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Auxin mediated gene expression in WT and arf2-6 mutant

ABSTRACT: Global gene expression data from 7-day old light-grown liquid cultured seedlings treated with or without auxin (5µM IAA) for 2 h. Columbia (WT) and Auxin response factor 2 (ARF2) T-DNA insertion mutant (arf2-6 ) were used for this study. Each experimental condition has three true replicates for a total of 12 hybridizations. Data analysis: Affymetrix GeneChip Microarray Suite version 5.0 software was used to obtain signal values for individual genes. The data files containing the probe level intensities (cell files) were used for background correction and normalization using the log2 scale robust multi-array analysis (RMA) procedure (Irizarry et al., 2003). The “R” environment (Ihaka and Gentleman, 1996) was used for running the RMA program. Data analysis and statistical extraction were performed using log2 converted expression intensity data within Microsoft Excel 98. Based on preliminary analysis, a hybridization signal less than 5.64385619 (= log2 50) was considered as background; all signals less than 5.64385619 were converted to 5.64385619 prior to further analysis. Keywords = Auxin Keywords = Auxin response factor Keywords: other

ORGANISM(S): Arabidopsis thaliana

PROVIDER: GSE631 | GEO | 2005/10/19

SECONDARY ACCESSION(S): PRJNA89755

REPOSITORIES: GEO

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Project description:The aim of this experiment was to decipher a transcriptional regulatory code of Arabidopsis Auxin Response Factor 2 (ARF2) and understand the molecular basis of ARF2’s role as a promoter of dark-induced senescence. A combination of computational tools and yeast one-hybrid experiments identified potential direct transcriptional regulators of ARF2 belonging to several TF families including AP2/ERFs and AREB/ABF/ABI5s. Using ARF2 promoter mutants in transcriptional activation and Y1H experiments, the binding sites of these TFs in the ARF2 promoter were identified. AP2/ERFs including ERF1, ERF15 and ORA59 transactivate the ARF2 promoter in Arabidopsis mesophyll protoplasts and this transactivation is positively regulated by ABA. Crucially, the positive effect of ABA on ERF1 transactivation of ARF2 relies on the functional ABRE motif in the ARF2 promoter and the presence of AREB/ABF/ABI5s (ABF3 and ABF4) suggesting the existence of combinatorial regulation of ARF2 by these direct regulators. ABF3 and ABF4 were unable to activate the ARF2 promoter on their own indicating the necessity of ERF1 as a coupling TF for AREB/ABF/ABI5-driven regulation of ARF2. We also show that MED25 (a subunit of the Mediator complex) is required for proper transcriptional regulation of ARF2 during senescence. med25 plants show decreased ARF2 mRNA levels during natural senescence and, similarly to arf2-6 mutants, exhibit a delayed dark-induced senescence phenotype. We show that MED25 is important for ERF1-, ERF15- and ORA59-dependent transactivation of ARF2 in Arabidopsis protoplasts and finally idenitfy genes differentially expressed in arf2-6 mutants compared to wildtype during dark-induced senescence. Hence, we elucidate a regulatory network around ARF2 identifying combinatorial transcriptional regulation of this gene during senescence as well as likely downstream targets. The combinatorial regulation, comprised of AP2/ERFs and AREB/ABF/ABI5s TFs, enables integration of at least two key hormone signaling pathways to drive expression of a key senescence regulator.

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Auxin mediated gene expression in WT and arf2-6 mutant

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