Evidence that localized energy coupling in thylakoids can continue beyond the energetic threshold onset into steady illumination.

Energy transduction from proton gradients into ATP formation in chloroplast thylakoids has been hypothesized to be driven equally efficiently by localized domain delta mu H+ or by a delocalized delta mu H+ (Beard, W. A. and Dilley, R. A. (1988) J. Bioenerg. Biomembr. 20, 129-154). An important question is whether the apparent localized protonmotive force energy coupling mode can be observed only in the dark-to-light transient in the flash excitation protocol commonly used, or whether the localized energy coupling gradient can be maintained under conditions of continuous illumination ATP formation. The assay in the previous work was to use permeable amines, added to thylakoids in the dark, and observe the effect of the amine on the length of the energization lag (number of single-turnover flashes) required to initiate ATP formation in the dark-to-light transition. Amine buffers delayed the ATP onset in high-salt-stored membranes but did not delay the onset with low salt-stored membranes. This work tested whether permeable amines show the different effects in low- or high-salt-stored thylakoids which had attained a steady-state ATP formation rate (in continuous light) for 20-40 s prior to adding the amine. Hydroxyethylmorpholine was the preferred amine for such experiments, a suitable choice inasmuch as it behaves similarly to pyridine in the flash-induced ATP formation onset experiments, but it permeates more rapidly than pyridine and it has a higher pKa, which enhances its buffering effects. With high-salt-stored thylakoids, 0.5 or 1.0 mM hydroxyethylmorpholine added after 40 s of continuous illumination caused a marked, but transient, slowing of the ATP formation rate, but little or no slowing of the rate was observed with low-salt-stored thylakoids (at similar phosphorylation rates for the two thylakoid samples). Those data indicate that in continuous illumination conditions the proton gradient driving ATP formation in thylakoids from the low-salt-stored treatment did not equilibrate with the lumen, but in thylakoids stored in high-salt the delta mu H+ freely equilibrated with the lumen. That suggestion was supported by measurement of the luminal pH under coupling conditions by the [14C]methylamine distribution method using low- or high-salt-stored thylakoids. Further supportive evidence was obtained from measuring the effect of permeable amine buffers on H+ uptake under coupled and basal conditions with both types of thylakoid.(ABSTRACT TRUNCATED AT 400 WORDS)