5-azacytidine-removal of the dark repression in plastid development of Euglena gracilis Klebs

Asynchronous, etiolated cells of Euglena gracilis, a light-dependent organism for plastid differentiation, were grown in complete darkness for 72 h in the presence or absence of 5 μg/mL 5-azaC, an inhibitor of DNA methylation. Plastid differentiation was followed by fluorescence and electron microscopy while the formation of Chl(ide) was ascertained by fluorimetric and HPLC analyses. It was found that in the presence of the compound, 20–40 % of the cells was induced to form differentiated plastids, with kinetics greatly comparable to that of the wild-type dark-grown Euglena exposed to light. Based on the immediate or light-induced red-fluorescence of the organelles viewed under UV light it was established that in about 10 % of the mentioned cells plastidogenesis reached the stage of Chl(ide) synthesis whereas in the other ones only the Chl(ide) precursors formed and a brief photoinduction was necessary for their assemblage. The removal of the dark repression of plastidogenesis operated by 5-azaC points to an involvement of DNA de(hypo)methylation in the expression of the genes responsible for plastid development as it occurs in other biological processes in both animals and plants. For the first time, Euglena gracilis was induced to form differentiated plastids in darkness.