QUENCHING ANALYSIS OF CHLOROPHYLL FLUORESCENCE BY THE SATURATION PULSE METHOD - PARTICULAR ASPECTS RELATING TO THE STUDY OF EUKARYOTIC ALGAE AND CYANOBACTERIA

The principles of chlorophyll fluorescence quenching analysis by the saturation pulse method are outlined with emphasis on particular aspects encountered in the study of eukaryotic algae and cyanobacteria. Major differences of these photosynthetic organisms with respect to higher plant leaves, for which quenching analysis originally was developed, are very rapid induction of O-2-dependent electron flow, close interaction between photosynthetic and respiratory metabolism, dark reduction of the plastoquinone pool and pronounced state transitions of energy distribution between the two photosystems. It is shown that the use of 25-50 ms pulses of saturating light for determination of maximal fluorescence yield is advantageous, in contrast to the 0.5-2 s pulses commonly used with higher plants. The shorter pulses are less invasive with respect to the induction of energising electron flow which can induce non-photochemical quenching and state changes. In particular, short saturation pulses are essential to study true dark changes of fluorescence yield. As an example, the induction of pronounced quenching of maximal fluorescence in Chlamydomonas by dark-anaerobic incubation is demonstrated. Analysis of the rapid rise kinetics upon onset of saturating light reveals two major phases, O-I-1 and I-1-I-2, with distinctly different properties. Arguments are put forward that an assessment of maximal fluorescence yield with single turnover saturating flashes is problematic, as there is a type of photochemical quenching, the elimination of which during the I-1-I-2 phase requires multiple turnovers at photosystem II. Furthermore, the variable fluorescence represented by the two phases is affected differently by non-photochemical quenching. It is shown that dark-anaerobic quenching in Chlamydomonas as well as state 2 quenching in Synechocystis are correlated with a preferential suppression of the I-1-I-2 phase. Experiments with Synechocystis are presented which demonstrate the potential of saturation pulse quenching analysis for the study of reversible state changes. The mutant M55 of Synechocystis 6803 appears to be ''locked'' in state 1.