The effect of permeant buffers on initial ATP synthesis by chloroplasts using rapid mix-quench techniques

The chemiosmotic hypothesis predicts that buffers which permeate chloroplast membranes should delay the formation of the proton gradient at the onset of illumination. If valinomycin and KCl are present to collapse the electrical potential as well, this delay should result in a lag in initial ATP synthesis. Using rapid-mix, acid-quench techniques, we have found that in light-driven ATP synthesis the permeant buffer imidazole does not increase the initial lag caused by the valinomycin-KCl pair. Similar results are obtained under methyl viologen or phenazine methosulfate/ascorbate-mediated photophosphorylation and are independent of the internal volume of the chloroplasts. Furthermore, we have observed that chloroplasts can synthesize significant amounts of ATP in darkness following an illumination period as short as 100 ms. This capacity for ATP synthesis in darkness after short pre-illumination periods is decreased in the presence of imidazole, and this may account for the apparent lags reported in earlier studies which have used rapid flash photophosphorylation in the presence of permeant buffers. The results of the present study argue that in chloroplasts, initial ATP synthesis and post-illumination ATP synthesis are driven by distinct components of the proton motive potential.