Adaptive modifications of the photosynthetic apparatus in Euglena gracilis Klebs exposed to manganese excess

Asynchronous cultures of wild-type Euglena gracilis were tested for their morphophysiological response to 10 mM MnSO4. Growth was only moderately slowed (15%), while oxygen evolution was never compromised. Inductively coupled plasma analyses indicated that the Mn cell content doubled with respect to controls, but no signs of localised accumulation were detected with X-ray microanalysis. Evident morphological alterations were found at the plastid level with transmission electron microscopy and confocal laser scanning microscopy. An increase in the plastid mass, accompanied by frequent aberrations of chloroplast shape and of the organisation of the thylakoid system, was observed. These aspects paralleled a decrease in the molar ratio of chlorophyll a to b and an increase in the fluorescence emission ratio of light-harvesting complex II to photosystem II, the latter evaluated by in vivo single-cell microspectrofluorimetry. These changes were observed between 24 and 72 h of treatment. However, the alterations in the pigment pattern and photosystem II fluorescence were no longer observed after 96 h of Mn exposure, notwithstanding the maintenance of the large plastid mass. The response of the photosynthetic apparatus probably allows the alga to limit the photooxidative damage linked to the inappropriately large peripheral antennae of photosystem II. On the whole, the resistance of Euglena gracilis to Mn may be due to an exclusion-tolerance mechanism since most Mn is excluded from the cell, and the small amount entering the organism is tolerated by means of morphophysiological adaptation strategies, mainly acting at the plastid level

Ageing of Euglena chloroplasts in vitro I. Variations in pigment pattern and in morphology

Isolated chloroplasts of Euglena gracilis Klebs were kept for 10 d in complete darkness at 4° C in a maintenance buffer (pH 7.5) without shaking. During incubation, the qualitative and quantitative changes in the pattern of photosynthetic pigments were evaluated by the combined use of spectrophotometry in the visible range of whole chloroplasts and their acetone extracts, of in vivo spectrofluorimetry and of reversed-phase HPLC. Microscopic and submicroscopic modifications were also followed by UV and transmission electron microscopy. The main findings were as follows: (1) a fast decay of all photosynthetic pigments, chiefly chlorophylls, not accompanied by evident signs of alteration of the thylakoid system during the first 5 d; (2) a higher stability of PSII compared to PSI and of antenna complexes compared to the relative reaction centres during the first 24-48 h; (3) a low accumulation of phaeoderivative compounds in spite of the marked decrease of chlorophyll content; (4) a lack of dephytylated compounds; (5) a quicker decay of the intensity of fluorescence emission with respect to the decreasing chlorophyll a content; and (6) a fast degradation of xanthophylls and ß-carotene with the consequent lack of defence from the ageing oxidative stresses. This accounts for the rapid loss of pigments, although the lack of other antioxidant defence mechanisms is not excluded. The characterization of some of the steps involved in plastid degradation may render this experimental model viable for further studies on plastid senescence, a multifactorial process still awaiting definite answers

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