of the protein but could exert a tight control of its activity by the intermediate of Mg(2+) and substrate concentrations and the levels of metabolites such as NADP(H), ATP, ADP, AMP. So, the regulation of the activity shown, in vitro, by energy charge and NADP(H) might be of physiological significance.

Bundle-sheath thylakoids from NADP-malic enzyme-type C4 plants require an exogenous electron donor for enzyme light activation.

Light activation of either NADP-malate dehydrogenase (EC 1.1.1.82) or fructose-1,6-bisphosphate phosphatase (EC 3.1.3.11) was assayed in a reconstituted chloroplastic, system comprising the isolated proteins of the ferredoxin-thioredoxin light-activation system and thylakoids from either mesophyll or bundle-sheath tissues of different C4 plants. While C4-plant thylakoids functionned almost equally well with C3-or C4-plant proteins, the photosyntem-II-deficient bundle-sheath thylakoids from the NADP-malic enzyme type, were unable to perform enzyme photoactivation unless supplemented with an electron donor to photosystem I. Bundle-sheath thylakoids isolated from plants showing no photosystem-II deficiency did not require such an addition. The results are discussed with respect to a possible requirement for a physiological reductant of ferredoxin for enzyme light activation in bundle-sheath, tissues.

Regulation of photosynthetic carbon assimilation at the cellular level: a review.

In green leaves and a number of algae, photosynthetically derived carbon is ultimately converted into two carbohydrate end-products, sucrose and starch. Drainage of carbon from the Calvin cycle proceeds via triose phosphate, fructose 6-phosphate and glycollate. Gluconeogenesis in photosynthetic cells is controlled by light, inorganic phosphate and phosphorylated sugars. Light stimulates the production of dihydroxyacetone phosphate, the initial substrate for sucrose and starch synthesis, and inhibits the degradative pathways in the chloroplast. Phosphate inactivates reactions of synthesis and activates reactions of degradation. Among the phosphorylated sugars a special role is allocated to fructose 2,6-bisphosphate, which is present in the cytoplasm at very low concentrations and inhibits sucrose synthesis directly by inactivating pyrophosphatedependent phosphofructokinase. The synthesis of sucrose plays a central role in the partitioning of photosynthetic carbon. The cytoplasmic enzymes, fructose bisphosphate phosphatase and sucrose phosphate synthase are likely key points of regulation. The regulation is carried out by several effector metabolites. Fructose 2,6-bisphosphate is likely to be the main coordinator of the rate of sucrose synthesis, hence of photosynthetic carbon partitioning between sucrose and starch.

Effect of Previous Nitrate Deprivation on (15)N-nitrate Absorption and Assimilation by Wheat Seedlings.

Nitrate uptake and assimilation were examined in intact 18 days old wheat (Triticum aestivum, cv Capitole) seedlings either permanently grown on nitrate (high-N seedlings) or N-stressed by transfer to an 0 N-solution for the final 7 days (low-N seedlings). The N-stressed seedlings were characterized by a lower organic N content (2.5 mg instead of 4.9 mg per seedling) and an increased root dry weight. The seedlings received (15)NO(3)K for 7 h in the light. Nitrate uptake was 2.8 times higher in low-N than in high-N seedlings. The assimilation rate was 35 and 16 µmol NO(3)(-)·(h-1)· g(-1) dry weight respectively. Partitioning of NO(3)(-) to reduction and assimilation was the very same in both kinds of seedlings. The results support the view that 50 % of the nitrate reduction in Triticum aestivum, cv Capitole could be achieved in the roots. The present observations are interpreted as evidence that factors closely associated with the seedling N-status may have a major role in regulating NO(3)(-) uptake and assimilation. In low-N seedlings, the high amount of carbohydrates in roots may add its stimulus to the specific inducing effect of nitrate whereas in high-N seedlings, excess of nitrate or amino-acids may set the pace by negative feedback control.

Nitrogen Isotope Fractionation Associated with Nitrate Reductase Activity and Uptake of NO(3) by Pearl Millet.

Nitrogen isotope fractionation by Pearl Millet (Pennisetum americanum L. and P. mollissimum L.) grown on nitrate was associated with nitrate reductase activity. Fractionation was evidenced at the step of nitrate reduction when the substrate-to-enzyme ratio was high (possibly saturating for the active sites of the nitrate reductase enzyme), for instance in young seedlings having a low nitrate reductase activity or in seedlings grown on high nitrate concentration.When the substrate concentration was low (and, hence, the active sites of the enzyme were possibly not saturated), the isotopic discrimination could only be associated with the uptake of nitrate into the cell. In that case, isotopic fractionation was null. It is concluded that the uptake of nitrate does not discriminate among nitrogen isotopes.

Light modulation of enzyme activity: activation of the light effect mediators by reduction and modulation of enzyme activity by thiol-disulfide exchange.

Light and dark modulation experiments with pea (Pisum sativum L.) chloroplast stromal fractions pretreated with dithiothreitol (to reduce protein disulfide bonds) or with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) (to block sulfhydryl groups) suggest that light modulation involves thiol-disulfide exchange on the modulatable stromal enzyme protein. Light-dependent reduction of DTNB involves a photosynthetic electron transport chain component located on the reducing side of photosystem I prior to ferredoxin; DTNB may be acting as a light effect mediator substitute. The thylakoid-bound light effect mediator system, then, in its light-activated reduced form probably catalyzes thiol-disulfide exchange reactions on stromal enzymes.

Relationship between the Level of Adenine Nucleotides and the Carboxylation Activity of Illuminated Isolated Spinach Chloroplasts: A Study with Antimycin A.

The changes in the levels of intact spinach (Spinacia oleracea L.) chloroplast adenine nucleotides during the time course of light-dependent CO(2) fixation were determined with respect to the effect of antimycin A. This study demonstrated that antimycin A lowered the rate of ATP formation during the induction period of carboxylation. While the steady state levels of ATP and the energy-charge value also decreased in the presence of antimycin, the concomitant increase of the CO(2) fixation activities insured higher ATP turnover rates. Changes in the labeling of CO(2) fixation products during the lag phase suggested a stepwise activation of the Calvin cycle, with fructose 1,6-diphosphate, and ribulose 5-phosphate kinase being activated before ribulose 1,5-diphosphate carboxylase. The possible mechanisms of the enhancement of CO(2) fixation activity by antimycin A in relation to its action on photophosphorylation during the lag phase are discussed.

Effect of antimycin a on photosynthesis of intact spinach chloroplasts.

Low concentrations (0.5-10 mum) of antimycin A were shown to increase the rate of CO(2) fixation, O(2) evolution and inorganic phosphate esterification in intact spinach (Spinacia oleracea) chloroplasts. The increase was highest when the light intensity was saturating. Stimulation was independent of the bicarbonate concentration and was accompanied by an enhancement in the synthesis of glycerate 3-phosphate with a decrease in dihydroxyacetone phosphate. The antibiotic decreased the Michaelis constant of the chloroplast but not of ribulose 1,5-diphosphate carboxylase for bicarbonate. It was suggested that antimycin A is affecting that portion (outer envelope) of the intact chloroplast which contains the enzyme mechanism for controlling the pace of CO(2) fixation.