Transcriptional Profiling Of Flower Buds In Three Types Of Male Sterile Lines Compared To Their Maintainer Line And Characterization Of Three Genes Involved In Pollen Development In Brassica Campestris L.

The application of male sterility becomes an important way in heterosis breeding to simplify the seed production procedures and reduce the production cost. The utilization of hererosis is prevalent in cruciferae crops breeding, and the male sterile line is a useful system for their hybrid seed production. However, the occurrence and the mechanism of plant male sterility still remain mysteries that have not been unveiled thoroughly. For male sterility mainly manifests in pollen abortion, to make clear of the whole process of pollen development and the mechanism underlined thus become the basis point for understanding of male sterility. Pollen development is regulated by a complex regulatory network that comprises a wide variety of genes. Transcriptional profiling on the mutant compared with its wild-type is a successful approach to investigate the dynamic gene expression during pollen development and to detect genes involved in this biological process, and ultimately to help to understand the characteristic of pollen development and the corresponding molecular mechanism. In this study, we established three different male sterile types, 'Aijiaohuang' genic male sterility AB line (ajhGMS 'Bcajh97-01A/B'), 'Polima' genic-cytoplasmic male sterility (polG-CMS 'Bcpol97-05A') and 'Ogura' cytoplasmic male sterility (oguCMS 'Bcogu97-06A') in Brassica campestris L. ssp. chinensis Makino, syn. B. rapa ssp. chinensis. These male sterile lines share a common maintainer line. Then, we conducted profiling comparisons between flower buds of these male sterile lines and their maintainer line using complementary deoxyribonucleic acid amplified fragment length polymorphism (cDNA-AFLP) technology together with ATH1 genome array analysis. By analyzing the changes in expression pattern of genes acting downstream due to the gene mutation in different male sterile plants, plenty of candidate genes involved in pollen development were identified, and thus laid a foundation for establishing the profiles of gene expression in different types of male sterile lines. After that, three genes were selected for further characterization, including two putative polygalacturonase (PG) genes, BcMF2 {Brassica campestris male fertility 2) and BcMF9 {Brassica campestris male fertility 9) which may be related to cell wall biosynthesis and regulation, and one novel gene, BcMFW {Brassica campestris male fertility 10) with unknown function. Anti-sense RNA or RNA interference (RNAi) technique were used to investigate the function of the three genes, and morphologic, molecular biology as well as cytology analysis were carried through in functional assignment. Comparison of gene structure, expression pattern and biological function of three members, BcMF2, BcMF6 {Brassica campestris male fertility 6) and BcMF9 in PG gene family were also performed in this study. The results obtained are as follows. (1) Observation on the pollen development in male sterile line 'Bcajh97-01A' suggests that it's a mutant of male meiosis cytokinesis in B. campestris, and the corresponding gene, MMC may encode a protein acting specifically in male meiosis. When observed the morphology and cytology character of the ajhGMS 'Bcajh97-01A/B' we found that the only detectable difference between the male sterile plants 'Bcajh97-01A' and the fertile plants 'Bcajh97-01B' was the formation of mature pollen grains. Just after telophase II, the cytokinesis of male meiosis is disturbed in 'Bcajh97-01A' anther, and the formation of intersporal wall does not occur, which in turn results in the failure of mature pollen formation. Genetics analysis indicated that the male sterility was controlled by a single recessive allele. These results suggest that 'Bcajh97-01A' plant is indeed a mutant of male meiosis cytokinesis, and the corresponding mutated gene MMC may be directly involved in male meiotic cytokinesis.(2) Investigations on the difference among the flower buds transcriptional profiles of male sterile lines compared with the common maintainer line both by cDNA-AFLP and ATH1 genome array analysis, show that gene expression in the flower buds of these various male sterile types are similar but not entirely identical. We conducted expression profiling comparisons between each male sterile line, ajhGMS 'Bcajh97-01A'.polG-CMS 'Bcpol97-05A' and oguCMS 'Bcogu97-06A' and the common maintainer line of B. campestris both by cDNA-AFLP analysis and hybridizing with the ATH1 genome array from Arabidopsis genome. The outcomes indicate that the mutation of the gene which controls the fertility of each plant have caused dramatic changes in expression pattern of genes acting downstream. Expression of numerous genes are blocked or weakened, otherwise that of some other genes are activated or enhanced. But gene expression changes in different male sterile line are not identical due to the different gene mutation. Compared to the maintainer line 'Bcajh97-01B', 93 genes are up-regulated and 158 are down-regulated in 'Bacjh97-01A', 174 genes are up-regulated and 212 are down-regulated in 'Bcpol97-05A' 196 genes are up-regulated and 242 are down-regulated in 'Bcogu97-06A', and thereinto, 37 genes down-regulated and 24 up-regulated simultaneously in three male sterile types are included. There are also many genes sharing common expression pattern between the male sterile lines, including 5 genes down-regulated and 24 up-regulated simultaneously in 'Bcajh97-01A' and 'Bcpol97-05A' 70 genes down-regulated and 9 up-regulated simultaneously in 'Bcajh97-01A' and 'Bcogu97-06A' 70 genes down-regulated and 45 up-regulated simultaneously in 'Bcpol97-05A' and 'Bcogu97-06A'(3) Function classification of differentially expressed genes reveals that most of the genes involved in pollen and/ or anther development are function-unknown. Expression profiling comparisons of different male sterile lines and their maintainer line increase the number of pollen and/ or anther-expressed and/ or specific gene. We classified the differentially expressed gene detected in the flower buds transcriptome of the male sterile lines and their common maintainer line into several categories, based on the putative function as well as gene ontology annotations derived from homologies. About 50% of these genes have an unknown or hypothetical function. On account of the fact that each of these male sterile plant only differed from the fertile plants line by the failure of pollen formation and anther development, we conclude that the differentially-expressed genes in the flower buds between the male sterile lines and the maintainer line are most likely to act in pollen and/ or anther development. Those differentially-expressed genes with unknown of hypothetical function may represent new candidate proteins involved in pollen and/ or anther development. If this is true, the number of genes expressed and/ or specifically expressed in pollen and anther will be increased to some extent for the identification of these genes.(4) Based on the function classification, it is found that pollen and/ or anther transcriptome from different plants with unlike genetic pattern possesses similar 'module' but unique composition. When classified the differentially-expressed genes into functional categories, we found that there were similar categories in three individual groups of differentially-expressed genes derived from the comparison between each male sterile line and its maintainer line. However, the proportions of the same category in three groups are not entirely identical. The major categories of genes down-regulated simultaneously in all the male sterile lines are involved in transporter and channel, protein metabolism, electron transport or energy pathways and defense mechanisms and stress response; up-regulated simultaneously in those are involved in transporter and channel, transcription and general metabolism. Further more, many similarities were found between the gene expression in pollen and/ or anther of 'Bcajh97-01A' and 'Bcogu97-06A' Genes related to cell wall biosynthesis and regulation is ranked in the first place of the down-regulated genes both in 'Bcajh97-01A' and 'Bcogu97-06A' and genes related to cytoskeleton and signal transduction also occupy a higher proportion. Otherwise, few genes involved in transcription have been detected in the down-regulated genes both in 'Bcajh97-01A' and 'Bcogu97-06A' In the up-regulated genes in both of these two transcriptome, those related to protein metabolism account for a higher proportion. The transcriptome composition features of flower buds in 'Bcajh97-01A' as well as in 'Bcogu97-06A' are more similar to that of Arabidopsis pollen. In contrast, there is lesser similarity between the transcriptome of flower buds of 'Bcpol97-05A' and the other two. The most distinguished difference is the proportion of genes involved in cell wall biosynthesis and regulation, cytoskeleton and signal transduction. These categories occupy higher proportion in the male sterile pollen of 'Bcpol97-05A' but a lower proportion in the fertile pollen of 'Bcpol97-05B' Thus it can be seen that though there is similar module in the pollen and/ or anther transcriptome of the male sterile line with different genetic pattern, each of them possesses unique composition.(5) Spatial and temporal expression pattern analysis demonstrates that each gene related to pollen and/ or anther development has a distinct expression dynamic. We investigated the expression pattern of 27 fragments derived from the transcriptome comparison above. Although most of the transcripts were first detected around the time of microspore mitosis and accumulate continuously as the pollen matured, each transcript had a unique expression level at different developmental stages. The expressions of some other fragments were detectable at the earlier stage of pollen development. These results from the expression pattern analysis validate the complexity of gene expression and regulation during pollen and/ or anther development, and demonstrate that activation or silencing and orderly changes of expression level are consistent with the requirements for normal development of pollen and/ or anther and thus for achieving their function. This study also made a relevant attempt to assign genes to different developmental stages of pollen and anther in B. campestris.(6) Expression pattern and functional analysis of BcMF2 indicate that it may encode a novel PG involved in pollen intine deposition. Spatial and temporal expression pattern analysis was performed for BcMF2, a gene encoding PG isolated from flower buds of 'Bcajh97-01B' previously. BcMF2 transcript was first detected in the flower buds at stage 3, which corresponded to the tetrad stage of microspore development, though it was comparatively weak. The signal then became stronger up to the flower buds at stage 4, which corresponded to the uninucleate stage, and then declined in the flower buds at stage 5 full of mature pollen grains. This expression pattern considered it an "early" gene in pollen. Functional analysis of BcMF2 by anti-sense technique revealed that it might be involved in pollen wall development. The germination of pollen from transgenic plants with anti-sense BcMF2 was affected. About 80% transgenic pollen formed a balloon-tipped pollen tube during in vitro germination. Though the pollen tube could penetrate through the stigma, it stopped growth in the style. Further investigation on the configuration and the development of transgenic pollen revealed that all the pollens were malformed, and the number of germination furrow as well as its distribution was anomalistic. The intine of transgenic pollen thickened unconventionally during pollen development. It's a hypothesis that the silence of BcMF2 may result in the disturbance in pectin metabolism in the pollen wall and in turn leads to the aborted intine and germination furrow. For there is no similarity found in sequence and expression pattern between BcMF2 and the known PG gene acting at the earlier stage, BcMF2 is considered as a novel gene encoding PG whose transcript is accumulated at the earlier stage.(7) Another PG gene named BcMF9 in B. campestris was isolated and characterized as a gene playing roles both in intine and exine formation. BcMF9 was amplified from a fragment, BBS13/BPO023 detected in the flower buds of 'Bcajh97-01B' Alignment against the published sequences revealed the presence of highly conserved domains and features among plant polygalacturonases in the deduced amino acid sequence of BcMF9, and molecular phylogenetic analysis put it into the cluster of pollen-expressed PG. BcMF9 was considered as a "late" gene in pollen, because its expression signal was first detectable in the microspore at the tetrad stage, and became stronger at the uninucleate stage and maintained till pollen matured. Its transcript was also detected in the tapetum cell from tetrad stage to the degeneration of tapetal cells. Functional analysis of BcMF9 by anti-sense technique revealed that it may play roles both in intine and exine formation. The in vitro germination percentage of transgenic pollen was dramatically reduced. About 81% pollen tubes burst during germination. The pollen tube could penetrate through the stigma, but it stopped growth in the style soon after that. Further investigation revealed that silence of BcMF9 led to the produce of malformed pollen with a flat meshwork on the face and anomalous germination furrows. The intine of transgenic pollen thickened unconventionally during the pollen development and at the late stage, the bacula and the tectum fell off from the exine, which subsequently resulted in the overflowing of tryphine. These results strongly indicate that BcMF9 may play an important role in pollen wall formation. Furthermore, we found that degeneration of tapetum was accelerated in the transgenic anther. Microspore development was normal before the abnormity in tepetum occurring, but soon after the abnormity occurring, it was defected in exine formation. We speculate that the silence of BcMF9 disturbes the normal process of programmed cell death (PCD) in the tapetal cells, and starts an alternative cell death process which accelerated the degeneration process. Break of the normal PCD in tapetum may alter the releasing of tapetal wall materials onto the pollen grains and thus affect the complete deposition of pollen wall. Alternatively, BcMF9 may act directly in the formation of pollen wall. It may be expressed in the tepetum and then is secreted into the locule and located on the microspore during the pollen development, for the putative signal peptide in BcMF9 protein and the unique expression pattern of BcMF9.(8) Comparison of BcMF2, BcMF6 and BcMF9, three members of the PG gene family reveals that they may play different role in pollen development. The gene structure, expression pattern, gene evolution and biological function of three PG genes, BcMF2, BcMF6 and BcMF9 were analyzed. Though it was found that these PG genes accumulated in the same developmental stages, the expression level in each stage and the dynamics of expression of them are different. The evolutions of them in family Cruciferae are not coincident, and the silences of them result in similar but not identical phenotype. These results illuminate that the function and the pattern of gene activity of BcMF2, BcMF6 and BcMF9 may be different.(9) A novel gene named BcMFlO predominantly expressed in flower buds of 'Bcajh97-01B' and ? 'Bcpol97-05A' but silenced in those of 'Bcajh97-01A' and 'Bcogu97-06A' was isolated and characterized. This gene was amplified from a fragment, BBS31I BPO079 detected in the comparison of flower buds transcriptome of male sterile lines and the maintainer line. Bioinformatical analysis of the sequence revealed several patterns which were involved in protein location, cell adhesion and signal transduction associated with cell proliferation, differentiation, and apoptosis. It is supposed that BcMF10 may represent a new candidate protein involved in signal transduction. RT-PCR and in situ hybridization was performed to investigate the expression pattern of BcMF10, and the result indicated it was an "early" gene in pollen. In 'Bcajh97-01B' BcMF10 has its transcript began to be detectable in pollen mother cell and tapetum at the early developmental stage, and became strong during the meiosis. Then, the expression decreased gradually up to the mature pollen. However, the location of its transcript has not changed. Functional analysis by RNAi technique revealed that silence of BcMF10 blocked the normal germination of pollen. About 75% transgenic pollens could not germinate in vitro, and 15.6% grew with a burst tip. But the burst pollen tube could grow to some extent and then spray the content, which distinguished it from the other burst tube observed generally with an immediate burst soon after germination. We suppose that BcMF10 may encode a protein synthesized in the tapetum and secreted out of the cell which plays a role in the secretion of nutriments or cell wall materials for the pollen grains.