Studies On The Function Of The NF-Y Transcription Factor Genes TaNFYB-B3 And TaNF-YB4 Of Wheat In Mediating Plant Osmotic Stress Tolerance

Nuclear factor Y(NF-Y)transcription factor plays an important role in mediating plant resistance to abiotic stresses,such as drought and salt stress,by regulating the transcription of dowastream stress-defensive genes.Based on the transcription factor genes TaNFYB-B3 and TaNF-YB4 the authors identified earlier,which belong to NF-YB family in wheat,the molecular characterization of the genes,expression pattern in response to drought stress,and the function of them in mediating plant stress tolerance were studied.The main results are as follows.1.The molecular characterization of the NF-YB family transcription factor genes TaNFYB-B3 and TaNF-YB4 in wheat were analyzed using the bioinformatic method.The full-length cdna sequence of TaNFYB-B3(accession number KM078739.1)was 624 bp,and its open reading frame was 624 bp,encoding a polypeptide chain with 207 amino acid residues.The formula of protein is C943H1468N278O311S12,in which contains 25 acidic amino acids(Asp+Glu)and 21 basic amino acids(Arg+Lys).The molecular weight of TaNFYB-B3 protein is 22.06 kD and its isoelectric point(pI)is 5.75.The protein contains a conserved H4 superfamily domain(aa73-aa267)similar to the NF-YB transcription factor family members.The results of phylogenetic analysis showed that TaNF-YB3 share high similiraties to the NF-Y family genes derived from species of Triticum aestivum,Hordeum vulgare,and Phyllostachys pubescens at nucleotide acid level.2.The full-length cdna sequence of TaNF-YB4(accession number AK457878.1)is 1018 bp,and its open reading frame is 411 bp,encoding a polypeptide chain with 136 amino acid residues.The formula of protein is C647H1044N178O210S8,in which contains 21 acidic amino acids(Asp+Glu)and 20 basic amino acids(Arg+Lys).The molecular weight of TaNF-YB4 protein is 14.93 kD and its isoelectric point(pI)is 5.65.The protein contains a conserved H4 superfamily domain(aa19-aa83)similar to the NF-YB transcription factor family members.TaNF-YB4 is highly homologous to the NF-YB transcription factor family genes derived from oil palm,pineapple,barley,common wheat,single grain wheat,and rice and millet.3.The expression patterns of TaNFYB-B3 and TaNF-YB4 genes were shown to be modified under stress conditions compared with those under normal growth conditions.The expression level of TaNF-YB4 gene in roots and leaves showed an upregulated trend with the treatment progression within 27 h drought and low-Pi conditions.Likewise,the gene expression level of TaNFYB-B3 in roots and leaves was increased after 27 h treatments of drought and salt stress conditions.These results indicated that the transcript showed a typical stress-induced expression pattern of the genes in roots and leaves.4.Compared with wild type control,the transgenic lines of TaNF-YB4 strain with overexpression and downregulation changed growth features under drought and low-Pi treatments,showing that the plant growth of the Sense line(Sen 1)increased the dry weight and leaf area.However,the growth of Anti lines(Anti-2)were alleviated on growth,showing decreased dry weight and leaf area.The observation on stomatal closure characterization indicated that the stomatal closure of Sen 1 was faster whereas that of Anti 2 was slower than that of control.In addition,under drought treatment,compared with the control,the photosynthetic capacity,soluble protein and soluble sugar content,and cell active oxygen homeostasis related parameters of sense lines were significantly improved.These results suggested that TaNF-YB4 regulated the physiological processes and biochemical metabolic pathways related to drought stress,and improved sugar and nitrogen accumulation,photosynthetic carbon assimilation and cellular active oxygen homeostasis,by which to playean important biological role in regulating plant drought stress resistance.5.Semi-quantitative expression analysis of the protective enzyme genes in transformed TaNF-YB4 tobacco lines under drought treatment was performed.Results showed that compared with wild type,the sense line(Sen 1)displayed elevated expression levels for genes of NtCAT1;2,POD1;3,POD4,MnSOD2,SOD2,PYL2,SRK2A-2,and SAPK1、Of which,the expression of NtSODl and NtSOD2 increased obviously.In contrast,the expression levels of them in antisense lines were decreased.These results indicated that TaNF-YB4 could enhance the expression and activity of protective enzyme genes by regulating the transcription of them,which contributes to the enhances ability of plants to osmotic stress resistance.6.In order to determine subcellular localization of TaNFYB-B3,the expression vector TaNFYB-B3-GFP fusion was constructed.Detection of the expression of the fusion in tobacco epidermal cells,it was found that the fluorescence signal derived from the fusion was located at the nucleus of the transformed tobacco epidermal cells,which was consistent with the regulatory function of the transcription factors in transcriptional regulation of downstream genes.To sum up,the molecular characterization of the wheat transcription factors TaNFYBB3 and TaNF-YB4 were analyzed and their functions were evaluated in this study,which provided a basis for improving the plant stress resistance for crop plants.