Room temperature microspectrofluorimetry as a useful tool for studying the assembly of the PSII chlorophyll-protein complexes in single living cells of etiolated Euglena gracilis Klebs during the greening process

The assembly kinetics of the PSII chlorophyll-protein complexes was followed during the greening of Euglena gracilis by microspectrofluorimetry in vivo, at room temperature, on single living cells. The study was correlated to micro- and submicroscopic events accompanying the proplastid to chloroplast transformation and with the immunolocalization of the LHCPII. Etiolated cells of Euglena gracilis were grown in darkness in Mego’s heterotrophic liquid medium under shaking at 25±1 °C. At the stationary phase of growth, they were exposed to continuous light (330 µmol m-2 s-1) for 72 h. The analyses were carried out on samples collected at different times of illumination. Microspectrofluorimetric data were recorded in the 620-780 nm range (excitation at 436 nm) and were resolved into Gaussian components corresponding to the reaction centres (RCII) and the inner antennae (CP43-47) of the PSII and LHCPII. From the RCII/CP43-47 and LHCPII/PSII ratios, it was inferred that (1) a disconnection between RCII and CP43-47 syntheses occurs during the lag phase of chloroplast differentiation, RCII being synthesized before the inner antennae. This results in the accumulation of uncoupled PSII Chl-protein complexes; (2) after lag phase, the RCII and CP43-47 syntheses are connected one to another; (3) the freshly synthesized LHCPII complexes are immediately assembled with the PSII, suggesting that the outer antennae always maintain the form bound to PSII. Micro- and submicroscopical observations and LHCPII immunolocalization were in agreement. These data suggest that microspectrofluorimetry may constitute a useful non-destructive tool for studying the assembly kinetics of PSII, under fully physiological life conditions.