Reversal of 3-(3,4-dichlorophenyl)-1,1-dimethylurea inhibition of carbon dioxide fixation in spinach chloroplasts and protoplasts by dicarboxylic acids

3-(3,4-Dichlorophenyl)-1,1-dimethylurea (DCMU) inhibition of (14)CO(2) fixation in isolated intact spinach (Spinacia oleracea L.) chloroplasts was reversed (by about 34%) by l-malate but not by oxaloacetate (OAA). However, OAA reversed the DCMU inhibition in spinach protoplasts indicating an extrachloroplastic enzyme requirement. Extrachloroplastic OAA reduction was coupled with external dihydroxyacetone phosphate (DHAP) oxidation, and the malate formed from such coupling might then enter the chloroplasts. Evidence was presented using ruptured protoplasts that the export of recently formed 3-phosphoglyceric acid (PGA) out of chloroplasts in exchange for external DHAP was reversed by excess OAA. The PGA/DHAP shuttle across the chloroplast envelope was found to be regulated by the external concentrations of DHAP and OAA.     

Regulation of Steady-State Photosynthesis in Isolated Intact Chloroplasts under Constant Light: Responses of Carbon Fluxes, Metabolite Pools and Enzyme-Activation States to Changes of Electron Pressure

The reactions of isolated intact spinach chloroplasts at saturatinglight and CO₂ to changes in steady-state electron flow werefollowed at the various stages of photosynthesis. Alterationsin the rate of electron flow were induced by the addition ofoxaloacetate (OAA), nitrite or methyl viologen (MV). Two typesof effect can be distinguished: (1) When a small fraction ofthe electrons produced are accepted by OAA or nitrite (up to20% of the electrons produced in the light), the activationstate of the NADP⁺-dependent malate dehydrogenase (NADP-MDH)was strongly decreased, whereas qP and the rate of O₂-productionwere increased. qN, the stromal metabolite pools and the [¹⁴C]-CO₂-fixationrate were only marginally influenced. (2) Higher amounts ofnitrite or MV decreased O₂ production and strongly inhibited[¹⁴C]CO₂ fixation. This treatment further increased the ATP/ADPratio, but had little effect on the NADPH + H⁺/NADP⁺ ratio.The stromal concentrations of 3PGA, DHAP and FBP, and the ratesof 3PGA and DHAP export were drastically changed. In particular,the DHAP/3PGA ratio increased, and the rate of 3PGA export wasdecreased by minor changes in the rate of electron flow. Additionof high amounts of nitrite or MV, but not of OAA decreased theactivation states of NADP-MDH and fructose 1,6-bisphosphatase(FBPase), while the activation states of NADP⁺-dependent glyceraldehyde3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK)remained unchanged under all conditions. (Received February 10, 1997; Accepted September 2, 1997)     

Relationships between Carbon Dioxide, Malate, and Nitrate Accumulation and Reduction in Corn (Zea mays L.) Seedlings

The observation that exposure of the leaf canopy to increasing concentrations of CO₂ (100-400 μl/l) decreases the influx of nitrate to the leaf blades, but not to the roots or stalks (largely leaf sheaths), was reconfirmed using ¹⁵NO₃⁻. Decreases in leaf nitrate supply were associated with decreases in induction of nitrate reductase, thus supporting the view that the influx of nitrate to a tissue is a major factor in regulation of the level of nitrate reductase. The whole plant ¹⁵N distribution data show that the CO₂ effects were due to decreased influx of nitrate into the leaf blade rather than CO₂-enhanced nitrate reduction. The decreases in nitrate accumulation by the leaf blade with increases in CO₂ concentration were only partially accounted for by differences in transpiration. Because the initial malate concentration of root tissue (detopped plants) had no subsequent effect on nitrate uptake, it seems unlikely that high levels of malate induced by CO₂ were responsible for the exclusion of nitrate from the leaf blades.     

Reduction of Nitrate via a Dicarboxylate Shuttle in a Reconstituted System of Supernatant and Mitochondria from Spinach Leaves

Substantial rates of nitrate reduction could be achieved with a reconstituted system from spinach leaves containing supernatant, mitochondria, NAD⁺, oxaloacetate (OAA), and an oxidizable substrate. Appropriate substrates were glycine, pyruvate, citrate, isocitrate, fumarate, or glutamate. The reduction of NO₃⁻ with any of the substrates could be inhibited by n-butyl malonate, showing that the transfer of reducing power from the mitochondria to the supernatant involved the malate exchange carrier. The addition of ADP to the reconstituted system decreased NO₃⁻ reduction and this decrease could be reversed by the addition of rotenone or antimycin A. The operation of the OAA/malate shuttle was achieved most quickly in the system when low concentrations (≤0.1 millimolar) of OAA were added. A corresponding increase in the lag time for the operation of the OAA/malate shuttle was observed when the OAA concentration was increased. Concentrations for half-maximal activity of OAA, glycine, NAD⁺, and NO₃⁻ in the reconstituted system were 42 micromolar, 0.5 millimolar, 0.25 millimolar, and 26 micromolar, respectively. The transfer of reducing power from the mitochondria to the soluble phase via the OAA/malate shuttle can not only provide NADH for cytoplasmic reduction but can also sustain oxidation of tricarboxylic cycle acids and the generation of α-ketoglutarate independently of the respiratory electron transport chain.     

Inhibited light activation of fructose and sedoheptulose bisphosphatase in spinach chloroplasts exposed to osmotic stress

The light activation of fructose-1,6-bisphosphatase (EC 3.1.3.11) and sedoheptulose-1,7-bisphosphatase (EC 3.1.3.37) was inhibited in isolated intact spinach (Spinacia oleracea L.) chloroplasts exposed to reduced osmotic potentials. Decreases in the velocity and magnitude of light activation correlated with the overall reduction in CO₂ fixation rates. Responses of osmotically stressed chloroplasts to both varying pH and exogeous dihydroxyacetone phosphate (DHAP) or 3-phosphoglycerete (PGA) were examined. In the presence of DHAP, the absolute rate of CO₂ fixation was increased and this increase was most pronounced at alkaline pH. Enhanced light activation of these enzymes was also observed under these conditions. However, in the presence of PGA, similar increases in photosynthetic rate and enzyme activation were not evident. Light-dependent stromal alkalization was unaffected by the stress treatments. Inhibition of light activation under hypertonic conditions is discussed in terms of substrate availability, possible alterations of the redox state of ferredoxin and associated electron carriers, and inhibited enzyme-enzyme or enzyme-substrate interactions involved in the light activation process.Abbreviations and symbols DHAP dihydroxyacetone phosphate - PGA 3-phosphoglycerate - _s osmotic potential     

Photoassimilation of CO2 by Isolated Bundle-Sheath Strands of Zea mays I. Stimulation of CO2 Assimilation by Adding Various Intermediates of the Photosynthetic Cycle; Evidence for a Deficient Photosystem II Activity

Isolated bundle-sheath (BS) strands from leaves of mature maize plants show enhanced rates of CO₂ fixation in the presence of reduced intermediates of the photosynthetic cycle (R5P, DHAP, FruDP.) 3PGA is the major labelled product of ¹⁴CO₂ fixation whatever the substrate added. CO₂ fixation is much lower with PGA than with reduced intermediates, suggesting a limited capacity of the cells to regenerate RuDP (the CO₂-acceptor) from PGA. These two experimental facts, which are characteristic features of bundle-sheath photosynthesis for maize (a species with agranal bundle-sheath chloroplasts) indicate that phaotosystem II activity is a limiting factor for the evolution of the bundle-sheath photosynthetic process. Nevertheless, a reducing capacity arises as proved by sensitivity of CO₂ fixation to DCMU, particularly when PGA is added to the bundle-sheath. PGA synthesis occurs, in the presence of non-limiting amounts of CO₂, according to the equation: RuDP + CO₂→ 2 PGA; the oxygen effect on ¹⁴CO₂ fixation, at lower CO₂ concentration, is interpreted as a dilution effect of the internal pool of ¹⁴CO₂ by unlabelled CO₂ generated by photorespiration.     

Photoassimilation of CO2 by Isolated Bundle-Sheath Strands of Zea mays

The role of malate decarboxylation as a source of CO₂ and NADPH for the evolution of photosynthesis of isolated maize bundle-sheath strands has been investigated. The bundle-sheath cells were supplied with malate plus NADP, in the presence of intermediates of the Calvin cycle to increase the rate of CO₂ fixation. The effects of malate addition on the rate of 3 phospoglycerate synthesis, with non-saturating concentrations of bicarbonate, can be explained by an increase of the cellular pool of CO₂ in the cells due to malate decarboxylation. The CO₂ reacting with RuDP to give phosphoglycerate corresponds effectively to carbon atom 4 of malate. Malate addition produces an enhancement of the rate of CO₂ incorporation which is much more important when the reducing power is the limiting process for the evolution of the Calvin cycle (with phosphoglycerate as added substrate and/or in the presence of DCMU. These results demonstrate the utilization of NADPH produced by malate decarboxylation for the regeneration of RuDP. NADPH can also reverse the reaction of malate decarboxylation and gives rise to a synthesis of malate by carboxylation of pyruvate. In contrast, the pattern of ¹⁴C distribution among compounds is not strongly modified by malate addition. This result suggests that PGA reduction in the whole leaf must occur also in mesophyll cells to allow correctregeneration of the reduced compounds of the photosynthetic cycle.     

Variation with age in the photosynthetic carbon fixation pattern by leaves of Amaranthus paniculatus and Oryza sativa: Change in the primary carboxylation but no shift from C4 or C3 pathway

Abstract The pattern of photosynthetic carbon fixation by leaves of Amaranthus paniculatus L. (a C₄ plant) and Oryza sativa L. (a C₃ plant) varied with age. Younger leaves of A. paniculatus incorporated ¹⁴CO₂ into malate and aspartate while senescent leaves fixed predominantly into phosphoglycerate (PGA) and sugar phosphates. Only developing leaves of O. sativa formed malate/aspartate whereas mature and senescent leaves produced PGA/sugar phosphates as the initial labelled products. Correspondingly the ratio of phosphoenolpyruvate/ribulose bisphosphate (RuBP) carboxylase activities was higher in younger leaves of A. paniculatus and developing leaves of O. sativa than in older leaves. However, pulse chase experiments revealed that the main donors of carbon to end products, irrespective of leaf stage, were C₄ acids and PGA in A. paniculatus and O. sativa respectively. The results suggest that although an apparent change from initial β-carboxylation to RuBP carboxylation occurs during leaf ontogeny in both the plants, the overall leaf photosynthesis remains C₄ or C₃. The high rate of ¹⁴CO₂ incorporation into PGA/sugar phosphates by senescent leaves of A. paniculatus is suggested to be partly due to the increased intercellular spaces in their mesophyll, allowing greater access of CO₂ directly to RuBP carboxylase in the bundle sheath.     

Compartmentation and export of 14CO2 fixation products in mesophyll protoplasts from the C4-plant Digitaria sanguinalis

Isolated mesophyll protoplasts, and protoplast extracts containing intact chloroplasts, from the C₄ species Digitaria sanguinalis have been used to study Compartmentation and export of C₄ acids, using different C₃ precursors as substrate for ¹⁴CO₂ fixation. Mg²⁺ was necessary for maximum ¹⁴CO₂ fixation rates with both protoplasts and protoplast extracts, whereas Mg²⁺ was inhibitory for oxaloacetate and phosphoglycerate reduction. This inhibition could be overcome by preincubating the materials in the light with excess of EDTA before addition of Mg²⁺. Under these conditions pyruvate as substrate for ¹⁴CO₂ fixation induced mainly malate formation, whereas phosphoglycerate as substrate induced oxaloacetate formation, indicating competition for available NADPH between oxaloacetate and phosphoglycerate reduction. Oxaloacetate could be exported from the protoplasts at rates comparable to the rates of ¹⁴CO₂ fixation in intact leaves (200 μmol/mg Chl × h). This product probably passed the plasma membrane by simple diffusion, whereas the export of malate and aspartate seemed to be regulated, with the size of the intraprotoplast pool being relatively independent of the export rate. It is concluded that transport via the plasma membrane-cell wall path may play a role in metabolite flow during photosynthesis in C₄ plants.     

Photosynthetic Carbon Fixation in Guard Cell Protoplasts of Vicia faba L. : Evidence from Radiolabel Experiments

Photosynthetic carbon fixation in guard cells was reexamined in experiments with highly purified guard cell protoplasts from Vicia faba L. irradiated with red light. The fate of ¹⁴CO₂ (4.8 microcuries of NaHCO₃; final concentration: 100 micromolar) supplied to these preparations was investigated with two-dimensional paper, and thin layer chromatography. Rates of CO₂ fixation were 5- to 8-fold higher in the light than in darkness. Separation of acid-stable products into water-insoluble, neutral, and anionic fractions showed that more radioactivity was incorporated into the neutral fraction in the light than in the dark. In the dark, malate and aspartate comprised 90% of the radiolabel found in the anionic fraction, whereas in the light, radioactivity was also found in 3-phosphoglyceric acid (PGA), sugar monophosphates, sugar diphosphates, and triose phosphates. Phosphorylated compounds contained up to 60% of the label in the light-treated anionic fraction. Phosphatase treatment and rechromatography of labeled sugar diphosphate showed the presence of ribulose, a specific metabolite of the photosynthetic carbon reduction pathway (PCRP). In time-course experiments, labeled PGA was detected within 5 seconds. With time, the percentage of label in PGA decreased and that in sugar monophosphate increased. We conclude that PGA is a primary carboxylation product of the PCRP in guard cells and that the activity of the PCRP, and phosphoenolpyruvate-carboxylase is metabolically regulated.     

CO2 and malate metabolism in starch-containing and starch-lacking guard-cell protoplasts

Isolated, purified mesophyll and guard-cell protoplasts of Vicia faba L. and Allium cepa L. were exposed to ¹⁴CO₂ in the light and in the dark. The guard-cell protoplasts of Vicia and Allium did not show any labeling in phosphorylated products of the Calvin cycle, thus appearing to lack the ability to reduce CO₂ photosynthetically. In Vicia, high amounts of radioactivity (35%) appeared in starch after 60-s pulses of ¹⁴CO₂ both in the light and in the dark. Presumably, the ¹⁴CO₂ is fixed into the malate via PEP carboxylase and then metabolized into starch as the final product of gluconeogenesis. This is supported by the fact that guard-cell protoplasts exposed to malic acid uniformly labeled with ¹⁴CO₂ showed high amounts of labeled starch after the incubation, whereas cells labeled with [4-¹⁴C]malate had minimal amounts of labeled starch (1/120).In contrast, the starch-deficient Allium, guard-cell protoplasts did not show any significant ¹⁴CO₂ fixation. However, adding PEP to an homogenate stimulated ¹⁴CO₂ uptake, thus supporting the interpretation that the presence of starch as a source of PEP is necessary for incorporating CO₂ and delivering malate. With starch-containing Vicia guard-cell protoplasts, the correlation between changes in volume and the interconversion of malate and starch was demonstrated. It was shown that the rapid gluconeogenic conversion of malate into starch prevents an increase of the volume of the protoplasts, whereas the degradation of starch to malate is accompanied by a swelling of the protoplasts.Abbreviations GCPs guard-cell protoplasts - MCPs mesophyll cell protoplasts - PEP phosphoenolpyruvate - DTT dithiothreitol - 3-PGA 3-phosphoglyceric acid - RiBP ribulose 1,5 bisphosphate - MDH malate dehydrogenase - MES 2-(N-morpholino)ethane sulfonic acid - CAM crassulacean acid metabolism     

Effect of Fusicoccin on Dark CO(2) Fixation by Vicia faba Guard Cell Protoplasts

When Vicia faba guard cell protoplasts were treated with fusicoccin, dark ¹⁴CO₂ fixation rates increased by as much as 8-fold. Rate increase was saturated with less than 1 micromolar fusicoccin. Even after 6 minutes of dark ¹⁴CO₂ fixation, more than 95% of the incorporated radioactivity was in stable products derived from carboxylation of phosphoenolpyruvate (about 50% and 30% in malate and aspartate, respectively). The relative distribution of ¹⁴C among products and in the C-4 position of malate (initially more than 90% of [¹⁴C]malate) was independent of fusicoccin concentration. After incubation in the dark, malate content was higher in protoplasts treated with fusicoccin. A positive correlation was observed between the amounts of ¹⁴CO₂ fixed and malate content.

It was concluded that (a) fusicoccin causes an increase in the rate of dark ¹⁴CO₂ fixation without alteration of the relative fluxes through pathways by which it is metabolized, (b) fusicoccin causes an increase in malate synthesis, and (c) dark ¹⁴CO₂ fixation and malate synthesis are mediated by phosphoenolpyruvate carboxylase.

     

Light dependent reduction of nitrate by pea chloroplasts in the presence of nitrate reductase and C4-dicarboxylic acids

In the presence of purified nitrate reductase (NR) and 1 mM NADH, illuminated pea chloroplasts catalysed reduction of NO₃^? to NH₃ with the concomitant evolution of O₂. The rates were slightly less than those for reduction of NO₂^? to NH₃ and O₂, evolution by chloroplasts in the absence of NR and NADH (ca 6 μg atoms N/mg Chl/hr). Illuminated chloroplasts quantitatively reduced 0.2 mM oxaloacetate (OAA) to malate. In the presence of an extrachloroplast malate-oxidizing system comprised of NAD-specific malate dehydrogenase (NAD-MDH), NAD, NR and NO₃^?, illuminated chloroplasts supported OAA-dependent reduction of NO₃^? to NH₃ with the evolution of O₂. The reaction did not proceed in the absence of any of these supplements or in the dark but malate could replace OAA. The results are consistent with the reduction of NO₃^?by reducing equivalents from H₂O involving a malate/OAA shuttle. The ratios for O₂, evolved: C₄-acid supplied and N reduced: C₄-acid supplied in certain experiments imply recycling of the C₄-acids.     

Effect of carbon dioxide on nitrate accumulation and nitrate reductase induction in corn seedlings

Exposure of the leaf canopy of corn seedlings (Zea mays L.) to atmospheric CO₂ levels ranging from 100 to 800 μl/l decreased nitrate accumulation and nitrate reductase activity. Plants pretreated with CO₂ in the dark and maintained in an atmosphere containing 100 μl/l CO₂ accumulated 7-fold more nitrate and had 2-fold more nitrate reductase activity than plants exposed to 600 μl/l CO₂, after 5 hours of illumination. Induction of nitrate reductase activity in leaves of intact corn seedlings was related to nitrate content. Changes in soluble protein were related to in vitro nitrate reductase activity suggesting that in vitro nitrate reductase activity was a measure of in situ nitrate reduction. In longer experiments, levels of nitrate reductase and accumulation of reduced N supported the concept that less nitrate was being absorbed, translocated, and assimilated when CO₂ was high. Plants exposed to increasing CO₂ levels for 3 to 4 hours in the light had increased concentrations of malate and decreased concentrations of nitrate in the leaf tissue. Malate and nitrate concentrations in the leaf tissue of seven of eight corn genotypes grown under comparable and normal (300 μl/l CO₂) environments, were negatively correlated. Exposure of roots to increasing concentrations of potassium carbonate with or without potassium sulfate caused a progressive increase in malate concentrations in the roots. When these roots were subsequently transferred to a nitrate medium, the accumulation of nitrate was inversely related to the initial malate concentrations. These data suggest that the concentration of malate in the tissue seem to be related to the accumulation of nitrate.     

Response of photosynthetic carbon assimilation in mesophyll protoplasts to restriction on mitochondrial oxidative metabolism: Metabolites related to the redox status and sucrose biosynthesis

The patterns of cellular metabolites related to redox status and sucrose biosynthesis in mesophyll protoplasts of pea (Pisum sativum L.) were examined in the absence or presence of oligomycin (inhibitor of oxidative phosphorylation) or antimycin A (inhibitor of cytochrome pathway) or salicylhydroxamic acid (SHAM) (inhibitor of alternative pathway). The increase on illumination in the rate of photosynthesis or cellular metabolites was more at optimal CO₂ (1.0 mM NaHCO₃) compared to that at limiting CO₂ (0.1 mM NaHCO₃). Furthermore, the inhibition of photosynthesis in presence of mitochondrial inhibitors was more pronounced at optimal CO₂ than that at limiting CO₂. There was a marked increase in steady-state levels of triose-P/PGA (phosphoglyceric acid) and glucose-6-phosphate (Glc-6-P) in the presence of oligomycin and antimycin A. In contrast, SHAM caused a marked increase in malate/OAA (oxaloacetate). We suggest that dissipation of excess redox equivalents generated in photosynthesis occurs through both cytochrome and alternative pathways, while sucrose biosynthesis is backed up by cytochrome pathway alone. Thus, mitochondrial respiration (through both cytochrome and alternative pathways of mitochondrial electron transport) optimizes chloroplast photosynthesis by modulating cellular metabolites related to both intracellular redox state and sucrose biosynthesis.     

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     

Low CO(2) Prevents Nitrate Reduction in Leaves

The correlation between CO₂ assimilation and nitrate reduction in detached spinach (Spinacia oleracea L.) leaves was examined by measuring light-dependent changes in leaf nitrate levels in response to mild water stress and to artificially imposed CO₂ deficiency. The level of extractable nitrate reductase (NR) activity was also measured. The results are: (a) In the light, detached turgid spinach leaves reduced nitrate stored in the vacuoles of mesophyll cells at rates between 3 and 10 micromoles per milligram of chlorophyll per hour. Nitrate fed through the petiole was reduced at similar rates as storage nitrate. Nitrate reduction was accompanied by malate accumulation. (b) Under mild water stress which caused stomatal closure, nitrate reduction was prevented. The inhibition of nitrate reduction observed in water stressed leaves was reversed by external CO₂ concentrations (10-15%) high enough to overcome stomatal resistance. (c) Nitrate reduction was also inhibited when turgid leaves were kept in CO₂-free air or at the CO₂-compensation point or in nitrogen. (d) When leaves were illuminated in CO₂-free air, activity of NR decreased rapidly. It increased again, when CO₂ was added back to the system. The half-time for a 50% change in activity was about 30 min. It thus appears that there is a rapid inactivation/activation mechanism of NR in leaves which couples nitrate reductase to net photosynthesis.     

Carbon dioxide assimilation in some aerial plant organs and tissues

Summary CO₂ fixation characteristics of a number of mature (but not senescing) tissues and organs (the outer layers of green pod and the seed testa of Vicia faba L.; the outer layers of green pod and seeds of Trigonella foenum-graecum L.; the outer layers of the green fruit of Lycopersicon esculentum Mill.) were studied and compared with their respective C₃ leaf characteristics. On a chlorophyll basis phosphoenolpyruvate carboxylase, malic enzyme (NADP) and malate dehydrogenase (NAD and NADP) acitivites were much higher in the non-leaf tissues (except for V. faba seed testa) than the leaf tissues. Generally, on a protein basis the differences were less significant. All tissues possessed ribulose-1.5-diphosphate carboxylase activity though there was great variation in activities both on a protein and chlorophyll basis. Protein: chlorophyll ratios varied greatly from tissue to tissue being lowest in the leaf tissue (11.5–14.0) and highest in V. faba seed testa (805.5). Chlorophyll a:b ratios were all between 2 and 3. ¹⁴CO₂ uptake in the dark by L. esculentum fruit slices was about 1/3 that in the light and the major, initially labelled product was malate both in the light and dark. Neither typical C₄-photosynthesis or crassulacean acid metabolism were exhibited by the non-leaf tissues and it was considered that the increased levels of certain enzyme activities were present to refix and recycle respired CO₂.Abbreviations PEP phosphoenolpyruvate - RuDP ribulose -1,5-, diphosphate - MDH malate dehydrogenase - CAM Crassulacean acid metabolism - OAA oxaloacetic acid     

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     

Characterization of the Formation and Distribution of Photosynthetic Products by Sedum praealtum Chloroplasts

Photoassimilation of ¹⁴CO₂ by intact chloroplasts from the Crassulacean acid metabolism plant Sedum praealtum was investigated. The main water-soluble, photosynthetic products were dihydroxyacetone phosphate (DHAP), glycerate 3-phosphate (PGA), and a neutral saccharide fraction. Only a minor amount of glycolate was produced. A portion of neutral saccharide synthesis was shown to result from extrachloroplastic contamination, and the nature of this contamination was investigated with light and electron microscopy. The amount of photoassimilated carbon partitioned into starch increased at both very low and high concentrations of orthophosphate. High concentrations of exogenous PGA also stimulated starch synthesis.

DHAP and PGA were the preferred forms of carbon exported to the medium, although indirect evidence suported hexose monophosphate export. The export of PGA and DHAP to the medium was stimulated by high exogenous orthophosphate, but depletion of chloroplastic reductive pentose phosphate intermediates did not occur. As a result only a relatively small inhibition in the rate of CO₂ assimilation occurred.

The rate of photoassimilation was stimulated by exogenous PGA, ribose 5-phosphate, fructose 1,6-bisphosphate, fructose 6-phosphate, and glucose 6-phosphate. Inhibition occurred with phosphoenolpyruvate and high concentrations of PGA and ribose 5-phosphate. PGA inhibition did not result from depletion of chloroplastic orthophosphate or from inhibition of ribulose 1,5-bisphosphate carboxylase. Exogenous PGA and phosphoenolpyruvate were shown to interact with the orthophosphate translocator.