Functional genomics analysis of the arabidopsis ABI5 bZIP transcription factor

During embryogenesis, the architecture of the plant and the food reserves for seed germination are established. Abscisic acid (ABA) regulates seed development and dormancy. It controls genes involved in stress responses. ABA-responsive basic leucine zipper (bZIP) transcription factors are identified by interaction with ABA responsive cis-regulatory elements. The transcription factor ABI5 is one of these. It regulates gene expression during embryogenesis and in response to ABA. An ABA-insensitive mutant, abi5-6, exhibits no gross morphological defects other than the effect on seed germination in the presence of ABA. Thus, microarray analysis was employed to search for molecular phenotypes. We used cDNA microarrays to analyze ABA regulated gene expression and the role of ABI5 in seedlings. 310 genes were identified as ABI5/ABA regulated genes. 161 of these genes were regulated by ABI5, and 134 of ABI5-regulated genes were co-regulated by ABA. Only a small number of genes altered expression in both Pro35S:ABI5 and abi5-6 genetic backgrounds indicating the preferential binding of the bZIP protein dimers to specific promoter sequences. To determine the optimal platform for identifying ABI5-regulated genes in seeds, a cDNA microarray, the Agilent Arabidopsis Oligo microarray, and the Affymetrix ATH1 arrays were tested. Cross platform comparisons utilized 4,518 genes present on all three platforms. The best correlation was between the Agilent and the Affymetrix results. Furthermore, the Affymetrix results correlated best with qRT-PCR validation data for selected genes. A small number of genes including AtCOR413 pm-1 showed a consistent expression pattern across the three platforms. A robust ABRE cis-regulatory element was identified in the promoter of AtCOR413 pm-1. Further studies showed binding of ABI5 to the promoter of AtCOR413 pm-1 by Electrophoretic Mobility Shift Assays (EMSA) and validated the expression of ABI5 and AtCOR413 pm-1 in abi5-6 seeds by qRT-PCR and RNA gel blot analysis. Transactivation assays using AtCOR413 pm-1 promoter:GUS fusions in Arabidopsis dry seed and seedlings revealed ABI5 acts as a negative regulator for AtCOR413 pm-1 in dry seeds, while other proteins may play major roles in regulating responses to ABA and low temperature (LT) in seedlings.