Relationships among violaxanthin deepoxidation, thylakoid membrane conformation, and nonphotochemical chlorophyll fluorescence quenching in leaves of cotton (Gossypium hirsutum L.)

The kinetics and temperature dependencies of development and relaxation of light-induced absorbance changes caused by deepoxidation of violaxanthin to antheraxanthin and zeaxanthin (deltaZ; peak at 506 nm) and by light scattering (deltaS; peak around 540 nm) as well as of nonphotochemical quenching of chlorophyll fluorescence (NPQ) were followed in cotton leaves. Measurements were made in the absence and the presence of dithiothreitol (DTT), an inhibitor of violaxanthin deepoxidase. The amount of NPQ was calculated from the Stern-Volmer equation. A procedure was developed to correct gross deltaS (deltaSg) for absorbance changes around 540 nm that are due to a spectral overlap with deltaZ. This protocol isolated the component which is caused by light-scattering changes alone (deltaSn). In control leaves, the kinetics and temperature dependence of the initial rate of rise in deltaSn that takes place upon illumination, closely matched that of deltaZ. Application of DTT to leaves, containing little zeaxanthin or antheraxanthin, strongly inhibited both deltaSn and NPQ, but DTT had no inhibitory effect in leaves in which these xanthophylls had already been preformed, showing that the effect of DTT on deltaSn and NPQ results solely from the inhibition of violaxanthin deepoxidation. The rates and maximum extents of deltaSn and NPQ therefore depend on the amount of zeaxanthin (and/or antheraxanthin) present in the leaf. In contrast to the situation during induction, relaxation of deltaZ upon darkening was much slower than the relaxation of deltaSn and NPQ. The relaxation of deltaSn and NPQ showed quantitatively similar kinetics and temperature dependencies (Q10 = 2.4). These results are consistent with the following hypotheses: The increase in lumen-proton concentration resulting from thylakoid membrane energization causes deepoxidation of violaxanthin to antheraxanthin and zeaxanthin. The presence of these xanthophylls is not sufficient to cause deltaSn or NPQ but, together with an increased lumen-proton concentration, these xanthophylls cause a conformational change, reflected by deltaSn. The conformational change facilititates nonradiative energy dissipation, thereby causing NPQ. Membrane energization is prerequisite to conformational changes in the thylakoid membrane and resultant nonradiative energy dissipation but the capacity for such changes in intact leaves is quite limited unless zeaxanthin (and/or antheraxanthin) is present in the membrane. The sustained deltaSn and NPQ levels that remain after darkening may be attributable to a sustained high lumen-proton concentration.