Molecular Mechanism Of Transcriptional Regulation Mediated By ROS-ABA Signaling Pathway In Arabidopsis Thaliana

COLD6, a drought and ABA sensitive gene in Arabidopsis, have been identified to interact physically with GPX3by using biochemical method. Bioinformatics analysis of the COLD6shows that it has no typical DNA binding sequences and may work as a transcriptional regulator. It is found that COLD6has two domains WWE and RST, which predicted to mediate protein-protein interaction. It is demonstrated that interaction of GPX3and COLD6were regulated by RST domain using the Yeast Two-hybrid. Furthermore we found a transcriptional factor HRF1that can interact physically with COLD6in nucleus investigated by biochemical analysis carried out in vitro and in vivo.GPX3::GFP and COLD6::GFP fusion proteins using transient expression in protoplast of mesophyll cells showed GPX3protein localized in the mitochondrial membrane and COLD6in the nucleus. It would thus appear not physically possible for COLD6interacting with GPX3in vivo in plants. Based on the results, we think that COLD6may move from nucleus to cytoplasm. An analysis of the COLD6amino acid sequence indicated that COLD6has not only three NLSs, but also a NES. Then we investigated the localization of COLD6under stress conditions such as ABA and H2O2. Guard cell and protoplasts transiently transformed with COLD6::GFP transgenic seedlings were exposed to either ABA or H2O2stress. Amazingly, when treated with ABA or H2O2after3h, the COLD6fusion proteins accumulated in not only the nucleus but also the cytoplasm near the cell periphery. It was further found that the movement from nucleus to cytoplasm was almost reversed by the addition of10mM ascorbic acid as a H2O2scavenger. These results suggested that H2O2participated in the migration of COLD6from nucleus to cytoplasm.By genetics and molecular biology methods, we constructed doble mutant gpx3/cold6、cold6/hrf1and triple mutant gpx3/cold6/hrf1and then tested the sensitivity of these mutants to ABA and H2O2. Treatment of WT and these mutants Arabidopsis seedlings with H2O2and ABA at a special concentration, After15days, the double mutant and triple mutant show hypersensitivity to H2O2and ABA, which exhibited slower growth and increased damage compared with the WT such as browning and bleaching leaves. Furthermore the double and trile mutants behaved just like the cold6single mutant and no additive effect was seen. gpx3single mutants show some sensitive to H2O2but not to lower concertration ABA. hrf1single mutants have no response to ABA and H2O2.In summary, we consider that GPX3-COLD6-HRF1pathway may be formed and regulate redox delay in Arabidopsis, which a complex combinded by GXP3with COLD6might sense and integrate the H2O2signal in the cytoplasm, and then transduce to the transcriptional factor HRF1in the nucleus by COLD6movement from cytoplasm to nucleus, which further regulate the expression of the downstream gene.