Proteomics Analysis Of Honglian-type Cytoplasmic Male Sterile Rice Mitochondria

Cytoplasmic male sterility (CMS) is an important phenomenon describing maternal inherited pollen abortion in the plant kingdom. Coupled with corresponding restorer line, CMS has been extensively used for hybrid seeds production in order to improve crops yields, so as to satisfy the growing demand for food supply in the world. Although quite a lot of mitochondrial genes have been identified as responsible for CMS, some of which have been verified through trangenic research, very little mechanism details are known. Honglian rice is a representative CMS type utilized in ternary hybrid rice seeds production, which have been planted up to6million hectares in China. In previous study, we idendified mitochondria-coded gene orfH79and showed that its protein ORFH79is responsible for Honglian CMS by transgenic approach. However, it remains unclear how the aberrant ORFH79organizes and functions in mitochondria, and also by which pathway ORFH79induces pollen abortion.In this study, we purified rice mitochondria from the etiolated seedlings from the sterile line Yuetai A (YtA) and its corresponding fertile maintainer line Yuetai B (YtB) using differential centrifugation and Percoll gradient. We conducted mass spectrometry quantification with a novel stable isotope dimethyl labeling in order to compare the mitochondria proteome. Meanwhile, blue native polyacrylamide gel electrophoresis (BN-PAGE) and2D BN/Tricine-SDS PAGE were employed to quantitatively compare the composition of mitochondria complexes. Proteomic research analysis idendified216rice mitochondrial proteins, including208proteins in total form,91proteins in asembled form and83proteins in common. The function of all the identified proteins were assigned into12functional categories, covering most mitochondrial functions. A heatmap was generated showing the number of identified peptides from each protein in the corresponding BN gel fraction where they were identified, indicating that proteins assembled in the same complex were concentrated in the same fraction, which suggested that the abundance of proteins in BN gels could represent the complexes they assembled. By investigating the correlation between proteins and the complexes they assembled, we find that most mitochondrial proteins were slightly up-regulated while the proteins actually assembled in complexes were reduced in the sterile line. This difference were especially typical in complex I and complex V which involve in oxidative phosphorylation system. Above results suggest a defect in mitochondrial complex assembling. ROS related and stress responsive proteins were also observed up-regulated genreally in the sterile line, which confirmed the sever oxidative stress in protein level. It is plausible to hypothesize that oxidative stress related to ROS leads to mitochondrial protein oxidation/damage which causes complexes’assembling defect.From our total mitochondrial proteome analysis, we only identified four proteins, as>2fold increased in the sterile line, including two proteins (Os07g46920.1, Os07g40250.1) up-regulated4to6folds. These two proteins are similar in protein sequence and both annotated as sex determination protein tasselseed-2(TS2). Their homolog in maize were reported to have concern with jasmonic acid (JA) biosynthesis. Absolute quantification of JA related metabolites indicated that hydroxy octadecatrienoic acid (13-HOTE) and cis-(+)-12-oxophytodienoic acid (OPDA) were significantly increased in seedlings and differentially regulated in tassels of YtA, which suggested that JA metabolism were abnormal during both vegetative and reproductive development stage in the sterile line. This discovery proposes a novel clue for the mechanism of CMS.Anti-ORFH79serum were produced with recombinant ORFH79proteins without N-terminal COX1-like sequence. We found membrane protein ORFH79could be solubilized with non-ionic detergent dodecylmaltoside (DDM). BN-PAGE coupled with Tricine SDS PAGE enabled us to find that ORFH79were associated with several large complexes ranged from～400kDa to～1.2MDa, which pointed out that CMS protein may function as complexes. Co-immunoprecipitation and comparison with proteomic results suggested that ORFH79may involve in the assembling of F0sector of complex V or several unknown complexes.