Measurements of variable chlorophyll fluorescence using fast repetition rate techniques: defining methodology and experimental protocols

We present a methodology, called fast repetition rate (FRR) fluorescence, that measures the functional absorption cross-section (sigmaPS II) of Photosystem II (PS II), energy transfer between PS II units (p), photochemical and nonphotochemical quenching of chlorophyll fluorescence, and the kinetics of electron transfer on the acceptor side of PS II. The FRR fluorescence technique applies a sequence of subsaturating excitation pulses ('flashlets') at microsecond intervals to induce fluorescence transients. This approach is extremely flexible and allows the generation of both single-turnover (ST) and multiple-turnover (MT) flashes. Using a combination of ST and MT flashes, we investigated the effect of excitation protocols on the measured fluorescence parameters. The maximum fluorescence yield induced by an ST flash applied shortly (10 &mgr;s to 5 ms) following an MT flash increased to a level comparable to that of an MT flash, while the functional absorption cross-section decreased by about 40%. We interpret this phenomenon as evidence that an MT flash induces an increase in the fluorescence-rate constant, concomitant with a decrease in the photosynthetic-rate constant in PS II reaction centers. The simultaneous measurements of sigmaPS II, p, and the kinetics of Q-A reoxidation, which can be derived only from a combination of ST and MT flash fluorescence transients, permits robust characterization of the processes of photosynthetic energy-conversion.