No abstract

Recent progress in chlorophyll fluorescence research is reviewed, with emphasis on separation of photochemical and non-photochemical quenching coefficients (qP and qN) by the 'saturation pulse method'. This is part of an introductory talk at the Wageningen Meeting on 'The use of chlorophyll fluorescence and other non-invasive techniques in plant stress physiology'. The sequence of events is investigated which leads to down-regulation of PS II quantum yield in vivo, expressed in formation of qN. The role of O2-dependent electron flow for ΔpH- and qN-formation is emphasized. Previous conclusions on the rate of 'pseudocyclic' transport are re-evaluated in view of high ascorbate peroxidase activity observed in intact chloroplasts. It is proposed that the combined Mehler-Peroxidase reaction is responsible for most of the qN developed when CO2-assimilation is limited. Dithiothreitol is shown to inhibit part of qN-formation as well as peroxidase-induced electron flow. As to the actual mechanism of non-photochemical quenching, it is demonstrated that quenching is favored by treatments which slow down reactions at the PS II donor side. The same treatments are shown to stimulate charge recombination, as measured via 50 μs luminescence. It is suggested that also in vivo internal thylakoid acidification leads to stimulation of charge recombination, although on a more rapid time scale. A unifying model is proposed, incorporating reaction center and antenna quenching, with primary control of ΔpH at the PS II reaction center, involving radical pair spin transition and charge recombination to the triplet state in a first quenching step. In a second step, triplet excitation is trapped by zeaxanthin (if present) which in its triplet excited state causes additional quenching of singlet excited chlorophyll.

A new measuring system for monitoring absorbance changes around 830 n m is described, which was developed by modification of a commercially available pulse modulation fluorometer. All modifications concern the emitter-detector unit of the fluorometer, such that only this unit needs to be exchanged when changing from fluorescence to absorbance measurements and vice versa. The new system is shown to be well-suited for measuring redox changes of P700, the reaction center of photosystem I, in intact leaves and isolated chloroplasts. The observed kinetic changes at 830 nm in response to single turnover or multiple turnover saturating flashes are practically identical to those previously measured around 700 nm . The signal/noise ratio is sufficiently high to give well-resolved kinetics without signal averaging. W h e n P700 is oxidized by far-red background light, valuable information on the state of the intersystem electron transport chain is given by the re-reduction kinetics induced by single or multiple turnover saturating flashes. Such measurements are facilitated by the use of poly-furcated fiberoptics. With intact leaves, almost identical responses are found when measuring through the leaf (transmission mode) or from the leaf surface (remission mode). Modulated chlorophyll fluorescence can be measured in parallel; application of saturation pulses for fluorescence quenching analysis produces transient P700 oxidation without oversaturating the measuring system. The information on the P700 redox state complements that obtained from fluorescence measurements, yielding a new practical tool in plant physiological research.

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.

The fluorescence rise kinetics in saturating light display two well separated components with largely different properties. The rapid rise from F0 to a first intermediate level, I1 is photochemically controlled, while the following phases leading to a secondary intermediate level, I2 and to a peak level, P, are limited by thermal reactions. Treatments which primarily affect components at the photosystem II donor side are shown to increase quenching at I1 and/or to suppress the secondary fluorescence rise to I2. Preillumination by single turnover saturating flashes causes I1- quenching oscillating with period-4 in dependence of flash number. It is suggested that this quenching correlates with (S2 + S3) states of the watersplitting enzyme system. Suppression of the secondary, I1 - I2 rise component is invariably found with treatments which lower electron donation rate by the watersplitting system and are known to favor the low potential form of cyt b 559. Three different mechanisms are discussed on the basis of which donor-side dependent quench­ing could be interpreted: 1) Non-photochemical quenching by accumulation of the P 680+ radical cation. 2) Dissipative photochemical quenching at a special population of PS II centers (β- or non- B centers) displaying low donor capacity and high rates of charge recombination. 3) Dissipative photochemical quenching via cyclic electron flow around PS II, involving alternate donors to P 680+ (like cyt b 559 or carotenoid in their low potential forms), which can compete when donation rate from the water splitting system is slowed down. The possibility of donor-side limitation also being involved in “energy dependent” quenching is discussed.