miRNAs are involved in the pollen development during the CMS occurrence in rice.miRNAs are 20–24 nt endogenously expressed small RNAs that play key roles in the regulation of many growth and developmental processes in plants. The knowledge on cytoplasmic male sterility (CMS) regulation by miRNAs in rice is rather limited. In this study, Illumina sequencing was employed to examine the expression profiles of rice anther miRNAs from the CMS line MeixiangA (MxA) and its maintainer line MeixiangB (MxB). A total of 518 known miRNAs and 144 novel miRNAs were identified during rice anther development. Based on the number of sequencing reads, a total of 24 miRNAs were discovered to be differentially expressed between MxA and MxB, and the results were partially validated by qRT-PCR. Among these, 16 miRNAs were decreased and 8 miRNAs were increased in MxA compared with MxB. Target prediction showed that they target genes encoding EF-hand family proteins, F-box domain-containing proteins, MYB transcription factors, PPR-containing proteins and transposons. The expression patterns for targets of osa-miR528, osa-miR5793, osa-miR1432, osa-miR159, osa-miR812d, osa-miR2118c, osa-miR172d and osa-miR5498 were selectively examined, and the results showed that there was a negative correlation on the expression patterns between miRNAs and their targets. These targets have previously been reported to be related with pollen development and male sterility, suggesting that miRNAs might act as regulators of CMS occurrence in rice anthers. Furthermore, miRNA editing events were observed. The U → C and U → A editing phenomenon was validated by molecular cloning and sequencing. These findings contribute to our understanding of the roles of miRNAs during anther development and CMS occurrence in rice.

•Cytological analysis indicates that the sterile line MxA is aborted at the early uninucleate stage.•We identify a homologous WA352 gene in the sterile line MxA.•Ultrastructural study indicates the mitochondria dysfunction in MxA.•Proteins expression patterns are explored using iTRAQ MS/MS method.•The ROS level in the anthers is higher in MxA than that in MxB.Cytoplasmic male sterility (CMS) is a widely observed phenomenon, which is especially useful in hybrid seed production. Meixiang A (MxA) is a new rice CMS line derived from a pollen-free sterile line named Yunnan ZidaoA (ZD-CMS). In this study, a homologous WA352 gene with variation in two nucleotides was identified in MxA. Cytological analysis revealed that MxA was aborted in the early uninucleate stage. The protein expression profiles of MxA and its maintainer line MeixiangB (MxB) were systematically compared using iTRAQ-based quantitative proteomics technology using young florets at the early uninucleate stage. A total of 688 proteins were quantified in both rice lines, and 45 of these proteins were found to be differentially expressed. Bioinformatics analysis indicated a large number of the proteins involved in carbohydrate metabolism or the stress response were downregulated in MxA, suggesting that these metabolic processes had been hindered during pollen development in MxA. The ROS (reactive oxygen species) level was increased in the mitochondrion of MxA, and further ultrastructural analysis showed the mitochondria with disrupted cristae in the rice CMS line MxA. These findings substantially contribute to our knowledge of pollen developmental defects in ZD-CMS rice line.Biological significanceMeixiangA (MxA) is a new type of rice CMS line, which is derived from pollen-free sterile line Yunnan ZidaoA. In this study, the cytological, molecular and proteomic approaches were used to study the characteristics of this new CMS line. Cytological study indicates the CMS line is aborted at the early uninucleate stage. A potential sterile gene ZD352 is identified in MxA, the protein product of which is mainly accumulated at the MMC/Meiotic stage. iTRAQ based proteomic analysis is performed to study the relevant proteins involved in the CMS occurance, 45 proteins are found to be significant differentially expressed and these proteins are involved in many cellular processes such as carbohydrate metabolism, stress response, protein synthesis. To our knowledge, this is the first report using the iTRAQ-labeled quantitative proteomic to study the protein expression variation during the abortion processes between a CMS line and its maintainer line. These results provide new insights on the CMS mechanisms of ZD-CMS rice line.

Rice glutelin is a multi-subunit storage protein and has high nutritional value. However, many glutelin subunits are still not identified by experiment approach. In this study, a novel subunit (OsGluBX) was discovered by sequence alignment in the UniProtKB database. And then, the OsGluBX of rice from japonica cv. Nipponbare and indica cv. 9311 were cloned and expressed in Escherichia coli system and further identified by Western blotting. The total storage proteins were extracted from the grains of Nipponbare and 9311, and the native OsGluBX were identified. The novel OsGluBX subunit was classified into the subfamily B based on its high homology to the subfamily B members and their immunoblotting identification against the subfamily-specific antibody. Furthermore, two-dimensional electrophoresis analysis showed similarity and difference of the entire glutelin profiles between the two subspecies. Moreover, the atomic coordinate of the OsGluBX was constructed based on homology modeling approach and refined by molecular dynamics simulations. The spatial conformation of the OsGluBX protein was stable during the simulation, and the obvious hydrogen bonds were observed to maintain the integrity and stability of the β-sheets region of the OsGluBX. Research into this novel OsGluBX subunit has improved our understanding of the glutelin family in rice.

Alpha-momorcharin (α-MC), a member of the ribosome-inactivating protein (RIP) family, has been used not only as antiviral, antimicrobial, and antitumor agents, but also as toxicant to protozoa, insects, and fungi. In this study, we expressed the protein in Escherichia coli Rosetta (DE3) pLysS strain and purified it by nickel–nitrilotriacetic acid affinity chromatography. A total of 85 mg of homogeneous protein was obtained from 1 l culture supernatant of Rosetta (DE3) pLysS, showing a high recovery rate of 73.9%. Protein activity assay indicated that α-MC had both N-glycosidase activity and DNA-nuclease activity, the former releasing RIP diagnostic RNA fragment (Endo’s fragment) from rice rRNAs and the latter converting supercoiled circular DNA of plasmid pET-32a(+) into linear conformations in a concentration-dependent manner. Specially, we found that α-MC could inhibit the mycelial growth of Fusarium solani and Fusarium oxysporum with IC50 values of 6.23 and 4.15 μM, respectively. Results of optical microscopy and transmission electron microscopy demonstrated that α-MC caused extensive septum formation, loss of integrity of the cell wall, separation of the cytoplasm from the cell wall, deformation of cells with irregular budding sites, and apoptosis in F. solani. Moreover, α-MC was active against Pseudomonas aeruginosa with an IC50 value of 0.59 μM. The α-MC protein carries a high potential for the design of new antifungal drugs or the development of transgenic crops resistant to pathogens.

The Hsp90 (for heat shock protein90) and the Sgt1 (for suppressor of the G2 allele of skp1) are widely distributed in animals, yeast, and plants. The former functions as molecular chaperon activating a series of client proteins, the latter functions as an adaptor protein participating in multiple biological processes such as immunity response through interactions with different protein complexes. In the present study, we have constructed a homology model of Hsp90-Sgt1 complex in rice based on a recently resolved structure from barley and Arabidopsis to explore its binding mechanisms and to understand the detailed interaction profile. A total of 20 ns explicit solvent molecular dynamics simulations combined with MM-GBSA computations and virtual alanine scanning were performed for the modeled complex. In the final structure, three strong salt bridges were found between OsHsp90 and OsSgt1, D217(OsHsp90) - K186(OsSgt1), D218(OsHsp90) - K237(OsSgt1) and K161(OsHsp90) - E239(OsSgt1). Besides, residue Y173 of OsSgt1 played a vital role in the interactions with OsHsp90, the detailed interactions were discussed. These results would help us understand the critical features determining the Hsp90-Sgt1 binding process.

Most mitochondrial proteins are synthesized in the cytosol as precursor and imported into the mitochondria by Tom complexes (translocase of outer membrane complexes). Knowledge of the binding mechanism between precursor and Tom20 in plants is very limited. Here, computational methods are employed to improve our understanding of the interactions between both molecules. To this end, we model mitochondrial superoxide dismutase precursor (pSOD) in complex with Tom20 in Oryza sativa (OsTom20). In a first stage, five main binding modes were generated using clustering analysis, energy minimization, and expert knowledge. In a second stage, the quality and validity of the resulting complexes is assessed by molecular dynamics (MD) simulations with a generalized Born solvation model. The change in binding free energies is estimated using a computational alanine scanning technique. We identified a particularly favorable complex between pSOD and OsTom20, exhibiting the lowest binding free energy among all candidates and correlating well with experimental data. Furthermore, three independent explicit solvent MD simulations of this structure, each of 100 ns duration, reveal that hydrophobic interactions occur between pSOD and OsTom20, in particular between L158 of pSOD and W81 of OsTom20, as evidenced by analysis of intermolecular distances and corresponding relative free energy landscapes. L158 is part of an interacting LRTLA motif. These results provide new insight into the structural basis and dynamics of precursor recognition by Tom20 in plant, and their generality is supported by sequence alignments with seven other plants.

On the base of construction of a new type of cytoplasmic male sterile (CMS) line ZidaoA, we analyzed the editing of transcripts of mitochondrial ATP synthase subunit 9 gene (atp9) from CMS line and its maintainer line. With PCR, RT-PCR, and direct sequencing, complete nucleotide sequences were determined for the mitochondrial atp9 gene and its cDNA from two lines of purple rice type rice: CMS line Ying xiang A and its maintainer line Ying xiang B. The atp9 transcript of Ying xiang A was shown to have no editing sites and the transcript of Ying xiang B was shown to have two editing sites with changes affecting the amino acid sequence of the protein product. The editing of the atp9 transcript from Ying xiang B was found to change an arginine codon into a termination codon, shortening the protein of Ying xiang B to the “standard” size. And the Ying xiang A transcript, which has no termination codon, cannot be translated to a normal protein. The results demonstrate the important role of RNA editing in the production of the functional ATP9 subunit and suggest that RNA editing could be likely associated with cytoplasmic male sterility.

Glutelin, a major protein in rice grains, is encoded by a multigene family. However, its protein composition is not well characterised. Here, we identified and characterised two novel glutelin subunits, GluBX and GluC. The individual glutelin subunits of japonica cv. Nipponbare and indica cv. 93-11 rice were analysed using 2-dimensional gel electrophoresis, LC–MS/MS, and Western blotting. Comparison of the glutelin profiles between three japonica and three indica cultivars indicated two distinct subunits (GluA-1 and GluA-3 isomers) and a distinction in the subunit composition (notably GluA-3 and Lys-rich GluB-1 components) of these two subspecies. Sequence alignment revealed different nutritional (Lys residues) and functional (Cys residues) characteristics between the type-A and type-B glutelin subfamilies. We also analysed amino acid and total protein contents of the grains in thirty-five cultivars, and we demonstrated that the Lys-rich glutelin composition of indica cultivars is superior to that of japonica cultivars. The Lys-rich and Cys-poor GluBX subunit is a native protein and is a high nutritional protein in grains. Our combined approaches for the identification of glutelin subunits have revealed the nutritional characteristics of individual subunits in rice, and this knowledge will provide new insights for improving grain quality during rice breeding.Highlights► Identification and characterisation of two novel subunits, GluBX and GluC. ► 2D-PAGE coupled LC–MS/MS and Western blot analysis of the entire glutelin profiles. ► Assessment of glutelin subunit composition in two rice subspecies. ► Glutelin Lys-rich components in indica cultivars are superior to those in japonica.

RNA editing plays an important role in the regulation of mitochondrial gene expression in flowering plants. In this study, we examined RNA editing of the mitochondrial genes cox2, atp6 and atp9 in five isonuclear alloplasmic male-sterile lines (IAMSLs) of rice to investigate whether different cytoplasmic types affect RNA editing. Although many editing sites were conserved among the three genes, we found that the editing efficiency of certain sites was significantly different between different IAMSLs or between IAMSLs and their corresponding cytoplasmic donor CMS lines. Furthermore, several editing sites were found to be either present or absent in certain IAMSLs and their corresponding CMS lines. These results indicate that nuclear loci, as well as unknown editing factors within the mitochondria of different cytoplasmic types, may be involved in RNA editing, and they suggest that RNA editing in plant mitochondria is affected by nucleo-cytoplasmic interactions.Highlights► RNA editing patterns of atp6, atp9 and cox2 were investigated in different CMS lines. ► Some novel editing sites were identified in the mitochondria of rice. ► RNA editing in rice mitochondria may be affected by nucleo-cytoplasmic interactions. ► RNA editing in ZD-type cytoplasm is different from that in other rice CMS lines. ► The “unedited” Atp9 proteins in Meixiang A may associate with ZD-type rice CMS.

The genetic diversity analysis of 90 barley samples, including 45 wild close relatives of barley from the Tibet region of China and 45 wild accessions from different countries throughout the Middle East, were carried out using ISSR and SSR markers. The results showed that Tibetan wild close relatives of barley had a higher genetic diversity than those from the Middle East. Ten ISSR primers amplified 91 allelic variants, of which 79 were polymorphic (86.81%), in the Tibetan genotypes and 82 allelic variants, of which 66 were polymorphic (80.49%), in the Middle East genotypes. Eleven primer pairs of SSR markers amplified 100 allelic variants among the Tibetan genotypes with 100 polymorphic bands (100%). Among the Middle East genotypes, 78 allelic variants were produced containing 77 polymorphic bands (98.72%). Moreover, the total gene diversity analysis (HT) values of Tibetan barley (0.227 for ISSRs and 0.126 for SSRs) were higher than those of Middle East (0.212 for ISSRs and 0.102 for SSRs). Cluster analysis of the ISSR and SSR results by the UPGMA method revealed two distinct groups correlated with the geographic origin of sampling. These results offer a new evidence for the theory of the origin of Hordeum vulgare L. To cite this article: A. Wang et al., C. R. Biologies 332 (2009).

MicroRNAs (miRNAs) are a new class of non-protein coding small RNAs that regulate gene expression at the post-transcriptional level in plants and animals. Although thousands of miRNAs were identified in many plant species, only 3 miRNAs have been reported in Lotus Japonicus, a model legume plant. In this study, 80 potential miRNA candidates were identified in 28 ESTs and 52 GSSs of L. japonicus using a homology-based computational analysis. A total of 735 miRNA targets were predicted and some of them encoded transcription factors as well as genes that function in stress response, signal transduction, methylation and others. Quantitative real-time PCR (qRT-PCR) analysis indicated that miR156a, miR160a and miR399a participated in seed germination of L. japonicus. GO and KEGG analysis suggested that the predicted miRNAs might target genes involved in lipid, nitrogen, starch sucrose metabolism and signal transduction.Highlights► Eighty potential miRNA candidates were identified in Lotus japonicus. ► Four sense and antisense miRNAs pairs belonged to four families of miR166, miR171, miR396, miR5025, respectively. ► The antisense miRNA Lja-miR171c was reverse complementary to the sense miRNA Lja-miR171a. ► qRT-PCR analysis indicated that miR156a, miR160a and miR399a may involve in seed germination of L. japonicus. ► We identified 735 potential targets, many of which involved in stress response, metabolism and signal transduction.