Mechanism Of Wheat Methionine Sulfoxide Reductase Genes TaMSRA4.1 In Abiotic Stress Response

Wheat is one of the important crops with the most extensive distribution,the larger amount of acreage,the larger production and the most abundance of processed products.It plays an important role in the development of modern agriculture.Wheat is a glycophyte limited to abiotic stress.With the growing scarcity of water resources and saline land desertification,it caused a great negative impact on the yield and quality of wheat by many abiotic stresses such as high salt and drought.Therefore,it is an important direction to identify new stress-tolerance genes to creat varieties of wheat in China in the future using conventional and modern biotechnologies.Based on the transcriptomic data of SR3 previously,it was found that some of methionine sulfoxide reductase(MSR)genes including TaMSRA4.1 were involved in abiotic stress response.In this psaper,TaMSRA4.1 gene cloned from wheat cv.SR3 were selected to further study.In our previous work,it was found that TaMSRA4.1 has the typical characteristics of MSRA genes.Its product located in chloroplasts and it was expressed in root,stem and highest in the leaf.It was shown that overexpression TaMSRA4.1 in Arabidopsis thaliana could increase the salt tolerance.On this basal work,its role in wheat and its mechanism in response to salt or drought stress were further discovered in this paper.The main results obtained are as follows:1.Analysis of the molecular structure and enzymatic properties ofTaMSRA4.1On the basis of previous research,it was found that TaMSRA4.1 is similar with MSRA4 from the other higher plants in sequence with three conservative function structure domains.In this paper,the enzymatic properties of TaMSRA4.1 in vitro was further studied.It was found that TaMSRA4.1 had a specific identification to S-type methionine sulfone and belongs to the MSRA family members.The expression pattern of TaMSRA4.1 gene under multi-stresses was analyzed by qRT-PCR.It was found that the expression level of TaMSRA4.1 under NaCl,PEG,ABA and H2O2 stress showed a certain rule,and could be induced by these stresses.2.The function analysis of TaMSRA4.1 geneYangmai 20,TaMSRA4.1 over-expressor in wheat and Arabidopsis over-expression lines,wild-type Col-0 and msra4 mutants were treated by different levels of NaCl and PEG,and their phenotypic changes were observed.Under control condition,there were no differences between Yangmai 20 and its TaOE lines.The TaOE lines grew much better than that of wild type with significantly longer total root length and higher chlorophyll content under100 mM NaCl or 10%PEG treatment.According to the ROS staining experiments,the ROS level were significantly lower in TaOE lines than the control under salt or drought stress.ROS scavenge enzyme activities were measured.It was found that CAT activity in TaOE lines was significantly higher than that in wild type.Similar phenotype results were found in A.thaliana materials.There were no obviously differences among wild type,AtOE lines and msra4 mutants in control condition.When treated with 100 mM NaCl or 200 mM Mannitol,the AtOE lines had significantly longer roots with better growth in compare with the wild type.While the growth of msra4 is obviously weaker than that of the wild type.At the same time,the survival rate experiments were carried out in soil under the sallt and drought treatment.The results showed that the survival rate of the AtOE lines was significantly higher than that of the other types of Arabidopsis,which showed that the OE lines had a stronger resistance to salt or drought stress.The water loss rate experiment showed that the leaves of AtOE lines had stronger water-retaining property.It was also found that the ROS level was significantly lower in AtOE lines compared to the wide-type by ROS staining experiments,and the AtOE lines had higher SOD and CAT enzyme activities,lower H2O2 and MDA content.The superiority of AtOE lines were more distinctly under salt or drought treatment,while the msra4 mutants showed the opposite phenotype.It was found that the marker genes of ROS signaling and the ROS generation were down-regulated while the ROS scavenging genes were shown up-regulated by qRT-PCR.Further,we found that the stomatal of AtOE lines were more sensitivity to ABA with smaller stomatal aperture.The endogenous ABA synthesis and downstream ABA-dependent stress-responsive pathway were significantly up-regulated by detected the relevant marker genes in ABA signaling pathway.The ABA content in AtOE lines were significantly higher than that in wild type,the results suggested that the ABA signal pathway were promoted in AtOE lines.We found that TaMSRA4.1 regulated ABA synthesis and ABA signaling pathways separately by adding ABA specific inhibitor Norflurazon and the phenotype experiment of TaMSRA4.1/aba2 line.3.The mechanism analysis of TaMSRA4.1 geneThrough bioinformatics prediction analysis,heme oxygenase HOI was predicted to be the potential interacting protein of MSRA4 in many Triticum species and soybean.We cloned the wheat Heme oxygenase TaHO1 and analyzed the gene structure of TaHO1.It were found that the TaHO1 protein can be divided into variable region(VD)and Heme Domain(Heme area),the TaHO1 protein was rich in Met residues(2.4%),and most of them were surface located,it conform to the characteristics of the MSR substrate.We confirmed that TaMSRA4.1 could interacted with TaHO1 by Y2H,BiFC and Co-IP experiments.Further analyses showed that the Heme Domain of TaHO1 contributed to the TaMSRA4.1 interaction.The substrate enzyme activity experiments in vitro showed that TaMSRA4.1 could reduce oxidized TaHO1,indicating that TaHO1 is the substrate of TaMSRA4.1.Recombinant proteins of wild type TaHO1,and mutants in which Met residues were replaced with another non-polar amino acid,Leu(TaHO1 M84L,TaHO1 M92L,TaHO1 M218L,TaHO1 M84/92L),were generated and purified.Upon treatment with HOC1 and restored by TaMSRA4.1,our observations suggest that the M84 and M92 residues of TaHO1 are important for maintaining enzymatic activities,and their non-functional oxidized states are probably important targets for protein repair by TaMSRA4.1.Pharmacological phenotype experiments and genetic experiments confirmed that TaMSRA4.1 plays a role in response to salt and drought stress through TaHO1.Take together,TaMSRA4.1 interacted with TaHO1 in structure,regarded TaHO1 as substrate in fuction and played an stress-tolerance role through TaHO1.Further,the phenotype experiments of Arabidopsis material showed that CO,one of the important products of HOI,was involved in TaMSRA4.1 salt or drought stress tolerance.This study demonstrated the function and mechanism of TaMSRA4.1 in stress-tolerance,which will provide new information for understanding the molecular basis of wheat tolerance,and a new source of resistance genes for wheat breeding.