The miR408a-BBP-LAC3/CSD1 module regulates anthocyanin biosynthesis mediated by crosstalk between copper homeostasis and ROS homeostasis during light induction in Malus plants.

INTRODUCTION: The expression of miR408 is affected by copper (Cu) conditions and positively regulates anthocyanin biosynthesis in Arabidopsis. However, the underlying mechanisms by which miR408 regulates anthocyanin biosynthesis mediated by Cu homeostasis and reactive oxygen species (ROS) homeostasis remain unclear in Malus plants. OBJECTIVES: Our study aims to elucidate how miR408a and its target, basic blue protein (BBP) regulate Cu homeostasis and ROS homeostasis, and anthocyanin biosynthesis in Malus plants. METHODS: The roles of miR408a and its target BBP in regulating anthocyanin biosynthesis, Cu homeostasis, and ROS homeostasis were mainly identified in Malus plants. RESULTS: We found that the BBP protein interacted with the copper-binding proteins LAC3 (laccase) and CSD1 (Cu/Zn SOD superoxide dismutase), indicating a potential crosstalk between Cu homeostasis and ROS homeostasis might be mediated by miR408 to regulate the anthocyanin accumulation. Further studies showed that overexpressing miR408a or suppressing BBP transiently significantly increased the expression of genes related to Cu binding and Cu transport, leading to anthocyanin accumulation under light induction in apple fruit and Malus plantlets. Consistently, opposite results were obtained when repressing miR408a or overexpressing BBP. Moreover, light induction significantly increased the expression of miR408a, CSD1, and LAC3, but significantly reduced the BBP expression, resulting in increased Cu content and anthocyanin accumulation. Furthermore, excessive Cu significantly increased the anthocyanin accumulation, accompanied by reduced expression of miR408a and Cu transport genes, and upregulated expression of Cu binding proteins including BBP, LAC3, and CSD1 to maintain the Cu homeostasis and ROS homeostasis in Malus plantlets. CONCLUSION: Our findings provide new insights into the mechanism by which the miR408a-BBP-LAC3/CSD1 module perceives light and Cu signals regulating Cu and ROS homeostasis, ultimately affecting anthocyanin biosynthesis in Malus plants.

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.