Function And Mechanism Of Wheat TaBRI1-B In Response To Alkaline Salt Or Osmotic Stress

Soil drought and salinization are widespread environmental limitation that affect the growth,development,and yield of cereal crop by reducing their photosynthetic capacity and water uptake,etc.As an important cereal crop,both growth and development of wheat are affected by various environmental stresses.Therefore,improving the stress tolerance of wheat is a key issue that we need to solve promptly.It was found that TaUGGT-B2,located in the endoplasmic reticulum and interacted with TaBRI1,played an prominent role in wheat stress tolerance.In order to analyze the role and mechanism of TaUGGT-B2 deeply,the function and mechanism of wheat BRI1 on stress tolerance were studied.TaBRI1 has three copies in wheat genome,locating on the A,B,and D sub-genomes,respectively.Due to the highest expression level of copy B,it was selected for the following experiments.TaBRI1-B,which protein located on the cell membrane,was expressed in roots,stems,and leaves of wheat,with the highest expression level in leaves.It was found that the expression of TaBRI1-B was downregulated by multi-inducers including PEG 6000(osmotic stress,drought simulation),alkaline carbonate(NaHCO3:Na2CO3=9:1,pH 8.9),H2O2,and ABA.TaBRI1-B VIGS lines(TaVIGS lines),overexpression lines(TaOE lines),and RNAi lines(TaRNAi lines)were constructed to study the function and mechanism of it in wheat response to osmotic or alkaline salt stress.The results showed that the TaOE lines had a better growth than the wild type ones;while the TaOE lines were more sensitive to both stresses than the wild type ones under stress conditions.Although the growth was inhibited compared to wild-type plants under no stress conditions,either TaRNAi lines or TaVIGS lines showed more tolerance to osmotic or alkaline salt stress.When BR inhibitor PPZ was added,the growth of TaOE lines was inhibited and their stress sensitive phenotypes were restored.However,when BL added,TaRNAi lines restored their growth indicating that BR could positively regulate plant growth but negatively regulate wheat stress tolerance.Because of the expression of TaBRI1-B induced by H2O2,the expression of ROS-related genes was investigated.It indicated that the expression of ROS scavenging genes decreased while the expression of ROS producing genes increased in TaVIGS lines compared to wild type plants under stress treatment.It was found that under stress conditions,the content of H2O2 in the leaves of TaOE lines was significantly higher than that of wild type and TaRNAi lines by quantitative analysis of H2O2.It showed that under stress conditions,the related enzyme activities in TaOE lines were significantly lower than those in wild type,while those in TaRNAi lines enhanced slightly compared to the wild type.The above results suggest that ROS content increased in TaOE lines while decreased in TaRNAi plants under both stress treatments.We then tested the content of soluble sugars and found that under normal conditions,there was no significant difference among various genotypes;under two stress conditions,the soluble sugar content in the TaOE lines significantly decreased compared to the wild-type,while in the TaRNAi lines increased.it showed that under stress conditions,compared to YM20,both grain size and TKW of TaOE lines were significantly inhibited,while either of them in TaRNAi lines was enhancer than those of YM20.Potential interaction proteins were obtained by screening a wheat cDNA library,then their interactions betweenTaBRI1-B and TaFRK-D or TaCOR410-B were verified by yeast two-hybrid(Y2H),and luciferase complementary assay(LCA).It indicated that TaBRI1-B could phosphorylate TaFRK-D in vitro,which may be a downstream protein of TaBRI1-B.The function of wheal TaFRK-D was further investigated in response to both stresses.It was found that the soluble sugar content in TaFRK-D VIGS lines was notably reduced and sensitive to stress compared to wild type.It showed that TaUGGT-B2 could glycosylate TaBRI1-B.not Tabril-B.which was obtained by site-directed mutagenesis to mutate the glycosylation site of TaBRI1-B.In summary,the above results indicated that TaBRI1-B net,atively regulated wheat stress tolerance by interacting with TaFRK-D to regulate soluble sugar content in response to stress,and it also regulated ROS content,but the mechanism is unknown;TaUGGT-B2 could glycosylate TaBRI1-B regulating,the balance of ER stress and wheat growth/development.The results of this experiment are helpful to understand stress tolerance mechanism and provide new gene target for wheat stress tolerance breeding.