Mechanism Of Wheat Methionine Sulfoxide Reductase TaMSRB3.1 In Response To Osmotic Stress

Drought is one of the mainly abiotic stressors in agricultural production.When subjected to drought stress,plant cell will produce a large amount of reactive oxygen species(ROS),which causes oxidative damage to macromolecules in cells.Wheat is sensitive under abiotic stress treatment resulting in excess ROS,which damages proteins in cells,especially proteins containing Met and Cys residues in the surface.The plant cell contains methionine sulfoxide reductase(MSR),which can reduce the oxidized Met.Although many studies have reported their functions in stress tolerance in other organisms,the anti-osmotic stress function and mechanism of TaMSRB3.1 of wheat have not been reported.TaMSRB3.1 gene from cv.SR3 with salt/drought tolerance will be systematically studied on its mechanism in response to osmotic stress.The main results obtained are as follows:1.Molecular characteristics of TaMSRB3.1Previous studies have shown that the TaMSRB3.1 gene sequence is highly similar to the MSRB family gene sequences in other plants.Their deduced amino acids contained four conserved functional domains and one of them at the C-terminus,the SelR domain is a characteristic sequence of the MSRB family.It was found that TaMSRB3.1 can specifically recognize and reduce R-type methionine sulfoxide in vitro,which proved that TaMSRB3.1 is a member of the MSRB family.TaMSRB3.1 is expressed in roots,stems and leaves,and has the highest expression in leaves.TaMSRB3.1 was localized in the chloroplast and its expression was up-regulated induced by ABA,PEG,and H2O2.2.The function and physiological mechanism of TaMSRB3.1Arabidopsis strains including Col-0,TaMSRB3.1 over-expression lines(OE),msrb3 mutant and the msrb3 mutant reversion were used and treated with mannitol and H2O2.The results showed that there were no significant phenotypic differences in different Arabidopsis lines under control conditions.Under the treatment of 100 mM,200 mM and 300 mM of mannitol and 1.0 mM,1.5 mM of H2O2,the OE lines grew better,the root length was significantly greater than Col-0,and the root length of the reversion was longer than that of Col-0 too,but the root length of msrb3 was significantly smaller than Col-0.Experiment on soil water loss survival rate showed that the survival rate of OE lines was significantly higher than that of Col-0,and msrb3 was the lowest.The water retention rate of OE lines was significantly higher than that of Col-0.and msrb3 was the lowest.These results indicated that overexpression of TaMSRB3.1 significantly enhances Arabidopsis resistance to osmotic stress.The ROS staining experiments showed that the ROS content in the OE lines was lower than that of Col-0.While the msrb3 was the highest.The quantitative analysis of H2O2 showed that the content of H2O2 in OE lines was significantly reduced,and the enzyme activities of SOD,CAT and POD,which are related to ROS elimination,also increased significantly.While the msrb3 had the lower enzyme activities than the wild type.It was also found that in AtOE lines,the marker genes of ROS generation such as RbohD and RbohF were down-regulated.While the expression of ROS scavenging enzymes CAT1,CAT3,FeSODl and Cu/ZnSOD3 was significantly up-regulated.These results show that TaMSRB3.1 overexpressing in Arabidopsis can regulate ROS balance.Further,it was found that,the stomatal of OE lines compared to the other types was more sensitivity to ABA with smaller stomatal aperture.Under the control conditions,the stomatal opening of each line of Arabidopsis was approximately the same.However,after adding ABA treatment,the stomatal opening of AtOE lines was significantly reduced,While the msrb3 has low sensitivity to ABA and small changes in stomatal opening.In addition,the expressions of endogenous synthetic genes NCED3,AAO2,ABA1 and downstream stress response genes RD29B and RD22 of ABA were found to be significantly up-regulated in the AtOE lines.It is indicated that overexpression of TaMSRB3.1 can increase the sensitivity of Arabidopsis stoma to ABA and it can also regulate ABA signaling pathways.3.The mechanism analysis of TaMSRB3.1 geneUsing bioinformatic analysis,it was predicted that heme oxygenase HOI is a potential interaction protein of MSRB3.TaHO1 is also located in the chloroplast as well as TaMSRB3.1.At the same time,the wheat TaHO1 protein contains about 2.4%of Met residues,and is mostly distributed on the surface of TaHO1,which is consistent with the characteristics of MSR potential substrates.We confirmed that TaMSRB3.1 could interacted with TaHO1 by Y2H,BiFC,Co-IP and genetic experiments and played an stress-tolerance role through TaHO1.