The members of the fourth subgroup of R2R3-MYB (Sg4 members) are well-known inhibitors of phenylpropanoid and lignin synthesis pathways. The C2 domain is closely related to the transcriptional inhibitory activity of Sg4 members. Phosphorylation modification enhances the transcriptional inhibitory activity of Sg4 members. Here, we identified a phosphorylation site on the C2 domain of CsMYB4a from tea plants (Camellia sinensis). A mitogen-activated protein kinase (MAPK), named CsMPK3-2, phosphorylated this site on the C2 domain of CsMYB4a. Further experiments revealed that phosphorylation of CsMYB4a weakened its ability to inhibit the gene expression of PAL, C4H, and 4CL in the phenylpropanoid pathway and activated the expression of transcription factor YABBY5, maintaining the adaxial–abaxial polarity of the leaf. Knocking out NtYAB5 in CsMYB4a transgenic tobacco partially repaired the leaf wrinkling phenotype caused by CsMYB4a. The C1 domain exhibited an activation function when the C2 domain of CsMYB4a was phosphorylated by CsMPK3-2, causing this reversal phenomenon. These results enrich our understanding of the regulatory diversity of Sg4 members.

Subgroup 4 (Sg4) members of the R2R3-MYB are generally known as negative regulators of the phenylpropanoid pathway in plants. Our previous research showed that a R2R3-MYB Sg4 member from Camellia sinensis (CsMYB4a) inhibits expression of some genes in the phenylpropanoid pathway, but its physiological function in the tea plant remained unknown. Here, CsMYB4a was found to be highly expressed in anther and filaments, and participated in regulating filament growth. Transcriptome analysis and exogenous auxin treatment showed that the target of CsMYB4a might be the auxin signal pathway. Auxin/indole-3-acetic acid 4 (AUX/IAA4), a repressor in auxin signal transduction, was detected from a yeast two-hybrid screen using CsMYB4a as bait. Gene silencing assays showed that both CsIAA4 and CsMYB4a regulate filament growth. Tobacco plants overexpressing CsIAA4 were insensitive to exogenous α-NAA, consistent with overexpression of CsMYB4a. Protein-protein interaction experiments revealed that CsMYB4a interacts with N-terminal of CsIAA4 to prevent CsIAA4 degradation. Knock out of the endogenous NtIAA4 gene, a CsIAA4 homolog, in tobacco alleviated filament growth inhibition and α-NAA insensitivity in plants overexpressing CsMYB4a. All results strongly suggest that CsMYB4a works synergistically with CsIAA4 and participates in regulation of the auxin pathway in stamen.