No abstract

An improved method is introduced for the determination of the quantum yield of photosystem I. The new method employs saturating light pulses with steep rise characteristics to distinguish, in a given physiological state, centers with an open acceptor side from centers with a reduced acceptor side. The latter do not contribute to PSI quantum yield ((I)~). Oxidation of P700 is measured by a rapid modulation technique using the absorbance change around 830 nm. The quantum yield ~1 is calculated from the amplitude of the rapid phase of absorbance change (AA; 830 nm) upon application of a saturation pulse in a given state, divided by the maximal AA (830 nm) which is induced by a saturation pulse with far-red background illumination. Using this technique, q~t can be determined even under conditions of acceptor-side limitation, as for example in the course of a dark-light induction period or after elimination of CO 2 from the gas stream. Thus determined ~ values display a close-to-linear relationship with those for the quantum yield of PSII (q~H) calculated from chlorophyll fluorescence parameters. It is concluded that the proposed method may provide new information on the activity of the PSI acceptot side and thus help to separate the effects of acceptorside limitation from those of cyclic PSI, whenever a nonlinear relationship between q~ll and the P700-reduction level is observed.

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.

Technical features of a novel multi-color pulse amplitude modulation (PAM) chlorophyll fluorometer as well as the applied methodology and some typical examples of its practical application with suspensions of Chlorella vulgaris and Synechocystis PCC 6803 are presented. The multi-color PAM provides six colors of pulse-modulated measuring light (peak-wavelengths at 400, 440, 480, 540, 590, and 625 nm) and six colors of actinic light (AL), peaking at 440, 480, 540, 590, 625 and 420–640 nm (white). The AL can be used for continuous illumination, maximal intensity single-turnover pulses, high intensity multiple-turnover pulses, and saturation pulses. In addition, far-red light (peaking at 725 nm) is provided for preferential excitation of PS I. Analysis of the fast fluorescence rise kinetics in saturating light allows determination of the wavelength- and sample-specific functional absorption cross section of PS II, Sigma(II)λ, with which the PS II turnover rate at a given incident photosynthetically active radiation (PAR) can be calculated. Sigma(II)λ is defined for a quasi-dark reference state, thus differing from σPSII used in limnology and oceanography. Vastly different light response curves for Chlorella are obtained with light of different colors, when the usual PAR-scale is used. Based on Sigma(II)λ the PAR, in units of μmol quanta/(m2 s), can be converted into PAR(II) (in units of PS II effective quanta/s) and a fluorescence-based electron transport rate ETR(II) = PAR(II) · Y(II)/Y(II)max can be defined. ETR(II) in contrast to rel.ETR qualifies for quantifying the absolute rate of electron transport in optically thin suspensions of unicellular algae and cyanobacteria. Plots of ETR(II) versus PAR(II) for Chlorella are almost identical using either 440 or 625 nm light. Photoinhibition data are presented suggesting that a lower value of ETR(II)max with 440 nm possibly reflects photodamage via absorption by the Mn-cluster of the oxygen-evolving complex.