We have tested the hypothesis that the promotion of flowering by prolonged exposure to low temperatures Many plants growing at high latitudes require exposure, as germinating seeds or vegetatively growing plants, to prolonged periods at low temperatures (vernalization) before they will initiate flowering. This ensures that flowering will occur in the warm days of spring and summer, which are favorable for pollination and seed development. Whereas some plants have an absolute requirement for vernalization, others, such as the late-flowering ecotypes of Arabidopsis, show a facultative requirement for vernalization, and will eventually flower even in the absence of a cold treatment (for review see ref. 1). The phenomenon of vernalization was first described in the middle of the 19th century (Kleppart, cited in ref.2), but only recently has a hypothesis suggesting a molecular mechanism for the low-temperature promotion of flowering been proposed (3).The cold treatment is perceived by cells, mitotically active at the time of treatment; early flowering results when these cells, or their descendants, form the floral͞inflorescence meristem, indicating that the vernalization signal is inherited mitotically rather than being transmitted from ''vernalized cells'' to the apex (4, 5). The vernalization signal is not transmitted to sexual progeny (1). The perceived parallels between the inheritance of the vernalization signal and of DNA methylation patterns led to the hypothesis that the vernalization response, in Arabidopsis and other plants, is mediated by changes in DNA methylation (3). Specifically it was proposed that the cold treatment results in demethylation of the promoter region(s) and subsequent activation of a gene or genes critical for initiating reproductive development. Mitotic inheritance of the vernalization signal is consistent with clonal inheritance of DNA methylation patterns (6), but it is now known that, in plants, methylation patterns may not be reset between generations (7-9), suggesting that factors other than DNA methylation may be involved in resetting the vernalization signal.Treatment of plants with the demethylating agent, 5-azacytidine (5-azaC) resulted in early flowering (3, 10). This earlyflowering response was restricted to plants that normally respond to vernalization, including certain late-flowering ecotypes and mutants of Arabidopsis and winter wheat. Spring wheats and other late-flowering Arabidopsis mutants that are insensitive to vernalization did not develop an early-flowering phenotype after 5-azaC treatment. These observations suggest that this response was specific to the vernalization-dependent pathway to flowering (3, 10, 11) rather than being a nonspecific effect of the treatment.In addition to causing demethylation, 5-azaC is a general inhibitor of transcription (12), so it is possible that the promotion of flowering by 5-azaC resulted from effects other than demethylation of DNA. To discriminate between these possibilities we have used Arabidopsis plants in which meth...