The MYB80 transcription factors of Arabidopsis and cotton: comparative studies in function and their utilization for the development of a novel reversible male sterility system

Submission note: A thesis submitted in total fulfilment of the requirements for the degree of Doctor of Philosophy to the Department of Botany, School of Life Sciences, Faculty of Science, Technology and Engineering, La Trobe University, Bundoora.

The Arabidopsis MYB80 transcription factor is required for normal pollen development. Inhibiting MYB80 expression results in complete male sterility and manipulating MYB80 expression provides a means to create hybrid vigour in crops such as cotton. Two highly similar MYB80 homologs were identified from cotton. Full length GhMYB80 driven by the AtMYB80 promoter rescued the atmyb80 mutant in Arabidopsis, restoring male fertility. When fused with the 32R (EAR) repressor motif, GhMYB80 (driven by its own promoter) resulted in male sterile Arabidopsis plants. Hence, GhMYB80 and AtMYB80 have similar functions in regulating pollen development. The GhMYB80 and AtMYB80 proteins possess four regions. Truncated constructs were used to determine which regions are required to rescue the atmyb80 mutant. Three highly conserved regions, namely the MYB domain, the adjacent 44-amino acid peptide and the C-terminus, when combined, were sufficient to restore male sterility. A novel EAR motif (N32R) was developed and shown to strongly interact with the TOPLESS corepressor in yeast. Complete male sterility of transgenic Arabidopsis plants was obtained by fusing AtMYB80 with the N32R motif. Using the yeast-two hybrid assay, the TPL N-terminus was shown to interact with EAR motifs. A novel fertility restorer was created using the TPL N-terminus as an anti-EAR domain together with the conserved AtMYB80 C-terminus. Fertility restoration occurred in the hybrid F1 plants. Finally, three AtMYB80 direct target genes, encoding a pectin methylesterase, a MYB transcription factor and a glyoxal oxidase (GLOX2), were identified using the chromatin immunoprecipitation (ChIP)-qPCR technique. Electrophoretic mobility shift assays confirmed that MYB80 bound to the MYB cis-elements in the GLOX2 promoter. The glox1glox2 double mutant was created and possessed a partially male sterile phenotype.