MiR399d and epigenetic modification comodulate anthocyanin accumulation in Malus leaves suffering from phosphorus deficiency

Inorganic phosphorus (Pi) deficiency induces anthocyanin accumulation in the leaves of some plant species; however, the molecular mechanisms underlying this phenomenon have not been well characterized. Here, we showed that microRNA399d (miR399d), high-affinity Pi transporter McPHT1;4, and McMYB10 are strongly induced in Malus leaves suffering from Pi deficiency. By culturing explants of transiently transformed plants in MS medium under conditions of Pi sufficiency and Pi deficiency, miR399d and McPHT1;4 were shown to play essential roles in the response to Pi deficiency and to play positive roles in the regulation of anthocyanin biosynthesis. Silencing of McHDA6 expression and treatment with the inhibitor trichostatin A suggested that the low expression of McHDA6 simultaneously reduced the transcription of McMET1 and decreased the methylation level of the McMYB10 promoter; however, the expression of McMYB10 and anthocyanin content were increased. Bimolecular fluorescence complementation and yeast two-hybrid assays revealed that McHDA6 binds directly to McMET1 through its BAH2 and DNMT1-RFD domains. Based on the results of our study, we propose a mechanism for the molecular regulation of anthocyanin biosynthesis, namely, the miR399d and epigenetic modification comodulation model, to explain the phenomenon in which leaves turn red under conditions of Pi deficiency.