Identification And Functional Characterization Of Myb Transcription Factor Genes TaODORANT1 And TaMyb1D In Wheat

Environmental stresses,such as drought,high salinity,and extreme temperatures,adversely affect plant growth,development,and productivity.To adapt to such environmental conditions,complex response mechanisms have been evolved in plants,including transcriptional regulation networks for the transduction of stress signals.The implementation of these intricate networks depends on the participation of various transcription factors(TFs),such as MYB,NAC,AP2/ERF,bZIP,bHLH,and WRKY.The MYB superfamily has been identified and analyzed in Arabidopsis and rice.Several lines of evidence demonstrate that numerous R2R3-MYB proteins function in response to abiotic stress.However,only a few MYB TFs from wheat(Triticum aestivum)have so far been characterized to participate in abiotic stress.Identifying potential stress response MYB genes and studing the mechanisms will lay the foundation for plant genetic improvement by genetic engineering.In this study,12 novel R2R3-MYB genes were identified and cloned from wheat.Based on the expression patterns of these novel genes in wheat under various abiotic stresses and signal molecule treatments,TaODORANT1 and TaMyb1 D were selected to further investigate their function in abiotic stress response.The main results are as follows:(1)A total of 12 R2R3-MYB genes were identified in wheat using bioinformatic methods.Then structures,conserved MYB domains,and evolutionary relationships of these genes were analyzed.(2)Twelve TaMYB genes were cloned from wheat(T.aestivum cv.Chinese Spring)in our study.The expression patterns of these novel genes in wheat under various abiotic stress and signal molecule treatments were examined by semi-quantitation RT-PCR.The result showed that a few genes of them were induced by one or more stress treatments.Among them,the expression levels of TaODORANT1 and TaMyb1 D were obviously induced by multiple stress treatments.Quantitation RT-RCR analysis showed that TaODORANT1 was up-regulated in wheat under PEG6000,NaCl,ABA,and H2O2 treatments;and TaMyb1 D was induced by PEG6000 and H2O2 treatments in wheat.These results indicated that TaODORANT1 and TaMyb1 D might be involved in the abiotic stress reaponses.(3)Transient expression of the fused protein in onion epidermal cells revealed that TaODORANT1-GFP and TaMyb1D-GFP were localized in the nucleus.Transactivation activity analysis indicated that TaODORANT1 had transactivation activity,which was attributed to the amino acid residues at C-terminal 171 to 265.In contrast,TaMyb1 D had no transactivation activity,and it may be a transcriptional repressor ascribed to presence of EAR-motif in its C-terminus.Transient expression assay indicated that TaMyb1 D repressed the promoter activities of OsCAD and TaCAD,and functioned as a transcriptional repressor.Binding activity assay showed that the fused protein TaODORANT1 could bind to type II MYB binding motif,while TaMyb1 D could bind to type I and AC MYB binding motif.(4)The phenotypic and physiological analyses of WT and TaODORANT1-overexpressing tabacco plants showed that TaODORANT1 enhanced plant tolerances to drought and salt stresses.Under drought stress,TaODORANT1 overexpressing transgenic tobacco plants exhibited higher relative water content and lower water loss rate.The status of stomatal closure was observed and the stomatal width:length ratios were measured under dehydration and exogenous ABA treatments.Result showed that the stomatal aperture of transgenic tobacco leaves was smaller than that of the WT.Under salt stress,Na+ concentration was significantly lower in TaODORANT1-overexpressing transgenic tobacco plants than WT.Under drought and salt stresses,the transgenic plants showed higher CAT and SOD enzyme activities but lower ion leakage,H2O2 and malondialdehyde contents.Moreover,TaODORANT1 overexpression up-regulated the expression levels of several ROS-and stress-related genes in response to both drought and salt stresses.These results demonstrated that TaODORANT1 positively regulates plant tolerances to drought and salt stresses.(5)The phenotypic and physiological analyses of WT and TaMyb1D-overexpressing tabacco plants showed that TaMyb1 D enhanced plant tolerances to drought and methyl viologen(MV)stresses.TaMyb1D-overexpressing transgenic tobacco plants exhibited higher relative water content and lower water loss rate under drought stress,as well as higher chlorophyll content in leaves under oxidative stress.Under drought and oxidative stresses,transgenic plants showed lower ion leakage and malondialdehyde and H2O2 contents.Moreover,TaMyb1 D up-regulated the expression levels of ROS-and stress-related genes in response to drought stress.These results demonstrated that overexpression of TaMyb1 D enhanced tolerances to drought and oxidative stresses in tobacco plants by activating the ROS-scavenging enzyme system and regulating the expressions of stress-related genes.Moreover,TaMyb1 D was also involved in process of plant development.Under normal condition,obvious phenotype differences were observed in mature leaves of transgenic plants.The mature leaves of transgenic plants presented darker green characterized by higher chlorophyll content and presented creases between the branched veins in comparison to that of WT.Overexpression of TaMyb1 D in tobacco plants repressed the expression of phenylpropanoid metabolism related genes and downregulated the accumulation of lignin in stems and flavonoids in leaves,thus affecting plant development under normal condition.