The Function And Regulation Of The Foxtail Millet DREB-type Transcriptional Factor SiARDP

Drought stress greatly impacts growth, and is one of the major environmental factors that limit crop production. Plants have evolved many molecular mechanisms to adapt and resist to drought stress. There are two main transcriptional signal pathways to response to drought stress:abscisic acid (ABA)-dependent and-independent signal pathway. Drought stress responsive signals are complex, and some cross-talks exist between two signal pathways.Foxtail millet, an ancient crop in China, provides rich nutrient elements and has excellent drought tolerance, and is an important strategic food crops in China. However the molecular mechanisms of drought stress in foxtail millet remain unknown. In this study, an ABA-responsive dehydration responsive element (DRE) binding protein gene, named SiARDP, was cloned from foxtail millet using a yeast one-hybrid screening assay. Northern blot and qRT-PCR assay showed that SiARDP induced by drought, high salt, low temperature stress and ABA treatment. The qRT-PCR assay showed that the transcriptional levels of SiARDP in leaf were higher than in root, stem and inflorescence. The SiARDP was located in the nucleus of foxtail millet protoplasts. Electrophoretic mobility shift assay (EMSA) showed that the fusion protein SiARDP-His could bind to the DRE core element, and the SiARDP had the ability of transcriptional activity in yeast. Heterologous expression of SiARDP in Arabidposis enhanced the tolerance to drought and high salt stresses. Under drought stress, transgenic Arabidposis accumulated more proline than the wild type plants, and under high salt stress, the electrolyte leakage of transgenic plants was much lower than that of wild type plants. Overexpression of SiARDP in foxtail millet enhanced tolerance to drought stress, and accumulated more proline. The qRT-PCR assay showed that the transcriptional levels of many stress-relevant genes with several DRE core elements in their promoter regions increased in transgenic foxtail millet. The data suggested that SiARDP responds to drought stress and regulates some functional genes to improve tolerance.Further analyses showed that two ABA-responsive element (ABRE) exist in the promoter region of SiARDP. We cloned two ABRE binding protein (AREB) genes from foxtail millet, named SiAREBl and SiAREB2. The qRT-PCR showed that SiAREB1and SiAREB2induced by drought, high salt stresses and ABA treatment. SiAREB1and SiAREB2located in nucleus of foxtail millet protoplasts and had the ability of transcriptional activation in yeast. The EMSA, yeast one hybird and ChIP assays shown that SiAREB1and SiAREB2could bind to the ABRE core element in SiARDP promoter region in vitro and vivo. Heterologous expression of SiAREB1and SiAREB2in Arabidopsis enhanced the tolerance to drought and high salt stresses and activated the transcription of the target genes. The point mutant and protein phosphorylation assay demonstrated that the ability of transcriptional activation of SiAREB2was regulated by phosphorylation.Taken together, SiARDP was DREB-type transcriptional factor and responsive to abiotic stress. SiAREB transcriptional factors regulated the expression of SiARDP under drought and salt stress in ABA-dependent pathway, but under low temperature other regulatory factors regulated SiARDP in ABA-independent pathway.