Regulation of NADP-malate dehydrogenase in C4 plants: relationship among enzyme activity, NADPH to NADP ratios, and thioredoxin redox states in intact maize mesophyll chloroplasts

NADP-malate dehydrogenase activity, the ratio of NADPH to NADP, and thioredoxin redox state in Zea mays chloroplasts were determined after various treatments. Following transfer from dark to light, NADP-malate dehydrogenase was activated more than 20-fold within 10 min while the proportion of pyridine nucleotide as NADPH increased from about 25 to 90%, and the proportion of thioredoxin in the reduced form increased from 20 to more than 90%, in less than 1 min. After transfer back to the dark, NADPH levels dropped very rapidly to the initial values recorded before illumination, while enzyme activity and reduced thioredoxin levels decreased more slowly. Addition of oxaloacetate or 3-phosphoglycerate to illuminated chloroplasts results in a decrease of about 70% in the activity of NADP-malate dehydrogenase, a 30% decrease in the level of NADPH, and a 25% decrease in the reduced thioredoxin content. Adding dihydroxyacetone phosphate and pyruvate had no effect. These results are considered in relation to the hypothesis that NADP-malate dehydrogenase activity in chloroplasts may be determined by factors regulating the ratio of NADPH to NADP as well as those influencing the redox state of thioredoxin.     

Regulation of C4 photosynthesis: regulation of pyruvate, Pi dikinase by ADP-dependent phosphorylation and dephosphorylation

Pyruvate, Pi dikinase in extracts of chloroplasts from mesophyll cells of Zea mays is inactivated by incubation with ADP plus ATP. This inactivation was associated with phosphorylation of a threonine residue on a 100 kDa polypeptide, the major polypeptide of the mesophyll chloroplast stroma, which was identified as the subunit of pyruvate, Pi dikinase. The phosphate originated from the beta-position of ADP as indicated by the labelling of the enzyme during inactivation in the presence of [beta-32P]ADP. During inactivation of the enzyme up to 1 mole of phosphate was incorporated per mole of pyruvate, Pi dikinase subunit inactivated. 32P label was lost from the protein during the Pi-dependent reactivation of pyruvate, Pi dikinase.     

Lipid peroxidation and the degradation of cytochrome P-450 heme

The enzyme content and functional capacities of mesophyll chloroplasts from Atriplex spongiosa and maize have been investigated. Accompanying evidence from graded sequential blending of leaves confirmed that mesophyll cells contain all of the leaf pyruvate, P_i dikinase, and PEP carboxylase activities and a major part of the adenylate kinase and pyrophosphatase. 3-Phosphoglycerate kinase, NADP glyceraldehyde-3-P-dehydrogenase, and triose-P isomerase activities were about equally distributed between mesophyll and bundle sheath cells but other Calvin cycle enzymes were very largely or solely located in bundle sheath cells. In A. spongiosa extracts of predominantly mesophyll origin the proportion of the released pyruvate, P_i dikinase, adenylate kinase, pyrophosphatase, 3-phosphoglycerate kinase, and NADP glyceraldehyde-3-P dehydrogenase retained in pelleted chloroplasts was similar but varied between 30 and 80% in different preparations. The proportion of these enzymes and NADP malate dehydrogenase recovered in maize chloroplast preparations varied between 15 and 35%. Washed chloroplasts retained most of the activity of these enzymes but ribulose diphosphate carboxylase and other Calvin cycle enzyme activities were undetectable. Among the evidence for the integrity of these chloroplasts was their capacity for light-dependent conversion of pyruvate to phosphoenolpyruvate and O₂ evolution when 3-phosphoglycerate or oxaloacetate were added. These results support our previous conclusions about the function of mesophyll chloroplasts in C₄-pathway photosynthesis and clearly demonstrate that they lack Calvin cycle activity.     

Influence of Oxygen and Temperature on the Dark Inactivation of Pyruvate, Orthophosphate Dikinase and NADP-Malate Dehydrogenase in Maize

The influence of oxygen and temperature on the inactivation of pyruvate, Pi dikinase and NADP-malate dehydrogenase was studied in Zea mays. O₂ was required for inactivation of both pyruvate, Pi dikinase and NADP-malate dehydrogenase in the dark in vivo. The rate of inactivation under 2% O₂ was only slightly lower than that at 21% O₂. The in vitro inactivation of pyruvate, Pi dikinase, while dependent on adenine nucleotides (ADP + ATP), did not require O₂.

The postillumination inactivation of pyruvate, Pi dikinase in leaves was strongly dependent on temperature. As temperature was decreased in the dark, there was a lag period of increasing length (e.g. at 17°C there was a lag of about 25 minutes) before inactivation proceeded. Following the lag period, the rate of inactivation decreased with decreasing temperature. The half-time for dark inactivation was about 7 minutes at 32°C and 45 minutes at 17°C. The inactivation of pyruvate, Pi dikinase in vitro following extraction from illuminated leaves was also strongly dependent on temperature, but occurred without a lag period. In contrast, NADP-malate dehydrogenase was rapidly inactivated in leaves (half-time of approximately 3 minutes) during the postillumination period without a lag, and there was little effect of temperature between 10 and 32°C. The results are discussed in relation to known differences in the mechanism of activation/inactivation of the two enzymes.

     

Control of the activation/inactivation of pyruvate, Pi dikinase from the C4 plant maize by adenylate energy charge, pyruvate, and analogs of pyruvate

Pyruvate, Pi dikinase, which is localized in the mesophyll chloroplasts of C4 plants, requires a high adenylate energy charge for conversion of the enzyme from the inactive to the active form. The inactivation process is favored by a low energy charge, being maximal at values below 0.7. Pyruvate and analogs of pyruvate, oxamate and oxalate, strongly inhibit the inactivation process at millimolar levels. The results suggest that light activation of the enzyme in vivo may be mediated by an increased adenylate energy charge in the chloroplast. Pyruvate may allow a higher steady-state level of activation to be achieved in vivo by inhibiting inactivation.     

Regulation of C4 photosynthesis: purification and properties of the protein catalyzing ADP-mediated inactivation and Pi-mediated activation of pyruvate,Pi dikinase

Pyruvate,Pi dikinase regulatory protein (PDRP) has been highly purified from maize leaves, and its role in catalyzing both ADP-mediated inactivation (due to phosphorylation of a threonine residue) and Pi-mediated activation (due to dephosphorylation by phosphorolysis) of pyruvate,Pi dikinase has been confirmed. These reactions account for the dark/light-mediated regulation of pyruvate,Pi dikinase observed in the leaves of C4 plants. During purification to apparent homogeneity the ratio of these two activities remained constant. The molecular weight of the native PDRP was about 180,000 at pH 8.3 and 90,000 at pH 7.5. Its monomeric molecular weight was 45,000. It was confirmed that inactive pyruvate,Pi dikinase free of a phosphate group on a catalytic histidine was the preferred substrate for activation. Michaelis constants for orthophosphate and the above form of active pyruvate,Pi dikinase were determined, as well as the mechanism of inhibition of the PDRP-catalyzed reaction by ATP, ADP, AMP, and PPi. For the inactivation reaction, Km values were 1.2 microM for the active pyruvate,Pi dikinase and 52 microM for ADP. CDP and GDP but not UDP could substitute for ADP. The inactivation reaction is inhibited by inactive pyruvate,Pi dikinase competitively with respect to both active pyruvate,Pi dikinase and ADP. Both the activation and inactivation reactions catalyzed by PDRP have a broad pH optimum between 7.8 and 8.3. The results are discussed in terms of the likely mechanism of dark/light regulation of pyruvate,Pi dikinase in vivo.     

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 Mg²⁺ 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.Abbreviations AEC adenylate energy charge - DTT dithiothreitol - FBPase fructose 1,6-bisphosphatase - Fd ferredoxin - FTR ferredoxin-thioredoxin reductase - NADP-MDH NADP-malate dehydrogenase - P glycolate-phosphoglycolate - P glycolate phosphatase-phosphoglycolate phosphatase - PSII photosystem II - PPDK pyruvate, Pi dikinase - Rubisco Ribulose 1,5-bisphosphate carboxylase/oxygenase     

Activation of NADP-Malate Dehydrogenase, Pyruvate,Pi Dikinase, and Fructose 1,6-Bisphosphatase in Relation to Photosynthetic Rate in Maize

The activity and extent of light activation of three photosynthetic enzymes, pyruvate,Pi dikinase, NADP-malate dehydrogenase (NADP-MDH), and fructose 1,6-bisphosphatase (FBPase), were examined in maize (Zea mays var Royal Crest) leaves relative to the rate of photosynthesis during induction and under varying light intensities. There was a strong light activation of NADP-MDH and pyruvate,Pi dikinase, and light also activated FBPase 2- to 4-fold. During the induction period for whole leaf photosynthesis at 30°C under high light, the time required to reach half-maximum activation for all three enzymes was only 1 minute or less. After 2.5 minutes of illumination the enzymes were fully activated, while the photosynthetic rate was only at half-maximum activity, indicating that factors other than enzyme activation limit photosynthesis during the induction period in C₄ plants.

Under steady state conditions, the light intensity required to reach half-maximum activation of the three enzymes was similar (300-400 microEinsteins per square meter per second), while the light intensity required for half-maximum rates of photosynthesis was about 550 microEinsteins per square meter per second. The light activated levels of NADP-MDH and FBPase were well in excess of the in vivo activities which would be required during photosynthesis, while maximum activities of pyruvate,Pi dikinase were generally just sufficient to accommodate photosynthesis, suggesting the latter may be a rate limiting enzyme.

There was a large (5-fold) light activation of FBPase in isolated bundle sheath strands of maize, whereas there was little light activation of the enzyme in isolated mesophyll protoplasts. In mesophyll protoplasts the enzyme was largely located in the cytoplasm, although there was a low amount of light-activated enzyme in the mesophyll chloroplasts. The results suggest the chloroplastic FBPase in maize is primarily located in the bundle sheath cells.

     

Role of Metabolites in the Reversible Light Activation of Pyruvate, Orthophosphate Dikinase in Zea mays Mesophyll Cells in Vivo

Whole leaf and mesophyll cell concentrations of pyruvate, phosphoenolpyruvate (PEP), ATP, and ADP were determined in Zea mays during the reversible light activation of pyruvate, orthophosphate dikinase in vivo. Mesophyll cell levels of the four metabolites were estimated by extrapolation from values in freeze-quenched leaf samples that were fractionated by differential filtration through nylon mesh nets (adapted from M Stitt, HW Heldt [1985] Planta 164: 179-188). During the 3 minutes required for complete light activation of dikinase, pyruvate levels in the mesophyll cell decreased (from 166 ± 15 to 64 ± 10 nanomoles per milligram of chlorophyll [nmol/mg Chl]) while PEP levels increased (from 31 ± 4 to 68 ± 4 nmol/mg Chl, with a transient burst of 133 ± 16 nmol/mg Chl at 1 minute). Mesophyll cell levels of ATP increased (from 22 ± 4 to 48 ± 3 nmol/mg Chl) and ADP levels decreased (from 16 ± 4 to 7 ± 6 nmol/mg Chl) during the first minute of illumination. Upon darkening of the leaf and inactivation of dikinase, pyruvate levels initially increased in the mesophyll (from 160 ± 30 to a maximum of 625 ± 40 nmol/mg Chl), and then slowly decreased to about the initial value in the light over an hour. PEP levels dropped (from 176 ± 5 to 47 ± 3 nmol/mg Chl) in the first 3 minutes and remained low for the remainder of the dark period. Mesophyll levels of ATP and ADP rapidly decreased and increased, respectively, about twofold upon darkening. The trends observed for these metabolite levels in the mesophyll cell during the light/dark regulation of pyruvate, orthophosphate dikinase activity suggest that pyruvate and PEP do not play a major role in vivo in regulating the extent of light activation (dephosphorylation) or dark inactivation (ADP-dependent threonyl phosphorylation) of dikinase by its bifunctional regulatory protein. While the changes in ADP levels appear qualitatively consistent with a regulatory role for this metabolite in the light activation and dark inactivation of dikinase, they are not of a sufficient magnitude to account completely for the tenfold change in enzyme activity observed in vivo.     

Nonaqueous purification of maize mesophyll chloroplasts

A nonaqueous fractionation method to obtain highly purified mesophyll chloroplasts from lyophilized leaves of Zea mays L. is described. The levels of several metabolites including pyruvate were determined in the purified mesophyll chloroplast fractions which were prepared from leaves exposed to different light intensities. The role of pyruvate in the regulation of pyruvate,Pi dikinase in these chloroplasts under different light intensities is discussed.     

The redox state of the NADP system in illuminated chloroplasts

Pyridine nucleotide levels were measured in intact spinach chloroplasts. The NADPH/NADP ratio was close to unity in darkened chloroplasts. On illumination, chloroplast NADP levels decreased rapidly. The decrease was more prominent at low than at high light intensities. In the presence of bicarbonate, NADP subsequently increased to reach a steady-state level. The kinetics of the increase were related in general, but not in detail, to the lag phase of photosynthesis. In the steady state, chloroplast NADP was sometimes, particularly during photosynthesis at high light intensities, less reduced in the light than in the dark. In the dark-light transition, phosphoglycerate reduction is driven by increases in the ratios NADPH/NADP and ATP/ADP. When photosynthesis accelerates after the initial lag phase, the NADPH/NADP ratio decreases and a high ratio of phosphoglycerate to triose phosphate becomes an important factor in driving carbon reduction. Under photosynthetic flux conditions, the redox state of the chloroplast NADP system appeared to be governed largely by the chloroplast ratio of phosphoglycerate to dihydroxyacetone phosphate and by the phosphorylation potential [ATP]/[ADP] [P_i]. The inhibitor of cyclic electron transport, antimycin A, increased reduction of the chloroplast NADP system. Even when reduction was almost complete in the presence of 5 μM antimycin A, photosynthesis was still significant at low light intensities. Electrons appeared to be effectively distributed between the cyclic electron-transport pathway and the noncyclic route to NADP at NADPH/NADP ratios as low as about 1. When bicarbonate was absent, the NADP system remained largely reduced in the light. The energy-transfer inhibitor, Dio-9, and uncouplers and agents which interfered with pH regulation of the Calvin cycle increased reduction of the NADP system while decreasing photosynthesis.     

Light Activation of Pyruvate,Pi Dikinase and NADP-Malate Dehydrogenase in Mesophyll Protoplasts of Maize : Effect of DCMU, Antimycin A, CCCP, and Phlorizin

Pyruvate,Pi dikinase (PPDK, EC 2.7.9.1) and NADP-malate dehydrogenase (MDH, EC 1.1.1.82) were activated in the light and inactivated following a dark treatment in mesophyll protoplasts of maize. DCMU (up to 33 micromolar), an inhibitor of noncyclic electron transport, inhibited activation of MDH much more strongly than it did PPDK. Antimycin A (6.6-33 micromolar), an inhibitor of cyclic photophosphorylation, inhibited the activation of PPDK (up to 61%), but had little or no effect on activation of MDH. Carbonyl cyanide m-chlorophenylhydrazone (0.2-2 micromolar) and nigericin (0.4 micromolar), uncouplers of photophosphorylation, inhibited activation of PPDK while stimulating the activation of MDH. Phlorizin (0.33-1.7 millimolar), an inhibitor of the coupling factor for ATP synthesis, strongly inhibited activation of PPDK but only slightly effected light activation of MDH. These results suggest that noncyclic electron flow is required for activation of NADP-MDH and that photophosphorylation is required for activation of PPDK.     

Effect of adenine nucleotides on the reaction catalyzed by pyruvate,orthophosphate dikinase in maize

The effect of adenine nucleotides in pyruvate, orthophosphate dikinase (EC 2.7.9.1, ATP, pyruvate, orthophosphate phosphotransferase)_was studied with the enzyme furified from maize, and with the enzyme obtained from mesophyll chloroplast extracts during assay in the direction of pyruvate conversion to phosphoenolpyruvate. (1) In studies with the purified enzyme, the relationship of initial velocity to ATP concentrations follows Michaelis-Menten kinetics, and the K_m value for ATP was 22.8 μM (± 5.1 μM, n = 5). (2) AMP was a competitive inhibitor with respect to ATP, and its K_i value was 35.8 μM (± μM, n = 4). There was no inhibition of catalysis by ADP up to a concentration of 460 μM. (3) The theoretical response of the enzyme to change in the adenylate energy charge was calculated from the kinetic constants for ATP and AMP. The experimentally obtained values were similar to the theoretical response when varying energy charge was generated by addition of appropriate amounts of ATP, ADP and AMP in assays with the purified enzyme. The response of the enzyme to energy charge at different pH values (pH 7.0, 7.5, and 8.0) was similar, although the activity of the enzyme at pH 7.0 was about 40% of that at pH 8.0. (4) When mesophyll chloroplast extracts of maize, which contain high levels of adenylate kinase, were used as the source of the enzyme and the adenylate energy charge was generated by addition of different concentrations of ATP and AMP, the influence on catalysis was similar to that with the purified enzyme. (5) The data show that the effect of varying energy chage on the activity of the dikinase is not typical of a U-type enzyme, in contrast to phosphoglycerate kinase (EC 2.7.2.3, ATP: 3-phospho-D-glycerate 1-phosphotransferase), which is more strongly regulated. (6) Evidence is presented for competition between the dikinase and phosphoglycerate kinase for ATP in mesophyll chloroplast extracts of maize. (7) When the effect of adenylate energy charge on the state of activation and the direct effect on catalysis of the dikanase are combined, the total capacity for catalysis is very dependent on the energy charge.     

Co-operative activation of chloroplast fructose-1,6-bisphosphatase by reductant,pH and substrate

Intact chloroplasts capable of high rates of photosynthesis fail to reduce CO₂ when illuminated in the absence of oxygen. While anaerobiosis limits proton gradient formation leading to ATP deficiency (Ziem-Hanck, U. and Heber, U. (1980) Biochim. Biophys. Acta 591, 266–274), light activation of fructose-1,6-bisphosphatase was also inhibited by anaerobiosis, whereas light activation of NADP-malate dehydrogenase was stimulated by anaerobiosis, indicating that reductant was still available for light activation. The chloroplast pool of NADP was largely reduced during illumination under anaerobiosis and electron transport to oxaloacetate was not inhibited by anaerobic conditions. Significant light activation of fructose-bisphosphatase was observed in anaerobic chloroplasts with 3-phosphoglycerate as substrate, but not with dihydroxyacetone phosphate (3-phosphoglycerate supports electron transport and hence proton gradient formation). In the absence of added substrates, illumination of anaerobic chloroplasts resulted in some light activation of fructose-bisphosphatase when the pH of the medium was increased. Under these conditions, light activation was stimulated by dihydroxyacetone phosphate. Dihydroxyacetone phosphate added together with oxaloacetate allowed light activation of fructose-bisphosphatase in anaerobic chloroplasts, while neither substrate added alone was effective. Formation of a transthylakoid proton gradient can therefore substitute for an alkaline suspension medium by causing an alkaline shift of the stromal pH on illumination. The data are interpreted as indicating that fructose-bisphosphatase, but not NADP-malate dehydrogenase, requires an alkaline pH and the presence of substrate for rapid reductive light activation and they bear on the interpretation of the lag observed in photosynthesis in chloroplasts and leaves on illumination after a prolonged dark period.     

Regulation of C4 photosynthesis: inactivation of pyruvate, Pi dikinase by ADP-dependent phosphorylation and activation by phosphorolysis

These studies provide information about the mechanism of the light/dark-mediated regulation of pyruvate, Pi dikinase (EC 2.7.9.1) in leaves. It is shown that inactivation is due to a phosphorylation of the enzyme from the beta-phosphate of ADP, and that activation occurs by phosphorolysis to remove the enzyme phosphate group. During ADP plus ATP-dependent inactivation of pyruvate, Pi dikinase in chloroplast extracts, 32P was incorporated into the enzyme from [beta-32P]ADP. Approximately 1 mol of phosphate was incorporated per mol of monomeric enzyme subunit inactivated. There was very little incorporation of label from ADP or ATP labeled variously in other positions with 32P or from the nucleotides labeled with 3H in the purine ring. Purified pyruvate, Pi dikinase was also labeled from [beta-32P]ADP during inactivation. In this system, phosphorylation of the enzyme required the addition of the "regulatory protein" shown previously to be essential for catalyzing inactivation and activation. During orthophosphate-dependent reactivation of pyruvate, Pi dikinase, it was shown that the enzyme loses 32P label and that pyrophosphate is produced. The significance of these findings in relation to regulation of the enzyme in vivo is discussed.     

Light and temperature dependence of the rate and degree of activation of pyruvate,Pi dikinase in vivo in maize

Activation of pyruvate,Pi dikinase by light was studied in leaf discs of maize which were illuminated for 1 h at light intensities ranging from approximately 3% to 50% of full sunlight and at temperatures of 10, 22.5, and 35°C. At the highest light intensity the degree of activation was similar and relatively independent of temperature between 10 and 35°C. Under low light the degree of activation was high at 10°C but decreased rapidly with increasing temperature. There was a similar effect of light and temperature on the activation of NADP-malate dehydrogenase.At low temperature, the rate of activation of pyruvate,Pi dikinase was relatively low and independent of the light intensity used and the rate of inactivation in the dark was extremely low. At high temperature, the rate of activation was high and dependent on the light intensity used while the rate of dark inactivation was also relatively high. The degree of activation is discussed in relation to the possible influence of light and temperature on the turnover between the active and inactive forms of pyruvate,Pi dikinase during illumination.This research was supported by the Japan-U.S. Cooperative Research Program (The Japan Society for the Promotion of Science, NFS Grant INT 78-17245), NSF Grant PCM 77-09284, by the Japanese Ministry of Education and by the College of Agriculture and Life Sciences, University of Wisconsin, Madison, Wisconsin.     

A high-activity ATP translocator in mesophyll chloroplasts of Digitaria sanguinalis, a plant having the C-4 dicarboxylic acid pathway of photosynthesis

The effect of exogenous adenine nucleotides on CO₂ fixation and oxygen evolution was studied with mesophyll protoplast extracts of the C₄ plant Digitaria sanguinalis. Exogenous ATP was found to stimulate the rate of pyruvate and pyruvate + oxalacetate induced CO₂ fixation, as well as reverse the inhibition of CO₂ fixation by carbonyl cyanide m-chlorophenyl hydrazone and several electron transport inhibitors. The ATP-dependent stimulation of CO₂ fixation varied from 40 to 70 μmol CO₂ fixed/mg chlorophyll per h, suggesting that ATP was crossing the chloroplast membranes at rates of 80–140 μmol/mg chlorophyll per h, since 2 ATP are required for each CO₂ fixed. Fixation of CO₂ could also be induced in the dark by exogenous ATP, in which case ADP accumulated outside the chloroplasts. This suggests that external ATP is exchanging for internal ADP. In contrast, ADP and AMP were found not to traverse chloroplast membranes, on the basis that neither nucleotide inhibited CO₂ fixation or stimulated oxygen evolution that was limited by available ADP for phosphorylation. Further evidence that ATP can enter the chloroplasts was obtained by direct measurements of the increase in ATP in the chloroplasts due to addition of exogenous ATP in the dark. These studies yielded minimal rates of ATP uptake on the order of 30–40 μmol/mg chlorophyll per h. It is suggested that a membrane translocator exists that specifically transports ATP into the chloroplasts in exchange for ADP. The significance of these findings are considered with respect to the C₄ pathway of photosynthesis.     

CO(2) Assimilation and Malate Decarboxylation by Isolated Bundle Sheath Chloroplasts from Zea mays

Conditions for optimal CO₂ fixation and malate decarboxylation by isolated bundle sheath chloroplasts from Zea mays were examined. The relative rates of these processes varied according to the photosynthetic carbon reduction cycle intermediate provided. Highest rates of malate decarboxylation, measured as pyruvate formation, were seen in the presence of 3-phosphoglycerate, while carbon fixation was highest in the presence of dihydroxyacetone phosphate; only low rates were measured with added ribose-5-phosphate. Chloroplasts exhibited a distinct phosphate requirement and this was optimal at a level of 2 millimolar inorganic phosphate in the presence of 2.5 millimolar 3-phosphoglycerate, dihydroxyacetone phosphate, or ribose-5-phosphate. Malate decarboxylation and CO₂ fixation were stimulated by additions of AMP, ADP, or ATP with half-maximal stimulation occurring at external adenylate concentrations of about 0.15 millimolar. High concentrations (>1 millimolar) of AMP were inhibitory. Aspartate included in the incubation medium stimulated malate decarboxylation and CO₂ assimilation. In the presence of aspartate, the apparent Michaelis constant (malate) for malate decarboxylation to pyruvate by chloroplasts decreased from 6 to 0.67 millimolar while the calculated V_max for this process increased from 1.3 to 3.3 micromoles per milligram chlorophyll. Aspartate itself was not metabolized. It was concluded that the processes mediating the transport of phosphate, 3-phosphoglycerate, and dihydroxyacetone phosphate transport on the one hand, and also of malate might differ from those previously described for chloroplasts from C₃ plants.     

Regulation of C4 photosynthesis: identification of a catalytically important histidine residue and its role in the regulation of pyruvate,Pi dikinase

These studies provide further information regarding the mechanism of the light/dark-mediated regulation of pyruvate,Pi dikinase in leaves. It is shown that a catalysis-linked phosphorylation of pyruvate,Pi dikinase can be demonstrated following incubation of the enzyme with [32P]phosphoenolpyruvate or [beta-32P]ATP plus Pi, that the enzyme-bound phosphate is located on a histidine residue, and that this phosphate is retained during ADP-mediated inactivation. Further evidence is provided that phosphorylation of this histidine is a prerequisite for ADP-mediated inactivation through phosphorylation of a threonine residue from the beta-phosphate of ADP. It is demonstrated that diethylpyrocarbonate (which forms a derivative with histidine residues) prevents [32P]phosphoenolpyruvate-dependent labeling (catalytic labeling) and [beta-32P]ADP-dependent labeling (inactivation labeling) of the enzyme. In addition, it is demonstrated that oxalate, an analog of pyruvate, competitively inhibits ADP-dependent inactivation with respect to ADP. The significance of these results is discussed with regard to the mechanism of regulation of pyruvate,Pi dikinase in vivo.