Carbon dioxide and nitrite photoassimilatory processes do not intercompete for reducing equivalents in spinach and soybean leaf chloroplasts

Previously, C Baysdorfer and JM Robinson (1985 Plant Physiol 77: 318-320) demonstrated that, in a reconstituted spinach chloroplast system, NADP photoreduction functioning at most maximal rate and reductant demand, was the successful competitor with NO₂⁻ photoreduction for reduced ferredoxin. This resulted in a repression of NO₂⁻ reduction until all NADP available had been almost totally reduced. Further experiments, employing isolated, intact spinach leaf plastids and soybean leaf mesophyll cells, were conducted to examine competition for reductant between CO₂ and NO₂⁻ photoassimilation, in situ. In isolated, intact plastid preparations, regardless of whether the demand for reductant by CO₂ photoassimilation was high (5 millimolar `CO<sub>2</sub>') with rates of CO<sub>2</sub> fixation in the range 40 to 90 micromoles CO<sub>2</sub> fixed per hour per milligram chlorophyll, low (0.5 millimolar `CO₂') with rates in the range 5 to 8 micromoles CO₂ per hour per milligram chlorophyll, or zero (no `CO<sub>2</sub>'), NO<sub>2</sub><sup>−</sup> photoreduction displayed equal rates in the range of 8 to 22 micromoles per hour per milligram chlorophyll. In the absence of `CO₂', but in the presence of saturating white light, 3-phosphoglycerate photoreduction at rates of 82 to 127 micromoles per hour per milligram chlorophyll did not repress, and occasionally stimulated concomitant rates of NO₂⁻ reduction which ranged from 23.4 to 38.5. Conversely, in plastid preparations, NO₂⁻ at levels of 50 to 100 micromolar, stimulated plastid CO₂ fixation when `CO<sub>2</sub>' was saturating with respect to carboxylation. Further, levels of NO<sub>2</sub><sup>−</sup> in the range 250 to 2500 micromolar, stimulated soybean leaf mesophyll cell net CO<sub>2</sub> fixation as much as 1.5-fold if `CO₂' was saturating with respect to CO₂ fixation. It appeared likely that, in high light in vivo, CO₂ and NO₂⁻ photoassimilatory processes are not forced to intercompete for reduced ferredoxin in the intact chloroplast.     

Photophosphorylation and Carbon Dioxide Fixation by Chloroplasts Isolated from Populus deltoides

A system has been developed for the isolation of photosynthetically active chloroplasts from leaves of Populus deltoides. A high proportion of the chloroplasts appeared intact. The maximum rates of different photosynthetic processes were as follows: CO₂ fixation 3.5 micromoles per milligram chlorophyll per hour, noncyclic ATP synthesis 10 micromoles per milligram chlorophyll per hour, and cyclic ATP synthesis 300 micromoles per milligram chlorophyll per hour.     

Relationship between Respiration and Photosynthesis in Guard Cell and Mesophyll Cell Protoplasts of Commelina communis L

A mass spectrometric method combining ¹⁶O/¹⁸O and ¹²C/¹³C isotopes was used to quantify the unidirectional fluxes of O₂ and CO₂ during a dark to light transition for guard cell protoplasts and mesophyll cell protoplasts of Commelina communis L. In darkness, O₂ uptake and CO₂ evolution were similar on a protein basis. Under light, guard cell protoplasts evolved O₂ (61 micromoles of O₂ per milligram of chlorophyll per hour) almost at the same rate as mesophyll cell protoplasts (73 micromoles of O₂ per milligram of chlorophyll per hour). However, carbon assimilation was totally different. In contrast with mesophyll cell protoplasts, guard cell protoplasts were able to fix CO₂ in darkness at a rate of 27 micromoles of CO₂ per milligram of chlorophyll per hour, which was increased by 50% in light. At the onset of light, a delay observed for guard cell protoplasts between O₂ evolution and CO₂ fixation and a time lag before the rate of saturation suggested a carbon metabolism based on phosphoenolpyruvate carboxylase activity. Under light, CO₂ evolution by guard cell protoplasts was sharply decreased (37%), while O₂ uptake was slowly inhibited (14%). A control of mitochondrial activity by guard cell chloroplasts under light via redox equivalents and ATP transfer in the cytosol is discussed. From this study on protoplasts, we conclude that the energy produced at the chloroplast level under light is not totally used for CO₂ assimilation and may be dissipated for other purposes such as ion uptake.     

Aerobic and anaerobic respiration in the intact spinach chloroplast

Aerobic and anaerobic chloroplastic respiration was monitored by measuring ¹⁴CO₂ evolution from [¹⁴C]glucose in the darkened spinach (Spinacia oleracea) chloroplast and by estimating the conversion of fructose 1,6-bisphosphate to glycerate 3-phosphate in the darkened spinach chloroplast in air with O₂ or in N₂ with nitrite or oxaloacetate as electron acceptors. The pathway of ¹⁴CO₂ evolution from labeled glucose in the absence and presence of the inhibitors iodoacetamide and glycolate 2-phosphate under air or N₂ were those expected from the oxidative pentose phosphate cycle and glycolysis. Of the electron acceptors, O₂ was the best (2.4 nanomoles CO₂ per milligram chlorophyll per hour), followed by nitrite and oxaloacetate. With respect to glycerate 3-phosphate formation from fructose 1,6-bisphosphate, methylene blue increased the aerobic rate from 3.7 to 5.4 micromoles per milligram chlorophyll per hour. A rate of 4.8 micromoles per milligram chlorophyll per hour was observed under N₂ with nitrite and oxaloacetate.     

Evidence for Chloroplastic Succinate Dehydrogenase Participating in the Chloroplastic Respiratory and Photosynthetic Electron Transport Chains of Chlamydomonas reinhardtii

A method for isolating intact chloroplasts from Chlamydomonas reinhardtii F-60 was developed from the Klein, Chen, Gibbs, Platt-Aloia procedure ([1983] Plant Physiol 72: 481-487). Protoplasts, generated by treatment with autolysine, were lysed with a solution of digitonin and fractionated on Percoll step gradients. The chloroplasts were assessed to be 90% intact (ferricyanide assay) and free from cytoplasmic contamination (NADP isocitrate dehydrogenase activity) and to range from 2 to 5% in mitochondrial contamination (cytochrome c oxidase activity). About 25% of the cellular succinate dehydrogenase activity (21.6 micromoles per milligram chlorophyll per hour, as determined enzymically) was placed within the chloroplast. Chloroplastic succinate dehydrogenase had a K_m for succinate of 0.55 millimolar and was associated with the thylakoidal material derived from the intact chloroplasts. This same thylakoidal material, with an enzymic assay of 21.6 micromoles per milligram chlorophyll per hour was able to initiate a light-dependent uptake of oxygen at a rate of 16.4 micromoles per milligram chlorophyll per hour when supplied with succinate and methyl viologen. Malonate was an apparent competitive inhibitor of this reaction. The succinate dehydrogenase activity present in the chloroplast was sufficient to account for the photoanaerobic rate of acetate dissimilation in H₂ adapted Chlamydomonas (M Gibbs, RP Gfeller, C Chen [1986] Plant Physiol 82: 160-166).     

Photosynthesis of Lipids from CO(2) in Spinacia oleracea

Young expanding spinach leaves exposed to ¹⁴CO₂ under physiological conditions for up to 20 minutes assimilated CO₂ into lipids at a mean rate of 7.6 micromoles per milligram chlorophyll per hour following a lag period of 5 minutes. Label entered into all parts of the lipid molecule and only 28% of the ¹⁴C fixed into lipids was found in the fatty acid moieties, i.e. fatty acids were synthesized from CO₂in vivo at a mean rate of 2.1 micromoles per milligram chlorophyll per hour. Intact spinach chloroplasts isolated from these leaves incorporated H¹⁴CO₃ into fatty acids at a maximal rate of 0.6 micromole per milligram chlorophyll per hour, but were unable to synthesize either the polar moieties of their lipids or polyunsaturated fatty acids. Since isolated chloroplasts will only synthesize fatty acids at rates similar to the one obtained with intact leaves in vivo if acetate is used as a precursor, it is suggested that acetate derived from leaf mitochondria is the physiological fatty acid precursor.     

Photosynthesis by isolated protoplasts, protoplast extracts, and chloroplasts of wheat: influence of orthophosphate, pyrophosphate, and adenylates

Protoplasts, protoplast extracts (intact chloroplasts plus extrachloroplastic material), and chloroplasts isolated from protoplasts of wheat (Triticum aestivum) have rates of photosynthesis as measured by light-dependent O₂ evolution of about 100 to 150 micromoles of O₂ per milligram of chlorophyll per hour at 20 C and saturating bicarbonate. The assay conditions sufficient for this activity were 0.4 molar sorbitol, 50 millimolar N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid KOH (pH 7.6), and 10 millimolar NaHCO₃ with protoplast, plus a requirement of 1 to 10 millimolar ethylenediaminetetraacetate (EDTA) and 0.2 to 0.5 millimolar inorganic orthophosphate (Pi) with protoplast extracts and chloroplasts. Protoplast extracts evolved approximately 6 micromoles of O₂ per milligram of chlorophyll before photosynthesis became largely dependent on exogenous Pi while photosynthesis by chloroplasts had a much stronger dependence on exogenous Pi from the outset.

Photosynthesis by chloroplasts from 6-day-old wheat plants under optimum levels of Pi was similar to that with the addition of 5 millimolar inorganic pyrophosphate (PPi) plus 0.2 millimolar adenosine-5′-diphosphate (ADP). Either PPi or ADP added separately inhibited photosynthesis. When chloroplasts were incubated in the dark for 2 to 6 minutes, photosynthesis was strongly inhibited by 5 millimolar PPi and this inhibiting was relieved by including adenosine-5′-triphosphate (ATP) or ADP (0.2 to 0.6 millimolar). Chloroplasts from 9-day-old wheat leaves were slightly less sensitive to inhibition by PPi and showed little or no inhibition by ADP.

Chloroplasts isolated from protoplasts and assayed with 0.3 millimolar Pi added before illumination have an induction time from less than 1 minute up to 16 minutes depending on the time of the assay after isolation and the components of the medium. In order to obtain maximum rates of photosynthesis and minimum induction time, NaHCO₃ and chelating agents, EDTA or PPi (+ATP), are required in the chloroplast isolation, resuspension and assay medium. With these inclusions in the isolation and resuspension medium the induction time decreased rapidly during the first 20 to 30 minutes storage of chloroplasts on ice. Requirements for isolating intact and photosynthetically functional chloroplasts from wheat protoplasts are discussed.

     

Photosynthetic and Carbohydrate Metabolism in Isolated Leaf Cells of Digitaria pentzii

Mesophyll cells and bundle sheath strands were isolated rapidly from leaves of the C₄ species Digitaria pentzii Stent. (slenderstem digitgrass) by a chopping and differential filtration technique. Rates of CO₂ fixation in the light by mesophyll and bundle sheath cells without added exogenous substrates were 6.3 and 54.2 micromoles of CO₂ per milligram of chlorophyll per hour, respectively. The addition of pyruvate or phosphoenolpyruvate to the mesophyll cells increased the rates to 15.2 and 824.6 micromoles of CO₂ per milligram of chlorophyll per hour, respectively. The addition of ribose 5-phosphate increased the rate for bundle sheath cells to 106.8 micromoles of CO₂ per milligram of chlorophyll per hour. These rates are comparable to those reported for cells isolated by other methods. The K_m(HCO₃⁻) for mesophyll cells was 0.9 mm; for bundle sheath cells it was 1.3 mm at low, and 40 mm at higher HCO₃⁻ concentrations. After 2 hours of photosynthesis by mesophyll cells in ¹⁴CO₂ and phosphoenolpyruvate, 88% of the incorporated ¹⁴C was found in organic acids and 0.8% in carbohydrates; for bundle sheath cells incubated in ribose 5-phosphate and ATP, more than 58% of incorporated ¹⁴C was found in carbohydrates, mainly starch, and 32% in organic acids. These findings, together with the stimulation of CO₂ fixation by phosphoenolpyruvate for mesophyll cells and by ribose 5-phosphate plus ATP for bundle sheath cells, and the location of phosphoenolpyruvate and ribulose bisphosphate carboxylases in mesophyll and bundle sheath cells, respectively, are in accord with the scheme of C₄ photosynthesis which places the Calvin cycle in the bundle sheath and C₄ acid formation in mesophyll cells.     

Phycobilisome-thylakoid Topography on Photosynthetically Active Vesicles of Porphyridium cruentum

Conditions are described for isolating functional phycobilisome-thylakoid vesicles from the red alga Porphyridium cruentum. Phycobilisome-thylakoid vesicles were prepared by brief sonication and centrifugation in a medium containing 0.5 molar sucrose, 0.5 molar potassium phosphate, and 0.3 molar sodium citrate (pH 7.0). They required ferricyanide as an oxidant and had O₂ evolution rates (about 450 micromoles O₂ per hour per milligram chlorophyll) higher than whole cells (about 250 micromoles O₂ per hour per milligram chlorophyll). Energy transfer to photosystem II chlorophyll was evident from a high F695 nanometer (−196 C) emission peak. Preparations could be stored for over 24 hours and were considerably more stable than those from the cyanobacterium Anabaena variabilis (Katoh T, E Gantt 1979 Biochim Biophys Acta 546: 383-393). In electron micrographs of negatively stained material, the active thylakoid vesicles were found covered by closely spaced phycobilisomes on their external surface. The phycobilisome number in negatively stained vesicles was 450 per square micrometer, which was in the same range as the 400 per square micrometer observed in surface sections. A cell containing 1.5 × 10⁻⁶ micrograms phycoerythrin and 1.3 × 10⁻⁶ micrograms chlorophyll was found to contain 5 to 7 × 10⁵ phycobilisomes on a thylakoid area of 1.1 to 1.6 × 10³ square micrometers.     

Transport of Glycerate across the Envelope Membrane of Isolated Spinach Chloroplasts

Uptake of d, l-glycerate into the chloroplast stroma has been studied using the technique of silicone oil filtering centrifugation. Glycerate uptake was 3 to 5 times higher in the light than in darkness, the stimulation by light being abolished by the proton ionophore carbonyl cyanide p-trifluoromethoxyphenyl hydrazone. The pH optimum for uptake was 7.0 at 2°C and 8.5 at 20°C, but at all pH values the rate of uptake was higher at 20°C than at 2°C. Uptake was concentration dependent, saturating above 8 millimolar glycerate. At 2°C, the K_m was 0.3 millimolar and the V_max was 13 micromoles per milligram of chlorophyll per hour. At 20°C initial rates of glycerate uptake were higher than 40 micromoles per milligram of chlorophyll per hour.     

Occurrence and Regulatory Properties of Uridine Diphosphatase in Fully Expanded Leaves of Soybean (Glycine max Merr.) and Other Species

High activities (100-200 micromoles UDP hydrolyzed per milligram chlorophyll per hour) of uridine-5′ diphosphatase (UDPase) have been identified in extracts of fully expanded soybean (Glycine max Merr.) leaves. In desalted crude extracts, UDPase activity was strongly inhibited by low concentrations of Mg:ATP (I₅₀ = 0.3 millimolar). Two forms of the enzyme were resolved by gel filtration on Sephadex G-150. The higher molecular weight form (UDPase I, about 199 kilodaltons by gel filtration) retained ATP sensitivity (I₅₀ = 0.3 millimolar), whereas the major, lower molecular weight form (UDPase II, about 58 kilodaltons) was markedly less sensitive to ATP inhibition (I₅₀ = 2.7-3.0 millimolar). Subsequent purification of UDPase I by ion-exchange chromatography on DEAE cellulose produced a lower molecular weight enzyme (about 74 kilodaltons by gel filtration) that had reduced ATP sensitivity similar to UDPase II. Ion-exchange chromatography of UDPase II did not alter molecular weight or ATP sensitivity. UDPase II, after the DEAE-cellulose step, was specific for nucleoside diphosphates. Maximum reaction velocity decreased in the following sequence; UDP > GDP > CDP. ADP was not a substrate for the enzyme. The reaction catalyzed was hydrolysis of the terminal-P of UDP to form UMP. The enzyme was stimulated by Mg²⁺ and the pH optimum was centered between pH 6.5 and 7.0. In a survey of various species, soybean cultivars had highest activities of apparent UDPase and other species ranged in apparent activity from 0 to 30 micromoles hydrolyzed per milligram chlorophyll per hour.     

Active CO(2) Transport by the Green Alga Chlamydomonas reinhardtii

Mass spectrometric measurements of dissolved free ¹³CO₂ were used to monitor CO₂ uptake by air grown (low CO₂) cells and protoplasts from the green alga Chlamydomonas reinhardtii. In the presence of 50 micromolar dissolved inorganic carbon and light, protoplasts which had been washed free of external carbonic anhydrase reduced the ¹³CO₂ concentration in the medium to close to zero. Similar results were obtained with low CO₂ cells treated with 50 micromolar acetazolamide. Addition of carbonic anhydrase to protoplasts after the period of rapid CO₂ uptake revealed that the removal of CO₂ from the medium in the light was due to selective and active CO₂ transport rather than uptake of total dissolved inorganic carbon. In the light, low CO₂ cells and protoplasts incubated with carbonic anhydrase took up CO₂ at an apparently low rate which reflected the uptake of total dissolved inorganic carbon. No net CO₂ uptake occurred in the dark. Measurement of chlorophyll a fluorescence yield with low CO₂ cells and washed protoplasts showed that variable fluorescence was mainly influenced by energy quenching which was reciprocally related to photosynthetic activity with its highest value at the CO₂ compensation point. During the linear uptake of CO₂, low CO₂ cells and protoplasts incubated with carbonic anhydrase showed similar rates of net O₂ evolution (102 and 108 micromoles per milligram of chlorophyll per hour, respectively). The rate of net O₂ evolution (83 micromoles per milligram of chlorophyll per hour) with washed protoplasts was 20 to 30% lower during the period of rapid CO₂ uptake and decreased to a still lower value of 46 micromoles per milligram of chlorophyll per hour when most of the free CO₂ had been removed from the medium. The addition of carbonic anhydrase at this point resulted in more than a doubling of the rate of O₂ evolution. These results show low CO₂ cells of Chlamydomonas are able to transport both CO₂ and HCO₃⁻ but CO₂ is preferentially removed from the medium. The external carbonic anhydrase is important in the supply to the cells of free CO₂ from the dehydration of HCO₃⁻.     

Effects of Light and Elevated Atmospheric CO(2) on the Ribulose Bisphosphate Carboxylase Activity and Ribulose Bisphosphate Level of Soybean Leaves

Soybean (Glycine max L. Merr. cv Bragg) was grown throughout its life cycle at 330, 450, and 800 microliters CO₂ per liter in outdoor controlled-environment chambers under solar irradiance. Leaf ribulose-1,5-bisphosphate carboxylase (RuBPCase) activities and ribulose-1,5-bisphosphate (RuBP) levels were measured at selected times after planting. Growth under the high CO₂ levels reduced the extractable RuBPCase activity by up to 22%, but increased the daytime RuBP levels by up to 20%.

Diurnal measurements of RuBPCase (expressed in micromoles CO₂ per milligram chlorophyll per hour) showed that the enzyme values were low (230) when sampled before sunrise, even when activated in vitro with saturating HCO₃⁻ and Mg²⁺, but increased to 590 during the day as the solar quantum irradiance (photosynthetically active radiation or PAR, in micromoles per square meter per second) rose to 600. The nonactivated RuBPCase values, which averaged 20% lower than the corresponding HCO₃⁻ and Mg²⁺-activated values, increased in a similar manner with increasing solar PAR. The per cent RuBPCase activation (the ratio of nonactivated to maximum-activated values) increased from 40% before dawn to 80% during the day. Leaf RuBP levels (expressed in nanomoles per milligram chlorophyll) were close to zero before sunrise but increased to a maximum of 220 as the solar PAR rose beyond 1200. In a chamber kept dark throughout the morning, leaf RuBPCase activities and RuBP levels remained at the predawn values. Upon removal of the cover at noon, the HCO₃⁻ and Mg²⁺-activated RuBPCase values and the RuBP levels rose to 465 and 122, respectively, after only 5 minutes of leaf exposure to solar PAR at 1500.

These results indicate that, in soybean leaves, light may exert a regulatory effect on extractable RuBPCase in addition to the well-established activation by CO₂ and Mg²⁺.

     

Oxygen exchange associated with electron transport and photophosphorylation in spinach thylakoids

O₂ uptake in spinach thylakoids was composed of ferredoxin-dependent and -independent components. The ferredoxin-independent component was largely 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) insensitive (60%). Light-dependent O₂ uptake was stimulated 7-fold by 70 μM ferredoxin and both uptake and evolution (with O₂ as the only electron acceptor) responded almost linearly to ferredoxin up to 40 μM. NADP⁺ reduction, however, was saturated by less than 20 μM ferredoxin. The affinity of O₂ uptake for for O₂ was highly dependent on ferredoxin concentration, with $\text{K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{(O}{}_2\text{)}$ of less than 20 μM at 2 μM ferredoxin but greater than 60 μM O₂ with 25 μM ferredoxin. O₂ uptake could be suppressed up to 80% with saturating NADP⁺ and it approximated a competitive inhibitor of O₂ uptake with a K_i of 8–15 μM. Electron transport in these thylakoids supported high rates of photophosphorylation with NADP⁺ (600 μmol ATP/mg Chl per h) or O₂ (280 μmol/mg Chl per h) as electron acceptors, with $\text{ATP}\text{2}\text{e}$ ratios of 1.15–1.55. Variation in $\text{ATP}\text{2}\text{e}$ ratios with ferredoxin concentration and effects of antimycin A indicate that cyclic electron flow may also be occurring in this thylakoid system. Results are discussed with regard to photoreduction of O₂ as a potential source of ATP in vivo.     

Some Enzymes and Properties of the Reductive Carboxylic Acid Cycle Are Present in the Green Alga Chlamydomonas reinhardtii F-60

The reductive carboxylic acid cycle, the autotrophic pathway of CO₂ assimilation in prokaryotes (photosynthetic and nonphotosynthetic autotrophic bacteria), was investigated in Chlamydomonas reinhardtii F-60, an algal mutant lacking a complete photosynthetic carbon reduction pathway (C₃) due to a deficiency in phosphoribulokinase. Evidence was obtained consistent with the presence of the reductive carboxylic acid cycle in F-60. This conclusion is based on the fact that: (a) acetate approximately doubled CO₂ fixation in whole cells (4 micromoles per milligram chlorophyll per hour) and in chloroplasts (32 nanomoles per milligram chlorophyll per hour); and (b) pyruvate synthase, α-ketoglutarate synthase, and ATP-citrate lyase, three indicators of the cycle, were found in cell-free extracts.     

Regulation by amino acids of photorespiratory ammonia and glycolate release from ankistrodesmus in the presence of methionine sulfoximine

Methionine sulfoximine induced release of ammonia from illuminated cells of Ankistrodesmus braunii (Naegeli) Brunnth, in normal air, but less in air enriched to 3% CO₂. In normal air, methionine sulfoximine also induced glycolate release. Addition of either glutamate, glycine, or serine suppressed glycolate release, whereas glutamate and glycine at the same time stimulated ammonia release. The results indicate that inhibition of glutamine synthetase and thereby inhibition of photorespiratory nitrogen cycling restricts the sink capacity for glycolate in the photorespiratory carbon cycle. An external supply of glutamate, glycine, or serine seems to stimulate glyoxylate transamination and thus partly restores the sink capacity. Calculations of total glycolate formation rates in air from glycolate and ammonia release rates in the presence of methionine sulfoximine and glutamate revealed values of approximately 20 micromoles glycolate per milligram chlorophyll per hour on the average. Similar calculations led to an estimated rate of photorespiratory ammonia release in air, in the absence of methionine sulfoximine, of about 10 micromoles per milligram chlorophyll per hour on the average, a value comparable to the primary nitrogen assimilation rate of 8 micromoles per milligram chlorophyll per hour.     

Isolation of mesophyll protoplasts from mature leaves of soybeans

A procedure based on a combined cellulase-Pectolyase Y-23 enzyme digestion and metrizamide-sorbitol gradient purification protocol was developed for isolating mesophyll protoplasts from mature leaves of soybean (Glycine max L. Merr.). Based on chlorophyll content, this procedure results in a 10 to 15% protoplast yield from fully expanded mature leaves and a 20 to 30% yield from young (expanding) leaves within 3 hours. Isolated protoplasts displayed high rates of HCO₃⁻-dependent photosynthesis; greater than 75 micromoles O₂ evolved per milligram chlorophyll per hour at 25°C. This photosynthetic rate is comparable to that of mesophyll cells isolated mechanically from the same leaves.     

Efficiency of hydrogen photoproduction by chloroplast-bacterial hydrogenase systems

A comparative study of H₂ photoproduction by chloroplasts and solubilized chlorophyll was performed in the presence of hydrogenase preparations of Clostridium butyricum. The photoproduction of H₂ by chloroplasts in the absence of exogenous electron donors, and with irreversibly oxidized dithiothreitol and cysteine, is thought to be limited by a cyclic transport of electrons wherein methylviologen short-circuits the electron transport in photosystem I. The efficiency of H₂ photoproduction by chloroplasts with ascorbate and NADPH is limited by a back reaction between light-reduced methylviologen and the oxidized electron donors. The use of a combination of electron donors (dithiothreitol and ascorbate), providing anaerobiosis without damage to chloroplasts, makes it possible to avoid consumption of reduced methylviologen for the reduction of oxidized electron donors and to exclude the short-circuiting of electron transfer. Under these conditions, photoproduction of H₂ was observed to occur with a rate of 350 to 400 micromoles H₂ per milligram chlorophyll per hour. In this case, the full electron-transferring capability of photosystem I (measured by irreversible photoreduction of methyl red or O₂) is used to produce H₂.     

On the Mechanism of Activation by Light of the NADP-dependent Malate Dehydrogenase in Spinach Chloroplasts

With intact spinach (Spinacia oleracea L. cv. Vital R) chloroplasts, the activity of the NADP-dependent malate dehydrogenase after activation by light was 30 micromoles of malate formed per milligram of chlorophyll per hour; an identical rate of O₂ evolution was obtained upon oxaloacetate reduction by the intact plastids. However, when the activity of NADP-dependent malate dehydrogenase was measured subsequently to maximal activation of the enzyme by dithiothreitol (DTT) an average rate of 113 micromoles per milligram of chlorophyll per hour was obtained. When membranes and stroma were separated after osmotic disruption of the chloroplasts, 28% of NADP-dependent malate dehydrogenase activity inducible by DTT was found with the membranes and 72% was found in the stromal fraction. The membrane-associated portion of the enzyme corresponds well with the activity achieved after activation by light. About 64% of an activator system was found to be associated also with the membrane fraction. Washing the membranes with buffer removed more activator than enzyme. However, both were removed almost completely by ethylenediaminetetraacetate. It was concluded that both a portion of the enzyme and the total activator system are associated with the chloroplast membranes in vivo and that the activator is more loosely bound than the enzyme. A model describing the partial activation of chloroplastic NADP-dependent malate dehydrogenase by light and the total activation by DTT is presented.     

Characteristics of the Mg-ATPase Activity Associated with the Membrane-Bound Maize Coupling Factor

The membrane-bound coupling factor of maize mesophyll thylakoids is a latent ATPase. Mg²⁺-ATPase activity can be induced in the light with either dithiothreitol or low concentrations of trypsin. Maize thylakoids that are activated with light plus trypsin exhibit considerably higher levels of activity in Na₂SO₃-dependent Mg²⁺-ATPase assays compared to thylakoids that are light and dithiothreitol activated (1400 micromoles per milligram of chlorophyll per hour versus 200 micromoles per milligram of chlorophyll per hour). Treatment with light and dithiothreitol or light plus trypsin were also required to demonstrate high levels of octyl glucoside-dependent Mg²⁺-ATPase activity in maize mesophyll thylakoids. Only small differences in octyl glucoside-dependent Mg²⁺-ATPase activity were observed in preparations that were activated in the light with either trypsin or dithiothreitol. Mg²⁺-ATPase activity can also be induced in maize mesophyll chloroplasts by illuminating intact preparations under appropriate conditions. Little or no ATPase activity was observed in the absence of illumination or in the presence of light plus methyl viologen. The active state decayed in the dark with a t_½ of 6 to 7 minutes at room temperature. Based on the effect of the thiol oxidant, o-iodosobenzoate, and the uncoupler, nigericin, on the kinetics of deactivation of ATPase activity in intact maize chloroplasts, it appears that the activation process requires a transmembrane proton gradient and reduction of a key disulfide bridge in the gamma of chloroplast coupling factor one.