QUANTITATIVE-ANALYSIS OF THE EFFECTS OF INTRATHYLAKOID PH AND XANATHOPHYLL CYCLE PIGMENTS ON CHLOROPHYLL-A FLUORESCENCE LIFETIME DISTRIBUTIONS AND INTENSITY IN THYLAKOIDS

The xanthophyll cycle-dependent dissipation of excitation energy in hi gher plants is one of the most important regulatory and photoprotectiv e mechanisms in photosynthesis. Using parallel time-resolved and pulse -amplitude modulation fluorometry, we studied the influence of the int rathylakoid pH and the xanthophyll cycle carotenoids on the PSII chlor ophyll (Chl) a fluorescence yield in thylakoids of Arabidopsis, spinac h, and barley. Increases in concentrations of dithiothreitol in thylak oids, which have a trans-thylakoid membrane pH gradient and are known to have decreased conversion of violaxanthin (V) to zeaxanthin (Z), le ad to (1) decreases in the fractional intensity of the similar to 0.5 ns Chl a fluorescence Lifetime (tau) distribution component and simult aneous increases in a 1.6-1.8 ns fluorescence component and (2) increa ses in the maximal fluorescence intensity. These effects disappear whe n the pH gradient is eliminated by the addition of nigericin. To quant itatively explain these results, we present a new mathematical model t hat describes the simultaneous effects of the chloroplast trans-thylak oid membrane pH gradient and xanthophyll cycle pigments on the PSII Ch l a fluorescence tau distributions and intensity. The model assumes th at (1) there exists a specific binding site for Z (or antheraxanthin, A) among or in an inner antenna complex (primarily CP29), (2) this bin ding site is activated by a low intrathylakoid pH (pK approximate to 4 .5) that increases the affinity for Z (or A), (3) about one Z or A mol ecule binds to the activated site, and (4) this binding effectively '' switches'' the fluorescence tau distribution of the PSII unit to a sta te with a decreased fluorescence tau and emission intensity (a ''dimme r switch'' concept). This binding is suggested to cause the formation of an exciton trap with a rapid intrinsic rate constant of heat dissip ation. Statistical analysis of the data yields an equilibrium associat ion constant, K-a, that ranges from 0.7 to 3.4 per PSII fur the proton ated/activated binding site for Z (or A). The model explains (1) the r elative fraction of the similar to 0.5 ns fluorescence component as a function of both Z and A concentration and intrathylalkoid pH, (2) the dependence of the ratio of F-m'/F-m on the fraction of the 0.5 ns flu orescence tau component (where F-m' and F-m are maximal fluorescence i ntensities in the presence and the absence of a pH gradient), and (3) the dependence of the ratio of F-m'/F-m on the concentration of Z and A and the intrathylakoid pH.