Cytosine methylation of the FWA promoter promotes direct in vitro shoot regeneration in Arabidopsis thaliana

ABSTRACT Epigenetic modifications within promoter sequences can act as regulators of gene expression. Shoot regeneration is influenced by both DNA methylation and histone methylation, but the mechanistic basis of this regulation is obscure. Here, we identified 218 genes related to the regeneration capacity of callus that were differentially transcribed between regenerable calli (RC) and non‐regenerable calli (NRC) in Arabidopsis thaliana. An analysis of the promoters of five of the differentially expressed genes (FWA, ACC1, TFL1, MAX3, and GRP3) pointed to an inverse relationship between cytosine methylation and transcription. The FWA promoter was demethylated and highly expressed in NRC, whereas it was methylated and expressed at low levels in RC. Explants of the hypomethylation mutants fwa‐1 and fwa‐2 showed strong levels of FWA expression and regenerated less readily than the wild type, suggesting that FWA inhibits direct in vitro shoot regeneration. WUSCHEL‐RELATED HOMEOBOX 9 (WOX9), which is required for shoot apical meristem formation, was directly repressed by FWA. Overexpressing WOX9 partly rescued the shoot regeneration defect of fwa‐2 plants. These findings suggest that cytosine methylation of the FWA promoter forms part of the regulatory system governing callus regenerability and direct in vitro shoot regeneration. DNA methylation influences shoot regeneration and methylation of the Arabidopsis FLOWERING WAGENINGEN promoter represses its expression, leading to higher expression of WUSCHELRELATED HOMEOBOX 9 and promotion of shoot regeneration, revealing a methylationbased regulatory pathway controlling shoot regeneration.