Functional Characterization of FLP, a MYB Transcription Factor Involved in Arabidopsis Stomatal Development

Abstract: Stomata are essential for plant growth. FLP and MYB88, two R2R3 MYB proteins, have been shown to redundantly regulate guard cell division and differentiation in the Arabidopsis stomatal cell lineage. Loss of FLP and MYB88 functions results in multiple clustered stomata as a consequence of extra guard cell divisions. However, the molecular mechanisms by which FLP and MYB88 participate in stomatal development remain unclear. To investigate this problem, I first showed that FLP and MYB88 are DNA-binding proteins with DNA-binding preferences distinct from other known MYB proteins. The flp-8 allele harboring a single amino acid mutation in the MYB domain lacks in vitro DNA-binding activity, indicating that the DNA-binding activity of FLP is essential for FLP function in stomatal development. I showed that the Cyclin-dependent kinase B1;1 (CDKB1;1) gene is a FLP/MYB88 direct target, and that its expression is repressed by FLP/MYB88. To fully understand how FLP/MYB88 regulate guard cell division and differentiation, I took a chromatin-immunoprecipitation coupled with tiling array hybridization (ChIP-chip) approach and identified several new FLP/MYB88 putative direct targets including multiple cell cycle genes. Based on the information gained from these experiments, I proposed a model in which FLP/MYB88 limit to one guard mother cell division to form just two guard cells in each stoma by inhibiting gene expression of some cell cycle genes. In addition, I characterized two alternatively spliced FLP transcripts in planta. The corresponding proteins have distinct DNA-binding and transcriptional activities. To attempt to uncover the mechanism driving these differences, I did structural prediction of exon 11, and a model is suggested. Having abnormal stomata, loss of FLP and MYB88 function results in more susceptible to drought and salt stress conditions compared to wild type plants. Gene expression studies showed that stress-induced genes have significantly lower expression in flp-1 myb88 compared to wild type plants, suggesting the importance of FLP/MYB88 in abiotic stress response. Together, these studies start to explore molecular function of FLP/MYB88 in Arabidopsis stomatal development and the biological significance of FLP/MYB88 under abiotic stress conditions.