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Metabolism and Enzymology

Choline Oxidation by Intact Spinach Chloroplasts ¹

MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, Michigan 48824

Plants synthesize betaine by a two-step oxidation of choline (choline betaine aldehyde betaine). Protoplast-derived chloroplasts of spinach (Spinacia oleracea L.) carry out both reactions, more rapidly in light than in darkness (AD Hanson et al. 1985 Proc Natl Acad Sci USA 82: 3678-3682). We investigated the light-stimulated oxidation of choline, using spinach chloroplasts isolated directly from leaves. The rates of choline oxidation obtained (dark and light rates: 10-50 and 100-300 nanomoles per hour per milligram chlorophyll, respectively) were approximately 20-fold higher than for protoplast-derived chloroplasts. Betaine aldehyde was the main product. Choline oxidation in darkness and light was suppressed by hypoxia. Neither uncouplers nor the Calvin cycle inhibitor glyceraldehyde greatly affected choline oxidation in the light, and maximal choline oxidation was attained far below light saturation of CO₂ fixation. The light stimulation of choline oxidation was abolished by the PSII inhibitors DCMU and dibromothymoquinone, and was partially restored by adding reduced diaminodurene, an electron donor to PSI. Both methyl viologen and phenazine methosulfate prevented choline oxidation. Adding dihydroxyacetone phosphate, which can generate NADPH in organello, doubled the dark rate of choline oxidation. These results indicate that choline oxidation in chloroplasts requires oxygen, and reducing power generated from PSI. Enzymic reactions consistent with these requirements are discussed.

¹ Supported by the United States Department of Energy under contract DE-AC02-76ERO-1338, by the United States Department of Agriculture under grant 87-CRCR-1-2460 from the Competitive Research Grants Office, and by grants from the State of Michigan REED-Biotechnology Program, from CIBA-GEIGY Corporation, and from the Beet Sugar Development Foundation. P. W. and C. L., respectively, received fellowships from the French Ministry of Foreign Affairs and CONACYT, Mexico. Michigan Agricultural Experiment Station Journal Article No. 12406.

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