The mechanisms behind how a transcription factor elicits a given phenotype can be complex. The aim of the research presented herein was to provide experimental evidence to characterise the upstream and downstream regulation of the Arabidopsis thaliana R2R3-MYB transcription factor, AtMYB61. To address these aims, three separate experiments were undertaken. First, three direct downstream target genes of AtMYB61 were predicted based on a two-stage complete transcriptome analysis, using publicly available microarray datasets in combination with a custom microarray dataset comparing the transcriptomes of WT, atmyb61 and 35S::MYB61 plants. These candidate target genes encode the following proteins: a KNOTTED1-like transcription factor, a caffeoyl-CoA 3-O-methyltransferase and a pectin-methylesterase. AtMYB61 bound the 5’ non-coding regulatory regions of these target genes, as determined by electrophoretic mobility shift assay. Second, the preferred DNA-binding sites of recombinant AtMYB61 protein were assessed with a cyclic amplification and selection of targets (CASTing) assay. Key interactions between amino acids in the AtMYB61 DNA-binding site and nucleotides in the preferred DNA targets were predicted by molecular modeling. While recombinant AtMYB61 was sufficient to drive gene expression from CASTing-identified target DNA sequences in yeast, it did so in a manner that was not entirely consistent with predicted DNA-binding affinities determined by a nitrocellulose filter binding assay. Finally, the molecular components that function upstream to modulate AtMYB61 expression were determined. AtMYB61 was determined to be de-repressed by sucrose in a mechanism involving its second intron. An over-represented motif was conserved within the second intron of Brassicaceae AtMYB61 homologues and this motif functioned as a binding target for a putative sugar-mediated repressor, as determined by EMSA. Putative AtMYB61 repressor proteins that bound this motif in the absence of sucrose were affinity purified and characterised using LC-MS/MS, and the proteins identified based on their MS fingerprints.

The mechanisms behind how a transcription factor elicits a given phenotype can be complex. The aim of the research presented herein was to provide experimental evidence to characterise the upstream and downstream regulation of the Arabidopsis thaliana R2R3-MYB transcription factor, AtMYB61. To address these aims, three separate experiments were undertaken. First, three direct downstream target genes of AtMYB61 were predicted based on a two-stage complete transcriptome analysis, using publicly available microarray datasets in combination with a custom microarray dataset comparing the transcriptomes of WT, atmyb61 and 35S::MYB61 plants. These candidate target genes encode the following proteins: a KNOTTED1-like transcription factor, a caffeoyl-CoA 3-O-methyltransferase and a pectin-methylesterase. AtMYB61 bound the 5’ non-coding regulatory regions of these target genes, as determined by electrophoretic mobility shift assay. Second, the preferred DNA-binding sites of recombinant AtMYB61 protein were assessed with a cyclic amplification and selection of targets (CASTing) assay. Key interactions between amino acids in the AtMYB61 DNA-binding site and nucleotides in the preferred DNA targets were predicted by molecular modeling. While recombinant AtMYB61 was sufficient to drive gene expression from CASTing-identified target DNA sequences in yeast, it did so in a manner that was not entirely consistent with predicted DNA-binding affinities determined by a nitrocellulose filter binding assay. Finally, the molecular components that function upstream to modulate AtMYB61 expression were determined. AtMYB61 was determined to be de-repressed by sucrose in a mechanism involving its second intron. An over-represented motif was conserved within the second intron of Brassicaceae AtMYB61 homologues and this motif functioned as a binding target for a putative sugar-mediated repressor, as determined by EMSA. Putative AtMYB61 repressor proteins that bound this motif in the absence of sucrose were affinity purified and characterised using LC-MS/MS, and the proteins identified based on their MS fingerprints.