The general principles involved in chlorophyll fluorescence quenching analysis by the saturation pulse method are presented, outlining the rationale for using the empirical fluorescence parameters Fv/Fm and Fv/Fm' as indices for the photosystem II (PSII) photochemical quantum yield, ΦII, in the dark-adapted or illuminated states, respectively. The relationship between ΦII and the quantum yield of photosynthetic electron transport is linear over a wide range of quantum flux densities. However, there is a fraction of PSII contributing approximately 30% to maximal quantum yield, which is closed at rather low quantum flux densities, while at the same time there is only a small drop in ΔF/Fm'. The details of Fm and Fm' determination by application of saturating light are critically examined, with emphasis on the situation in algae where the fluorescence rise to the peak leLel is followed by a rapid decline. For this purpose, the rapid induction kinetics upon onset of strong continuous illumination are investigated. Dark-adapted samples show two distinct intermediate fluorescence levels, I1 and I2, in the polyphasic rise from the O to the P level. The I1 level separates a biphasic 'photochemical' rise, which also can be induced by a saturating single turnover flash, from several 'thermal' phases, induction of which requires multiple turnovers at PSII. Arguments are put forward favouring the I2 level for assessment of Fm or Fm', on which calculation of Fv/Fm or ΔF/Fm' is based. It is shown that although an assessment based on the I1 level, as practised by the so-called pump- and-probe method, does lead to a consistent underestimation of ΔF/Fm, in many cases similar information as with I2 determination is obtained.

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The relationship between the empirical fluorescence index ΔF/Fm' and the quantum yield of linear electron flow, Φ(s), was investigated in isolated spinach thylakoids. Conditions were optimised for reliable determination of ΔF/Fm' and Φ(s) with methyl viologen or ferricyanide as electron acceptors under coupled and uncoupled conditions. Ascorbate in combination with methyl viologen was found to stimulate light-induced O2-uptake which is not reflected in ΔF/Fm' and interpreted to reflect superoxide reduction by ascorbate. In the absence of ascorbate, the plot of ΔF/Fm' vs. Φ(s) was mostly linear, except for the range of high quantum yields, i.e. at rather low photon flux densities. With ferricyanide as acceptor, use of relatively low concentrations (0.1-0.3 mM) was essential for correct Fm'-determinations, particularly under uncoupled conditions. Under coupled and uncoupled conditions the same basic relationship between ΔF/Fm' and Φ(s) was observed, irrespective of Φ(s) being decreased by increasing light intensity or by DCMU-addition. The plots obtained with methyl viologen and ferricyanide as acceptors were almost identical and similar to corresponding plots reported previously by other researchers for intact leaves. It is concluded that the index ΔF/Fm' can be used with isolated chloroplasts for characterisation of such types of electron flow which are difficult to assess otherwise, as e.g. O2 dependent flux. The origin of the 'non-linear' part of the relationship is discussed. An involvement of 'inactive' PS II centers with separate units and inefficient QA-QB electron transfer is considered likely.

The pH-dependence of light-driven O2-reduction in intact spinach chloroplasts is studied by means of chlorophyll fluorescence quenching analysis and polarographic O2-uptake measurements. Most experiments are carried out in presence of KCN, which blocks activities of Calvin cycle, ascorbate peroxidase and superoxide dismutase. pH is varied by equilibration with external buffers in presence of nigericin. Vastly different pH-optima for O2-dependent electron flow are observed in the presence and absence of the redox catalyst methyl viologen. Both fluorescence quenching analysis and O2-uptake reveal a distinct pH 5 optimum of O2-reduction in the absence of methyl viologen. In the presence of this catalyst, O2-reduction is favoured in the alkaline region, with an optimum around pH 8, similar to other types of Hill reaction. It is suggested that in the absence of methyl viologen the extent of irreversibility of O2-reduction is determined by the rate of superoxide protonation. This implies that O2-reduction takes place within the aprotic phase of the thylakoid membrane and that superoxide-reoxidation via oxidized PS I donors competes with protonation. Superoxide protonation is proposed to occur at the internal surface of the thylakoid membrane. There is no competition between superoxide reoxidation and protonation when in the presence of methyl viologen the site of O2-reduction is shifted into the protic stroma phase. In confirmation of this interpretation, fluorescence measurements in the absence of KCN reveal, that non-catalysed O2-dependent electron flow is unique in beingstimulated by the transthylakoidal pH-gradient. On the basis of these findings a major regulatory role of O2-dependent electron flow under excess light conditions is postulated.