Investigations on the Reaction Pattern of Photosystem II in Leaves fromArabidopsis thalianaby Time-Resolved Fluorometric Analysis

Abstract

The transients of normalized fluorescence yield induced by an actinic laser flash in dark adapted leaves of Arabidopsis thaliana plants were measured with new equipment, that was developed as part of this work and permits the covarage of a wide time domain of 8 decades from 100 ns to 10 s. The raw data obtained were processed and analyzed within the framework of the "3-quencher" model with Q(A) as photochemical and P680(+)(*) and (3)Car as nonphotochemical quenchers. Comparative measurements with hydroxylamine treated PS II membrane fragments from spinach revealed that the widely used "dogma"of virtually identical efficiency of photochemical (Q(A)) and nonphotochemical (P680(+)(*)) quenching has to be revised: the constant of the latter exceeds that of the former by a factor of about 2. As a consequence, the probability of recombination between P680(+)(*) and Q(A)(-) and its kinetics have to be explicitly taken into account for the interpretation of flash induced fluorescence yield transients. The analysis of the experimental data within this extended "3-quencher" model reveals that a fully consistent description is achieved for the data gathered from measurements with intact leaves from wild type plants excited with actinic laser flashes of different energies (number of photons per flash and unit area). On the basis of these results it is shown that, in dark adapted leaves excited with a single laser flash, P680(+)(*) is predominantly (about 80% of the total reaction) reduced by Y(Z) via nanosecond kinetics and Q(A)(-) reoxidation is dominated by a kinetics of about 150 mus that are ascribed to PS II complexes with the Q(B) site occupied by PQ. The excess of excited chlorophyll singlet states decays to a significant extent via the carotenoid "triplet valve"with transient population of (3)Car. The present data provide the basis for analyses of A. thaliana mutants with modified lipid content and composition. The results of these investigations are described in an accompanying report (Steffen, R., Kelly, A. A., Huyer, J., Dormann, P., and Renger, G. (2005) Investigations on the reaction pattern of photosystem II in leaves from Arabidopsis thaliana wild type plants and mutants with genetically modified lipid content, Biochemistry 44, 3134-3142).

PS II model-based simulations of single turnover flash-induced transients of fluorescence yield monitored within the time domain of 100 ns–10 s on dark-adapted Chlorella pyrenoidosa cells

“…Special experimental setups are used to monitor PF(t) and FI(t). The time course of PF(t) is mostly determined by using the technique of time-correlated single photon counting (TCSPC) (Schatz et al 1988;Roelofs et al 1992;Renger et al 1995;Vasil'ev et al 1996;Bergmann et al 1998) whereas a great variety of methods is applied for monitoring FI(t): samples are excited either by continuous light at different intensities (Ireland et al 1984;Renger and Schulze 1985;Bulychev et al 1987;Neubauer and Schreiber 1987;Baake and Shloeder 1992;Strasser et al 1995Strasser et al , 2004Bulychev and Vredenberg 2001;Chemeris et al 2004;Lazar 2006) or by light pulses of different duration and intensities (Schreiber et al 1986;Schreiber and Krieger 1996;Christen et al 1999Christen et al , 2000Goh et al 1999;Steffen et al 2001Steffen et al , 2005a, with or without the background of continuous light.…”

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

“…5 and 6), a sigmoidal rise of the MTF-induced fluorescence response curve. The sigmoidicity arises from the fact that the variable fluorescence Fv is controlled by the rate constant of light excitation k L and of release of donor side quenching k s 1 : Characteristics of donor side quenching can be read from the rise and decay kinetics of quenching release in ultra short single turnover excitations (Butler 1972;Mauzerall 1972;Steffen 2003;Steffen et al 2005;Belyaeva et al 2006). The 'degree' of sigmoidicity of F(t) is determined by the ratio between k L and k s 1 (Figs.…”

“…This increase has been ascribed to the release of photochemical quenching by Q A due to its reduction to Q A - (Duysens and Sweers 1963). The kinetics of the ns-STF-induced variable fluorescence in intact cells and chloroplasts have shown a biphasic rise in the 0.1-100 ls and a multiphasic recovery in the 0.05-10 4 ms time range, respectively (Mauzerall 1972;Steffen et al 2005;Belyaeva et al 2006). The kinetic pattern of the recovery phase is similar to that of STF-and TTF-induced responses (Nedbal et al 1999;Vredenberg et al 2006; with a major fast phase in the 0.05-1 ms time range ascribed to quenching recovery in association with Q A -reoxidation in Q B reducing RCs.…”

“…If it is assumed, following evidence derived from ns laser-induced F(t) kinetics discussed earlier, that the fluorescence yield in y 1 (= [Y Z + P Phe Q A -]) does not differ much (Steffen et al 2005), if at all, from that of y 0 due to effective DSQ, one expects that the relative fluorescence induction curve rFv(t)(= [F(t) -Fo]/(Fm -Fo)) will coincide with y 2 (t), i.e., rFv(t) = y 2 (t). It is obvious from Eq.…”

Fluorescence Emission from the Photosynthetic Apparatus

“…Further, the measured F o is not a constant and it in no way can faithfully stand for the low limit of PS II Chl a fluorescence because, in addition to fluorescence emitted by active PS II complexes, F o includes emissions from inactive (or Q B -nonreducing) PS II complexes (Zhu et al, 2005;Vredenberg, 2008) and from PS I (Papageorgiou, 1975;Briantais et al, 1986;Pfündel, 1998;Gitelson et al, 1999;Peterson et al, 2001;Schreiber, 2004;Steffen et al, 2005). Other causes that do affect the measured value of F o include: (i) de-quenching because of dark reduction of Q A and the PQ pool by metabolites Briantais et al, 1986;Haldimann and Tsimilli-Michael, 2005;Hohmann-Marriott et al, 2010) and the attendant fluorescence lowering by the state 1 → 2 transition (Malkin et al, 1980); (ii) quenching by transmembrane electrochemical gradients (DpH + DY; Papageorgiou and Govindjee, 1967;Krause et al, 1982;Kramer et al, 2004;Krause and Jahns, 2004;Vredenberg, 2004;Vredenberg and Prasil, 2009); (iii) Metal ion-effected redistribution of EE between photosystems (Murata, 1969); (iv) Quenching by zeaxanthin (xanthophyll cycle; Gilmore et al, 1998;Golan et al, 2004;Holt et al, 2004), as well as by lutein (see e.g., Matsubara et al, 2011), and (v) unregulated quenching by molecular oxygen (Papageorgiou et al, 1972).…”