Mechanism of Non-Photochemical Chlorophyll Fluorescence Quenching. I. The Role of De-Epoxidised Xanthophylls and Sequestered Thylakoid Membrane Protons as Probed by Dibucaine

Abstract

Dibucaine reportedly inhibits the light-induced transthylakoid proton gradient of chloroplasts without inhibiting energy-dependent non-photochemical chlorophyll fluorescence quenching (Laasch, H. and Weis, E. (1989). Photosynthesis Research 22, 137-146). We show that dibucaine can inhibit fluorescence quenching, depending on the de-epoxidation state of the xanthophyll cycle. Whereas dibucaine (20-40 μM) had little effect on fluorescence quenching in pre-illuminated-type thylakoids (loaded with zeaxanthin and antheraxanthin), it strongly inhibited quenching in dark-adapted-type thylakoids (no preinduction of de-epoxidation). Dibucaine inhibited lumen acidification similarly in both types of thylakoids and also the induction of violaxanthin de-epoxidation in dark-adapted thylakoids. Thus dark-adapted and pre-illuminated thylakoids differed in de-epoxidation states and their suspectibility to dibucaine inhibition of fluorescence quenching corresponded to this difference. The mechanism of inhibition of de-epoxidation by dibucaine is unclear. It could be due to the inhibition of lumen acidification but an inhibition of the violaxanthin available for de-epoxidation is not excluded. High dibucaine concentrations inhibited de-epoxidase activity directly. Dibucaine inhibition of fluorescence quenching, however, is not limited to the inhibition of de-epoxidation. Small but clear effects on fluorescence quenching were present in thylakoids even with de-epoxidation preinduced. Moreover, thylakoids with preinduced de-epoxidation were more resistant to dibucaine inhibition of fluorescene quenching when poised by salt treatments for proton partitioning into membrane-sequestered domains than when poised for proton partitioning into delocalised domains. We conclude that non-photochemical quenching of chlorophyll fluorescence depends on both de-epoxidised xanthophylls and sequestered proton domains in the thylakoid membranes