Mechanism of Non-Photochemical Chlorophyll Fluorescence Quenching. II. Resolution of Rapidly Reversible Absorbance Changes at 530 Nm and Fluorescence Quenching by the Effects of Antimycin, Dibucaine and Cation Exchanger, A23187

Abstract

The putative relationship between the light-induced absorbance increase at 530 nm (ΔA₅₃₀), the so-called light-scattering change, and non-photochemical chlorophyll fluorescence quenching (NPQ) was examined by the effect of inhibitors. Antimycin at a low concentration (350 nM) completely inhibited fluorescence quenching while only partially inhibiting A₅₃₀. This effect was independent of the mode of thylakoid energisation and preinduction of violaxanthin de-epoxidation. Dibucaine at 20 FM abolished NPQ but had little effect on ΔA₅₃₀. Moreover, the light-induced ΔA₅₃₀ signal was present even in the absence of de-epoxidised xanthophylls. The cation exchanger A23187 blocked the development of NPQ as well as relaxed fluorescence quenching at steady state without involving a major portion of ΔA₅₃₀. Thus, the relationship between energy-dependent A₅₃₀ changes and fluorescence quenching was non-linear under all conditions tested. The light-induced absorbance increase at 530 nm, therefore, is insufficient for NPQ. The differential effects of inhibitors are explained schematically, depicting three phases for NPQ: (a) formation of zeaxanthin and antheraxanthin by the xanthophyll cycle; (b) formation of a state reflected by A₅₃₀ that is induced by the transthylakoid ApH, possibly involving aggregation of LHCII; and (c) fluorescence quenching by the combined effect of both steps and by the H+-cation exchange properties of thylakoid membranes.