Anthocyanins are the primary determinants of floral pigmentation in Chrysanthemum ×morifolium, and ethylene acts as a key regulator of their biosynthesis. Although the ethylene-mediated regulatory circuitry is functionally important, its underlying mechanism in chrysanthemum has remained unclear. In this study, we identified CmMYB4 as a transcriptional repressor that directly suppresses the expression of key anthocyanin biosynthetic genes, including CmDFR (dihydroflavonol 4-reductase), CmUFGT (flavonoid 3-O-glucosyltransferase), and Cm3MaT (anthocyanin 3-O-glucoside-6″-O-malonyltransferase). Time-ordered gene co-expression network analysis comparing the transcriptomes of CmMYB4-overexpressing and control plants, together with molecular biology experimental results, further revealed CmERF3 (ethylene response factor 3) as a hierarchical upstream regulator of anthocyanin biosynthesis. Exogenous ethylene treatment induced CmERF3 expression while reducing anthocyanin accumulation. Subsequent functional characterization showed that the overexpression of CmERF3 suppresses anthocyanin biosynthesis in both tobacco and chrysanthemum by directly activating CmMYB4 and repressing CmDFR, CmUFGT, and Cm3MaT. Collectively, these findings revealed that ethylene inhibits anthocyanin biosynthesis through a CmERF3−CmMYB4−LBGs (late biosynthetic genes) regulatory module. This study not only elucidates the molecular mechanism governing ethylene-mediated anthocyanin inhibition but also provides new perspectives for the molecular engineering of ornamental traits in chrysanthemum.

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