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The relaxation of the non-photochemical quenching of chlorophyll fluorescence has been investigated in cells of the green alga Dunaliella following illumination. The relaxation after the addition of DCMU or darkening was strongly biphasic. The uncoupler NH4Cl induced rapid relaxation of both phases, which were therefore both energy-dependent quenching, qE. The proportion of the slow phase of qE increased at increasing light intensity. In the presence of the inhibitors rotenone and antimycin the slow phase of qE was stabilised for in excess of 15 min. NaN3 inhibited the relaxation of almost all the qE. The implications of these results are discussed in terms of the interpretation of the non-photochemical quenching of chlorophyll fluorescence in vivo and the mechanism of qE.

The quantitative relationship between energy-dependent quenching of chlorophyll fluorescence (qE) and trans-thylakoid pH difference (ΔpH, estimated with 9-aminoacridine) was compared in chloroplasts from leaves preilluminated at low oxygen in the absence of CO2 with chloroplasts from leaves darkened under the same conditions. The extent of both phenomena was varied by changes in actinic light intensity. Chloroplasts from preilluminated leaves contained high levels of zeaxanthin (up to 15% of total carotenoid) and were capable of forming qE at lower ΔpH values than chloroplasts from dark-adapted leaves, which lacked zeaxanthin. Infusion of dithiothreitol into leaves prior to preillumination prevented the light-induced formation of zeaxanthin; chloroplasts prepared from these leaves showed the same relationship between qE and ΔpH observed in chloroplasts fom dark-adapted leaves. The rate of appearance and disappearance of the change in relationship between qE and ΔpH upon leaf preillumination and darkening was closely matched by the kinetics of synthesis and degradation of zeaxanthin. The quantitative relationship between qE and quenching of the dark-level of fluorescence (F0) was very similar in chloroplasts from preilluminated or dark-adapted leaves, containing disparate amounts of zeaxanthin. In both sets of chloroplasts, qE was inhibited by antimycin A. These latter observations suggest that the formation of qE involves mechanistically similar features in chloroplasts containing or lacking zeaxanthin. Overall, the data are discussed in terms of action of zeaxanthin as a ‘quenching amplifier’, functioning physiologically to allow qE formation in the absence of high ΔpH potentially prohibitive to high rates of CO2 fixation.