Studies On NF-Y Type Transcription Factor Genes In Regulating Plant Drought Tolerance And Related Physiological Mechanisms In T.aestivum

It is of great practical significance to enhance the ability of wheat plant to resist drought adversity.Transcription factors,the important regulatory factors in plant species,play important biological roles in mediating plant growth and development and in resisting abiotic stresses.In this study,three wheat NF-Y transcription factor genes referred to as TaNF-YA7,TaNF-YB2,and TaNF-YB11,the molecular characteristics of them,their abilities to mediate plant resistance to drought stress,and their related physiological and molecular mechanisms have been studied.The main results are as follows:1.TaNF-YA7,a gene belonging to the NF-YA subfamily,was located on chromosome 5B.The gene contained an open reading frame with a length of 621 bp,encoding 206-aa polypeptide with a protein molecular mass of 22.15 kDa,and an isoelectric point(pI)of 7.90.The TaNF-YA7 protein contains a conserved HAP2 domain(100 aa to 161 aa).TaNF-YB2,a gene belonging to the NF-YB subfamily,is located on chromosome 2B with an open reading frame length of 675 bp,encoding 225-aa polypeptide with molecular mass of 23.38 kDa and isoelectric point(pI)of 5.74,containg the typical conformation of NF-YB and conserved domain.TaNF-YB11,a member of the NF-YB subfamily,is located on chromosome 1A,with an open reading frame length of 411 bp,encoding 136 amino acids with a molecular mass of 14.93 kDa and an isoelectric point(pI)of 5.65.TaNF-YB11 has a highly sequence similarities to the NF-YB family genes of some different plant species.These gene translation products were sorted by endoplasmic reticulum and localized in the nucleus.2.The transcripts of TaNF-YA7,TaNF-YB2,and TaNF-YB11 in the root and leaf tissues of wheat seedlings showed significant responses to drought stress,as shown by the fact that under 27 h drought treatment,the expression levels of TaNF-YA7,TaNF-YB2,and TaNF-YB11 were continuously up-regulated as the drought treatment progression.After 27 hours of drought treatment,the expression levels of the above genes in root and leaf tissues were gradually restored as the recovery treatment progressed.This indicates that the above genes in the NF-Y family of wheat tested exhibit significant response to drought stress at the transcriptional level.Using a cis-acting element prediction tool for gene promoters,it was identified that the NF-Y gene promoters contain the elements of ABRE,MYB,MYC,MBS,MYB recognition site,and AAGAA motif,which are involved in the response of genes to drought stress.Further,promoter deletion fragments were used to drive the expression of the reporter gene(glucosidase gene GUS)and GUS histochemical staining was performed,which confirmed the function of above elements in regulating gene transcription in response to drought stress.3.Under drought treatment,compared with the wild type control,the plant growth potential of lines with overexpression TaNF-YA7,TaNF-YB2,and TaNF-YB11 was significantly enhanced,and the biomass in them was improved.Among them,the biomass of TaNF-YA7 increased by 1.9 times,the biomass of TaNF-YB2 increased by 1.4 times,and the biomass of TaNF-YB11 increased by 1.6 times.On the contrary,the growth potential of the lines with knockdown expression of above genes deteriorated,and the biomass in them significantly decreased.The results showed that the NF-Y transcription factor gene of wheat tested played an important role in mediating plant drought resistance.4.Under drought treatment,compared with WT,the stomatal closure rates in lines with NF-Y gene overexpression were increased in response to drought stress(1 hour).Under water deficit treatment(3 h),the water holding capacity of isolated leaves in lines with NF-Y gene overexpression increased with the treatment process.Under drought treatment,the photosynthetic carbon assimilation ability of the above gene sense lines improved,proline and soluble sugar content increased,cell protective enzyme activity increased,and malondialdehyde(MDA)content decreased.This indicates that TaNF-YA7,TaNF-YB2,and TaNF-YB11 enhance the ability of plants to resist drought stress by regulating the physiological and biochemical processes related to stress response.5.Compared with the wild type,transcripts of distinct genes of key enzymes(Δ1-pyrroline-5-carboxylate synthetase,P5CS)regulating proline biosynthesis and some members of SOD,CAT and POD families were increased in TaNF-YA7,TaNF-YB2 and TaNF-YB11 lines.The P5CS gene,SOD gene,CAT gene and POD gene of TaNF-YA7 lines were TaP5CS2,TaSOD3,TaCATl and TaPOD4,respectively.The P5CS,SOD,CAT and POD genes of TaNF-YB2 lines were TaP5CS2,TaSOD1,TaCAT5 and TaPOD5,respectively.The P5CS,SOD,CAT and POD genes of TaNF-YB11 lines were TaP5CS2,TaSOD2 and TaCAT3,respectively.The proline content and plant biomass of the TaP5CS2 lines with knockdown expression decreased under drought treatment.6.Compared with WT treated by drought,964 differentially expressed genes(DEGs)were identified in TaNF-YA7 overexpression lines,including 678 up-regulated genes and 286 down-regulated genes.926 differentially expressed genes(DEGs)were identified in TaNF-YB11 overexpression lines,including 614 up-regulated genes and 312 down-regulated genes.GO and KEGG analysis of the identified DEGs showed that the above genes belonged to the GO groups of "biological process"," cellular component" and"molecular function" and KEGG biochemical pathways such as "abiotic response","oxidative stress defense","ion transport and photosynthesis","cellular processes","environmental information processing","genetic information processing","metabolism"and " organismal systems".7.Protein interaction analysis showed that there was protein-protein interaction occurrence between TaNF-YA7,TaNF-YB2 and TaNF-YC7,and protein interaction between TaNF-YA2 and TaNF-YB11 and TaNF-YC3.This indicates that the above NF-Y proteins are involved in the construction of heterotrimer TaNF-YA7/TaNF-YB2/TaNF-YC7 and TaNF-YA2/TaNF-YB11/TaNF-Y C3,respectively,which are nvolved in the transcriptional regulation of drought stress responsive genes.8.Regression analysis showed that the expression levels of TaNF-YA7,TaNF-YB2,TaNF-YB11 and TaP5CS2 were highly correlated with the yield of water-saving wheat varieties at maturity stage.These genes could be used as molecular evaluation indices for selecting water-saving and drought-resistant wheat varieties.In summary,this study revealed that wheat NF-Y transcription factor genes,namely TaNF-YA7,TaNF-YB2,and TaNF-YB11 are typical in drought stress response.Overexpression of these genes confers plants enhanced ability to resist drought stress by regulating the physiological and biochemical processes related to osmotic stress responses.It can be used as a molecular index for selecting and evaluating water-saving cultivated wheat varieties.