Functional diversity of subgroup 5 R2R3-MYBs promoting proanthocyanidin biosynthesis and their key residues and motifs in tea plant

The tea plant (Camellia sinensis) is rich in polyphenolic compounds. Particularly, flavan-3-ols and proanthocyanidins (PAs) are essential for the flavor and disease-resistance property of tea leaves. The fifth subgroup of R2R3-MYB transcription factors comprises the primary activators of PA biosynthesis. This study showed that subgroup 5 R2R3-MYBs in tea plants contained at least nine genes belonging to the TT2, MYB5, and MYBPA types. Tannin-rich plants showed an expansion in the number of subgroup 5 R2R3-MYB genes compared with other dicotyledonous and monocot plants. The MYBPA-type genes of tea plant were slightly expanded. qRT–PCR analysis and GUS staining analysis of promoter activity under a series of treatments revealed the differential responses of CsMYB5s to biotic and abiotic stresses. In particular, CsMYB5a, CsMYB5b, and CsMYB5e responded to high-intensity light, high temperature, MeJA, and mechanical wounding, whereas CsMYB5f and CsMYB5g were only induced by wounding. Three genetic transformation systems (C. sinensis, Nicotiana tabacum, and Arabidopsis thaliana) were used to verify the biological function of CsMYB5s. The results show that CsMYB5a, CsMYB5b, and CsMYB5e could promote the gene expression of CsLAR and CsANR. However, CsMYB5f and CsMYB5g could only upregulate the gene expression of CsLAR but not CsANR. A series of site-directed mutation and domain-swapping experiments were used to verify functional domains and key amino acids of CsMYB5s responsible for the regulation of PA biosynthesis. This study aimed to provide insight into the induced expression and functional diversity model of PA biosynthesis regulation in tea plants.