CO2-dependent Oxygen Research Articles

Contribution of lowered unsaturation levels of chloroplast lipids to high temperature tolerance of photosynthesis in Chlamydomonas reinhardtii

A mutant of Chlamydomonas reinhardtii designated as hf-9 is impaired in fatty acid desaturation of chloroplasts, and showed lowered unsaturation levels of chloroplast lipids, as compared with the parent (Sato et al., Eur. J. Biochem., 230 (1995) 987–993). The effects of temperature on photosynthesis were compared between hf-9 and the parent for investigation whether or not unsaturation levels of chloroplast lipids are correlated with the thermal properties of photosynthesis. Growth rates determined by turbidity were higher in the parent than in hf-9at both 10 and 24°C, while similar for the parent and hf-9 at 39°C. The cells grown at 24°C revealed that both activities of CO 2-dependent oxygen evolution and photosystem II were higher in the parent than in hf-9 in the range between 7 and 40°C. In contrast, hf-9 surpassed the parent in both activities at 45°C. Optimal temperatures for both activities were at around 35°C and 40°C in the parent and hf-9, respectively. Incubation of the cells at 41 and 45°C demonstrated that the activity of photosystem II in hf-9 was more tolerant to the high temperatures than that in the parent. These results suggest that lowered unsaturation levels of chloroplast lipids constributed to high temperature tolerance of photosystem II, and eventually to that of photosynthesis.

Enhanced susceptibility of photosynthesis to high leaf temperature in triazine-resistant Solanum nigrum L. Evidence for photosystem II D 1 protein site of action

We have used nuclearly isogenic lines of Solanum nigrum, that differ genetically in the triazine-resistance trait, to investigate the mechanistic basis for the greater susceptibility of photosynthesis in resistant biotypes to elevated leaf temperature. Based on measurement of CO 2-dependent oxygen evolution by leaf discs, of electron transfer partial reactions in isolated thylakoid membranes, and of chlorophyll fluorescence induction parameters of both leaves and thylakoid membranes, we conclude that: (1) The single base substitution in the chloroplast psb A gene that is responsible for triazine resistance is almost certainly also responsible for the decreased tolerance to high temperature; (2) The high temperature-induced inhibition of photosynthesis in the resistant biotype is caused by an inhibition of electron transfer between the primary and secondary quinone acceptors of photosystem II; (3) The enhanced temperature susceptibility is likely to be a significant contributing factor in the competitive disadvantage of triazine-resistant biotypes.

Effects of dimethazone (FMC 57020) on chloroplast development: II. Pigment synthesis and photosynthetic function in cowpea ( Vigna unguiculata L.) primary leaves

The herbicidal action of dimethazone [FMC 57020; 2-(2-chlorophenyl) methyl-4,4-dimethyl-3-isoxalidinone] on cowpea ( Vigna unguiculata L.) primary leaves was studied. Seeds were imbibed in 0.5 m M herbicide for 1 day and then seedlings were grown in darkness. In 6-day-old, etiolated seedlings, there was no effect of the herbicide on protochlorophyllide accumulation or on phototransformation of protochlorophyllide to chlorophyllide, however, the Shibata shift was greatly slowed. Accompanying this was a delay in phytylation of chlorophyllide. Protochlorophyllide resynthesis in a dark period after phototransformation of existing protochlorophyllide in etiolated tissue was also slowed by dimethazone. In the light, carotenoid accumulation and chlorophyll accumulation were slowed by the herbicide, resulting a pale green appearance of the leaves. The capacity for CO 2-dependent oxygen evolution or FeCN-dependent oxygen evolution did not develop in dimethazone-treated tissue during 24 hr of light exposure. In situ measurement of variable fluorescence and cytochrome f photooxidation/dark reduction indicated that some cyclic electron transport developed very slowly in dimethazone-treated plants. No effect of the herbicide was found on either FeCN-dependent oxygen evolution or variable fluorescence in fully greened tissues. Dimethazone was concluded to have an effect on chloroplast development rather than a direct effect on photosynthesis.

Chloroplast phosphoproteins: Distribution of phosphoproteins within spinach chloroplasts

The distribution of phosphoproteins within spinach chloroplasts was studied. Intact chloroplasts with good rates of CO 2-dependent oxygen evolution were fed [γ- 32P]ATP and then separated into stroma and membrane fractions. Only one major labelled stroma protein was identified by gel electrophoresis/autoradiography, with a mol. wt. of 66 000. The membranes were separated into envelopes and thylakoid fractions. Three labelled proteins were separated by gel electrophoresis in the envelope with mol. wt. of 50 500, 29 000 and 13 000.

Induction of a Mehler reaction in chloroplast preparations by methyl viologen and by ferredoxin: Effects on photosynthesis by intact chloroplasts

CO 2-dependent oxygen evolution in illuminated spinach chloroplast preparations was found to be inhibited by addition of methyl viologen or ferredoxin. Under the conditions described, and in the absence of ribose-5-phosphate, the K i for inhibition by methyl viologen was 0.4 μM and the K i for inhibition by ferredoxin was 13 μM. The effect of osmotic shock on chloroplast oxygen uptake in the Mehler reaction implies that at the concentrations used neither methyl viologen nor ferredoxin freely penetrates the envelope of intact chloroplasts. The observed inhibition of CO 2-dependent oxygen evolution is therefore attributed in each case to a Mehler reaction in the broken chloroplasts of the preparations. Inhibition by methyl viologen (0.8 μM) and by ferredoxin (20 μM) could be reversed by ascorbate and by catalase. As reported [1] for flavin mononucleotide H 2O 2 is likely to be the chief inhibitory agent. The difference in mechanism of autoxidation between methyl viologen and ferredoxin is therefore less important in this system than is their common production of H 2O 2.

Induction of a Mehler reaction in chloroplast preparations by flavin mononucleotide: Effects on photosynthesis by intact chloroplasts

Addition of flavin mononucleotide (FMN) to spinach chloroplast preparations containing intact chloroplasts was found to result in inhibition of photosynthetic CO 2-dependent oxygen evolution. Since the inhibition could be reversed by catalase, it is attributed primarily to production of H 2O 2 in an FMN-mediated Mehler reaction which occurs in the broken chloroplasts of the preparations. Ascorbate and superoxide dismutase also reversed the inhibition though to a lesser extent. The inhibition was also offset by the presence of ribose-5-phosphate. The complete dependence of these effects on the presence of FMN is consistent with the view that intact chloroplasts do not themselves produce inhibitory concentrations of H 2O 2 under conditions optimal for photosynthetic CO 2-fixation.

Inhibition of CO 2 fixation by adenosine 5′-diphosphate and the role of phosphate transport in isolated pea chloroplasts

CO 2-dependent oxygen evolution by isolated pea ( Pisum sativum cv Massey Gem) chloroplasts was inhibited by ADP. AMP inhibited oxygen evolution to a greater extent than ADP while no inhibition was observed with ATP. Oxygen evolution with ADP or AMP was more sensitive to phosphate inhibition than in the absence of added adenine nucleotides. Photosynthetic intermediates (ribose 5-phosphate or 3-phosphoglycerate) partially relieved the inhibition by ADP. Pyrophosphate also inhibited oxygen evolution, yet, with PP 1 plus ADP, oxygen evolution was stimulated. Inhibition of the phosphate transporter using p-chloromercuriphenylsulfonic acid did not reverse ADP inhibition but the subsequent reversal of ADP inhibition by PP 1 was prevented. Pyrophosphate relieved inhibition of oxygen evolution by P i both in the presence and in the absence of added adenine nucleotides. Uptake of ADP into pea chloroplasts apparently results in an inhibition of 3-phosphoglycerate kinase, and the subsequent accumulation of 3-phosphoglycerate and decrease in internal phosphate make the chloroplasts highly susceptible to inhibition by phosphate. We propose that PP i enters chloroplasts via the phosphate transporter and is hydrolyzed internally by the chloroplast inorganic pyrophosphatase. The increase in internal phosphate concentration would prevent loss of photosynthetic intermediates from the chloroplast, under these conditions, and thus stimulate oxygen evolution.

Uptake of ATP analogs by isolated pea chloroplasts and their effect on CO 2 fixation and electron transport

1. The ATP analog, adenylyl-imidodiphosphate rapidly inhibited CO 2-dependent oxygen evolution by isolated pea chloroplasts. Both α, β- and β, γ-methylene adenosine triphosphate also inhibited oxygen evolution. The inhibition was relieved by ATP but only partially relieved by 3-phosphoglycerate. Oxygen evolution with 3-phosphoglycerate as substrate was inhibited by adenylyl-imidodiphosphate to a lesser extent than CO 2-dependent oxygen evolution. The concentration of adenylyl-imidodiphosphate required for 50% inhibition of CO 2-dependent oxygen evolution was 50 μM. 2. Although non-cyclic photophosphorylation by broken chloroplasts was not significantly affected by adenylyl-imidodiphosphate, electron transport in the absence of ADP was inhibited by adenylyl-imidodiphosphate to the same extent as by ATP, suggesting binding of the ATP analog to the coupling factor of phosphorylation. 3. The endogenous adenine nucleotides of a chloroplast suspension were labelled by incubation with [ 14C]ATP and subsequent washing. Addition of adenylyl-imidodiphosphate to the labelled chloroplasts resulted in a rapid efflux of adenine nucleotides suggesting that the ATP analog was transported into the chloroplasts via the adenine nucleotide translocator. 4. It was concluded that uptake of ATP analogs in exchange for endogenous adenine nucleotides decreased the internal ATP concentration and thus inhibited CO 2 fixation. Oxygen evolution was inhibited to a lesser extent in spinach chloroplasts which apparently have lower rates of adenine nucleotide transport than pea chloroplasts.

CO 2 reduction by intact chloroplasts under a diminished proton gradient

9-Aminoacridine has been used to monitor the intrathylakoid pH of photo-synthetically competent intact chloroplasts. Values obtained from 9-aminoacridine accumulation in the chloroplasts must be corrected for light-dependent binding of 9-aminoacridine to the thylakoid membranes. During nitrite reduction by intact chloroplasts, the intrathylakoid proton concentration increased. It decreased somewhat during CO 2 reduction. However, low concentrations of uncoupling amines such as NH 3 or cyclohexylamine, which rapidly penetrated the chloroplast envelope and decreased the intrathylakoid proton concentration, failed to reduce, and actually stimulated, rates of CO 2-dependent oxygen evolution even under rate-limiting light. In contrast, low concentrations of carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) or nigericin, which inhibited CO 2 reduction, even appeared to increase the intrathylakoid proton concentration. As indicated by measurements of the 515 nm signal of the chloroplasts, the light-induced membrane potential was not much affected by low concentrations of the uncoupling amines, but was decreased by FCCP and by high concentrations of the amines. Even in the presence of high concentrations of NH 4Cl, ATP/ADP ratios of illuminated chloroplasts remained far above the ratios observed in the dark. In contrast, low concentrations of FCCP were sufficient to reduce ATP/ADP ratios to the dark value even under high intensity illumination. The observations are difficult to explain within the framework of the chemiosmotic hypothesis as presently discussed.

Photosynthesis in a reconstituted chloroplast system from spinach. Some factors affecting CO 2-dependent oxygen evolution with fructose-1, 6-bisphosphate as substrate

When envelope-free spinach chloroplasts are incubated with stromal protein, catalytic NADP, catalytic ADP, radioactive bicarbonate and fructose 1,6-bisphosphate, 14CO 2 fixation starts immediately upon illumination but oxygen evolution is delayed. The delay is increased by the addition of fructose 6-phosphate and by a variety of factors known (or believed) to increase fructose bisphosphatase activity (such as dithiothreitol, more alkaline pH, higher [Mg] and antimycin A). Conversely, the lag can be decreased or eliminated by the addition of an ATP-generating system. Bearing in mind the known inhibition, by ADP, of sn-phospho-3-glycerate (3-phosphoglycerate) reduction it is concluded that the lag in O 2 evolution results from the production of ribulose 5-phosphate from fructose bisphosphate and that this in turn inhibits the reoxidation of NADPH by adversely affecting the ADP/ATP ratio. The results are discussed in their relation to the mode of action of antimycin A and to regulation of the reductive pentose phosphate pathway.