Effect of Osmotic Stress on Photosynthesis Studied with the Isolated Spinach Chloroplast

The effect of increasing assay medium sorbitol concentration from 0.33 to 1.0 molar on the photosynthetic reactions of intact and broken spinach ( Spinacia oleracea L. var. Long Standing Bloomsdale) chloroplasts was investigated by monitoring O 2 evolution supported by the addition of glyceric acid 3-phosphate (PGA), oxaloacetic acid (OAA), 2,5-dimethyl- p -benzoquinone, and 2,6-dichlorophenolindophenol or as O 2 uptake with methyl viologen as acceptor. Uncoupled 2,6-dichlorophenolindophenol-supported whole chain electron transport (photosystems I and II) was inhibited from the 0.33 molar rate by 14% and 48.6% at 0.67 and 1.0 molar sorbitol in the intact chloroplast and by only 0.4% and 25.0% in the broken chloroplast preparation. Whole chain electron flow from water to other oxidants (OAA, methyl viologen) was also inhibited at increased osmoticum in intact preparations while electron flow from water to methyl viologen, ferricyanide, and NADP in broken preparations did not demonstrate the osmotic response. Electron transport to 2,5-dimethyl- p -benzoquinone (photosystem II) from H 2 O and to methyl viologen (photosystem I) from 3,3′-diaminobenzidine were found to be unaffected by osmolarity in both intact and broken preparations. The stress response was more pronounced (26-38%) with PGA as substrate in the presence of 0.67 molar sorbitol than the inhibition found with uncoupled and coupled linear electron flow. In addition, substrate availability and ATP generated by cyclic photophosphorylation evaluated by addition of Antimycin A were found not to be mediating the full osmotic inhibition of PGA-supported O 2 evolution. In a reconstituted (thylakoids plus stromal protein) chloroplast system to which a substrate level of PGA was added, O 2 evolution was only slightly (7.8%) inhibited by increased osmolarity (0.33-0.67 molar sorbitol) indicating that the level of osmotic inhibition above that contributed by adverse effects on electron flow can be attributed to the functioning of the photosynthetic carbon reduction cycle within the intact chloroplasts.