Based upon analysis of data obtained from theATH1 microarrays, a cDNA that was highly induced afterdrought treatment, was isolated from Arabidopsis seedlings.RT-PCR and Quantitative Real-Time (QRT)-PCR experi-ments showed that expression level of the gene increasedsignificantly upon drought, UV, abscisic acid, high salinityand salicylic acid treatments. It was classified as a DREBsubfamily member based on multiple sequence alignmentand phylogenetic characterization. Since it encoded a proteinwith a typical ERF/AP2 DNA-binding domain and containeda glutamine-rich region near its N terminus, we named itQRAP2 (for glutamine-rich AP2). Gel retardation assay re-vealed that QRAP2 was able to form a specific complex withthe previously characterized DRE element while did notshow any affinity to the GCC box or the mutant DRE box.When fused to the GAL4 DNA-binding domain, either full-length QRAP2 or its N-terminus functioned effectively as atrans-activator in the yeast one-hybrid assay with its C-ter-minus completely inactive. Our data indicate that QRAP2could be a new member of the AP2/EREBP transcriptionfactor family involved in activation of down-stream targetgenes in response to environmental stress, especially underdrought conditions.
AP2/EREBP transcription factors (TFs) play very important roles in plant development, hormonal regulation and stress response. Upon genome-wide cDNA cloning, phylogenetic and expression pattern analyses of this plant specific TF family, we found that three of the members including At1g71450, At1g50680 and At5g13910, were likely involved in responses to ABA, cold and salt. Complementary DNAs containing putative full-length ORFs of these three TFs were obtained and fused individually to the GAL4 DNA-binding domains. All the 3 genes functioned effectively as trans-activators using yeast one-hybrid assays. RT-PCR experiments showed that the At1g71450 gene was induced by ABA and low temperature; the At1g50680 gene was responsive to quite a few stress conditions, but especially to freezing temperature; and the At5g13910 gene was induced by high salt treatment, drought and ethyl-ene. By searching the ABRC T-DNA insertion mutant stocks, we obtained knockout lines for these TFs. Homozygous ko1 (At1g71450) plants showed a hypersensitive response to ABA during seed germina-tion and also in stomata movement. Homozygous ko2 (At1g50680) plants showed a significant reduc-tion in plant freezing tolerance compared to the wild type after chilling treatment. Homozygous ko3 (At5g13910) were less tolerant to high salinity than wild type plants. Our data suggest that At1g71450 is a negative regulator in ABA signaling, while At1g50680 and At5g13910 are positive regulators in cold and salt stress responses, respectively.
