Stimulation of ammonia and 2-oxoglutarate-dependent o(2) evolution in isolated chloroplasts by dicarboxylates and the role of the chloroplast in photorespiratory nitrogen recycling

Intact chloroplasts isolated from spinach (Spinacia oleracea L.) leaves showed a light-dependent O(2) evolution (5.5 +/- 0.75 micromoles per milligram chlorophyll per hour) when supplied with ammonia and 2-oxoglutarate. This (ammonia, 2-oxoglutarate)-dependent O(2) evolution was stimulated 2- to 4-fold by the dicarboxylates, malate, succinate, fumarate, glutarate, and l-tartarate. Evolution of O(2) in the presence of malate was dependent on the presence of both 2-oxoglutarate and NH(4)Cl; malate with only either 2-oxoglutarate and NH(4)Cl alone did not support O(2) evolution. Furthermore, in the presence of malate, the amount of O(2) evolved was solely dependent on the amount of NH(4)Cl or 2-oxoglutarate added and malate did not affect the ratio of O(2) evolved to NH(4)Cl or 2-oxoglutarate consumed. Studies with inhibitors (2-(3,4-dichlorophenyl)-1,1-dimethyl urea, methionine sulfoximine, and azaserine) indicated that the above activity was directly linked to glutamine synthetase and glutamate synthase activity in the chloroplast and was not caused by the metabolism of malate. The V(max)/2 of (ammonia, 2-oxoglutarate)-dependent O(2) evolution was reached at 32 micromolar NH(4)Cl and 6 millimolar (approximately) 2-oxoglutarate in the absence of malate, and at 22 micromolar NH(4)Cl and 73 micromolar 2-oxoglutarate when malate (3 millimolar) was present.Intact chloroplasts isolated from pea (Pisum sativum) leaves also showed a stimulation of (ammonia, 2-oxoglutarate)-dependent O(2) evolution by malate. However glutamine was required for this activity even though glutamine with only either NH(4)Cl or 2-oxoglutarate did not respond to malate stimulation.The measured rates of (ammonia, 2-oxoglutarate)-dependent O(2) evolution in isolated spinach chloroplasts in the presence of malate were about 19.5 +/- 4.5 micromoles O(2) evolved per milligram chlorophyll per hour. This is adequate to sustain photorespiratory NH(3) recycling and the refixation of NH(3) arising from NO(3) under ambient conditions in the light. The role of the chloroplast in photorespiratory NH(3) recycling and the nature of the associated transport of 2-oxoglutarate into the chloroplast is discussed.     

Polarographic Study of Dicarboxylic-Acid-dependent Export of Reducing Equivalents from Illuminated Chloroplasts

Isolated pea chloroplasts, prepared by differential centrifugation, catalyzed O₂ evolution in the light in the presence of 0.03 to 3 millimolar malate, 0.12 to 1.2 millimolar NAD, 4 millimolar pyruvate and exogenous NAD-malate dehydrogenase and lactate dehydrogenase. The reaction, which did not occur in the absence of any one of these factors, was accompanied by the consumption of pyruvate; the ratio of O₂ evolved to pyruvate consumed was <0.5. When 0.1 millimolar [¹⁴C]malate was supplied most of the ¹⁴C label was recovered as malate. At low concentrations of malate (<0.1 millimolar), the ratio of O₂ evolved to malate supplied was greater than 0.5.     

Photosynthesis in phosphoenolpyruvate carboxykinase-type C4 plants: photosynthetic activities of isolated bundle sheath cells from Urochloa panicoides

A method has been developed for rapidly preparing bundle sheath cell strands from Urochloa panicoides, a phosphoenolpyruvate (PEP) carboxykinase-type C4 plant. These cells catalyzed both HCO3(-)- and oxaloacetate-dependent oxygen evolution; oxaloacetate-dependent oxygen evolution was stimulated by ATP. For this activity oxaloacetate could be replaced by aspartate plus 2-oxoglutarate. Both oxaloacetate- and aspartate plus 2-oxoglutarate-dependent oxygen evolution were accompanied by PEP production and both were inhibited by 3-mercaptopicolinic acid, an inhibitor of PEP carboxykinase. The ATP requirement for oxaloacetate- and aspartate plus 2-oxoglutarate-dependent oxygen evolution could be replaced by ADP plus malate. The increased oxygen evolution observed when malate plus ADP was added with oxaloacetate was accompanied by pyruvate production. These results are consistent with oxaloacetate being decarboxylated via PEP carboxykinase. We suggest that the ATP required for oxaloacetate decarboxylation via PEP carboxykinase may be derived by phosphorylation coupled to malate oxidation in mitochondria. These bundle sheath cells apparently contain diffusion paths for the rapid transfer of compounds as large as adenine nucleotides.     

Control of electron flow in intact chloroplasts by the intrathylakoid pH,not by the phosphorylation potential

In the presence of nitrite or oxaloacetate, intact chloroplasts evolved oxygen at a significant rate for the initial 1 to 2 min of illumination. Subsequently, oxygen evolution was suppressed progressively. The suppressed oxygen evolution was stimulated strikingly by NH₄Cl. The results indicate that coupled electron flow in intact chloroplasts is controlled in the light, and the control is released by NH₄Cl. However, at low concentrations, NH₄Cl was not an effective uncoupler of photophosphorylation in intact chloroplasts. Intrachloroplast ATP levels and ATP/ADP ratios were not significantly influenced by NH₄Cl. In contrast, the quenching of 9-aminoacridine fluorescence, which can be used to indicate the intrathylakoid pH in intact chloroplasts, was reduced drastically even by low concentrations of NH₄Cl. This suggests that the chloroplast phosphorylation potential is not in equilibrium with the proton gradient. In coupled chloroplasts, the intrathylakoid pH was lower in the light with nitrite than with oxaloacetate as electron acceptor. Electron flow was also more effectively controlled in chloroplasts illuminated with nitrite than with oxaloacetate. It is concluded that the intrathylakoid pH, not the phosphorylation potential, is a factor in the control of the rate of electron flow in intact chloroplasts.Abbreviations CCCP carbonylcyanide-m-chlorophenylhydrazone - OAA oxalo-acetate - MES 2-(N-morpholino)-ethanesulfonic acid - HEPES N-2-hyroxyethylpiperazine-N-2-ethanesulfonic acid Postal address     

Influence of antimycin a and uncouplers on anaerobic photosynthesis in isolated chloroplasts

Anaerobiosis depresses the light- and bicarbonate-saturated rates of O(2) evolution in intact spinach (Spinacia oleracea) chloroplasts by as much as 3-fold from those observed under aerobic conditions. These lower rates are accelerated 2-fold or more by the addition of 1 mum antimycin A or by low concentrations of the uncouplers 0.3 mm NH(4)Cl or 0.25 mum carbonyl cyanide m-chlorophenylhydrazone. Oxaloacetate and glycerate 3-phosphate reduction rates are also increased by antimycin A or an uncoupler under anaerobic conditions. At intermediate light intensities, the rate accelerations by either antimycin A or uncoupler are inversely proportional to the adenosine 5'-triphosphate demand of the reduction process for the acceptors HCO(3) (-), glycerate 3-phosphate, and oxaloacetate. The acceleration of bicarbonate-supported O(2) evolution may also be produced by adding an adenosine 5'-triphosphate sink (ribose 5-phosphate) to anaerobic chloroplasts. The above results suggest that a proton gradient back pressure resulting from antimycin A-sensitive cyclic electron flow is responsible for the depression of light-saturated photosynthesis under anaerobiosis.     

A polarographic study of glutamate synthase activity in isolated chloroplasts

Illuminated pea (Pisum sativum) chloroplasts actively catalyzed (glutamine plus alpha-ketoglutarate)-dependent O(2) evolution (average of 12 preparations 10.6 mumole mg chlorophyll per hour). The reaction was specific for glutamine and alpha-ketoglutarate; concentrations of 0.2 mm alpha-ketoglutarate and 0.6 mm glutamine, respectively, effected half-maximum rates of O(2) evolution. The reaction was inhibited by 3-(3,4-dichlorophenyl)-1-1-dimethylurea and did not occur in the dark. After osmotic shock chloroplasts did not catalyze O(2) evolution. The reaction was inhibited by azaserine and glutamate but not by 10 mm ammonia, 2.5 mm methionine sulfoximine, or 5 mm amino-oxyacetate; addition of amino-oxyacetate together with aspartate inhibited O(2) evolution. Arsenate (3 mm) enhanced O(2) evolution. The highest molar ratio for O(2) evolved per mole of alpha-ketoglutarate supplied was 0.40; the corresponding values for glutamine in the absence and presence of 3 mm arsenate were 0.20 and 0.24, respectively. The (glutamine plus alpha-ketoglutarate)-dependent O(2) evolution is attributed to photosynthetically coupled glutamate synthase activity and the activity is sufficient to account for the assimilation of inorganic nitrogen. The low molar ratio for glutamine is discussed.Chloroplasts also catalyzed (aspartate plus alpha-ketoglutarate)-dependent O(2) evolution but this reaction was inhibited by 5 mm amino-oxyacetate and it was insensitive to azaserine and methionine sulfoximine. This reaction was attributed to transaminase and photosynthetically coupled malate dehydrogenase activities.     

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

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

Biosynthesis of Sulfoquinovosyldiacylglycerol in Higher Plants: The Incorporation of SO(4) by Intact Chloroplasts in Darkness

Intact spinach chloroplasts incorporated ³⁵SO₄²⁻ into sulfoquinovosyldiacylglycerol in the dark at rates equivalent to those previously reported for illuminated chloroplasts provided that either ATP itself or an ATP-generating system was added. No additional reductant was necessary for SQDG synthesis by chloroplasts. The optimal concentration of ATP was between 2 and 3 millimolar. Rates of synthesis up to 2.6 nanomoles per milligram chlorophyll per hour were observed. UTP, GTP, and CTP could not substitute for ATP. Incubation of UTP with ATP (1:1) stimulated synthesis of sulfoquinovosyldiacylglycerol. No additional stimulation of the reaction was observed upon addition of other nucleoside triphosphates with ATP. For the generation of ATP in the chloroplast, addition of dihydroxyacetone phosphate alone did not promote synthesis of sulfoquinovosyldiacylglycerol, but in combination with inorganic phosphate and oxaloacetate, rates of synthesis up to 3.2 nanomoles per milligram chlorophyll per hour were observed. Dark synthesis was optimal in the presence of 2 millimolar dihydroxyacetone phosphate, 2 millimolar oxaloacetate, and 1 millimolar KH₂PO₄.     

Regulation of hepatic gluconeogenesis in the guinea pig by fatty acids and ammonia.

Octanoate and L-palmitylcarnitine inhibited the synthesis of P-enolpyruvate from alpha-ketoglutarate and malate by isolated guinea pig liver mitochondria. A 50% reduction in P-enolpyruvate formation was obtained with 0.1 to 0.2 mM octanoate or with 0.06 to 0.10 mM L-palmitylcarnitine. At these concentrations, oxidative phosphorylation remained intact and only much higher concentrations of fatty acids altered this process. The addition of NH4Cl in the presence of malate and increasing concentrations of alpha-ketoglutarate (or vice versa) enhanced the formation of glutamate, aspartate, and P-enolpyruvate. The addition of increasing concentrations of NH4Cl in the presence of fixed amounts of malate and alpha-ketoglutarate had a similar effect. Furthermore, the inhibition of P-enolpyruvate synthesis by fatty acids and the reduction of the acetoacetate to beta-hydroxybutyrate ratio were reversed by the addition of NH4Cl. Cycloheximide, which blocks energy transfer at site 1 of the respiratory chain, decreased P-enolpyruvate formation. When cycloheximide and either octanoate or L-palmitylcarnitine were added together, there was an even greater reduction in P-enolpyruvate synthesis from either malate or alpha-ketoglutarate than was noted with either fatty acid alone. Since cycloheximide lowers the rate of ATP synthesis this may in turn reduce P-enolpyruvate formation by a mechanism independent of changes in the mitochondrial NAD+/NADH ratio caused by fatty acids. In the isolated perfused liver metabolizing lactate, the inhibitory effect of octanoate on gluconeogenesis was partially relieved by the addition of 1 mM NH4Cl, but remained unchanged in the presence of 2 mM NH4Cl, despite a highly oxidized NAD+/NADH ratio in the mitochondria. In contrast to glucose synthesis, urea formation was markedly increased during the infusion of 1 mM as well as 2 mM NH4Cl. After cessation of NH4Cl infusion, there was an increase in glucose production, to a rate as high as that observed in the absence of octanoate. This increase was accompanied by the disappearance of alanine, aspartate, and glutamate which had been stored in the liver during NH4Cl infusion. Urea synthesis also decreased progressively. These results indicate that gluconeogenesis in guinea pig liver is regulated, in part, by alterations in the mitochondrial oxidation-reduction state. However, the modulation of this effect by changing the concentrations of intermediates of the aspartate aminotransferase reaction indicates competition for oxalacetate between the aminotransferase reaction and P-enolpyruvate carboxykinase.     

Inhibition and uncoupling of photophosphorylation in isolated chloroplasts by organotin, organomercury and diphenyleneiodonium compounds

1. Trialkyltin, triphenyltin and diphenyleneiodonium compounds inhibited ADP-stimulated O(2) evolution by isolated pea chloroplasts in the presence of phosphate or arsenate. Tributyltin and triphenyltin were the most effective inhibitors, which suggests a highly hydrophobic site of action. Phenylmercuric acetate was a poor inhibitor of photophosphorylation, which suggests that thiol groups are not involved. 2. Triethyltin was a potent uncoupler of photophosphorylation by isolated chloroplasts in media containing Cl(-), but had little uncoupling activity when Cl(-) was replaced by NO(3) (-) or SO(4) (2-), which are inactive in the anion-hydroxide exchange. It is suggested that uncoupling by triethyltin is a result of the Cl(-)-OH(-) exchange together with a natural uniport of Cl(-). Tributyltin, triphenyltin and phenylmercuric acetate had low uncoupling activity, probably because in these compounds the uncoupling activity is partially masked by inhibitory effects. 3. At high concentrations the organotin compounds caused inhibition of electron transport uncoupled by carbonyl cyanide m-chlorophenylhydrazone or NH(4)Cl. At these high concentrations the organotin compounds may be producing a detergent-like disorganization of the membrane structure. In contrast, diphenyleneiodonium sulphate inhibited uncoupled electron transport at low concentrations; however, this inhibition is less than the inhibition of photophosphorylation, which suggests that the compound also inhibits the phosphorylation reactions as well as electron transport. 4. The effects of these compounds on basal electron transport were complex and depended on the pH of the reaction media. However, they can be explained on the basis of three actions: inhibition of the phosphorylation reactions, uncoupling and direct inhibition of electron transport. 5. The inhibition of cyclic photophosphorylation in the presence of phenazine methosulphate by diphenyleneiodonium sulphate shows that it inhibits in the region of photosystem 1.     

Regulation of Photosynthetic Electron Transport in Intact Spinach Chloroplasts: II. MECHANISM OF SALT-INDUCED INCREASE IN OXALOACETATE PHOTOREDUCTION

The main focus of this study was to determine the mechanism by which certain exogenous monovalent salts stimulate rates of net O₂ evolution linked to oxaloacetate reduction in intact spinach chloroplasts. The influence of salts on the dicarboxylate translocator involved in the transport of oxaloacetate and on the activity and activation of the chloroplast enzyme NADP-malate dehydrogenase, which mediates electron transport to oxaloacetate, was examined. High concentrations of KCl (155 millimolar) increased the apparent K_m for oxaloacetate but did not significantly alter the maximal velocity of uptake. Likewise, external salts (KCl, MgCl₂, or KH₂PO₄) had minimal effects on the magnitude of light activation of NADP-malate dehydrogenase. In contrast, measurements of chloroplast NADP-malate dehydrogenase activity (after release by osmotic shock) showed a marked dependence on salt concentration. Rates were stimulated approximately 2-fold by both monovalent (optimally 75 millimolar) and divalent (optimally 20 millimolar) salts. It was inferred that the salt-induced increase in net rates of O₂ evolution linked to oxaloacetate reduction is due, at least in part, to stimulation of NADP-malate dehydrogenase caused by monovalent cation permeability of the chloroplast inner envelope membrane.     

A Two-Translocator Model for the Transport of 2-Oxoglutarate and Glutamate in Chloroplasts during Ammonia Assimilation in the Light

This study examines the transport of 2-oxoglutarate (2-OG) and other dicarboxylates during ammonia assimilation in illuminated spinach chloroplasts. The transport of all dicarboxylates examined was strongly inhibited by NH₄Cl preincubation in the light. Treatment with NH₄Cl caused a rapid depletion of the endogenous glutamate pool and a corresponding increase in endogenous glutamine content. The inhibition of transport activity by NH₄Cl was apparently linked to its metabolism in the light because inhibition of glutamine synthetase activity by the addition of l-methionine sulfoximine or carbonylcyanide-m-chlorophenylhydrazone abolished this affect. Measurements of endogenous metabolite pools showed that malate was most rapidly exchanged during the uptake of all exogenous dicarboxylates examined. Depending on the exogenous substrates used, the apparent half-times of efflux measured for endogenous malate, aspartate and glutamate were 10, 10 to 30, and 15 to 240 seconds, respectively. The transport of 2-OG was also inhibited by malate. But chloroplasts preincubated with malate in the presence or absence of NH₄Cl were found to have high transport activity similar to untreated chloroplasts. A two-translocator model is proposed to explain the stimulation of 2-OG transport as well as the stimulation of (NH₃, 2-OG)-dependent O₂ evolution by malate (KC Woo, CB Osmond 1982 Plant Physiol 69: 591-596) in isolated chloroplasts. In this model the transport of 2-OG on the 2-OG translocator and glutamate on the dicarboxylate translocator is coupled to malate counter-exchange in a cascade-like manner. This results in a net 2-OG/glutamate exchange with no net malate transport. Thus, during NH₃ assimilation the transport of 2-OG into and the export of glutamate out of the chloroplast occurs via the 2-OG and the dicarboxylate translocators, respectively.     

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.     

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     

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.     

Availability of monovalent and divalent cations within intact chloroplasts for the action of ionophores nigericin and A23187

1. A23187 will uncouple electron transport by broken chloroplasts in a divalent cation dependent manner provided that they have been treated with a low concentration of EDTA.2. A23187 stimulates oxaloacetate-dependent oxygen evolution and inhibits phosphoglycerate reduction by intact chloroplasts isolated in a cation-free medium whereas the full effect of nigericin was dependent on the presence of external K⁺.3. Uncoupling of oxaloacetate reduction by A23187 in intact chloroplasts is inhibited by EDTA and this effect is overcome by excess Mg²⁺.4. The results suggest that divalent and not monovalent cations are available for collapsing the light-induced H⁺ gradient within the intact organelle.     

光系统Ⅱ颗粒的毫秒延迟荧光的研究

本文主要报导了具有放氧活性的光系统Ⅱ(PSⅡ)颗粒的毫秒延迟荧光(ms-DF)的特性以及NH_4Cl对它的调节作用.     

Inhibition of 3-Phosphoglycerate-Dependent O(2) Evolution by Phosphoenolpyruvate in C(4) Mesophyll Chloroplasts of Digitaria sanguinalis (L.) Scop

The effects of phosphoenolpyruvate (PEP), inorganic phosphate (Pi), and ATP on 3-phosphoglycerate (PGA)-dependent O₂ evolution by chloroplasts of Digitaria sanguinalis (L.) Scop. (crabgrass) were evaluated relative to possible mechanisms of PEP transport by the C₄ mesophyll chloroplast. Crude and Percoll purified chloroplast preparations exhibited rates of PGA-dependent O₂ evolution in the range of 90 to 135 micromoles O₂ per milligram chlorophyll per hour, and up to 180 micromoles O₂ per milligram chlorophyll per hour at optimal Pi concentrations (approximately 0.2 millimolar at 9 millimolar PGA). Higher concentrations of Pi were inhibitory. PEP inhibited O₂ evolution (up to 70%) in both chloroplast preparations when the PEP to PGA ratio was high (i.e. 9 millimolar PEP to 0.36 millimolar PGA). Usually no inhibition was seen when the PEP to PGA ratio was less than 2. PEP acted as a competitive inhibitor and, at a concentration of 9 millimolar, increased the apparent K_m (PGA) from 0.15 to 0.53 millimolar in Percoll purified chloroplasts. A low concentration of PGA and high ratio of PEP to PGA, which are considered unphysiological, were required to detect any inhibition of O₂ evolution by PEP. Similar results were obtained from crude versus Percoll purified preparations. Neither the addition of Pi nor ATP could overcome PEP inhibition. As PEP inhibition was competitive with respect to PGA concentration, and as addition of ATP or Pi could not prevent PEP inhibition of PGA-dependent O₂ evolution, the inhibition was not due to PEP exchange of adenylates or Pi out of the chloroplast. Analysis of the effect of Pi and PEP, separately and in combination, on PGA-dependent O₂ evolution suggests interactions between PEP, Pi, and PGA on the same translocator in the C₄ mesophyll chloroplast. C₃ spinach chloroplasts were also found to be sensitive to PEP, but to a lesser extent than crabgrass chloroplasts. The apparent K_i values (PEP) were 3 and 21 millimolar for crabgrass and spinach, respectively.     

Influence of Applied NaCl on Crassulacean Acid Metabolism and Ionic Levels in a Cactus, Cereus validus

To determine possible physiological responses to salinity, seedlings of Cereus validus Haworth, a cactus from Salinas Grandes, Argentina, were treated with up to 600 millimolar NaCl for up to 16 days when they were about 9 months old and 100 millimeters tall. Salt stress decreased stem biomass, e.g. it was 19.7 grams for controls and 11.4 grams for plants treated with 400 millimolar NaCl for 14 days. Nocturnal CO₂ uptake in these obligate Crassulacean acid metabolism (CAM) plants was inhibited 67% upon treatment with 400 millimolar NaCl for 14 days (controls, 181 millimoles CO₂ per square meter), while nocturnal accumulation of malate was inhibited 49% (controls, 230 millimoles malate per square meter). The larger accumulation of malate as compared to uptake of atmospheric CO₂ suggests that internal CO₂ recycling occurred during the dark period. Such recycling was lower in the controls (~20%) than in the NaCl-treated plants (~50%). The nocturnal increase in malate and titratable acidity depended on the total daily photosynthetically active radiation available; measurements suggest a quantum requirment of 26 photons per malate. As NaCl in the medium was increased to 600 millimolar in daily increments of 50 millimolar, Na and Cl concentrations in the roots increased from about 7 to 100 millimolar, but K concentration in the cell sap remained near 26 millimolar. Concomitantly, concentrations of Na and Cl in the shoots increased from 8 to 17 millimolar and from 1 to 7 millimolar, respectively, while the K concentration increased about 16 to 60 millimolar. In plants maintained for 14 days at 500 millimolar NaCl, the root levels of Na and Cl increased to 260 millimolar, the shoot levels were about 60 millimolar, and the stem bases began to become necrotic. Such Na retention in the roots together with the special possibilities of carbon reutilization given by CAM are apparently survival mechanisms for the temporarily saline conditions experienced in its natural habitat.