JrMYB44 is required for the accumulation of polyphenols and contributes to drought tolerance in Juglans regia

Juglans regia, an important economic tree species, is planted all over the world, and drought is one of the crucial factors limiting its growth and development. The various polyphenol content in walnut plants constitutes one of the material bases for the differences in stress resistance among various germplasms. However, the molecular mechanism underlying stress response mediated by polyphenol -dependent pathways remains unclear. v-Myb avian myeloblastosis viral oncogene homolog (MYB) protein of transcription factors play important regulatory roles in the process of plant stress responses. Previously, we identified JrMYB44 could be involved in osmotic stress response in walnut. In this study, we confirmed that the drought resistance of four walnut cultivars ('Chandler', 'Xiangling', 'Xilin2' and 'Xifu1') is positively correlated with the accumulation of polyphenols. The content and component changes of polyphenols in JrMYB44 overexpression (OE) and suppression (SE) lines in both walnut and Arabidopsis thaliana demonstrated that JrMYB44 positively regulated polyphenols accumulation. The variation of JrMYB44 expression and polyphenol levels under drought treatment indicated significant correlation between JrMYB44-induced drought tolerance and polyphenol accumulation, which was involved in reactive oxidative species (ROS) balance. The differentially expressed genes (DEGs) between OE and WT implied that JrMYB44 could positively activate downstream genes to participate in the drought stress response. Yeast one-hybrid (Y1H), transient GUS expression assay and dual-luciferase reporter assay (DLR) confirmed that JrMYB44 could recognize downstream JrWRKY7 and JrDREB2A, two transcription factors previously reported to be involved in drought response. Meanwhile, it was confirmed by Y2H, GST-pull down and luciferase complementation imaging assay (LCI) that JrMYB44 could interact with JrMYC2 and JrDof1, another two previously reported potential drought response regulators. Collectively, these results indicated that JrMYB44 could activate JrWRKY7, JrDREB2A and interact with JrMYC2 and JrDof1 to promote walnut polyphenol accumulation and improve drought resistance in a ROS dependent manner.