Apple MdbHLH4 promotes the flowering transition through interactions with FLOWERING LOCUS C and transcriptional activation of FLOWERING LOCUS T

Apple (Malus domestica) is one of the most widely cultivated fruit trees in the world and plays a central role in the agricultural industry and the economy. Flowering is a multi-path synergistic process that helps plants achieve high quality and yield in a multilateral environment. Here, overexpressing MdbHLH4 promoted the flowering transition in Arabidopsis, while myc70, a mutant of the MdbHLH4 Arabidopsis homolog, only exhibited a late flowering phenotype under short days. Moreover, the late flowering myc70 mutant phenotype was attenuated by ectopic expression of MdbHLH4. The myc70 mutant recovered from late flowering under short days by exogenous application of gibberellic acid (GA)4+7. The expression of GA biosynthesis-related genes was upregulated and that of GA catabolic genes was downregulated in the apical meristems of the Arabidopsis MdbHLH4 transgenic lines compared with the wild-type, and the opposite was observed in the myc70 mutant. Furthermore, yeast-twohybrid (Y2H) assays revealed that MdbHLH4 and DELLA family proteins interacted with the C-terminal domain of MdFLC (FLOWERING LOCUS C) protein, suggesting potential competition between MdbHLH4 and MdDELLAs to interact with MdFLC in apples. Additionally, MdbHLH4 directly bound to the MdFT (FLOWERING LOCUS T) promoter and activated MdFT expression; thus promoting the floral transition. Our results suggest that MdbHLH4 is a key gene involved in plant flowering under short day conditions by regulating GA biosynthesis and signaling as well as the expression of MdFT, which provides new insight into the apple flowering mechanism.