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.