CcMYB12 Positively Regulates Flavonoid Accumulation during Fruit Development in <i>Carya cathayensis</i> and Has a Role in Abiotic Stress Responses

Flavonoid, an important secondary metabolite in plants, is involved in many biological processes. Its synthesis originates from the phenylpropane metabolic pathway, and it is catalyzed by a series of enzymes. The flavonoid biosynthetic pathway is regulated by many transcription factors, among which MYB transcription factors are thought to be key regulators. Hickory (Carya cathayensis) is an economic forest tree species belonging to the Juglandaceae family, and its fruit is rich in flavonoids. The transcriptome of exocarp and seed of hickory has previously been sequenced and analyzed by our team, revealing that CcMYB12 (CCA0691S0036) may be an important regulator of flavonoid synthesis. However, the specific regulatory role of CcMYB12 in hickory has not been clarified. Through a genome-wide analysis, a total of 153 R2R3-MYB genes were identified in hickory, classified into 23 subclasses, of which CcMYB12 was located in Subclass 7. The R2R3-MYBs showed a differential expression with the development of hickory exocarp and seed, indicating that these genes may regulate fruit development and metabolite accumulation. The phylogenetic analysis showed that CcMYB12 is a flavonol regulator, and its expression trend is the same as or opposite to that of flavonol synthesis-related genes. Moreover, CcMYB12 was found to be localized in the nucleus and have self-activation ability. The dual-luciferase reporter assay demonstrated that CcMYB12 strongly bonded to and activated the promoters of CcC4H, CcCHS, CcCHI, and CcF3H, which are key genes of the flavonoid synthesis pathway. Overexpression of CcMYB12 in Arabidopsis thaliana could increase the content of total flavonoids and the expression of related genes, including PAL, C4H, CHS, F3H, F3’H, ANS, and DFR, in the flavonoid synthesis pathway. These results reveal that CcMYB12 may directly regulate the expression of flavonoid-related genes and promote flavonoid synthesis in hickory fruit. Notably, the expression level of CcMYB12 in hickory seedlings was significantly boosted under NaCl and PEG treatments, while it was significantly downregulated under acid stress, suggesting that CcMYB12 may participate in the response to abiotic stresses. The results could provide a basis for further elucidating the regulation network of flavonoid biosynthesis and lay a foundation for developing new varieties of hickory with high flavonoid content