On the chlorophyll a fluorescence yield in chloroplasts upon excitation with twin turnover flashes (TTF) and high frequency flash trains

Abstract

Chlorophyll fluorescence is routinely taken as a quantifiable measure of the redox state of the primary quinone acceptor Q A of PSII. The variable fluorescence in thylakoids increases in a single turnover flash (STF) from its low dark level F o towards a maximum F m STF when Q A becomes reduced. We found, using twin single turnover flashes (TTFs) that the fluorescence increase induced by the first twin-partner is followed by a 20-30% increase when the second partner is applied within 20-100 ls after the first one. The amplitude of the twin response shows a period-of-four oscillation associated with the 4-step oxidation of water in the Kok cycle (S states) and originates from two different trapped states with a life time of 0.2-0.4 and 2-5 ms, respectively. The oscillation is supplemented with a binary oscillation associated with the two-electron gate mechanism at the PSII acceptor side. The F(t) response in high frequency flash trains (1-4 kHz) shows (i) in the first 3-4 flashes a transient overshoot 20-30% above the F m STF = 3*F o level reached in the 1st flash with a partial decline towards a dip D in the next 2-3 ms, independent of the flash frequency, and (ii) a frequency independent rise to F m = 5*F o in the 3-60 ms time range. The initial overshoot is interpreted to be due to electron trapping in the S 0 fraction with Q B -nonreducing centers and the dip to the subsequent recovery accompanying the reoxidation of the double reduced acceptor pair in these RCs after trapping. The rise after the overshoot is, in agreement with earlier findings, interpreted to indicate a photo-electrochemical control of the chlorophyll fluorescence yield of PSII. It is anticipated that the double exciton and electron trapping property of PSII is advantageous for the plant. It serves to alleviate the depression of electron transport in single reduced Q B -nonreducing RCs, associated with electrochemically coupled proton transport, by an increased electron trapping efficiency in these centers.Keywords Chlorophyll a Á Fluorescence quenching Á Q B -nonreducing center Á Single turnover excitation Á Twin turnover excitation Á Three state trapping mechanism (TSTM)

Analysis of initial chlorophyll fluorescence induction kinetics in chloroplasts in terms of rate constants of donor side quenching release and electron trapping in photosystem II

“…It has been evidenced (Vredenberg et al 2006) that the b-(So) fraction in dark-adapted preparations is populated with Q B -nonreducing RCs. These have been shown to become double reduced upon twin (TTF) excitation (Vredenberg et al 2007). It has been assumed, and justification for this can be read from the bottom curve in Fig.…”

“…5, that in control chloroplasts the contribution of double reduction in the b-fraction to the fluorescence kinetics in the 0-0.2 ms time range is negligible. In the presence of DCMU, both fractions b and (1 -b), have become Q B -nonreducing and susceptible to double reduction in a second excitation (Vredenberg et al 2006(Vredenberg et al , 2007. The double reduction will contribute to the kinetics of the initial F(t) rise in the presence of DCMU.…”

“…The equation above illustrates and would allow quantification of the amply documented four periodic (Schreiber and Neubauer 1987;Kolber et al 1998;Shinkarev 2004) and binary oscillation (Bowes and Crofts 1980;Shinkarev 2004) of the STF-induced variable fluorescence in relation to those of the rate (constants) of Y Z + reduction (k1) and Q A -oxidation (kAB), respectively. For example, if one assumes nFv STF = 2 (Vredenberg et al 2007), k si = k 1 = 10, 3, 1, and 30 ms -1 for OEC in state S = S 1 , S 2 , S 3 and S 4 (0), respectively (Babcock 1987) and kAB oscillating between 4 and 2 ms -1 (Bowes andCrofts 1987, Vredenberg et al 2006) one would get the following oscillating F(t)/F0 pattern 0.9, 0.6, 0.2, and 1.6 for a dark-adapted system with 100% S 1 and no misses and double hits. This pattern with minima at STF number 3, 7, etc.…”

“…1, second row) has been interpreted as evidence that this rise DSQ, is the reflection of the release of donor side quenching (Hiraki et al 2004;Vredenberg 2004). The interpretation of dealing with DSQ that is caused by Y Z + is supported by the observations that the amplitude of STF-and TTF-induced variable fluorescence responses is characterized by a period-of-four oscillation pattern associated with the four-step oxidation of water via the S-states of the OEC that are oxidized by Y Z + (Schreiber and Neubauer 1988;Kolber et al 1998;Shinkarev 2004;Vredenberg et al 2006Vredenberg et al , 2007. However, it is important to keep in mind that it is the rate constant of the release rather than the identity of the quencher associated with DSQ which determines the kinetics in the 0-0.2 ms time range of the OJDIP induction curve.…”

“…These studies among others have yielded information on rate constants and turnover of the primary quinone acceptor Q A of PSII, and on heterogeneity and intersystem energy transfer (connectivity) among PSII units. Application of dedicated new photometric technologies (Schreiber 1986;Kolber et al 1998;Nedbal et al 2000) and of appropriate powerful routines in mathematical software have promoted the possibilities to resolve fluorescence responses in single (STF), twin (TTF), and multi turnover (MTF) excitations with higher time resolution and accuracy (Vredenberg et al 2006(Vredenberg et al , 2007.…”