REPRESSOR OF SILENCING 1 Regulates the Expression of NICOTINAMIDASE 3 in Response to Abscisic Acid in Arabidopsis

DNA methylation is a key epigenetic mark that regulates various biological processes such as gene imprinting, transposon silencing, and X chromosome inactivation. REPRESSOR OF SILENCING 1 (ROS1), a bifunctional DNA glycosylase/lyase that directly removes 5-methylcytosine from DNA in Arabidopsis, can contribute to active DNA demethylation. Here, we showed that the ros1 mutants displayed hypersensitive phenotypes in response to abscisic acid (ABA) during seed germination and root elongation. To better understand the function of ROS1 in regulating the expressions of ABA-responsive genes associated with changes in global DNA methylation patterning, we performed the transcriptome and DNA methylome analyses. We identified a total of 30 commonly downregulated genes in ros1-3 and ros1-4 mutants upon ABA treatment, and nearly 70 percent of these genes were proximal to hypermethylated regions compared with wild type (wt). This result indicates that expressions of some ABA-responsive genes are epigenetically regulated via ROS1-mediated DNA demethylation. Most notable among them is NICOTINAMIDASE 3 (NIC3) which encodes a catalytic enzyme that converts nicotinamide into nicotinic acid. In ros1 mutants, the promoter region of NIC3 was hypermethylated, which resulted in decreased ABA-inducible expression. The nic3 mutants exhibited ABA hypersensitivity, and furthermore, the ABA hypersensitive phenotypes of ros1 mutants were rescued by ectopic expression of NIC3. This finding suggests that NIC3 expression via ROS1-initiated active DNA demethylation is critical for ABA responses, and thereby the availability of nicotinamide can be dynamically regulated under rapid environmental changes.