Kinetics of the oxygen evolution step in plants determined from flash-induced chlorophyll t a fluorescence

Photosystem II (PS II) of plants and cyanobacteria, which catalyzes the light-induced splitting of water and the release of oxygen, is the primary source of oxygen in the earth atmosphere. When activated by short light flashes, oxygen release in PS II occurs periodically with maxima after the third and the seventh flashes. Many other processes, including chlorophyll (Chl) t a fluorescence, are also modulated with period of four, reflecting their sensitivity to the activity of Photosystem II. A new approach has been developed for the analysis of the flash-induced fluorescence of Chl t a in plants, which is based on the use of the generalized Stern–Volmer equation for multiple quenchers. When applied to spinach thylakoids, this analysis reveals the presence of a new quencher of fluorescence whose amplitude is characterized by a periodicity of four with maxima after the third and the seventh flashes, in phase with oxygen release. The quencher appears with a delay of 0.5 ms followed by a rise time of 1.2–2 ms at pH 7, also in agreement with the expected time for oxygen evolution. It is concluded that the quencher is a product of the reaction leading to the oxygen evolution in PS II. The same quenching activity, maximal after the third flash, could be seen in dark adapted leaves, and provides the first fully time-resolved measurement of the kinetics of the oxygen evolution step in the leaf. Thus, the non-invasive probe of Chl t a fluorescence provides a new and sensitive method for measuring the kinetics of oxygen evolution with potential for use in plants and cyanobacteria t in vivo.