- OsMSRA4.1 and OsMSRB1.1 are constitutively expressed in all organs and can be induced by various stress conditions. Overexpression of either OsMSRA4.1 or OsMSRB1.1 in yeast enhanced cellular resistance to oxidative stress. In addition, OsMSRA4.1-overexpressing transgenic rice plants also showed enhanced viability under salt treatment.OsMSRA4.1 plays an important role in stress responses.
- Cloning of OsMSRA4.1 cDNAs:
Total RNA from rice leaves was extracted using the guanidinium isocyanate/acidic phenol method as described by Chomczynski and Sacchi. cDNA synthesis and RTPCR were performed using a kit. The OsMSRA4.1 gene-specific primers were 5'-TCGATGCCTCCTCTCCTCG-3'� and 5'-AGGATCCTTCACCCGTAGCAACGGAT-3' The OsMSRA4.1 cDNAs was cloned into the yeast expression vector p181AINE and the constructs were transformed into yeast using the lithium acetate method.
To test whether OsMSRs are involved in stress responses, transgenic rice plants harboring OsMSRA4.1 were generated. Positive transgenic lines (line 9 and line 43) were confirmed by Northern and Western analyses and selected for further analysis(Fig. 1a).
- Overexpression of OsMSRA4.1 enhances yeast tolerance to H2O2 stress, and that the OsMSRA4 can compensate for a deficiency in MSRA.
- To better understand the functions, the expression patterns of OsMSRA4.1 was examined by RT-PCR in difierent tissues. The results revealed that OsMSRA4.1 was constitutively expressed in all the tissues including roots, stems, leaves, Xowers and callus.
- To investigate the transcription inducibility of OsMSRs, Northern blot analysis was performed under various stress conditions.
OsMSRA4.1 expression was enhanced apparently by salt, mannitol, cold and high temperatures, and a slight increase was observed at the 48 h time point after ABA treatment. The expression of OsMSRA4.1 was also induced by MV treatment at 1 h and subsequently declined.
- OsMSRA4.1 and OsMSRB1.1 were able to reduce both the free MetSO and protein-bound-like MetSO substrates in the presence of
the DTT reducing system. In addition, the proteins showed higher catalytic activities with dabsyl-MetSO than free MetSO as a substrate. Transgenic rice plants harboring OsMSRA4.1 showed no phenotypic diVerence from wildtype under normal growth condition (Fig. 1b). However, when 3-week-old transgenic lines and the controls were treated with 100 mM NaCl for 2 weeks, all non-transgenic control rice plants wilted during extended treatment and showed salt-induced leaf curling, while OsMSRA4.1 transgenic plants grew much better than the control plants(Fig. 1c). the photosynthetic rate of OsMSRA4.1-overexpressing lines was 17–27% higher than that of wild-type plants under salt stress condition(Fig. 1d), which suggesting that overexpression of OsMSRA4.1 reduced the severity of the stress-induced impact on the photosynthesis.As shown in Fig. 1e and f, the relative electrolyte leakage values of OsMSRA4.1 transgenic lines were much lower(17–32%) than that of wild-type plants under salt treatment for 2 days, and MDA content in OsMSRA4.1 transgenic lines is also lower than wild-type, indicating that transgenic lines were more tolerant to salt stress than wild-type. Under normal conditions, there is no obvious diVerence on relative conductance and MDA between transgenic plants and wild-type.
- Subcellular localization and in vitro activity assay revealed that both OsMSR proteins are targeted to the chloroplast and have MSR activity.
- Based on the prediction software (TargetP), OsMSRA4, OsMSRB1, and OsMSRB3 all have N-terminal extensions which are predicted to be chloroplast transit peptides. OsMSRA5 is predicted to be localized to a secretory pathway. OsMSRA2.1, OsMSRA2.2, and OsMSRB5 are possibly restricted to cytosol (Table 1), but the exact subcellular localization remains to be experimentally investigated.
- Using the blastP search engine in the NCBI database, seven MSR genes were found in rice genome (Table 1). To avoid confusion,
Sequence alignment revealed that OsMSRA2.1, OsMSRA2.2, OsMSRA4, and OsMSRA5 belong to the MSRA, and OsMSRB1, OsMSRB3, and OsMSRB5 belong to the MSRB (Table 1). Accession numbers and chromosome locations of rice MSR gene family are listed in Table 1. Two transcripts (OsMSRA4.1/2 and OsMSRB1.1/2) with diVerent lengths for OsMSRA4 and OsMSRB1 were found, respectively, probably resulted from alternative splicing.
- Multiple alignment of OsMSRA sequences revealed that high similarity was found among OsMSRA2.1, OsMSRA2.2, and OsMSRA4. Three of the OsMSRA proteins contain one highly conserved cysteine residue housed in the GCFWG motif, while OsMSRA5 possesses a serine residue instead of this cysteine residue. OsMSRB sequences are also highly conserved and all OsMSRBs have four additional conserved cysteine residues that are organized in two CXXC motifs (two cysteines separated by two amino acid residues) which were found to coordinate structural Zinc.
- OsMSRA4.1 and OsMSRB1.1 orthologs in Arabidopsis：AtMSRA4 and AtMSRB1.
- Based on sequence alignments, on construction of unrooted phylogenetic trees and on some biochemical results, two MSRA subgroups can be distinguished. They differ essentially in the number and in the position of the cysteines involved in catalysis and enzyme
regeneration, but also in the subcellular localization. MSRA5 isoforms constitute an independent subgroup, while other proteins, either cytosolic or chloroplastic, are grouped into the same clad (Fig. 2). When looking at sequence homology, the overall identities range from 55 to 66% for MSRA5 proteins, from 55 to 93% for MSRA1-4, but only from 21 to 34% between MSRA5 proteins and other MSRAs. From the phylogenetic tree shown in Fig. 2, it appears that the chloroplastic MSRB1 isoforms constitute a distinct subgroup, while all other MSRB are grouped together.
In rice genome, MSR is encoded by a multigene family with at least seven members. The relatively large number of MSR genes in plants is particularly signiWcant. In Arabidopsis, the number of genes encoding for MSRs is even larger with 14 members, when compared to 9 in poplar and 7 in rice whereas generally mammals, yeast, and E. coli possess 2–4 MSR genes.
Labs working on this gene
- State Key Laboratory of Plant Genomics, National Centre for Plant Gene Research (Beijing), Institute of Genetics and Developmental Biology, Chinese Academy of Sciences (CAS), 100101 Beijing, China
- Graduate School of the Chinese Academy of Sciences, 100039 Beijing, China
- ↑ 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 Guo X, Wu Y, Wang Y, et al. OsMSRA4. 1 and OsMSRB1. 1, two rice plastidial methionine sulfoxide reductases, are involved in abiotic stress responses[J]. Planta, 2009, 230(1): 227-238.
- ↑ 2.0 2.1 2.2 Rouhier N, Dos Santos C V, Tarrago L, et al. Plant methionine sulfoxide reductase A and B multigenic families[J]. Photosynthesis research, 2006, 89(2-3): 247-262.
- ↑ Romero H M, Berlett B S, Jensen P J, et al. Investigations into the role of the plastidial peptide methionine sulfoxide reductase in response to oxidative stress in Arabidopsis[J]. Plant physiology, 2004, 136(3): 3784-3794.
- ↑ Dos Santos C V, Cuiné S, Rouhier N, et al. The Arabidopsis plastidic methionine sulfoxide reductase B proteins. Sequence and activity characteristics, comparison of the expression with plastidic methionine sulfoxide reductase A, and induction by photooxidative stress[J]. Plant physiology, 2005, 138(2): 909-922.
- ↑ Rouhier N, Dos Santos C V, Tarrago L, et al. Plant methionine sulfoxide reductase A and B multigenic families[J]. Photosynthesis research, 2006, 89(2-3): 247-262.