Isolation of Bundle Sheath Cell Chloroplasts from the NADP-ME Type C(4) Plant Zea mays: Capacities for CO(2) Assimilation and Malate Decarboxylation

Bundle sheath chloroplasts have been isolated from Zea mays leaves by a procedure involving enzymic digestion of mechanically prepared strands of bundle sheath cells followed by gentle breakage and filtration. The resulting crude chloroplast preparation was enriched by Percoll density layer centrifugation to yield intact chloroplasts (about 20 micrograms chlorophyll per 10-gram leaf tissue) with high metabolic activities. Based on activities of marker enzymes in the chloroplast and bundle sheath cell extracts, the chloroplasts were essentially free of contamination by other organelles and cytoplasmic material, and were generally about 70% intact. Chlorophyll a/b ratios were high (about 10). With appropriate substrates these chloroplasts displayed high rates of malate decarboxylation, measured as pyruvate formation, and CO₂ assimilation (maximum rates approximately 5 and 3 micromoles per minute per milligram chlorophyll, respectively). These activities were light dependent, linear for at least 20 minutes at 30°C, and displayed highest rates at pH 8.0. High metabolic rates were dependent on addition of an exogenous source of carbon to the photosynthetic carbon reduction cycle (3-phosphoglycerate or dihydroxyacetone phosphate) and a nucleotide (ATP, ADP, or AMP), as well as aspartate. Generally, neither malate decarboxylation nor CO₂ assimilation occurred substantially in the absence of the other activity indicating a close relationship between these processes. Presumably, NADPH required for the photosynthetic carbon reduction cycle is largely supplied during the decarboxylation of malate by NADP-malic enzyme. The results are discussed in relation to the role of bundle sheath chloroplasts in C₄ photosynthesis by species of the NADP-malic enzyme type.     

Evidence for Endogenous Cyclic Photophosphorylation in Intact Chloroplasts: CO(2) Fixation with Dihydroxyacetone Phosphate

This study examines the capacity of intact spinach (Spinacia oleracea L.) chloroplasts to fix ¹⁴CO₂ when supplied with Benson-Calvin cycle intermediates in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). Under these conditions, substantial ¹⁴CO₂ fixation occurred in the light but not in the dark when either dihydroxyacetone phosphate, ribulose 5-phosphate, fructose 6-phosphate, or fructose bisphosphate was added. The highest rate of ¹⁴CO₂ fixation (20-40 micromoles per milligram chlorophyll per hour) was obtained with dihydroxyacetone phosphate. In contrast, no ¹⁴CO₂ fixation occurred when 3-phosphoglycerate was used. ¹⁴CO₂ fixation in the presence of dihydroxyacetone phosphate and DCMU was inhibited by carbonylcyanide m-chlorophenylhydrazone, dl-glyceraldehyde, and pyridoxal 5′-phosphate. Low concentrations of O₂ (25-50 micromolar) stimulated ¹⁴CO₂ fixation, but the activity decreased with increasing O₂ concentrations. The fixation of ¹⁴CO₂ in the presence of DCMU and dihydroxyacetone phosphate was also observed in maize bundle sheath cells. These results provide direct evidence for cyclic photophosphorylation in intact chloroplasts. The activity measured is adequate to support all the extra ATP requirements for maximum rates of photosynthesis in these intact chloroplasts.     

Aspartate stimulation of malate decarboxylation in Zea mays bundle sheath cells: possible role in regulation of C4 photosynthesis.

Aspartate stimulated by as much as three fold the rate of malate decarboxylation by Zea mays bundle sheath cells. Both the basal and aspartate stimulated rates of malate decarboxylation were light-dependent. Stimulation appeared to be due to aspartate as such, rather than depending on aspartate metabolism, and was due partly to a reduction in the malate concentration required for maximum decarboxylation and partly to an increased maximum velocity of decarboxylation. The extractable activities of NADP malic enzyme, glyceraldehyde phosphate dehydrogenase, and 3-phosphoglycerate kinase recoverable from cells were not increased by preincubating cells with aspartate, and aspartate did not affect the activity of these enzymes in cell-free extracts. It is suggested that aspartate may influence the transport of either malate into or pyruvate out of bundle sheath chloroplasts.     

Transport of Phosphoenolpyruvate by Chloroplasts from Mesembryanthemum crystallinum L. Exhibiting Crassulacean Acid Metabolism

Chloroplasts from CAM-Mesembryanthemum crystallinum can transport phosphoenolpyruvate (PEP) across the envelope. The initial velocities of PEP uptake in the dark at 4°C exhibited saturation kinetics with increasing external PEP concentration. PEP uptake had a V_max of 6.46 (±0.05) micromoles per milligram chlorophyll per hour and an apparent K_mpep of 0.148 (±0.004) millimolar. The uptake was competitively inhibited by Pi (apparent K_i = 0.19 millimolar), by glycerate 3-phosphate (apparent K_i = 0.13 millimolar), and by dihydroxyacetone phosphate, but malate and pyruvate were without effect. The chloroplasts were able to synthesize PEP when presented with pyruvate. PEP synthesis was light dependent. The prolonged synthesis and export of PEP from the chloroplasts required the presence of Pi or glycerate 3-phosphate in the external medium. It is suggested that the transport of pyruvate and PEP across the chloroplasts envelope is required during the gluconeogenic conversion of carbon from malate to storage carbohydrate in the light.     

Effects of the relative extrachloroplastic concentrations of inorganic phosphate, 3-phosphoglycerate, and dihydroxyacetone phosphate on the rate of starch synthesis in isolated spinach chloroplasts

The effect of external inorganic phosphate (Pi) on starch synthesis in isolated spinach (Spinacia oleracea American Hybrid No. 424) chloroplasts in the presence of millimolar concentrations of 3-phosphoglycerate (PGA) and/or dihydroxyacetone phosphate (DAP) was examined. Whereas CO₂ fixation was relatively constant as the ratio of the external phosphate to the PGA + DAP varied from 1:3 to 3:1, starch synthesis varied from 17% to 2% of the CO₂ fixation rate. With DAP alone, maximal starch synthesis was about 10% of the CO₂ fixation rate. The data demonstrate that the Pi/(PGA + DAP) ratio in the cytoplasm of plant cells could serve to regulate the flow of newly fixed carbon into starch without alterations in the rate of CO₂ fixation.     

The Involvement of Aspartate and Glutamate in the Decarboxylation of Malate by Isolated Bundle Sheath Chloroplasts from Zea mays

Aspartate or glutamate stimulated the rate of light-dependent malate decarboxylation by isolated Zea mays bundle sheath chloroplasts. Stimulation involved a decrease in the apparent K_m (malate) and an increased maximum velocity of decarboxylation. In the presence of glutamate other dicarboxylates (succinate, fumarate) competitively inhibited malate decarboxylation by intact chloroplasts with respect to malate with an apparent K_i of about 6 millimolar. For comparison the K_i for inhibition of nicotinamide adenine dinucleotide phosphate-malic enzyme from freshly lysed chloroplasts by these dicarboxylates was 15 millimolar. A range of compounds structurally related to aspartate stimulated malate decarboxylation by intact chloroplasts. K_a values for stimulation at 5 millimolar malate were 1.7, 5, and 10 millimolar for l-glutamate, l-aspartate, and β-methyl-dl-aspartate, respectively. Certain compounds, notably cysteic acid, which stimulated malate decarboxylation by intact chloroplasts inhibited malate decarboxylation by nicotinamide adenine dinucleotide phosphate-malic enzyme obtained from lysed chloroplasts and assayed under comparable conditions. It was concluded that aspartate, glutamate, and related compounds affect the transport of malate into the intact chloroplasts and that malate translocation does not take place on the general dicarboxylate translocator previously reported for higher plant chloroplasts.     

Effects of glycidate on carbon dioxide fixation with isolated spinach chloroplasts

Glycidate (2,3-epoxypropionate) stimulated CO₂ fixation in isolated spinach chloroplasts up to 100%. In the presence of glycidate the initial lag phase was abolished and the chloroplasts exported mainly 3-phosphoglycerate instead of dihydroxyacetone phosphate.     

C4-Dicarboxylic acid metabolism in bundle-sheath chloroplasts,mitochondria and strands of Eriochloa borumensis Hack., a phosphoenolpyruvate-carboxykinase type C4 species

C₄-acid metabolism by isolated bundlesheath chloroplasts, mitochondria and strands of Eriochloa borumensis Hack., a phosphoennolpyruvate-carboxykinase (PEP-CK) species, was investigated. Aspartate, oxaloacetate (OAA) and malate were decarboxylated by strands with several-fold stimulation upon illumination. There was strictly light-dependent decarboxylation of OAA and malate by the chloroplasts, but the chloroplasts did not decarboxylate aspartate in light or dark. PEP was a primary product of OAA or malate decarboxylation by the chloroplasts and its formation was inhibited by 3-(3,4-dichlorophenyl)-1, 1-dimethylurea or NH₄Cl. There was very little conversion of PEP to pyruvate by bundle-sheath chloroplasts, mitochondria or strands. Decarboxylation of the three C₄-acids by mitochondria was light-independent. Pyruvate was the only product of mitochondrial metabolism of C₄-acids, and was apparently transaminated in the cytoplasm since PEP and alanine were primarily exported out of the bundle-sheath strands. Light-dependent C₄-acid decarboxylation by the chloroplasts is suggested to be through the PEP-CK, while the mitochondrial C₄-acid decarboxylation may proceed through the NAD-malic enzyme (NAD-ME) system. In vivo both aspartate and malate are considered as transport metobolites from mesophyll to bundle-sheath cells in PEP-CK species. Aspartate would be metabolized by the mitochondria to OAA. Part of the OAA may be converted to malate and decarboxylated through NAD-ME, and part may be transported to the chloroplasts for decarboxylation through PEP-CK localized in the chloroplasts. Malate transported from mesophyll cells may serve as carboxyl donor to chloroplasts through the chloroplastic NAD-malate dehydrogenase and PEP-CK. Bundle-sheath strands and chloroplasts fixed ¹⁴CO₂ at high rates and exhibited C₄-acid-dependent O₂ evolution in the light. Studies with 3-mercaptopicolinic acid, a specific inhibitor of PEP-CK, have indicated that most (about 70%) of the OAA formed from aspartate is decarboxylated through the chloroplastic PEP-CK and the remaining (about 30%) OAA through the mitochondrial NAD-ME. Pyruvate stimulation of aspartate decarboxylation is discussed; a pyruvate-alanine shuttle and an aspartate-alanine shuttle are proposed between the mesophyll and bundle-sheath cells during aspartate decarboxylation through the PEP-CK and NAD-ME system respectively.Abbreviations CK carboxykinase - -Kg -ketoglutarate - ME malic enzyme - 3-MPA 3-mercaptopicolinic acid - OAA oxaloacetate - PEP phosphoenolpyruvate - R5P ribose-5-phosphate     

Rates and properties of endogenous cyclic photophosphorylation of isolated intact chloroplasts measured by CO2 fixation in the presence of dihydroxyacetone phosphate

1. Dihydroxyacetone phosphate in concentrations ? 2.5 mM completely inhibits CO₂-dependent O₂ evolution in isolated intact spinach chloroplasts. This inhibition is reversed by the addition of equimolar concentrations of P_i, but not by addition of 3-phosphoglycerate. In the absence of P_i, 3-phosphoglycerate and dihydroxyacetone phosphate, only about 20% of the ¹⁴C-labelled intermediates are found in the supernatant, whereas in the presence of each of these substances the percentage of labelled intermediates in the supernatant is increased up to 70–95%. Based on these results the mechanism of the inhibition of O₂ evolution by dihydroxyacetone phosphate is discussed with respect to the function of the known phosphate translocator in the envelope of intact chloroplasts.2. Although O₂ evolution is completely suppressed by dihydroxyacetone phosphate, CO₂ fixation takes place in air with rates of up to 65μ mol · mg^?1 chlorophyll · h^?1. As non-cyclic electron transport apparently does not occur under these conditions, these rates must be due to endogenous pseudocyclic and/or cyclic photophosphorylation.3. Under anaerobic conditions, the rates of CO₂ fixation in presence of dihydroxyacetone phosphate are low (2.5–7 μmol · mg^?1 chlorophyll · h^?1), but they are strongly stimulated by addition of dichlorophenyl-dimethylurea (e.g. 2 · 10^?7 M) reaching values of up to 60 μmol · mg^?1 chlorophyll · h^?1. As under these conditions the ATP necessary for CO₂ fixation can be formed by an endogenous cyclic photophosphorylation, the capacity of this process seems to be relatively high, so it might contribute significantly to the energy supply of the chloroplast. As dichlorophenyl-dimethylurea stimulates CO₂ fixation in presence of dihydroxyacetone phosphate under anaerobic but not under aerobic conditions, it is concluded that only under anaerobic conditions an “overreduction” of the cyclic electron transport system takes place, which is removed by dichlorophenyl-dimethylurea in suitable concentrations. At concentrations above 5 · 10^?7 M dichlorophenyl-dimethylurea inhibits dihydroxyacetone phosphate-dependent CO₂ fixation under anaerobic as well as under aerobic conditions in a similar way as normal CO₂ fixation. Therefore, we assume that a properly poised redox state of the electron transport chain is necessary for an optimal occurrence of endogenous cyclic photophosphorylation.4. The inhibition of dichlorophenyl-dimethylurea-stimulated CO₂ fixation in presence of dihydroxyacetone phosphate by dibromothymoquinone under anaerobic conditions indicates that plastoquinone is an indispensible component of the endogenous cyclic electron pathway.     

The effect of dihydroxyacetone phosphate and 3-phosphoglycerate on O2 evolution and on the levels of ATP,ADP and Pi in isolated intact chloroplasts

Addition of dihydroxyacetone phosphate (2.5 mM) or 3-phosphoglycerate (2.5 mM) to a suspension of isolated intact chloroplasts, which contains P_i only in low concentrations (0.2 mM) leads to a competitive inhibition of P_i uptake in the light. In consequence, the ATP/ADP ratio is strongly decreased. The rate of O₂ evolution is also reduced under these conditions, but the degree of inhibition is much higher after addition of dihydroxyacetone phosphate than after addition of 3-phosphoglycerate. Therefore, besides the competitive inhibition of P_i uptake, additional effects of dihydroxyacetone phosphate and 3-phosphoglycerate on O₂ evolution and CO₂ fixation of isolated intact chloroplasts must occur, which are discussed.     

Nitrite Reduction in Reconstituted and Whole Spinach Chloroplasts during Carbon Dioxide Reduction

Nitrite reduction in either whole, isolated spinach chloroplasts (Spinacia oleracea L.) or in reconstituted spinach chloroplasts is stimulated by a short period of photosynthetic CO₂ fixation in the light prior to nitrite addition. With reconstituted chloroplasts, a similar stimulation can be obtained in nitrite reduction without CO₂ fixation by the addition of dihydroxyacetone phosphate or fructose 6-phosphate. Specific intermediate metabolites of the photosynthetic carbon reduction cycle may have a regulatory role in nitrite reduction in chloroplasts in the light.     

Purification and characterization of pea chloroplastic phosphoriboisomerase

Pea (Pisum sativum L.) chloroplastic phosphoriboisomerase (EC 5.3.1.6) can be purified to apparent homogeneity in less than 2 days time with a 53% yield. Important steps in the purification include heat treatment and pseudoaffinity chromatography on Red H-3BN Sepharose. The purified isomerase has a subunit molecular mass of 26.4 kD. The N-terminal sequence has been determined through 34 residues. pH optima are 7.8 (ribose-5-phosphate) and 7.7 (ribulose-5-phosphate); K_m values are 0.9 millimolar (ribose-5-phosphate) and 0.6 millimolar (ribulose-5-phosphate). The enzyme is inhibited by erythrose-4-phosphate, sedoheptulosebisphosphate, glyceraldehyde-3-phosphate, and 3-phosphoglycerate at concentrations close to those found in photosynthesizing chloroplasts. Countercurrent phase partitioning experiments indicate that the pea chloroplastic phosphoriboisomerase interacts physically with phosphoribulokinase.     

Photosynthesis in Isolated Chloroplasts of the Crassulacean Acid Metabolism Plant Sedum praealtum

Intact chloroplasts were isolated from protoplasts of the Crassulacean acid metabolism plant Sedum praealtum D.C. Typical rates of CO₂ fixation or CO₂-dependent O₂ evolution ranged from 20 to 30 micromoles per milligram chlorophyll per hour and could be stimulated 30 to 50% by several Calvin cycle intermediates. The pH optimum for CO₂ fixation was 7.0 to 7.6 with considerable activity as low as pH 6.4. Low concentrations of orthophosphate (Pi) (optimum 0.4 millimolar) stimulated photosynthesis while high concentrations (5 millimolar) caused some inhibition. Both CO₂ fixation and CO₂-dependent O₂ evolution exhibited a relatively long lag phase (4 to 6 minutes) which remained constant between 0.4 to 5 millimolar Pi. The lag phase could be decreased by addition of dihydroxyacetone-phosphate or ribose 5-phosphate. Further results are presented which suggest these chloroplasts have a functional phosphate translocator.     

Changes in Levels of Intermediates of the C(4) Cycle and Reductive Pentose Phosphate Pathway under Various Concentrations of CO(2) in Maize Leaves

The rate of CO₂ assimilation and levels of metabolites of the C₄ cycle and reductive pentose phosphate pathway in an attached leaf of maize (Zea mays L) were measured over a range of intercellular CO₂ concentration (Ci) of 10 to 190 microliters per liter. The CO₂ assimilation rate was saturated at a Ci of around 175 microliters per liter. The levels of ribulose 1,5-bisphosphate and fructose 1,6-bisphosphate decreased substantially with increasing Ci. The levels of 3-phosphoglycerate, phosphoenolpyruvate (PEP), and pyruvate increased with increasing Ci. The level of dihydroxyacetone phosphate increased moderately from Ci of 10 microliters per liter to 20 to 50 microliters per liter and stayed almost constant over the rest of the range of Ci investigated. The levels of fructose 6-phosphate did not show any significant changes over the range of Ci. The levels of glucose 6-phosphate decreased slightly with increasing Ci. Although photosynthetically inactive pools of malate, asparate, and alanine could mask real changes in levels of the photosynthetically active pools of these compounds, the apparent levels of these compounds and the total amount of intermediates in the C₄ cycle (malate, aspartate, pyruvate, PEP, and alanine) increased with increasing Ci. The results suggest that there is carbon input into the C₄ cycle from the reductive pentose phosphate pathway which increases the level of total intermediates of the C₄ cycle with increasing Ci.     

Adenine Nucleotide Levels, the Redox State of the NADP System, and Assimilatory Force in Nonaqueously Purified Mesophyll Chloroplasts from Maize Leaves under Different Light Intensities

Recently, a nonaqueous fractionation method of obtaining highly purified mesophyll chloroplasts from maize leaves was established. This method is now used to determine adenine nucleotide levels, the redox states of the NADP system, Pi levels and dihydroxyacetone phosphate/3-phosphoglycerate ratios in mesophyll chloroplasts of Zea mays L. leaves under different light intensities. The sum of the ATP, ADP, and AMP levels was estimated to be 1.4 millimolar and the ATP/ADP ratio was 1 in the dark and 2.5 to 4 in the light. The adenine nucleotides were equilibrated by adenylate kinase. The total concentration of NADP(H) in the chloroplasts was 0.3 millimolar in the dark and 0.48 millimolar in the light. The ratio of NADPH/NADP was 0.1 to 0.18 in the dark and 0.23 to 0.48 in the light. The Pi level was estimated to be 20 millimolar in the dark and 10 to 17 millimolar in the light. The 3-phosphoglycerate reducing system was under thermodynamic equilibrium in the light. The calculated assimilatory forces were 8 per molar and 40 to 170 per molar in the dark and the light, respectively. There was no relationship between the degree of activation of pyruvate, Pi dikinase, and adenylate energy charge, or ATP/ADP ratio or ADP level under various light intensities. Only a weak relationship was found between the degree of activation of NADP-malate dehydrogenase and the NADPH/NADP ratio or NADP(H) level with increasing light intensity. A possible regulatory mechanism which is responsible for the regulation of activation of pyruvate,Pi dikinase and NADP-malate dehydrogenase is discussed.     

Properties of α-glucan phosphorylase from pea chloroplasts

A partially purified preparation of α-glucan phosphorylase was obtained from chloroplasts of Pisum sativum by ion-exchange chromatography and gel filtration. The preparation, in which no other enzyme that metabolized starch or glucose 1 -phosphate could be detected, was characterized. The optimum for phosphorolysis was pH 7.2; at pH 8.0 the activity was reduced by 50%. The preparation showed normal hyperbolic kinetics with the substrates, and catalysed the formation of [¹⁴C]glucose 1-phosphate from ¹⁴C-labelled starch grains from pea chloroplasts. None of the following, generally at 5 and 10 mM, significantly altered the rate of phosphorolysis: glucose, fructose, sucrose, fructose 6-phosphate, fructose 1,6-bisphosphate, dihydroxyacetone phosphate, 3-phosphoglycerate, 2-phosphoglycerate, phosphoenolpyruvate, pyruvate, ATP, ADP, AMP, 6-phosphogluconate, 2-phosphoglycollate, Mg²⁺, dithiothreitol. However, phosphorolysis was inhibited by ADPglucose. Measurements of ADPglucose in leaves and in isolated chloroplasts showed that none could be detected in the dark and suggested that the concentration in the light was high enough to cause a modest inhibition of the phosphorylase. The control of the breakdown of chloroplast starch is discussed.     

Photosynthetic Properties of Chloroplasts from Chlamydomonas reinhardii

Chloroplasts isolated from synchronous cultures of the unicellular green alga Chlamydomonas reinhardii, SAG 11-32/b (−), fix CO₂ at rates between 25 and 50 micromoles per milligram chlorophyll per hour. The upper value is approximately half of the rate of the intact cell.

During storage in the dark on ice, the chloroplast preparation loses 30 to 50% of its CO₂ fixing capability per hour. Under reducing conditions (+ 1 millimolar dithiothreitol), this loss of activity is about twice as fast. The same reducing conditions stimulate CO₂ fixation in the light.

High concentrations of inorganic phosphate (>2 millimolar) inhibit CO₂ fixation. This inhibition is overcome by the addition of glycerate 3-phosphate. It is concluded that chloroplasts from C. reinhardii possess a higher plant type phosphate translocator. With respect to dependency upon light intensity, pH and Mg²⁺ concentration, the results were similar to that reported for chloroplasts from higher plants. However, in contrast to higher plant chloroplasts, maximum CO₂ fixation is observed at the relatively low osmotic concentration of 0.12 molar mannitol in the reaction buffer.

     

Antimycin A Stimulation of Rate-limiting Steps of Photosynthesis in Isolated Spinach Chloroplasts

Changes in levels of metabolites in isolated spinach (Spinacia oleracea) chloroplasts seen upon addition of antimycin A suggest that the activities of enzymes mediating several regulated reactions are affected. Apparently, the presence of added antimycin A does not increase the level of CO₂ in the chloroplasts, nor does it stimulate CO₂ fixation by increasing the level of the carboxylation substrate, ribulose-1,5-diphosphate. Rather, it appears that antimycin A increases CO₂ fixation rate by indirectly stimulating the enzyme, ribulose-1,5-diphosphate carboxylase (E.C. 4.1.1.39), which mediates the carboxylation of ribulose-1,5-diphosphate to give 3-phosphoglycerate. Another rate-limiting enzyme of the reductive pentose phosphate cycle, hexose diphosphatase (E.C. 3.1.3.11), seems also to be stimulated. The synthesis of polysaccharides (mostly starch) seems also to be stimulated. These results are interpreted as indicating that antimycin A addition enhances the general activation of those enzymes which already are activated during photosynthesis but are inactive in the dark. The ratio of adenosine triphosphate-adenosine diphosphate under conditions of photosynthesis was only moderately decreased in the presence of antimycin A, perhaps accounting in part for an observed increase in accumulation of 3-phosphoglycerate as compared with dihydroxyacetone phosphate. No significant effect on movement of metabolites from the chloroplast to the medium was seen.     

The roles of malate and aspartate in C4 photosynthetic metabolism of Flaveria bidentis (L.)

In C₄ grasses belonging to the NADP-malic enzyme-type subgroup, malate is considered to be the predominant C₄ acid metabolized during C₄ photosynthesis, and the bundle sheath cell chloroplasts contain very little photosystem-II (PSII) activity. The present studies showed that Flaveria bidentis (L.), an NADP-malic enzyme-type C₄ dicotyledon, had substantial PSII activity in bundle sheath cells and that malate and aspartate apparently contributed about equally to the transfer of CO₂ to bundle sheath cells. Preparations of bundle sheath cells and chloroplasts isolated from these cells evolved O₂ at rates between 1.5 and 2 mol · min^–1 · mg^–1 chlorophyll (Chl) in the light in response to adding either 3-phosphoglycerate plus HCO ₃ ^– or aspartate plus 2-oxoglutarate. Rates of more than 2 mol O₂ · min^–1 · mg^–1 Chl were recorded for cells provided with both sets of these substrates. With bundle sheath cell preparations the maximum rates of light-dependent CO₂ fixation and malate decarboxylation to pyruvate recorded were about 1.7 mol · min^–1 · mg^–1 Chl. Compared with NADP-malic enzyme-type grass species, F. bidentis bundle sheath cells contained much higher activities of NADP-malate dehydrogenase and of aspartate and alanine aminotransferases. Time-course and pulse-chase studies following the kinetics of radiolabelling of the C-4 carboxyl of C₄ acids from ¹⁴CO₂ indicated that the photosynthetically active pool of malate was about twice the size of the aspartate pool. However, there was strong evidence for a rapid flux of carbon through both these pools. Possible routes of aspartate metabolism and the relationship between this metabolism and PSII activity in bundle sheath cells are considered.Abbreviations DHAP dihydroxyacetone phosphate - NADP-ME(-type) NADP-malic enzyme (type) - NADP-MDH NADP-malate dehydrogenase - OAA oxaloacetic acid - 2-OG 2-oxoglutarate - PEP phosphoenolpyruvate - PGA 3-phosphoglycerate - Pi orthophosphate - Ru5P ribulose 5-phosphate     

C(4) Photosynthesis: Light-dependent CO(2) Fixation by Mesophyll Cells, Protoplasts, and Protoplast Extracts of Digitaria sanguinalis

Mesophyll cells, protoplasts, and protoplast extracts of Digitaria sanguinalis were used for comparative studies of light-dependent CO₂ fixation. CO₂ fixation was low without the addition of organic substrates. Pyruvate, oxaloacetate, and 3-phosphoglycerate induced relatively low rates (10 to 90 μmoles/mg chlorophyll·hr) of CO₂ fixation when added separately. However, a highly synergistic relationship was found between pyruvate + oxaloacetate and pyruvate + 3-phosphoglycerate for inducing light-dependent CO₂ fixation in the mesophyll preparations. Highest rates of CO₂ fixation were obtained with protoplast extracts. Pyruvate, in combination with oxaloacetate or 3-phosphoglycerate induced light-dependent rates from 150 to 380 μmoles of CO₂ fixed/mg chlorophyll·hr which are equivalent to or exceed reported rates of whole leaf photosynthesis in C₄ species. Concentrations of various substrates required to give half-maximum velocities of CO₂ fixation were determined, with the protoplast extracts generally saturating at the lowest substrate concentrations. Chloroplasts separated from protoplast extracts showed little capacity for CO₂ fixation. The results suggest that CO₂ fixation in C₄ mesophyll cells is dependent on chloroplasts and extrachloroplastic phosphoenolpyruvate carboxylase.