MECHANISM OF NONPHOTOCHEMICAL CHLOROPHYLL FLUORESCENCE QUENCHING .1. THE ROLE OF DE-EPOXIDIZED XANTHOPHYLLS AND SEQUESTERED THYLAKOID MEMBRANE PROTONS AS PROBED BY DIBUCAINE

Dibucaine reportedly inhibits the light-induced transthylakoid proton gradient of chloroplasts without inhibiting energy-dependent non-photo chemical chlorophyll fluorescence quenching (Laasch, H. and Weis, E. ( 1989). Photosynthesis Research 22, 137-146). We show that dibucaine ca n inhibit fluorescence quenching, depending on the de-epoxidation stat e of the xanthophyll cycle. Whereas dibucaine (20-40 mu M) had little effect on fluorescence quenching in pre-illuminated-type thylakoids (l oaded with zeaxanthin and antheraxanthin), it strongly inhibited quenc hing in dark-adapted-type thylakoids (no preinduction of de-epoxidatio n). Dibucaine inhibited lumen acidification similarly in both types of thylakoids and also the induction of violaxanthin deepoxidation in da rk-adapted thylakoids. Thus dark-adapted and pre-illuminated thylakoid s differed in de-epoxidation states and their suspectibility to dibuca ine inhibition of fluorescence quenching corresponded to this differen ce. The mechanism of inhibition of de-epoxidation by dibucaine is uncl ear. It could be due to the inhibition of lumen acidification but an i nhibition of the violaxanthin available for de-epoxidation is not excl uded. High dibucaine concentrations inhibited de-epoxidase activity di rectly. Dibucaine inhibition of fluorescence quenching, however, is no t limited to the inhibition of de-epoxidation. Small but clear effects on fluorescence quenching were present in thylakoids even with de-epo xidation preinduced. Moreover, thylakoids with preinduced deepoxidatio n 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 delo calised domains. We conclude that non-photochemical quenching of chlor ophyll fluorescence depends on both de-epoxidised xanthophylls and seq uestered proton domains in the thylakoid membranes.