Dependence of energization of thylakoids on frequency of exciting flashes in intact chloroplasts

We investigated the frequency-dependence of the flash-induced electrochromic absorbance change, ΔA515, and of the pH-indicating absorbance change of neutral red in isolated intact chloroplasts. The energization pattern of thylakoids depended strongly on the frequency (f) of the exciting flashes, tested between 0.05 and 2 s(-1). When the frequency was increased from 0.1 to 1 s(-1) the total initial change and the slow rise of ΔA515 decreased by about 30% and 70%, respectively, and both the slow rise and decay were considerably accelerated. These changes were fully reversible, even after prolonged excitation at 1 s(-1), if the frequency was decreased again to 0.1 s(-1). Accumulation of an appreciable transmembrane electric field strength could not be detected in any of our experiments, at high frequency, since the decay of ΔA515 was considerably accelerated when the frequency was increased. In contrast, ΔpH significantly increased at higher frequencies of the exciting flashes. In the steady-state (after about 100 flashes) ΔpH was about 0.5-0.8 pH unit higher than in the dark or at low frequencies. In the presence of nigericin or dithionite, both of which prevented accumulation of protons in the lumen, the total initial change in ΔA515 at f=1 s(-1) relative to that at f=0.1 s(-1) decreased to a similar extent as in the control. The proportion of the slow rise relative to the initial amplitude, however, did not decrease. Our data support the suggestion that ΔpH controls the amplitude of the slow rise of ΔA515. However, contrary to a previous statement (B. Bouges-Bouquet (1981) Biochim. Biophys. Acta 535, 327-340), we show that the ΔpH effect cannot be accounted for by variation of the rate of this kinetic component of ΔA515.