To address the role of DNA methylation in higher plants, genes encoding cytosine methyltransferases (MTases) in the model plant system Arabidopsis thaliana were cloned by homology and used in a reverse genetic approach to inhibit the process of DNA methylation in planta. MTases were shown to be encoded by a multigene family in Arabidopsis; the Arabidopsis MET1 gene exhibited a high degree of similarity with other known eukaryotic MTase genes. Expression of MET1 as an antisense RNA in transgenic plants resulted in a 34 to 71% reduction in total genomic cytosine methylation in both repetitive DNA and single-copy gene sequences. Demethylation induced developmental effects including altered heterochrony, changes in meristem identity and organ number, and female sterility, and prolonged both vegetative and reproductive phases of development. The late flowering phenotype resulting from demethylation was shown to segregate in MET1 antisense testcross progeny in a dominant fashion independent of the MET1 antisense transgene; late flowering segregated as a single semidominant trait in demethylated lines lacking the transgene. Demethylated plants were late flowering under both long and short day growth conditions, and vernalization did not attenuate the late flowering phenotype. Double mutant analyses between MET1 antisense and lines with mutations in the APETALA1, LEAFY, and TERMINAL FLOWER 1 loci, implicated in the control of inflorescence and floral meristem determinacy, indicated that methylation acts either independently or upstream of TFL1 and LFY, but may serve as a direct regulator of AP1. Considered together, these findings implicate DNA methylation in the establishment or maintenance of epigenetic developmental states in the meristem, and suggest methylation acts within a specific genetic pathway as a key regulator in the process of control of phase transition in higher plants.
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