The Mechanisms Of Male Sterility Of New Ogura CMS RC₇ In Chinese Cabbage

Chinese cabbage (Brassica campestris L. ssp. pekinensis) origins China, with significant heterosis, and cytoplasmic male sterility (CMS) is the best material of Chinese cabbage hybrids. To clarify the male sterility mechanism, we research cytoplasmic male sterility RC₇ and its maintainer line at flower morphology, microstructure and ultrastructure, isozyme, physiological and molecular levels in different development stage of anther and leaves. The results were as follows:1. Floral morphology show that: CMS RC₇ flower spreading and petal, sepal, anther, anther filament less than B₇, stigma and ovary larger than the maintainer. RC₇ that has stable sterility with 100% sterile plant ratio and sterile degree, mature anther dehiscence or part of the crack without pollen, normal nectary perfect pistil function and strong bearing ability. In order to understand the mechanism of male sterile line RC₇ and its maintainer line of Chinese cabbage, the microsporogenesis and male gametophyte developments with their relativity to the morphologies of flower buds at different developmental stages were microscopically examined. It was shown that, the microspore mother cells perform the simultaneous type of meiosis, and the microspores are arranged in a tetrahedral shape in the tetrad, and the tapetum of the Maintaine Line belongs to glandular type. The male sterile RC₇ appeared abortion signs in the tetrad type, and abortioned in uninucleate stage, which is belong to uninucleate abortion type. The reason for the abortion was possibly that the tapetum cells were subjected to hypertrophy via vacuolation so that the microspore were extruded and broken into pieces, so the bicellular pollen grains could not form.2. Cytoplasmic male sterile (CMS) line RC₇ of Chinese cabbage produced mature anthers without pollen. To understand the mechanisms for this, ultrastructural changes during development of microspores were investigated using the fixation and post-fixation method, in the present study. In the anthers of RC₇ line, development of microspores was not affected at the early tetrad stage. During ring-vacuolated period, some big large vacuoles appeared in tapetum cells, making the tapetum cells enlarger and extend to anther sac center during monocyte-period. Meanwhile the tapetum degenerated as the microspores aborted, resulting in pollen-deficient anthers. As a result, the locules collapsed and the anther shriveled. In pollen walls the callose was degraded, abnormal deposits of electrodense material gave rise to irregular spike-shaped structures, rather than the characteristic rod-like shape of the B₇ bacula. At the mitosis I microspore stage, the tapetum cells contained multiple plastids with numerous small spherical plastoglobuli, and lipid bodies, which were released into the anther cavity following lysis of the tapetum cell.3. In order to study the relationship between cytoplasmic male sterile line and reactive oxygen species metabolism in CMS RC₇, the activities of antioxidant enzymes was examined. Results showe the male sterile line, in which superoxide anion radical formation rate, contents of hydrogen peroxide (H₂O₂) and malonadehyde (MDA) was higher than the maintainer line, superoxide enzyme (SOD) activities was higher than the maintainer line, peroxidase (POD) higher than the maintainer line in mono-nucleus microspore stage and binucleate pollen stage. Catalase (CAT) was lower than the maintainer line. (APX) activities was higher than the maintainer line in mono-nucleus microspore flower buds and before mono-nucleus microspore flower buds, was significant lower than the maintainer line in binucleate pollen stage. In male sterile lines leaves of flowering stage, superoxide anion radical formation rate, contents of hydrogen peroxide (H₂O₂) and malonadehyde (MDA), peroxidase (POD) and superoxide enzyme (SOD) activities were higher than maintainer lines.4. In order to make further study on the biochemical mechanism of CMS RC₇, CMS RC₇ and its maintainers were used as the raw material to comparatively analyze the contents of soluble sugar, reducing sugar, soluble protein and free proline in their leaves and flower buds. The results showed that soluble sugar, reducing sugar, soluble protein contents was no significant difference between RC₇ and B₇ in Chinese cabbage vegetative growth stage. Soluble sugar, reducing sugar and soluble protein contents in the male sterility line was significantly lower than its maintainer in microspore development process. Free proline contents in the male sterility line was significantly higher than its maintainer in six-leaves stage, flowering stage leaves and small flower buds stage, was lower than that of its maintainer line in rosette stage leaves, mono-nucleus microspore flower buds and binucleate pollen stage. Free proline contents decreased in RC₇ line and increased in maintainer line in microspore development process.5. By means of anatomy and vertical slab polyacrylamide gel electrophoresis,cytology characteristics and perioxidase (POD), catalase (CAT), esterase(EST), and amylase(AMY), ATP and SOD isoenzymes of CMS RC₇ and its maintainers have been analysed. The results showed that difference of the number and intensity of the bands in POD and AMY zymograms was observed in flower buds at different stages of flower development between RC₇ and B₇. For CAT and EST zymogram, the maintainer line B₇ had two more band than that of the cytoplasmic male sterility line RC₇ in the binucleate pollen stage. The different expressions of CAT and EST zymogram between RC₇ and B₇ occurred at the same stage of cytological abortion. ATP zymograms were significantly different in the whole flower developmen process, the number of the bands are more in B₇ than RC₇. SOD zymograms were no significant difference between RC₇ and B₇.6. The gene expression in flower buds of CMS line and itsmaintainer line was compared by means of cDNA-AFLP, a total of 23 differential transcript-derived fragments (TDFs) were identified and sequenced. 11 TDFs were expressed specifically in CMS line, whereas10 could be detected only in the maintainer line, 2 TDFs were expressed both two lines. The analysis of sequence homology and functional classification indicated that H1 TDFs coming from CMS line shared high identities with Photoassimilate-responsive protein of Arabidopsis thaliana, H29 TDFs shared high identities with calcineurin-like phosphoesterase family of Arabidopsis thaliana. 10 TDFs from maintainer line were highly homologous to known sequences. These genes might be related to glucose metabolism, protein synthesis and transport, ethylene-inducible gene, electron transport and energy pathway, unknown or putative protein and so on. Eighteen ESTs were submitte to GenBank. They were: GT968₇₇3,GT968₇86,GT968₇8₇,GT968₇88,GT968₇89,GT968₇90,GT968₇₇4,GT968₇₇5,GT968₇₇6,GT968₇₇₇,GT968₇₇8,GT968₇₇9,GT968₇80,GT968₇81,GT968₇82,GT968₇83,GT968₇84,GT968₇85.7. One TDFs were selected and got full-length cDNA. Firstly, a differential TDF (Transcript derived fragments) EST H9 from the RNA of male sterile line RC₇ and its maintainer line of Chinese cabbage by cDNA-AFLP analysis was used as a querying probe to blast homologous ESTs sequences in the GenBank database, we spliced the homologous EST sequences assembled from GenBank, and gained an 5′sequence. Then, the primer of 3′was designed, the full length cDNA ofα- carbonate dehydratase 3 gene in Chinese cabbage was gained through RACE-PCR amplifying, and sequencing and registrated as accession number of GU143061 in GenBank, named BrACA3,which is 998 bp length and coded 2₇0 amino acids. Homologous analysis showed that amino acids sequence derived from it had ₇8% identity withα- carbonate dehydratase 3 (ACA3) in Arabidopsis. Amino acid sequence analysis showed that the ACA3 is a transmembrane protein and there is a signal peptide sequence between 19 and 20, there are serine, threonine and tyrosine phosphorylation sites. ACA3 gene in Chinese cabbage was not expressed as bud aborting, and be expressed in large flower buds of B₇.