DNA hypomethylation enables the transcriptional repressor SlSPL-CNR to control fruit flavor ester biosynthesis.

Reduction of DNA methylation has traditionally been associated with gene activation. Here, we show that DNA hypomethylation permits the binding of a transcriptional repressor, leading to gene silencing. In tomato, the SQUAMOSA PROMOTER BINDING PROTEIN-LIKE TF SlSPL-CNR exhibits methylation-sensitive DNA binding and preferentially occupies unmethylated GTACGG motifs. During fruit ripening, DEMETER-LIKE 2 (SlDML2)-mediated DNA demethylation at the alcohol acyltransferase 1 (SlAAT1) promoter allows SlSPL-CNR binding, which in turn represses SlAAT1 expression and thereby modulates the biosynthesis of ester metabolites-key components of fruit flavor. Structural analysis reveals that cytosine methylation introduces a steric clash with Gln94 in the SBP domain of SlSPL-CNR, explaining its methylation sensitivity. CRISPR knockout of SlSPL-CNR de-represses SlAAT1 and increases ester accumulation, confirming its inhibitory role. Importantly, this methylation-sensitive binding is conserved across SBP domain proteins from rice, maize, and tomato. Our findings reveal a mechanism in which DNA hypomethylation facilitates repressor recruitment, establishing a regulatory logic linking epigenetic dynamics to metabolic control in plants.