Phosphorylation of MYB75 transcription factor by MAP kinases in Arabidopsis thaliana

The Arabidopsis transcription factor MYB75 has been described in the literature as a positive transcriptional regulator of anthocyanin biosynthetic genes. More recently, MYB75 was shown to also negatively regulate lignin and other secondary cell wall biosynthetic genes. MYB75 has two canonical MAP kinase phosphorylation sites, at threonines 126 and 131; we wanted to explore the possibility that MYB75 is regulated post translationally through phosphorylation by MPKs. In our lab, Dr. Yonge found that MYB75 could be phosphorylated in vitro by MPK3, MPK4, MPK6 and MPK11, almost exclusively at threonine 131. Subsequently, I demonstrated that MYB75 interacts in vitro with a large number of Arabidopsis MAP kinases, suggesting that it could be a target for multiple MPKs. We decided to explore how phosphorylation affects MYB75 function, in terms of protein-protein interaction, protein turnover and localization, and describe the impact of this putative phosphorylation event on the ability of MYB75 to drive transcription of target genes. For this purpose we used point mutants, MYB75T131A and MYB75T131E to mimic permanently non-phosphorylated and phosphorylated versions of MYB75, respectively. While protein localization and protein-protein interactions of MYB75 were not strongly impacted in the point mutants, several in vivo experiments indicated that MYB75T131E is more labile than either MYB75WT or MYB75T131A, suggesting that phosphorylation at T-131 negatively affects protein stability. Over-expressor lines, 35Spr:3xHA:MYB75 gene, as well as inducible DEXpr:3xHA:MYB75 gene lines were used, to analyze MYB75 protein dynamics in vivo and to assess the ability of each phosphovariant to drive anthocyanin production. Arabidopsis plants over-expressing MYB75WT, MYB75T131A or MYB75T131E displayed increased anthocyanin production, compared to Col-0 WT, suggesting that phosphorylation status at T-131 does not affect this aspect of MYB75 function. However, HPLC analysis revealed quantitative differences between the biochemical species of anthocyanins and flavonols that accumulate in different phosphovariant over-expressing plants. Transcriptome analysis revealed that MYB75T131E is a more potent regulator of gene expression. This finding, along with distinct developmental differences between MYB75 phosphovariants, suggest that phosphorylation at T-131 could affect the ability of MYB75 to drive the expression of a broader spectrum of genes than previously described in the literature. Supplementary materials available at: http://hdl.handle.net/2429/66660Science, Faculty ofBotany, Department ofGraduat