Wake Forest University Winston Salem, North Carolina
Body of Abstract: Auxin modulates root development, regulating primary root elongation and the formation of lateral roots and root hairs. Auxin remodels development through the activation of Auxin Response Factors (ARFs) that turn on transcriptional networks. To refine the gene regulatory network downstream of auxin, we performed a transcriptomic analysis of Arabidopsis roots over a broad time course of treatment with indole-3-acetic acid (IAA). The resulting data revealed ARF19 was rapidly induced by auxin and was expressed in tissues where auxin remodels development. We employed RNA-Seq to predict ARF19-regulated genes by examining the transcriptome of arf19 and wild-type in the presence and absence of IAA. To determine which genes were directly targeted by ARF19, we looked for consensus binding sites for ARF19 in their promoters. These promoter regions with predicted AuxRE sites were cloned in front of YFP or RFP genes and the gene fusions were transformed into yeast cells expressing ARF19. Direct binding of ARF19 to these promoters turns on transcription and drives fluorescent protein synthesis in yeast, which was quantified via confocal microscopy, a fluorescent plate reader, and flow cytometry. Once we resolved a yeast-confirmed list of direct targets, we examined the function of ARF19 in the roots over a range of IAA concentrations. We determined that arf19-1 plants were resistant to the root hair inhibition seen at high levels of IAA in the wild type. Mutants in genes that are ARF19 targets were examined for similar changes in root hair auxin responses, suggesting ARF19’s gene regulatory network controls auxin-responsive root hair growth and development. Confirming direct targets of ARF19 allowed us to gain a greater understanding of the gene regulatory networks by which auxin modulates root development.