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

Abstract

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.

Transcript level coordination of carbon pathways during silicon starvation‐induced lipid accumulation in the diatom Thalassiosira pseudonana

“…Parameters investigated were genome‐wide transcript abundance (using both microarrays and RNA‐Seq), cell cycle progression and growth, lipid content (lipophilic dyes and fatty acid methyl ester analysis), pigment concentrations, shifts in photophysiology (photosynthesis–irradiance ( P – I ) curves and fast repetition rate fluorometry), and chloroplast features using imaging flow cytometry. Details of these methods in addition to which biochemical and physiological parameters were evaluated for a given experiment can be found in the supporting information (Supporting Information Methods S1; Table S1; Folch et al ., 1957; Platt et al ., 1975; Lewis & Smith, 1983; Benjamini & Hochberg, 1995; Kolber et al ., 1998; Arrigo et al ., 1999; Hildebrand & Dahlin, 2000; Zapata et al ., 2000; Dodds et al ., 2005; Shrestha et al ., 2012; Kim et al ., 2013; Love et al ., 2014). …”

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

“…Other electron transport intermediates at the acceptor and donor sides of PSII have been proposed as additional functional quenchers like Phe (Phe) (Klimov et al 1977;Vredenberg 2000), reduced forms of the secondary quinone acceptor Q B - (Samson et al 1999;Yaakoubd et al 2002, Schreiber 2002 and Q B 2- (Zhu et al 2005), plastoquinone (PQ) (Vernotte et al 1979), oxidized primary (P 680 + ) (Butler 1972), and secondary donor (Y z + ) (Vredenberg et al 2001) or side products like triplet carotenoids (car T ) (Steffen et al 2001). Their quenching has been discussed in relation to the seeming paradox that the release of fluorescence quenching F v in a single turnover flash (STF) in which all Q A is reduced, is substantially below that measured in multi-turnover light pulses (MTF) (Kramer et al 1995;Samson and Bruce 1996;Kolber et al 1998;Vasilev and Bruce 1998;Samson et al 1999;Koblizek et al 2001;Vredenberg et al 2005). The documented effect of electric fields on the fluorescence yield (Vos et al 1991;van Gorkom 1996;Vredenberg 2004) is assumed to be comparatively small in the time domain <2 ms, and is not considered here.…”

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

“…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.…”

“…In general, Fv/Fm-values around 0.8 (Fm/ Fo * 5) are considered to be representative of high performance and competence of the PSII machinery in leaves, green cells, and isolated chloroplasts. The availability of detection methods with improved sensitivity and time resolution has greatly contributed to identification of fluorescence characteristics and parameters (Schreiber 1983(Schreiber , 1986Bolhar-Nordenkampf et al 1989;Renger et al 1995;Schreiber et al 1995;Strasser et al 1995;Reifarth et al 1997;Kolber et al 1998;Nedbal et al 1999Nedbal et al , 2000. Estimation of initial fluorescence yield and of rise kinetics with much higher precision and time resolution has become possible.…”

“…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.…”