Molecular Characterization And Physiological Function Of Methionine Sulfoxide Reductase Gene Family In Wheat

Reactive oxygen species (ROS) would be generated during the course of normal aerobic metabolism, cellular metabolism in response to different environmental stresses, and pathogen attacks. It can cause detrimental effects on biomolecules, such as DNA, proteins, and lipids, and this process can possibly eventually lead to the oxidative destruction of the cell. Although most of organisms have evolved both enzymatic and nonenzymatic scavenging systems to eliminate ROS, there are still some ROS leaky, resulting in the oxidation of biological macromolecules. Several amino acid residues of proteins especially the sulfur-containing amino acids, cysteine (Cys) and methionine (Met) are susceptible to oxidation by various forms of ROS. The ROS-mediated oxidation of Met residues leads to a mixture of the S-and R-epimers of methionine sulfoxide (MetSO), which alters the activity and conformation of various proteins. However, these alterations can be reversed by means of peptide methionine sulfoxide reductases (MSRs). There are two major types of MSR, MSRA and MSRB, which are specific for the S-and R-diastereoisomers of MetSO, respectively. MSRA and MSRB have been identified virtually in many organisms including bacteria, yeast, mammals and plants. Compared to other organisms, plants are characterized by the presence of a large MSR gene family. There are5MSRA and9MSRB orthologs were identified in Arabidopsis, and at least4MSRA and3MSRB orthologs exist in rice. MSR may play more complex roles in plant than in other organisms, because of their multiple copy number and different cellular compartmentation.Wheat is one of the most important crops worldwide, but its growth and productivity are adversely affected by abiotic stresses such as drought and salt. Therefore, it is critical to enhance wheat stress tolerance for coping with these adverse stimuli and improving its productivity. In our previous work, a new somatic hybrid introgression line Shanrong No.3(SR3) was generated by somatic hybridization between Triticum aestivum cv. JN177and Agropyron elongatum, the latter is a salt and drought tolerant grass. Then the suppressive subtractive hybridization (SSH) technique was used to construct a salt-stress responsive SSH cDNA library of SR3and JN177wheat, which was used as a platform for identifying genes related to stress-resistance in wheat.So far, most of functional characterization about stress-tolerance of plant MSRs has been carried out in Arabidopsis and rice, but no data is available related to the wheat MSR gene family. In this study, we initially searched the full-length cDNA sequences published in the web NCBI using MSRs of Arabidopsis and rice, and found eight wheat orthologs. Detailed bioinformatics analyses for the eight genes have been carried out. We found that one is highly homologous to AtMSRA4with MSRA conserved domain, and named TaMSRA4. The other seven are similar to AtMSRB with MSRB conserved domain. Seven wheat MSRBs belong to three different subfamilies, entitled TaMSRB1subfamily including TaMSRB1.1and TaMSRB1.2, TaMSRB2subfamily including TaMSRB2.1, TaMSRB2.2and TaMSRB2.3, TaMSRB3subfamily including TaMSRB3.1and TaMSRB3.2. There is a high degree of similarity in their conserved domain between the TaMSRs and their homoeologous genes of Arabidopsis and rice. Sequences belong to the same subfamily are highly conserved, differences between each other only in a few amino acid sites. Eight TaMSRs were predicted to be located in distinct subcellular compartments by bioinformatic analysis. Members of TaMSRB2subfamily were predicted to be located in cytoplasm, while members of TaMSRA4, TaMSRB1and TaMSRB3subfamilies were predicted to be restricted in the chloroplast, these results exhibited a little divergence to each AtMSR ortholog. To explore genes’function of wheat MSR, we selected two genes belonging to different subfamilies, TaMSRB2.1and TaMSRB3.1for further research, of which the TaMSRB2.1exhibited notable changes in transcriptional level revealed from the microarray results. RT-PCR analysis showed that the expression of them were enhanced by salt and PEG during a short period of time. The two genes also have different expression patterns in different tissues. We cloned the two genes from the full-length cDNA library of SR3. Subcellular localization experiments showed that their exact subcellular localizations are consistent with prediction by bioinformatic analysis. Then we constructed their over-expression vectors and transformed them into Arabidopsis thaliana. Root-length phenotype experiments were pertbrmed under various stress conditions. It was shown that over-expression of TaMSRB2.1in Arabidopsis increased resistance to NaC1and mannitol treatments apparently, while there was no obvious difference between the transgenic lines and the wild when they were treated with H2O2or ABA. For TaMSRB3.1, the transgenic lines exhibited significant resistance to NaCl, H2O2, ABA, and high concentrations of mannitol. The transgenic lines of TaMSRB3.1also showed higher livability than the wild type in the water control experiment. TaMSRB2.1and TaMSRB3.1were suggested to play an important role to defense against abiotic stresses, and may be useful for application in crop improvement.