The role of pH in the regulation of carbon fixation in the chloroplast stroma. Studies on CO2 fixation in the light and dark

1. The pH in the stroma and in the thylakoid space has been measured in a number of chloroplast preparations in the dark and in the light at 20 °C. Illumination causes a decrease of the pH in the thylakoid space by 1.5 and an increase of the pH in the stroma by almost 1 pH unit.2. CO₂ fixation is shown to be strongly dependent on the pH in the stroma. The pH optimum was 8.1, with almost zero activity below pH 7.3. Phosphoglycerate reduction, which is a partial reaction of CO₂ fixation, shows very little pH dependency.3. Low concentrations of the uncoupler m-chlorocarbonylcyanide phenylhydrazone (CCCP) inhibit CO₂ fixation without affecting phosophoglycerate reduction. This inhibition of CO₂ fixation appears to be caused by reversal of light induced alkalisation in the stroma by CCCP.4. Methylamine has a very different effect compared to CCCP. Increasing concentrations of methylamine inhibit CO₂ fixation and phosphoglycerate reduction to the same extent. The light induced alkalisation of the stroma appears not to be significantly inhibited by methylamine, but the protons in the thylakoid space are neutralized. The inhibition of CO₂ fixation by higher concentrations of methylamine is explained by an inhibition of photophosphorylation. It appears that methylamine does not abolish proton transport.5. It is shown that intact chloroplasts are able to fix CO₂ in the dark, yielding 3-phosphoglycerate. This requires the addition of dihydroxyacetone phosphate as precursor of ribulosemonophosphate and also to supply ATP, and the addition of oxaloacetate for reoxidation of the NADPH in the stroma.6. Dark CO₂ fixation in the presence of dihydroxyacetone phosphate and oxaloacetate has the same pH dependency as CO₂ fixation in the light. This demonstrates that CO₂ fixation in the dark is not possible, unless the pH in the medium is artificially raised to pH 8.8.7. It is shown that pH changes occurring in the stroma after illumination are sufficient to switch CO₂ fixation from zero to maximal activity. This offers a mechanism for light control of CO₂ fixation, avoiding wasteful CO₂ fixation in the dark.     

Light-dependent changes of the Mg2+ concentration in the stroma in relation to the Mg2+ dependency of CO2 fixation in intact chloroplasts.

(1) Light-dependent changes of the Mg2+ content of thylakoid membranes were measured at pH 8.0 and compared with earlier measurements at pH 6.6. In a NaCl and KCl medium, the light-dependent decrease in the Mg2+ content of the thylakoid membranes at pH 8.0 is found to be 23 nmol Mg2+ per mg chlorophyll, whereas in a sorbitol medium it is 83 nmol Mg2+ per mg chlorophyll. (2) A light dependent increase in the Mg2+ content of the stroma was detected wjem chloroplasts were subjected to osmotic shock, amounting to 26 nmol/mg chlorophyll. Furthermore, a rapid and reversible light-dependent efflux of Mg2+ has been observed in intact chloroplasts when the divalent cation ionophore A 23 187 was added, indicating a light-dependent transfer of about 60 nmol of Mg2+ per mg chlorophyll from the thylakoid membranes to the stroma. (3) CO2 fixation, but not phosphoglycerate reduction, could be completely inhibited when A 23 187 was added to intact chloroplasts in the absence of external Mg2+. If Mg2+ was then added to the medium, CO2 fixation was restored. Half of the maximal restoration was achieved with about 0.2 mM Mg2+, which is calculated to reflect a Mg2+ concentration in the stroma of 1.2 mM. The further addition of Ca2+ strongly inhibits CO2 fixation. (4) The results suggest that illumination of intact chloroplasts causes an increase in the Mg2+ concentration of 1-3 mM in the stroma. Compared to the total Mg2+ content of chloroplasts, this increase is very low, but it appears to be high enough to have a possible function in the light regulation of CO2 fixation.     

The mechanism of the control of carbon fixation by the pH in the chloroplast stroma. Studies with nitrite-mediated proton transfer across the envelope.

1. CO2 fixation of intact spinach chloroplasts is inhibited by nitrite in a pH-dependent mode. At pH 7.3 in the medium 1 mM NaNO2 and at pH 7.9 5 mM NaNO2 were required for 50% inhibition. 2. The addition of nitrite leads to an acidificiation in the stroma. It appears that nitrite renders the envelope permeable for protons resulting in a breakdown of the pH gradient between the external space and the stroma. 3. In view of earlier results on the pH sensitivity of C02 fixation it is concluded that this pH shift in the stroma is responsible for the observed inhibition of CO2 fixation by nitrite. 4. Octanoate and to some extent also high concentrations of bicarbonate and acetate have a similar effect as nitrite in inhibiting CO2 fixation through an acidification in the stroma. 5. The levels of the intermediates of the CO2 fixation cycle were measured. A strong rise of the levels of fructose- and sedoheptulose biphosphates and a concomitant decrease of the corresponding monophosphates was observed during inhibition of CO2 fixation. It appears that the enzymatic steps of the CO2 fixation cycle responsible for the overall inhibition of CO2 fixation caused by lowering of the H+ concentration in the stroma are fructose- and sedopheptulose bisphosphatase. These two enzymes have an important function in the light regulation of CO2 fixation.     

On the light dependence of Fatty Acid synthesis in spinach chloroplasts

The capacity of intact chloroplasts to synthesize long chain fatty acids from acetate depends on the stroma pH in Spinacia oleracea, U. S. hybrid 424. The pH optimum is close to 8.5. Lowering of the stroma pH leads to a reduction of acetate incorporation but does not suffice to eliminate fatty acid synthesis completely. Chain elongation from palmitic to oleic acid shows the same pH dependence. Fatty acid synthesis is activated in the dark upon the simultaneous addition of dihydroxyacetone phosphate and orthophosphate supplying ATP and oxaloacetate for reoxidation of NADPH in the stroma. Under these conditions both dark fatty acid synthesis and synthesis of oleate from palmitate show the same pH dependence as in the light. Dark fatty acid synthesis is further stimulated by increasing the stromal Mg²⁺ concentration with the ionophore A 23187. In contrast to CO₂ fixation, dark fatty acid synthesis is considerably reduced by dithiothreitol (DTT). This observation may be due to an acetyl-CoA deficiency, caused by a nonenzymic acylation of DTT, and a competition for ATP between DTT-activated CO₂ fixation and fatty acid synthesis. Because d,l-glyceraldehyde as inhibitor of CO₂ fixation compensates the DTT effect on dark fatty acid synthesis, reducing equivalents may be involved in the light dependence of acetate activation.     

Evidence of a Low Stromal Mg Concentration in Intact Chloroplasts in the Dark: I. STUDIES WITH THE IONOPHORE A23187

The loss of Mg(2+) upon the addition of the ionophore A23187 in the dark was prevented by less than 0.1 millimolar MgCl(2) with intact chloroplasts suspended in a sorbitol medium, but required 1 to 3 millimolar MgCl(2) if the chloroplasts were in a K(+) -gluconate medium. Measurements of stromal pH in the dark indicated that, in the K(+) -gluconate medium, the stromal pH is nearly the same as that of the medium, whereas in the sorbitol medium it is much more acidic as reported previously. These observations suggest that the free Mg(2+) concentration in the stroma in the dark is between 1 and 3 millimolar. Other experiments on the inihibition by A23187 of CO(2) fixation in the light and in a system capable of catalyzing CO(2) fixation in the dark, and on the Mg(2+) binding properties of thylakoid membranes, are consistent with this conclusion. The results provide further support for the hypothesis that light-induced Mg(2+) concentration changes occur in the stroma that are important in the light-dark regulation of CO(2) fixation.     

Synthesis and hydrolysis of ATP by intact chloroplasts under flash illumination and in darkness.

ATP concentrations were measured in isolated intact spinach chloroplasts under various light and dark conditions. The following results were obtained: (1) Even in darkened chloroplasts and in the absence of exogenous substrates, ATP levels in the chloroplast stroma were significant. They decreased on addition of glycerate, phosphoglycerate or dihydroxyacetone phosphate. When dihydroxyacetone phosphate and oxaloacetate were added together, ATP levels increased in darkened chloroplasts owing to substrate level phosphorylation. (2) Under illumination with saturating single turnover flashes, oxygen evolution in the presence of phosphoglycerate, whose reduction requires ATP, was no lower on a unit flash basis at the low flash frequency of 2 Hz than at higher frequencies. Quenching of 9-aminoacridine fluorescence, which indicates the formation of a proton gradient in intact chloroplasts, decreased with decreasing flash frequencies, until there was no significant fluorescence quenching at a flash frequency of about 2 Hz. In contrast to intact chloroplasts, broken chloroplasts did not phosphorylate much ADP at the low flash frequency of 2 Hz. (3) Flashing at extremely low frequencies (0.2 Hz) caused ATP hydrolysis rather than ATP synthesis in intact chloroplasts. At higher flash frequencies, synthesis replaced hydrolysis. Still, even at high frequencies (10 Hz), the first flashes of a series of flashes given after a long dark time always decreased chloroplast ATP levels. From these results, it is concluded that the enzyme, which mediates ATP synthesis in the light, is inactive in darkened intact chloroplasts. Its light activation can be separated from the formation of the high energy condition, which results in ATP synthesis. After its activation, the enzyme catalyzes a reversible reaction.     

Flexibility of coupling and stoichiometry of ATP formation in intact chloroplasts.

Since coupling between phosphorylation and electron transport cannot be measured directly in intact chloroplasts capable of high rates of photosynthesis, attempts were made to determine ATP/2 e ratios from the quamdum requirements of glycerate and phosphoglycerate reduction and from the extent of oxidation of added NADH via the malate shuttle during reduction of phosphoglycerate in light. These different approaches gave similar results. The quantum requirement of glycerate reduction, which needs 2 molecules of ATP per molecule of NADPH oxidized was found to be pH-dependent. 9-11 quanta were required at pH 7.6, and only about 6 at pH 7.0. The quantum requirement of phosphoglycerate reduction, which consumes ATP and NADPH in a 1/1 ratio, was about 4 both at pH 7.6 ant at 7.0. ATP/2 e ratios calculated from the quantum requirements and the extent of phosphoglycerate accumulation during glycerate reduction were usually between 1.2 and 1.4, occasionally higher, but they never approached 2. Although the chloroplast envelope is impermeable to pyridine nucleotides, illuminated chlrooplasts reduced added NAD via the malate shuttle in the absence of electron acceptors and also during the reduction of glycerate or CO2. When phosphoglycerate was added as the substrate, reduction of pyridine-nucleotides was replaced by oxidation and hydrogen was shuttled into the chloroplasts to be used for phosphoglycerate reduction even under light which was rate-limiting for reduction. This indicated formation of more ATP than NADPH by the electron transport chain. From the rates of oxidation of external NADH and of phosphoglycerate reduction at very low light intensities ATP/2e ratios were calculated to be between 1.1 and 1.4. Fully coupled chloroplasts reduced oxaloacetate in the light at rates reaching 80 and in some instances 130 mumoles times mg-1 chlorophyll times h-1 even though ATP is not consumed in this reaction. The energy transfer inhibitor phlorizin did not significantly suppress this reduction at concentrations which completely inhibited photosynthesis. Uncouplers stimulated oxaloacetate reduction by factors ranging from 1.5 to more than 10. Chloroplasts showing little uncoupler-induced stimulation of oxaloacetate reduction were highly active in photoreducing CO2. Measurements of light intensity dependence of quantum requirements for oxaloacetate reduction gave no indication for the existence of uncoupled or basal electron flow in intact chloroplasts. Rather reduction is brought about by loosely coupled electron transport. It is concluded that coupling of phosphorylation to electron transport in intact chloroplasts is flexible, not tight. Calculated ATP/2e ratios were obtained under con a decreENG     

Synthesis and hydrolysis of ATP by intact chloroplasts under flash illumination and in darkness

ATP concentrations were measured in isolated intact spinach chloroplasts under various light and dark conditions. The following results were obtained: (1) Even in darkened chloroplasts and in the absence of exogenous substrates, ATP levels in the chloroplast stroma were significant. They decreased on addition of glycerate, phosphoglycerate or dihydroxyacetone phosphate. When dihydroxyacetone phosphate and oxaloacetate were added together, ATP levels increased in darkened chloroplasts owing to substrate level phosphorylation. (2) Under illumination with saturating single turnover flashes, oxygen evolution in the presence of phosphoglycerate, whose reduction requires ATP, was no lower on a unit flash basis at the low flash frequency of 2 Hz than at higher frequencies. Quenching of 9-aminoacridine fluorescence, which indicates the formation of a proton gradient in intact chloroplasts, decreased with decreasing flash frequencies, until there was no significant fluorescence quenching at a flash frequency of about 2 Hz. In contrast to intact chloroplasts, broken chloroplasts did not phosphorylate much ADP at the low flash frequency of 2 Hz. (3) Flashing at extremely low frequencies (0.2 Hz) caused ATP hydrolysis rather than ATP synthesis in intact chloroplasts. At higher flash frequencies, synthesis replaced hydrolysis. Still, even at high frequencies (10 Hz), the first flashes of a series of flashes given after a long dark time always decreased chloroplast ATP levels.From these results, it is concluded that the enzyme, which mediates ATP synthesis in the light, is inactive in darkened intact chloroplasts. Its light activation can be separated from the formation of the high energy condition, which results in ATP synthesis. After its activation, the enzyme catalyzes a reversible reaction.     

Light-dependent changes of the Mg concentration in the stroma in relation to the Mg dependency of CO2 fixation in intact chloroplasts

(1) Light-dependent changes of the Mg²⁺ content of thylakoid membranes were measured at pH 8.0 and compared with earlier measurements at pH 6.6. In a NaCl and KCl medium, the light-dependent decrease in the Mg²⁺ content of the thylakoid membranes at pH 8.0 is found to be 23 nmol Mg²⁺ per mg chlorophyll, whereas in a sorbitol medium it is 83 nmol Mg₂₊ per mg chlorophyll.

(2) A light dependent increase in the Mg²⁺ content of the stroma was detected when chloroplasts were subjected to osmotic shock, amounting to 26 nmol/mg chlorophyll. Furthermore, a rapid and reversible light-dependent efflux of Mg²⁺ has been observed in intact chloroplasts when the divalent cation ionophore A 23 187 was added, indicating a light-dependent transfer of about 60 nmol of Mg²⁺ per mg chlorophyll from the thylakoid membranes to the stroma.

(3) CO₂ fixation, but not phosphoglycerate reduction, could be completely inhibited when A 23 187 was added to intact chloroplasts in the absence of external Mg²⁺. If Mg²⁺ was then added to the medium, CO₂ fixation was restored. Half of the maximal restoration was achieved with about 0.2 mM Mg²⁺, which is calculated to reflect a Mg²⁺ concentration in the stroma of 1.2 mM. The further addition of Ca²⁺ strongly inhibits CO₂ fixation.

(4) The results suggest that illumination of intact chloroplasts causes an increase in the Mg²⁺ concentration of 1–3 mM in the stroma. Compared to the total Mg²⁺ content of chloroplasts, this increase is very low, but it appears to be high enough to have a possible function in the light regulation of CO₂ fixation.     

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 greater than or equal to 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 Pi, but not by addition of 3-phosphoglycerate. In the absence of Pi, 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 65 mu mol-mg1 chlorophyll-h1. 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 mumol-mg1 chlorophyll-h1), but they are strongly stimulated by addition of dichlorophenyl-dimethylurea (e.g. 2-10(-7) M) reaching values of up to 60 mumol-mg1 chlorophyll-h1. 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 t-at 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 phoshate by dibromothymoquinone under anaerobic conditions indicated that plastoquinone is an indispensible component of the endogenous cyclic electron pathway.     

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.     

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.     

Fructose-and sedoheptulosebisphosphatase. The sites of a possible control of CO2 fixation by light-dependent changes of the stromal Mg2+ concentration

1. The enzymatic steps of the CO₂ fixation cycle responsible for the overall inhibition of CO₂ fixation caused by the lowering of the Mg²⁺ concentration in the stroma were investigated. For this the Mg²⁺ concentration in the stroma was decreased by addition of the ionophore A 23187, and the levels of the intermediates of the CO₂ fixation cycle in the stroma of intact chloroplasts were assayed by ion exchange chromatography.2. The addition of the ionophore caused an increase of NADPH, ATP, fructose- and sedoheptulosebisphosphate and a dramatic decrease of phosphoglycerate in the stroma. These changes were reversed by the addition of Mg²⁺ and again affected by a subsequent addition of Ca²⁺. Ribulosebisphosphate and pentosemonophosphate levels in the stroma were only a little affected under these different conditions.3. The increase of the NADPH and ATP reflects the decreased utilization of these compounds due to the overall inhibition of CO₂ fixation. As phosphoglycerate and triosephosphate appear to be in near equilibrium with NADPH and ATP, the decrease of phosphoglycerate seems to be a consequence of the changes in the nucleotide levels.4. The rapid increase of fructose- and sedoheptulosebisphosphate after the addition of the ionophore A 23187 clearly demonstrates that the overall inhibition of CO₂ fixation caused by lowering the stromal Mg²⁺ is due to the inhibition of the hydrolysis of these sugar bisphosphates. It is concluded that the activities of fructose- and sedoheptulosebisphosphatase can be controlled by light dependent changes of the stromal Mg²⁺ concentration.     

Influence of light on the heat sensitivity of the photosynthetic apparatus in isolated spinach chloroplasts

The most heat-sensitive functions of chloroplasts in Spinacia oleracea L. including the stromal carboxylation reaction, the light-induced electrical field gradient across the thylakoid membrane, as well as the overall photosynthetic CO₂ fixation were less affected by heat if chloroplasts were heated in the light: 50% inactivation occurred around 35°C in the dark and around 40°C in the light. Relative low light intensities were sufficient to obtain optimal protection against heat. In contrast, the light-induced ΔpH across the thylakoid membrane, the photophosphorylation, and the photochemical activity of photosystem II which were less sensitive to heat in the dark (50% inactivation above 40°C) were not protected by light. Photosystem II even was destabilized somewhat by light.

The effect of light on the heat sensitivity of the water-splitting reaction was dependent on the pH in the medium. Protection by light only occurred at alkaline pH, in which case heat sensitivity was high (50% inactivation at 33°C in the dark and at 38°C in the light). Protection was prevented by uncouplers. At pH 6.8 when the heat sensitivity was low in any case (50% inactivation at 41°C in the dark), light had no further protecting effect.

Protection by light has been discussed in terms of light-induced transport of protons from the stroma to the thylakoid space and related ion fluxes.

     

pH Changes in the Cytoplasm of the Blue-Green Alga Anacystis nidulans Caused by Light-dependent Proton Flux into the Thylakoid Space

The pH in the cytoplasmic and thylakoid spaces of the blue-green alga, Anacystis nidulans, has been determined in the light and in the dark by uptake of 5,5-dimethyloxazolidine-2,4-dione and methylamine into the sucrose-impermeable ³H-H₂O space, as measured by silicon layer filtering centrifugation.     

Factors influencing dark nitrogen fixation in a blue-green alga.

Nitrogen-fixing activity declines first rapidly and then more gradually when Anabaenopsis circularis is transferred from light into dark conditions. The rate and duration of dark acetylene reduction (nitrogen fixation) depend upon conditions prevailing during the preceding light period. Factors (such as light intensity, CO2 concentration, and supply of glucose), which in the light affect photosynthesis and the accumulation of reserve carbon, have a profound effect on dark nitrogen fixation. Glucose greatly promotes nitrogen fixation in the light and supports prolonged nitrogenase activity in the dark. The results suggest that heterotrophic nitrogen fixation by blue-green algae in the field may be important both under light and dark conditions.     

A possible ribosomal-directed regulatory system in Euglena gracilis. Carbon dioxide fixation

It was shown that cycloheximide inhibits CO(2) fixation in Euglena cells in the dark, but no effect of chloramphenicol was found. The light-dependent CO(2) fixation was inhibited by chloramphenicol and by large amounts of cycloheximide, but was stimulated by small amounts of cycloheximide. The presence of the stimulatory concentration of cycloheximide abolished the inhibition effect of high concentrations of chloramphenicol. The results indicate that the light-dependent CO(2) fixation is controlled by a repression-derepression regulatory system, which seems to be independent of chlorophyll synthesis.     

Rb+ uptake by isolated pea mesophyll protoplasts in light and darkness

Rb⁺ uptake into protoplasts isolated from the mesophyll of Pisum sativum L. cv. Dan has been followed at intervals of a few minutes in the light and in the dark. The progress curve for uptake in the dark decreased in slope after about 7 min; in the light, by contrast, the slope increased. This effect was more pronounced at pH 7 than at pH 5.5. The pH profile for uptake in the dark rose with increasing pH: in the light the profile flattened, or even fell somewhat, between pH 5.5 and pH 6.5, then rose again. In the dark the proton uncoupler carbonyl cyanide m-chlorphenylhydrazone (CCCP) had little or no effect, either at pH 5.5 or at pH 7.4; in the light CCCP was strongly inhibitory, particularly at pH 7.4. Increasing concentrations of CCCP produced progressively more and more severe inhibition in the light, but in the dark produced a slight rise in uptake. The ATPase inhibitors quercetin, rutin and diethyl-stilbestrol, as well as arsenate, all depressed uptake in the light, particularly at higher pH Dark uptake was sensitive only at pH 5.5, not at pH 7.4. In marked contrast to the case of methyl-3 glucose, where protoplasts which were switched from light to dark took up sugar at the accelerated light rate for the first 7 min in the dark, a switch to darkness produced a Rb⁺ uptake rate below that for protoplasts held continuously in the dark. It is inferred that the mechanism of Rb⁺ uptake does not involve proton cotransport. Information regarding the membrane potential was obtained by following the distribution of tetraphenyl phosphonium (TPP⁺) between protoplasts and medium. The potential was more negative in the light than in the dark. It was also more negative at pH 7 than at pH 5 both in the light and in the dark. Treatment with CCCP produced no appreciable depolarization within the first 20 min, indicating thet the CCCP inhibition of Rb⁺ uptake in the light cannot be ascribed to a reduction in potential. An ATP-fueled K⁺ porter, or K⁺-H⁺ antiporter, seems the most likely explanation. The maintenance of the rising pH profile in the dark, despite the presence of a CCCP concentration which drastically inhibits light uptake, suggests that the profile does not depend on the operation of the proton pump.     

Stimulation by Light of Rapid pH Regulation in the Chloroplast Stroma in Vivo as Indicated by CO2 Solubilization in Leaves

Leaves of Brassica oleracea, Helianthus annuus, and Nicotiana rustica were exposed for 20 s to high concentrations of CO2. CO2 uptake by the leaf, which was very fast, was measured as a transient increase in the concentration of oxygen. Rapid solubilization of CO2 in excess of that which is physically dissolved in aqueous phases is proposed to be caused by bicarbonate formation in the stroma of chloroplasts, which contain carbonic anhydrase. On this basis, pH values and bicarbonate accumulation in the chloroplast stroma were calculated. Buffer capacities were far higher than expected on the basis of known concentrations in the chloroplast stroma. Moreover, apparent buffer capacities increased with the time of exposure to high CO2, and they were higher when the measurements were performed in the light than in the dark. During prolonged exposure of leaves to 16% CO2, calculated bicarbonate concentrations in the chloroplast stroma exceeded 90 mM in the dark and 120 mM in the light. The observations are interpreted as indicating that under acid stress protons are rapidly exported from the chloroplasts in exchange for cations, which are imported. The data are discussed in terms of effective metabolic pH control by ion transport, first across the chloroplast envelope and, then, across the tonoplast of leaf mesophyll cells. The direct involvement of the vacuole in the regulation of the chloroplast pH in leaf cells is suggested.