Photosynthetic Response to Alkaline pH in Anabaena variabilis

The rate of O₂ evolution and alkalization of the medium in low CO_2⁻ grown Anabaena variabilis was observed as affected by the pH in the medium. Both rates are severely inhibited by pH values higher than 9.5, but the latter is more sensitive to this treatment. This finding, as well as the lag observed in alkalization of the medium, but not in O₂ evolution, following the addition of HCO₃⁻ indicates that the transport of HCO₃ and OH⁻ (or H⁺) are not compulsorily coupled. The inhibition of photosynthesis by strongly alkaline pH is attributed to an alteration of the internal pH and, hence, the rate of carboxylation. This conclusion is supported by data showing that the rate of O₂ evolution is affected by pH more strongly at saturating [HCO₃⁻] than at limiting [HCO₃⁻]. Also, the rate of O₂ evolution at saturating light intensity is affected by pH more strongly than is the initial slope of the curve against light intensity or the rate of dark respiration.     

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.     

Photoautotrophic growth of soybean cells in suspension culture: I. Establishment of photoautotrophic cultures

Highly chlorophyllous photomixotrophic callus was visually selected from callus originating from soybean (Glycine max (L.) Merr. var. Corsoy) cotyledon. Suspension cultures initiated from this callus became photoautotrophic under continuous light with an atmosphere of 5% CO₂ (balance air). Dry weight increases of 1000 to 1400% in the 2-week subculture period have been observed. The cellular Chl content ranged from 4.4 to 5.9 micrograms per milligram dry weight which is about 75 to 90% of the Chl content in soybean leaves under equivalent illumination (300 micro-Einsteins per square meter per second).

No growth can be observed in the dark in sucrose-lacking medium or in the presence of 0.5 micromolar 3-(3,4-dichlorophenyl)-1,1-dimethylurea, a concentration which does not inhibit heterotrophic growth (on sucrose). Photoautotrophic growth has an absolute requirement for elevated CO₂ concentrations (>1%). During the 14-day subculture period, growth (fresh weight and dry weight) is logarithmic. Photosynthesis quickly increases after day 4, reaching a peak of 83 micromoles CO₂ incorporated per milligram Chl per hour while dark respiration decreases 90% from day 2 to day 6. The pH of the growth medium quickly drops from 7.0 to 4.5 before slowly increasing to 5.0 by day 14. At this pH range and light intensity (200-300 microEinsteins per square meter per second), no O₂ evolution could be detected although at high pH and light intensity O₂ evolution was recorded.

     

Modulation of chloroplast phosphofructokinase by NADPH : a mechanism for linking light to the regulation of glycolysis

Phosphofructokinase has been partially purified from spinach (Spinacia oleracea) chloroplasts and studied from the standpoint of light/dark regulation. At concentrations reported to occur physiologically, NADPH effected a sharp inhibition of the enzyme by: (a) lowering its affinity (increasing the apparent K_m) for both of its substrates, ATP and fructose 6-phosphate; and (b) lowering its V_max. Inhibition by NADPH was independent of pH and was observed both at pH 7.9 (pH of chloroplast stroma in the light) and pH 7.0 (stromal pH in the dark). The results are consistent with the conclusion that NADPH provides a mechanism for linking light to the modulation of phosphofructokinase activity and thereby to the regulation of glycolysis in chloroplasts.     

Changes in Parameters of Dark Respiration in Ascophyllum nodosum Induced by Different Salinities

The changes in the rates of O₂ consumption and CO₂ evolution by Ascophyllum nodosum thalli in the dark were studied during a 12 day incubation at 34, 20, and 10 salinities as well as at different pH. Depending on the initial environmental pH, the algae demonstrated antipodal functional responses to the same salinities and, as a consequence, different capacity to overcome hypoosmotic stress. In addition, we observed a more pronounced effect of pH on the rate of O₂ consumption than on the rate of CO₂ evolution. Simultaneous desalination and acidification of the medium (pH 7–8) increased O₂ consumption in the dark, which activated oxidative processes in the cells and increased their energy status. Forced pH stabilization (8.2) of the medium coupled with its desalination inhibited dark respiration of algae, which, in our opinion, decreased their energy status. Specific manifestation of hypoosmotic stress and adaptation in A. nodosum are discussed considering the assumed differences in algal energy status.     

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.     

The effect of pH on the inorganic carbon source for photosynthesis in the seagrass Zostera muelleri irmisch ex aschers

The ability of the seagrass Zostera muelleri Irmisch ex Aschers. to use HCO₃⁻ as well as CO₂ for photosynthesis was investigated by measuring photosynthetic O₂ evolution over a range of pH values. It was found that the apparent K_m CO₂ fell from 0.128 mM at pH 7.9 to 0.016 mM at pH 9.1 indicating that HCO₃⁻ as well as CO₂ may act as a substrate for photosynthesis.The true K_m CO₂ could not be determined due to inhibition of photosynthesis at pHs less than 7.8 K_m CO₂ must be at least 0.128 mM, the apparent K_m at pH 7.9, and is probably of the order of 0.200 mM CO₂, the same as that reported for other marine plants. K_m HCO₃⁻¹ is about 20 mM when CO₂-dependent photosynthesis is minimal. Such a high K_m HCO₃⁻ resembles values reported for freshwater, rather than marine plants.Photosynthetic O₂ evolution is not saturated with respect to total inorganic carbon in natural seawater (pH 8.2). It is suggested that the distinctive shoulder from pH 8.1 to 8.5 in the pH profile of photosynthetic O₂ evolution at a constant concentration of inorganic carbon is caused by an effect of pH on HCO₃⁻ uptake. The effect of pH on HCO₃⁻ uptake was determined by constructing a pH profile of photosynthesis at constant HCO₃⁻ concentration, and subtracting the estimated contribution of CO₂ to photosynthesis from this rate. The resultant curve has a maximum at pH 8.4 and declines sharply at pHs less than 8.     

Circadian photosynthetic reductant flow in the dinoflagellate Lingulodinium is limited by carbon availability

Circadian rhythms are the observed outputs of endogenous daily clocks and are thought to provide a selective advantage to cells adapted to daily light/dark cycles. However, the biochemical links between the clock and the overt rhythms in cell physiology are generally not known. Here, we examine the circadian rhythm in O₂ evolution by cultures of the dinoflagellate Lingulodinium, a rhythm previously ascribed to rhythmic electron flow through photosystem II. We find that O₂ evolution rates increase when CO₂ concentrations are increased, either following addition of DIC or a rapid decrease in culture pH. In medium containing only nitrate as an electron acceptor, O₂ evolution rates mirror the circadian rhythm of nitrate reductase activity in the cells. Furthermore, competition between photosynthetic electron flow to carbon and to nitrate varies in its relative efficiency through the day–night cycle. We also find, using simultaneous and continuous monitoring of pH and O₂ evolution rates over several days, that while culture pH is normally rhythmic, circadian changes in rates of O₂ evolution depend not on the external pH but on levels of internal electron acceptors. We propose that the photosynthetic electron transport rhythm in Lingulodinium is driven by the availability of a reductant sink.     

Glucose Respiration in the Intact Chloroplast of Chlamydomonas reinhardtii

Chloroplastic respiration was monitored by measuring ¹⁴CO₂ from ¹⁴C glucose in the darkened Chlamydomonas reinhardtii F-60 chloroplast. The patterns of ¹⁴CO₂ evolution from labeled glucose in the absence and presence of the inhibitors iodoacetamide, glycolate-2-phosphate, and phosphoenolpyruvate were those expected from the oxidative pentose phosphate cycle and glycolysis. The K_m for glucose was 56 micromolar and for MgATP was 200 micromolar. Release of ¹⁴CO₂ was inhibited by phloretin and inorganic phosphate. Comparing the inhibition of CO₂ evolution generated by pH 7.5 with respect to pH 8.2 (optimum) in chloroplasts given C-1, C-2, and C-6 labeled glucose indicated that a suboptimum pH affects the recycling of the pentose phosphate intermediates to a greater extent than CO₂ evolution from C-1 of glucose. Respiratory inhibition by pH 7.5 in the darkened chloroplast was alleviated by NH₄Cl and KCl (stromal alkalating agents), iodoacetamide (an inhibitor of glyceraldehyde 3-phosphate dehydrogenase), or phosphoenolpyruvate (an inhibitor of phosphofructokinase). It is concluded that the site which primarily mediates respiration in the darkened Chlamydomonas chloroplast is the fructose-1,6-bisphosphatase/phosphofructokinase junction. The respiratory pathways described here can account for the total oxidation of a hexose to CO₂ and for interactions between carbohydrate metabolism and the oxyhydrogen reaction in algal cells adapted to a hydrogen metabolism.     

Light Stimulation of Ethylene Release from Leaves of Gomphrena globosa L

The effect of light and CO₂ on both the endogenous and 1-aminocyclopropane-1-carboxylic acid (ACC)-dependent ethylene evolution from metabolically active detached leaves and leaf discs of Gomphrena globosa L. is reported. Treatment with varying concentrations of ACC did not appear to inhibit photosynthesis, respiration, or stomatal behavior. In all treatments, more ethylene was released into a closed flask from ACC-treated tissue, but the pattern of ethylene release with respect to light/dark/CO₂ treatments was the same.

Leaf tissue in the light with a source of CO₂ sufficient to maintain photosynthesis always generates 3 to 4 times more ethylene than tissue in the dark. Conversely, the lowest rate of ethylene release occurs when leaf tissue is illuminated and photosynthetic activity depletes the CO₂ to the compensation point. Ethylene release in the dark is also stimulated by CO₂ either added to the flask as bicarbonate or generated by dark respiration. Ethylene release increases dramatically and in parallel with photosynthesis at increasing light intensities in this C₄ plant. Ethylene release appears dependent on CO₂ both in the light and in the dark. Therefore, it is suggested that the important factor regulating the evolution of ethylene gas from leaves of Gomphrena may be CO₂ metabolism rather than light per se.

     

The role of Hox hydrogenase in the H2 metabolism of Thiocapsa roseopersicina

The purple sulfur phototrophic bacterium Thiocapsa roseopersicina BBS synthesizes at least three NiFe hydrogenases (Hox, Hup, Hyn). We characterized the physiological H₂ consumption/evolution reactions in mutants having deletions of the structural genes of two hydrogenases in various combinations. This made possible the separation of the functionally distinct roles of the three hydrogenases. Data showed that Hox hydrogenase (unlike the Hup and Hyn hydrogenases) catalyzed the dark fermentative H₂ evolution and the light-dependent H₂ production in the presence of thiosulfate. Both Hox⁺ and Hup⁺ mutants demonstrated light-dependent H₂ uptake stimulated by CO₂ but only the Hup⁺ mutant was able to mediate O₂-dependent H₂ consumption in the dark. The ability of the Hox⁺ mutant to evolve or consume hydrogen was found to depend on a number of interplaying factors including both growth and reaction conditions (availability of glucose, sulfur compounds, CO₂, H₂, light). The study of the redox properties of Hox hydrogenase supported the reversibility of its action. Based on the results a scheme is suggested to describe the role of Hox hydrogenase in light-dependent and dark hydrogen metabolism in T. roseopersicina BBS.     

Light-Stimulated Changes in the Acidity of Suspensions of Oat Protoplasts: Dependence upon Photosynthesis

Light induced an alkalinization and stimulated a subsequent acidification of the medium surrounding oat (Avena sativa L. cv Garry) leaf protoplasts. Blue light was less effective than would be predicted from photosynthetic action spectra. Nonetheless, 3-(3,4-dichlorophenyl)-1,1-dimethylurea prevented alkalinization and reduced acidification to the dark rate for protoplast suspensions exposed to all light regimes tested.

Alkalinization increased in parallel with initial rates of O₂ evolution as the quantum flux density of white light was raised to 75 microeinsteins per square meter per second. Alkalinization was accompanied by a decrease in the CO₂ content of the medium; therefore, it was attributed to photosynthetically induced CO₂ uptake. The effect of CO₂ depletion on the acidity of the medium appeared to be mainly restricted to the first 15 minutes of exposure to light. Consequently, subsequent pH changes primarily reflected a constant net proton efflux. Acidification occurred in the dark, but rates of acidification increased in response to increased light approximately in parallel with changes in a concomitant net O₂ efflux. The results indicated that protoplasts could acidify the medium in response to nonphotosynthetic activity, but that photosynthesis mediated light stimulation of acidification.

     

Light/dark labeling differences in chloroplast membrane polypeptides associated with chloroplast coupling factor 0

The fluorogenic reagent fluorescamine has been used to determine the labeling patterns of Type C spinach chloroplast membrane polypeptides. Membrane polypeptides labeled with fluorescamine were detected by scanning high resolution sodium dodecyl sulfate polyacrylamide gradient slab gels for fluorescence emission.Three membrane polypeptides show a decrease in the extent of labeling when chloroplast membranes are labeled in the light compared to when they are labeled in the dark. These polypeptides have apparent molecular weights of 32 000, 23 000 and 15 000.The decrease in labeling observed in the light is abolished or reduced by treatments which inactivate the light-generated transmembrane pH gradient. CF₁-depleted chloroplasts show neither a light-activated pH gradient nor a light/dark difference in labeling of these three polypeptides. Both a light-activated pH gradient and light/dark differences in labeling are observed in CF₁-depleted chloroplasts which have been treated with N,N′-dicyclohexylcarbodiimide.The same ammonium sulfate fractions of a 2% sodium cholate extract, which are believed to be enriched in the membrane-bound sector of the chloroplast ATPase (CF_o) are also found to be enriched in the 32 000, 23 000 and 15 000 molecular weight polypeptides. The three polypeptides are believed to be components of CF_o, and the light/dark labeling differences may indicate conformational changes within CF_o. Such conformational changes may reflect a mechanism which couples light-generated proton gradients to ATP synthesis.     

Interactions between Glucose and Inorganic Carbon Metabolism in Chlorella vulgaris Strain UAM 101

Chlorella vulgaris strain UAM 101 has been isolated from the effluent of a sugar refinery. This alga requires glucose to achieve maximal growth rate even under light saturating conditions. The growth rate of cultures grown on light + CO₂ + glucose (3.16 per day) reaches the sum of those grown on light + CO₂ (1.95 per day) and on dark + glucose (1.20 per day). Unlike other Chlorella strains, uptake of glucose (about 2 micromoles per milligram dry weight per hour) was induced to the same extent in the light and dark and was not photosensitive. The rate of dark respiration was not affected by light and was strongly stimulated by the presence of glucose (up to about 40% in 4 hours). The rate of photosynthetic O₂ evolution was measured as a function of the CO₂ concentration. These experiments were conducted with cells which experienced different concentrations of CO₂ or glucose during growth. The maximal photosynthetic rate was inhibited severely by growing the cells in the presence of glucose. A rather small difference in the apparent photosynthetic affinity for extracellular inorganic carbon (from 10-30 micromolar) was found between cells grown under low and high CO₂. Growth with glucose induced a reduction in the apparent affinity (45 micromolar) even though cells had not been provided with CO₂. Experiments performed at different pH values indicate CO₂ as the major carbon species taken from the medium by Chlorella vulgaris UAM 101.     

Light modulation of glucose-6-phosphate dehydrogenase: partial characterization of the light inactivation system and its effects on the properties of the chloroplastic and cytoplasmic forms of the enzyme

Light inactivation of glucose-6-phosphate dehydrogenase within the pea (Pisum sativum L.) leaf chloroplast has a narrow pH optimum between 7.2 and 7.4 and is NADP-sensitive. The pH optimum for dark activation is slightly lower. Inactivation apparently results in a simple decrease in maximal velocity of the chloroplastic and cytoplasmic forms of the enzyme with no concomitant change in pH optimum or K_m (glucose 6-phosphate).     

Gas Exchange Characteristics of the Submerged Aquatic Crassulacean Acid Metabolism Plant, Isoetes howellii

The submerged aquatic plant Isoetes howellii Engelmann possesses Crassulacean acid metabolism (CAM) comparable to that known from terrestrial CAM plants. Infrared gas analysis of submerged leaves showed Isoetes was capable of net CO₂ uptake in both light and dark. CO₂ uptake rates were a function of CO₂ levels in the medium. At 2,500 microliters CO₂ per liter (gas phase, equivalent to 1.79 milligrams per liter aqueous phase), Isoetes leaves showed continuous uptake in both the light and dark. At this CO₂ level, photosynthetic rates were light saturated at about 10% full sunlight and were about 3-fold greater than dark CO₂ uptake rates. In the dark, CO₂ uptake rates were also a function of length of time in the night period. Measurements of dark CO₂ uptake showed that, at both 2,500 and 500 microliters CO₂ per liter, rates declined during the night period. At the higher CO₂ level, dark CO₂ uptake rates at 0600 h were 75% less than at 1800 h. At 500 microliters CO₂ per liter, net CO₂ uptake in the dark at 1800 h was replaced by net CO₂ evolution in the dark at 0600 h. At both CO₂ levels, the overnight decline in net CO₂ uptake was marked by periodic bursts of accelerated CO₂ uptake. CO₂ uptake in the light was similar at 1% and 21% O₂, and this held for leaves intact as well as leaves split longitudinally. Estimating the contribution of light versus dark CO₂ uptake to the total carbon gain is complicated by the diurnal flux in CO₂ availability under field conditions.     

O(2) uptake in the light in chlamydomonas: evidence for persistent mitochondrial respiration

The nature of the process responsible for the stationary O₂ uptake occurring in the light under saturating CO₂ concentration in Chlamydomonas reinhardii has been investigated. For this purpose, a mass spectrometer with a membrane inlet system was used to measure O₂ uptake and evolution in the algal suspension. First, we observed that the O₂ uptake rate was constant (about 0.5 micromoles of O₂ per milligram chlorophyll per minute) during a light to dark transition and was not affected by 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Salicylhydroxamic acid had no effect on O₂ uptake in the dark or in the light, but was found to have the same inhibitory effect either in the dark or in the light when added to cyanide-treated algae. The stimulation of the O₂ uptake rate due to the uncoupling effect of carbonyl cyanide m-chlorophenylhydrazone was about the same in the dark or in the light. From these results, we conclude that mitochondrial respiration is maintained during illumination and therefore is not inhibited by high ATP levels. Another conclusion is that in conditions where photorespiration is absent, no other light-dependent O₂ uptake process occurs. If Mehler reactions are involved, in Chlamydomonas, under conditions where both photosynthetic carbon oxidation and reduction cycles cannot operate (as in cyanide-treated algae), their occurrence in photosynthesizing algae either under saturating CO₂ concentration or at the CO₂ compensation point appears very unlikely. The comparison with the situation previously reported in Scenedesmus (R. J. Radmer and B. Kok 1976 Plant Physiol 58: 336-340) suggests that different O₂ uptake processes might be present in these two algal species.     

Carbon dioxide fixation in the light and in the dark by isolated spinach chloroplasts

Factors affecting CO₂ fixation in the spinach (Spinacia oleracea) chloroplast were investigated. Free magnesium ions are shown to be highly inhibitory for photosynthetic CO₂ fixation in isolated intact spinach chloroplasts. The pH optimum for CO₂ fixation is about 8.5 but is dependent upon the reaction medium. Conditions are defined under which chloroplasts illuminated in the absence of CO₂ accumulate ribulose 1,5-diphosphate, and fix CO₂ in a subsequent dark period when high magnesium ion concentrations are provided. The regulation of photosynthetic CO₂ assimilation by these factors is discussed.     

H(2) metabolism in photosynthetic organisms: I. Dark h(2) evolution and uptake by algae and mosses

Dark H₂ metabolism was studied in marine and fresh water red algae, the green alga, Chlamydomonas, and mosses. A time variable and temperature-sensitive anaerobic incubation was required prior to H₂ evolution. H₂ evolution was sensitive to disalicylidenepropanediamine. An immediate H₂ uptake was observed in these algae. Immediate dark H₂ uptake but no evolution was observed in the mosses.