Functional Characterization Of Soybean Transcription Factor GmNF-Y1 And GmDi19-5 In Response To Abiotic Stresses

Aboitic stresses,such as drought and salinity,influence plant growth and severely limits-crop productivity.Therefore,understanding the molecular mechanisms and isolating genes of abiotic stress responses is necessary for genetic improvement of stress tolerance in crop.NF-Y is a heterotrimeric transcription factor comprised of three distinct subunits:NF-YA,NF-YB and NF-YC.Drought-induced proteins(Di19)are zinc finger transcription factors with two conserved C2H2 zinc finger domains.Several studies showed that NF-Y and Di 19 transcription factors might act as switches in the complex regulatory networks controlling abiotic stress processes.However,the mechnism of NF-Y and Di19 in response to abiotic stress has not been well established.In this paper,we characterized the functions of two GmNF-Y1 and GmDi19-5 by using biochemical,molecular genetic approaches.Furthmore,we screened the interaction proteins of GmNF-Y1 and identified the interaction between GmNF-Y1 and candidate proteins.The main results are as follows:1.Functional identification of GmNF-Y1:In this study,a NF-Y gene was isolated from soybean.The deduced GmNF-Y1 protein contained conserved core regions of NF-Y.GmNF-Y1 was highly expressed in leaf and cotyledon.GmNF-Y1 was upregulated by drought,H2O2 and ABA treatments.Subcellular localization showed that GmNF-Y1 was mainly localized at nucleus and plasma membrane.Overexpression of GmNF-Y1 obviously enhanced drought,osmotic and oxidative tolerance in transgenic Arabidopsis and wheat.Further analysis showed that a number of stress responsive genes were induced in GmNF-Y1 transgenic plants.Physiological and biochemical assayes showed that improved abiotic stress tolerance of GmNF-Y1 transgenic Arabidopsis and wheat was through reducing membrane damage in transgenic plants,with lesser malondialdehyde(MDA),but higher relative water content(RWC)and catalase(CAT)and peroxidase(POD)activities than wild type(WT)under stress conditions.2.Identification of the interaction between GmNF-Y1 and other candidate proteins:Four candidate interaction proteins with GmNF-Y1 were screened out from cDNA library constructed from drought and salt treated soybean seedlings by means of yeast two hybrid These proteins included NF-YC subunit NF-YZ,heat shock protein 40(HSP40),Jun activation domain-binding protein 1(JAB 1)and drought-induced protein Di 19-5.Bimolecular fluorescence complementation(BiFC)and pull down assayes demonsrated that all candidate proteins interacted with GmNF-Y1.In addition,a heterotrimer of GmNF-YX/NF-Y1/NF-YZ was identified by yeast three-hybrid system.3.Functional identification of GmDi19-5:A stress-induced gene,named GmDi19-5 was also cloned from soybean cDNA library,which contained two unusual Cys2-His2(C2H2)putative zinc finger domains.A total of seven soybean Di19 genes has been identified and characterized on the base of phylogenetic relationship,structural diversity,conserved protein motifs,hydrophilicity profiles,phosphorylation sites,chromosomal location and promoter regions.All seven proteins share high identity with other known Di19 proteins.Of them,GmDi19-5 showed the highest expression level among seven Di19 genes,under various stimuli.The dehydration-or salt-induced transcription of GmDi19-5 was blocked by Fluridone,an inhibitor of ABA biosynthesis,or DMTU,the scavenger of H2O2.GUS expression driven by the GmDi19-5 promoter was induced by salt,drought,oxidative and ABA stresses mainly at the vascular bundles,young leaves and roots.Subcellular localization showed that GmDi19-5 was localized at nucleus.Overexpression of GmDi19-5 in Arabidopsis increased their sensitivity under salt,drought,oxidative and ABA stress treatments and activated the expression of stress-associated genes.In conclusion,GmNF-Y1 conferred drought,osmotic and oxidative stress tolerance through regulating the expression of stress-related genes and enhancing the antioxidant system.GmDi19-5 acted as a negative regulator in response to salt and drought stresses by regulating ABA-and SOS-mediated signal pathways.These results provided gene resources,genetic materials and theory basis for improving abotic stress tolerance in crops.