Drought Resistance And Low Nitrogen Tolerance Evaluation And Mechanism Analysis Of GmDREB1/3 Transgenic Wheat

Drought is major abiotic stresses to wheat production,and averagely 30%of the global wheat production area per year are approximately affected by drought.Except to drought stress,nitrogen deficiency is another major abiotic stresses to wheat growth in many arid areas.To date,much research about abiotic stress resistance in plants has focused on a single type of resistance,such as drought or low nitrogen stress,no genes have been identified to be simultaneously resistant to both drought and low nitrogen stress.While it is notable that most of the studies examining the function of drought or nitrogen stress-related genes have been conducted in greenhouses,rather than across multi-plot demonstration for several years in field trials that could evaluate breeding utilization prospect.In this study,we overexpressed two DREB-like transcription factor from soybean(Glycine max),GmDREB1 and GmDREB3 in four popular commercial wheat varieties(Jimail9,Jimai20,Shi4185 and Jimai22),conducted an extensive series of multi-site for several years drought and low nitrogen stress experiments which revealed that transgenic expression of GmDREB1/3 confers tolerance to drought in wheat.Our studies also revealed that transgenic expression of GmDREB3 confers tolerance to low nitrogen stress in wheat,and we further analyzed the mechanism of drought resistance and low nitrogen tolerance of transgenic wheat.The main results of this research are summarized below:1.The results of drought resistance experiment showed that under indoor drought treatment conditions,the respective overexpression lines exhibited significantly higher survival rates,leaves of the overexpression lines had lower water loss rates than that of WT plants.Analysis of data from a continuous three-year experiment at multiple sites showed that the yields of the GmDREB1 transgenic wheat lines were significantly increased by 4.79-18.43%,and the yield of GmDREB3 transgenic wheat increased by 5.30-13.96%compared to the WT under limited irrigation condition(P<0.5).Under the no-irrigation condition,the average yield of GmDREB3 transgenic wheat were significantly increased compared to the WT by 7.70-11.39%(P<0.05).Under well-irrigated conditions,the yield of the transgenic lines were not significantly different from those of the WT.We analyzed the three major yield components and found that the most obvious difference was a significantly increased in the number of spikes in the transgenic lines vs.the wild-type plants as grown under LIR conditions(P<0.05);No significance effect on other agronomical character was found.The results of values calculated for four indexes(SSI,TOL,STI,and "b" analysis)indicated that the transgenic lines have high stress tolerance relative to the WT plants generally,we did note differences between the transgenic lines in the distinct genetic backgrounds.And a water use efficiency(WUE)analysis conducted at the Shijiazhuang site revealed that the WUE of overexpression lines plants was significantly(P<0.05)higher than WUE of WT plants,averaging a 10-20%increase in this trait.2.The result of analysis on the physiological mechanism of drought resistance showed that:GmDREB1 transgenic lines have improved photosynthesis and have enhance drought resistance from reduced MDA content and relative electrolytic leakage(REL),increasing proline content,chlorophyll content,GS activity,and stability of the rubisco large subunit under the LIR conditions.We examined morphological changes in detail and found the root fresh weights and dry weights of transgenic lines were significantly higher than those of the WT plants under drought stress(P<0.05).We also found that the mean root and stele diameters of the transgenic plants under drought stress conditions were significantly larger than those of the WT plants(P<0.05).Compared with WT,transgenic wheat had developed deep roots and had better root activity.3.Analysis on the molecular mechanism(s)that influence the enhanced drought tolerance of the GmDREB1 transgenic plants:RNA-seq analysis results indicated that some of the up-regulated genes were clustered into stress response-related terms such as 'response to stimulus' and 'signaling'.Notably,RNA-seq data also showed some COMT(caffeic acid O-methyltransferase)and TDC(tryptophan decarboxylase)and SNAT(serotonin N-acetyltransferase),which encode three additional melatonin biosynthesis-related proteins,were also up-regulated in the transgenic line.The expression levels of COMT,TDC,and SNAT in roots and leaves were up-regulated in the transgenic line by qPCR,the changes in melatonin synthesis in roots and leaves that we observed may be consistent with the up-regulated expression of these genes.And melatonin application significantly increased plant height,root growth,and total biomass of transgenic wheat under PEG treatment.DREB transcription factors may affect plant drought resistance by regulating melatonin synthesis,which may be a new response mechanism to water stress in plants.In addition,many genes related to plant photosynthesis,such as the WHAB1.6 gene,were up-regulated in transgenic wheat,which was consistent with the phenotype of high photosynthesis in GmDREB1 transgenic wheat.Thus,our results indicated that the increased drought tolerance resulting from the overexpression of GmDREB1 in transgenic wheat plants under drought stress may be mediated by regulating expressing of melatonin-and photosynthetic-related signaling pathway genes.4.We measured the physiological characteristics related to drought resistance capabilities of GmDREB3 transgenic wheat.The results showed that in the rainproof shelter,compared to the WT plants the overexpression lines showed significantly lower malondialdehyde(MDA)concentrations and relative electrolyte leakage values,and showed higher proline content,soluble protein content,chlorophyll content,and peroxidase(POD)activity in the flag leaves at the heading,at anthesis,and during the grain filling stages.In the field conditions,we found that under the limited irrigation condition the transgenic lines had higher photo synthetic capacity(higher photosynthetic rates and ?PS? values),proline content,soluble sugar content,superoxide dismutase(SOD)activity,Peroxidase(POD)activity,and catalase(CAT)activity than WT plants.We also detected lower malondialdehyde(MDA)content in both the flag leaf and the inverted second leaf in these plants.Collectively,these results from both rainproof shelter and in-field growth experiments indicated that wheat plants overexpressing GmDREB3 exhibited improved drought tolerance,likely resulting from increased photo synthetic capacity,and more active antioxidant metabolism compared to WT plants.5.Notably,GmDREB3 transgenic wheat also showed significant improvement in the ability to tolerate low nitrogen stress.We investigated the performance of the transgenic lines and WT plants under low nitrogen stress in the greenhouse,the transgenic lines had maximum per plant biomass,significantly longer root lengths,plant heights,shoot and root fresh weights,shoot and root dry weights than WT plants under low N conditions.We detected no significant differences under normal condition.We measured the nitrate flux rates of roots,which revealed that the transgenic lines had significantly higher nitrate influx rates than that WT plants.We investigated N use-related traits after harvest and found that the transgenic lines were significantly increased compared to WT by 10.00-40.20%and 16.20-36.00%in root N and grain N accumulation traits under low N conditions,respectively(P<0.05).In two consecutive growing seasons,the grain yield of the three transgenic lines significantly increased by 6.33-22.53%compared with WT plants.We also analyzed the three major yield components in more mature plants and found that the most obvious difference was the significantly increased spike number in the transgenic lines vs.the wild-type WT plants as grown under low N condition.Transgenic expressing GmDREB3 prolonged leaf functional duration and improved photosynthesis under the low N condition.6.Analysis on the molecular mechanism(s)that influence the enhanced drought and nitrogen tolerance of the GmDREB3 transgenic plants:ChIP-seq data showed GmDREB3-mediated significant induction of the transcription of several known LN-and drought-related transcription factor genes positioned within these DERs,including NRT2.5(high-affinity nitrate transporter 2.5),LEA(Late embryogenesis abundant),EREBP1(Ethylene-responsive transcription factor),and WRKY46(WRKY DNA-binding protein 46).Moreover,in light of our finding about the increased spike number per m2,it was conspicuous that our ChIP DEGs included the TB1 locus(Teosinte branched 1,a TCP family transcription factor that is a negative regulator of lateral branching).QPCR and transient luciferase reporter transcriptional activation assays indicated that GmDREB3 specifically activated the expression of the Luc gene as driven by the native promoters of TaNRT2.5 and TaLEA,both of which contain DRE/CBF elements.Collectively,the results of the greenhouse and multi-year multipoint drought resistance field experiments indicated that GmDREBl/3 can improve the drought resistance of wheat in different genetic backgrounds and can improve tolerance to low nitrogen stress.Through examination of various physiological indicators and expression profile analysis,the mechanism of GmDREB1/3 for improving the stress resistance of transgenic wheat was analyzed.These results provide a theoretical basis for using GmDREB1/3 to carry out crop molecular breeding,establishes a new idea for the simultaneously confers tolerance to drought and low nitrogen stress in a cereal crop.