Effect of 5-azacytidine treatment on germination and Mutator transposable element activity --Sally Otto and Virginia Walbot Seeds were imbibed in water, 1 mM or 10 mM 5-azacytidine. In animals, this nucleoside analog inhibits DNA methylation, but its effects in plant cells are not known. We found that seed germination was reduced by approximately 50% in the 10 mM 5-azacytidine treatment:

Water Control         1 mM         10 mM
    91.7                         91.7             49.1

Because germination does not require DNA or RNA synthesis, this finding suggests that 5-azacytidine acts as a metabolic poison in a pathway outside of macromolecular nucleic acid synthesis. Interestingly, once germination was complete, plant viability, morphology and fertility were unaffected by the 5-azacytidine seed treatment. We had hypothesized that this analog might result in novel morphology by causing demethylation of the genome. Brown et al. (Theor. Appl. Genet. 78:321, 1989) recently found that 5-azacytidine treatment also had remarkably few effects in plant tissue cultures.

We also tested whether 5-azacytidine affected the level of activity of Mutator lines carrying the bz2-mu1 reporter allele by scoring somatic instability in the progeny of the treated seed. Active lines were not stimulated to a significant extent, and inactive lines were not reactivated by analog treatment. Weakly active materials -- those with only a few spots per kernel and a lower than expected frequency of spotted kernels per ear--showed a dose-dependent rate of reactivation after analog treatment. In the water control 0.64% progeny kernels were spotted, while 1.77% of progeny kernels were spotted in the 1 mM 5-azacytidine treatment group, and 2.16% of progeny kernels were spotted from the 10 mM treatment group. This dependence of Mutator reactivation on 5-azacytidine treatment was significant at p<0.05, but not dramatic. Similarly, Martin et al. recently reported (EMBO J. 8:997, 1989) that Tam3 activity is only rarely modulated by 5-azacytidine treatment.

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