Function Of Wheat Zinc Finger Transcription Factors TaZFP593 And TaSAP1-1 On Mediating Tolerance To Abiotic Stresses

Zinc finger transcription factors play important roles in mediating responses of plants to abiotic stresses through regulating downstream genes.In this study,a C2H2-type zinc finger TF gene and a A20/AN1-type zinc finger TF gene in wheat respectively designated as TaZFP593 and TaSAP1-1 were identified.TaZFP593 contains a C2H2 zinc domain.Under nitrogen deprivation,the expression levels of TaZFP593 in wheat leaf and root were increased and reached the maximum at 27 h.In addition,the transcript abundance of TaZFP593 was shown to be enhanced along with the decreased level of nitrogen across the concentration regime examined,and the induced expression of TaZFP593 under nitrogen deprivation condition returned to original abundance once the wheat seedlings were retreated by a recovery nitrogen treatment.Using vermiculite as culture material combined with nutrient solution with different nitrogen concentration,transgenic tobacco lines overexpressing TaZFP593(Line1 and Line3)were used to characterize the function of TaZFP593 on mediating tolerance to nitrogen deprivation.Phenotype identification analysis suggests overexpression of TaZFP593 in tobacco confer improved tolerance to nitrogen deprivation.The transgenic plants of Line1 and Line3 exhibited enlarged plant,increased root number and biomass,etc.Further studies showed NtPIN3,which play importmen roles in mediating cellular efflux of auxin via polar transportation and modulates initiation and elongation of the primary and lateral roots,was up regulated under nitrogen deprivation in the transgenic lines compared with WT.And similar with the above changes,two nitrate transporters,NtNRT1-2-t and NtNRT2;2 were also up regulated in the same state.These regulations were in accordance with the better root characteristics of transgenic lines plants.Analyses on the cell protective enzyme(SOD,POD,and CAT)indicated that transgenic lines overexpressing TaZFP593 enhanced the enzymatic activities of SOD,POD and CAT but decreased the content of MDA.Expression analysis revealed that the transcript abundance of several members including NTCAT1,NTCAT3,NTPOD1.NtPOD1;7 and NTPOD2;1 were up regulated in the transgenic lines after nitrogen deprivation.However,enzymatic activityand expression level of NR,NIR and GS,three enzymes related with nitrogen assimilation,performanced no significant difference between transgenic lines and WT.These results indicated TaZFP593 may increase tolerance to nitrogen deprivation through regulation of multiple pathways,including changed root morphology,nitrogen absorption and ROS detoxification.TaSAP1-1 contains typical A20/AN1 zinc domain.Phylogenetic analysis revealed that TaSAP1-1 shares high similarity to a SAP family member TaSAP1 which also belongs to wheat species.Under abiotic stresses including phosphorus deprivation and NaCl,PEG,ABA treatment,the expressions of TaSAP1-1 were all up regulated in wheat roots and leaf with a little different in expression patterns.The results indicates TaSAP1-1 participate in multiple stress signaling network.Based on DNA recombinant technology and gene genetic transformation via Agrobacterium-tumefaciens mediated approach,the binary expression plasmids harboring TaSAP1-1 in both sense and antisense orientation were constructed,and the transgenic tobacco plant lines were generated.Combined cultivation ways of solid agar,hydroponic and vermiculite systems,the phenotype analysis of WT and transgenic lines under Pi deprivation and salt stress were conducted.The results showed that the transgenic lines improved seedling growth under above two stresses.Further studies showed that phosphorus transporter genes for trial in this study were up regulated under Pi deprivation in transgenic lines compared with WT.While after NaCl treatment,overexpressing TaSAP1-1 reduced the stomatal opening and increased the gene expression of the ABA receptors,such as NtSARK and NtSAPK for trial.These indicated that TaSAP1-1 may improve adaptation to above stresses by regulating gene expression related with transport of Pi or ABA signaling pathway.Furthermore,the binary expression plasmids harboring TaSAP1-1 and TaAMTR respectively fused GFP were constructed,which layed a foundation for the study of subcellular localization.The expression vectors pGADT7-TaSAP1-1/TaAMTR and pGBKT7-TaSAP1-1/TaAMTR were also constructed,and further study using yeast double hybridization technology showed interactions exist between TaSAP1-1 and TaAMTR.In view of the complexity of regulatory roles of SAP transcription factors,the molecular mechanism of TaSAP1-1 responding to Pi deprivation and salt stress remains to be further studied.