MEASUREMENTS OF VARIABLE CHLOROPHYLL FLUORESCENCE USING FAST REPETITION RATE TECHNIQUES - DEFINING METHODOLOGY AND EXPERIMENTAL PROTOCOLS

We present a methodology, called fast repetition rate (FRR) fluorescen ce, that measures the functional absorption cross-section (sigma(PS II )) Of Photosystem II (PS II), energy transfer between PS II units (p), photochemical and nonphotochemical quenching of chlorophyll fluoresce nce, 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 fl uorescence 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 th e effect of excitation protocols on the measured fluorescence paramete rs. The maximum fluorescence yield induced by an ST flash applied shor tly (10 Cls to 5 ms) following an MT flash increased to a level compar able to that of an MT flash, while the functional absorption cross-sec tion 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 P S II reaction centers. The simultaneous measurements of sigma(PS) (II) , p, and the kinetics of Q(A)(-) reoxidation, which can be derived onl y from a combination of ST and MT flash fluorescence transients, permi ts robust characterization of the processes of photosynthetic energy-c onversion. (C) 1998 Elsevier Science B.V. All rights reserved.