Deregulation of light-induced plastidogenesis in etiolated Euglena gracilis klebs treated with DNA hypermethylating 3 '-azido-3 '-deoxythymidine

The effect of the DNA hypermethylating 3'-azido-3'-deoxythymidine (AZT) on plastidogenesis was investigated on asynchronous, etiolated Euglena gracili s exposed to continuous light for 72 h. Based on (i) fluorescence and elect ron microscope aspects of the plastids, (ii) cytochemical localizations of functional PSI and PSII, (iii) immunolocalization of LHCII apoproteins, and (iv) spectrophotometric and HPLC analyses of the photosynthetic pigments, it was found that AZT, at a dose of 5 muM which does not encroach on cell v iability and only slightly interferes with cell proliferation, causes profo und alterations of plastids in 10 - 15% of the cells, without apparently af fecting the rest of the cell population. Proplastid-to-chloroplast transfor mation slowed in this cell sub-population and occurred without the normal c o-regulation that characterizes the process in wild type Euglena. In partic ular, thylakoid assembly was partially prevented while building materials c ontinued to be synthesized and accumulated in the stroma. Chlorophylls were over-synthesized, while carotenoids, phaeophytin and phylloquinone dramati cally diminished. The assemblage of PSI and PSII was inhibited. The externa l envelopes and thylakoid membranes were frequently damaged. Plastids, whos e total volume increased with respect to the controls, were often malformed and interconnected. Their fluorescence under UV light declined quickly. Th e fact that AZT acts specifically at the plastid level, causing effects tha t are greatly opposed to those previously observed in the alga treated with the DNA demethylating 5-azacytidine, points to a relationship between DNA methylation and plastid differentiation in Euglena.