Characterization of an Electron Transport Pathway Associated with Glucose and Fructose Respiration in the Intact Chloroplasts of Chlamydomonas reinhardtii and Spinach.
The role of an electron transport pathway associated with aerobic carbohydrate degradation in isolated, intact chloroplasts was evaluated. This was accomplished by monitoring the evolution of (14)CO(2) from darkened spinach (Spinacia oleracea) and Chlamydomonas reinhardtii chloroplasts externally supplied with [(14)C]fructose and [(14)C]glucose, respectively, in the presence of nitrite, oxaloacetate, and conventional electron transport inhibitors. Addition of nitrite or oxaloacetate increased the release of (14)CO(2), but it was shown that O(2) continued to function as a terminal electron acceptor. (14)CO(2) evolution was inhibited up to 30 and 15% in Chlamydomonas and spinach, respectively, by 50 mum rotenone and by amytal, but at 500- to 1000-fold higher concentrations, indicating the involvement of a reduced nicotinamide adenine dinucleotide phosphate-plastoquinone oxidoreductase. (14)CO(2) release from the spinach chloroplast was inhibited 80% by 25 mum 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone. (14)CO(2) release was sensitive to propylgallate, exhibiting approximately 50% inhibition in Chlamydomonas and in spinach chloroplasts of 100 and 250 mum concentrations, respectively. These concentrations were 20- to 50-fold lower than the concentrations of salicylhydroxamic acid (SHAM) required to produce an equivalent sensitivity. Antimycin A (100 mum) inhibited approximately 80 to 90% of (14)CO(2) release from both types of chloroplast. At 75 mum, sodium azide inhibited (14)CO(2) evolution about 50% in Chlamydomonas and 30% in spinach. Sodium azide (100 mm) combined with antimycin A (100 mum) inhibited (14)CO(2) evolution more than 90%. (14)CO(2) release was unaffected by uncouplers. These results are interpreted as evidence for a respiratory electron transport pathway functioning in the darkened, isolated chloroplast. Chloroplast respiration defined as (14)CO(2) release from externally supplied [1-(14)C]glucose can account for at least 10% of the total respiratory capacity (endogenous release of CO(2)) of the Chlamydomonas reinhardtii cell.
Singh
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Institute for Photobiology of Cells and Organelles, Brandeis University, Waltham, Massachusetts 02254.
Chen
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Gibbs
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