Methyl salicylate-induced CitERF92 promotes the accumulation of PMFs in citrus

Polymethoxylated flavones (PMFs) are a class of flavonoid compounds predominantly present in citrus and are renowned for their extensive biological activity (antiobesity, antioxidant and cardiovascular protection). Recent advancements have elucidated the biosynthetic pathway of PMFs, although the transcriptional regulatory mechanisms involved are not yet fully understood. In this study, we used PMF-rich Bingtangcheng ( Citrus sinensis Osbeck) fruit (BTC) and treated it with exogenous methyl salicylate (MeSA). Our study demonstrates that MeSA treatment dynamically regulates the biosynthesis of PMF monomers in citrus peels during low-temperature storage (10 °C), with 0.2 mmol · L -1 MeSA combined with 14-day storage effectively enhancing PMF accumulation. The transcriptomic analyses demonstrated that MeSA treatment activated the expression of essential genes associated with the PMF biosynthetic pathway, including FNSII , F3'H , and COMT . Additionally, MeSA also activated the biosynthesis and signaling pathways of other phytohormones. A transcription factor, CitERF92 , from the ERF family B3 group, was significantly induced by MeSA and strongly correlated with the expression of CitFNSII-1 . Dual-luciferase assays and electrophoretic mobility shift assays demonstrated that CitERF92 could bind to and significantly activate the promoter activity of CitFNSII-1 . Subsequent transient overexpression experiments confirmed that CitERF92 markedly increased the content of PMF monomers in the BTC peel. These results reveal the CitERF92-CitFNSII-1 module as a novel regulator of PMF biosynthesis, enhancing the understanding of the regulatory network that controls PMF production in citrus.