Reversal of quinone-induced chlorophyll fluorescence quenching

We have used two methods to investigate the reversibility of the interaction of substituted quinones with the thylakoid membrane of plant chloroplasts. Treatment of chloroplasts with added quinones lowers the room-temperature Photosystem II chlorophyll fluorescence intensity by variable amounts depending on the identity and concentration of the quinone. The extent of restoration of the chlorophyll fluorescence level is used as a measure of the effectiveness of the reversal technique. One reversal method involves the addition of thiols to quinone-treated chloroplasts to alter the quinone in a chemical way via a nucleophilic 1,4-Michael addition. In general, the modified quinones exhibit a lower affinity for the thylakoid membrane, as evidenced by an accompanying increase in chlorophyll fluorescence. The thiol concentrations necessary for quenching reversal are found to be in the order [dithiothreitol] less than [2-mercaptoethanol] less than [glutathione]. The second reversal method examines the extent to which added quinones can be removed from thylakoid membranes using a concentration gradient established by resuspension of quinone-treated chloroplasts in quinone-free media. The results further support the reversible nature of the quinone inhibition and indicate that the extent of recovery is dependent upon the degree of fluorescence inhibition originally induced by the added quinone.