MdSPL6 regulates the accumulation of anthocyanins in apple leaves induced by low temperature by activating the promoters of anthocyanin-related genes

Abstract

Anthocyanin accumulation in apple leaves is tightly regulated by environmental and hormonal signals. However, the mechanisms underlying cold perception in apple leaves remain poorly understood. These mechanisms mediate the crosstalk between low temperature and hormonal signals, which together regulate the anthocyanin biosynthetic pathway. In this study, we used apple (Malus domestica) cultivars ‘Royalty’, ‘Gala’, and ‘Orin’ as experimental materials. Through transcriptional expression analysis, downstream target gene screening, genetic functional validation and other molecular approaches, we aimed to dissect the molecular mechanism underlying the synergistic regulation of anthocyanin accumulation in apple leaves by low temperature and hormone signaling pathways. Here, we demonstrate that the transcription factor MdSPL6 plays a pivotal role in promoting anthocyanin biosynthesis in response to low–temperature and hormonal signaling. Expression analysis in Malus domestica ‘Royalty’ revealed that MdSPL6 is strongly induced in both leaves and calli under low temperature, 16 °C, accompanied by increased anthocyanin accumulation. Hormonal treatments revealed that methyl jasmonate (MeJA) significantly upregulates MdSPL6 expression and anthocyanin levels, with a synergistic effect observed under combined MeJA and low–temperature conditions. DNA affinity purification sequencing (DAP–seq) identified MdSPL6 target genes enriched in metabolic pathways including flavonoid biosynthesis. MdSPL6 directly binds and activates the promoters of flavonoid biosynthesis genes, as confirmed by yeast one–hybrid and dual–luciferase assays. Both transient expression in apple leaves and transformation in apple calli showed that MdSPL6 enhances red pigmentation and upregulates anthocyanin biosynthetic genes under low–temperature stress. Furthermore, miR156a may act as a negative regulator of MdSPL6 and thereby participate in low–temperature–induced anthocyanin accumulation. Together, our findings suggest that MdSPL6 acts as a central integrator of low temperature and hormonal signals to activate anthocyanin biosynthesis in apple leaves, providing new insights into the environmental regulation of pigmentation in woody plants.