Excitation and emission wavelength dependence of fluorescence spectra in whole cells of the cyanobacterium Synechocystis sp. PPC6803: Influence on the estimation of Photosystem II maximal quantum efficiency.

The fluorescence emission spectrum of Synechocystis sp. PPC6803 cells, at room temperature, displays: i) significant bandshape variations when collected under open (F) and closed (F) Photosystem II reaction centre conditions; ii) a marked dependence on the excitation wavelength both under F and F conditions, due to the enhancement of phycobilisomes (PBS) emission upon their direct excitation. As a consequence: iii) the ratio of the variable and maximal fluorescence (F/F), that is a commonly employed indicator of the maximal photochemical quantum efficiency of PSII (Φ), displays a significant dependency on both the excitation and the emission (detection) wavelength; iv) the F/F excitation/emission wavelength dependency is due, primarily, to the overlap of PSII emission with that of supercomplexes showing negligible changes in quantum yield upon trap closure, i.e. PSI and a PBS fraction which is incapable to transfer the excitation energy efficiently to core complexes. v) The contribution to the cellular emission and the relative absorption-cross section of PSII, PSI and uncoupled PBS are extracted using a spectral decomposition strategy. It is concluded that vi) Φ is generally underestimated from the F/F measurements in this organism and, the degree of the estimation bias, which can exceed 50%, depends on the measurement conditions. Spectral modelling based on the decomposed emission/cross-section profiles were extended to other processes typically monitored from steady-state fluorescence measurements, in the presence of an actinic illumination, in particular non-photochemical quenching. It is suggested that vii) the quenching extent is generally underestimated in analogy to F/F but that viii) the location of quenching sites can be discriminated based on the combined excitation/emission spectral analysis.