Expression And Functional Charscterization Of Four Polysaccharide Metabolism Genes Related To Pollen Wall Development In Brassica Campestris Ssp. Chinensis

The elaborately sculpted pollen wall plays important role not only in the process of pollen development but also in the fertilization, and contributes to the male sterility and pollen recognition. Pollen development, a process stemming from anther cell division and differentiation leading to male meiosis, as well as pollen wall, relies on the functions of numerous cell wall metabolism-related genes from both the microspore itself and sporophytic anther tissues including the tapetum. The entire dynamic regulatory network of pollen wall synthesis involves a considerable amount of genes. Revealing the functions of pollen wall development-related genes and the regulation relation among key genes in this process thus become the basis point for understanding of plant sexual reproduction, and not only provide valuable insight regarding male fertility, but also help to obtain high crop yield and improve reproductive efficiency with essential theoretical and applicable meaning. In our previous study, a genic male sterility (GMS) system named ’Bcajh97-01A/B’ which was constructed in Chinese cabbage-pak-choi(Brassica campestris L. ssp. chinensis Makino, syn. B. rapa ssp. chinensis) was used. Two classical arabinogalactan protein (AGP) genes, Brassica campestris Male Fertility8(BcMF8) and Brassica campestris Male Fertility18(BcMF18), were isolated and characterized, as well as two pectin methylesterase (PME) genes, Brassica campestris Male Fertility23a (BcMF23a) and Brassica campestris Male Fertility23b (BcMF23b). The structure and expression pattern of these genes were analyzed. In addition, antisense RNA technology was used to knock down the RNA levels of BcMF8and BcMF18individually and both. Meanwhile, BcMF18was overexpressed in Arabidopsis thaliana. The morphological, molecular biological and cytological aspects of the antisense RNA transgenic plants and the overexpression plants were characterized to understand the functional mechanism of the genes in pollen development and pollen tube growth. The results and conclusions obtained are summarized as follows.(1) BcMF8, a putative classical arabinogalactan protein-encoding gene, contributed to pollen wall development, aperture formation, and pollen tube growth. Results of our previous work showed that BcMF8is pollen-specific, and expressed at the onset of uninucleate stage to the mature pollen stage. Real-time RT-PCR and in situ hybridization showed that BcMF8also expressed in the pollen tube and the stigma of the pollinated pistil. Prokaryotic expression and western blot were used to ensure that BcMF8could encode a putative AGP of approximately20kDa, which was soluble.The results of subcellular localization indicated that BcMF8protein was secreted, and exhibited intracellular and extracellular localization. The functional interruption of BcMF8by antisense RNA technology resulted in slipper-shaped and bilaterally sunken pollen with abnormal intine development and aperture formation. The inhibition of BcMF8led to the decrease in in vitro pollen germination percentage. Even in the germinated pollen, the pollen tubes were unstable, abnormal-shaped, and burst more frequently, which was in accord with the in vivo pollen germination arrest at the stigma surface and retarded pollen tube growth in the stylar transmitting tissues.The phenotypic defects of antisense BcMF8RNA lines suggested a crucial function of BcMF8in pollen wall formation and pollen tube growth, modulating the physical nature of the pollen wall and in helping in maintain the integrity of the pollen tube wall matrix.(2) BcMF18, a putative classical arabinogalactan protein-encoding gene, was related to intine formation. Full length cDNA and DNA of BcMF18were isolated using homologous cloning. Structure and the deduced amino acid analysis indicated that BcMF18possessed the features of classical AGPs. RT-PCR and in situ hybridization showed that BcMF18was pollen-specific, whose transcript started to be expressed at the uninucleate stage and maintained throughout to the pollen at pollination stage, suggesting that BcMF18belonged to the class of’late’pollen genes. Prokaryotic expression and western blot were used to ensure that BcMF18could encode a putative AGP of approximately20kDa, which was soluble. The results of subcellular localization indicated that BcMF18protein was secreted, and exhibited intracellular and extracellular localization. Overexpression of BcMF18in Arabidopsis thaliana led to shorter mature siliques and severe sterility compared with the wild-type plants. Approximately50%of pollen in overexpression plants were shrunken and smaller, and could not germinate to produce pollen tube. Starting from the binucleate stage, collapsed pollen in overexpression plants was aborted with no content and nucleus. The intine layer at binucleate stage was constructed first, but immediately disappeared after degradation, with no cellulose deposition, while the basic structure of the exine layer was normal. Mature siliques of antisense BcMF18transgenic plants were shorter than those of control plants, and fertility frequencies were lower too. The functional interruption of BcMF18by antisense RNA technology resulted in the collapse of pollen with no content and nucleus. The intine layer of antisense BcMF18transgenic pollen at binucleate stage could not be formed with no cellulose deposition, while the basic structure of the exine layer was normal. These results indicated that BcMF18was involved in microspore development and may affect pollen intine formation, possibly by participating in cellulose deposition.(3) BcMF8and BcMF18, with similar but not the same expression pattern, were functionally redundant in the process of tapetum programmed cell death (PCD) but not in the pollen development. BcMF8and BcMF18shared52.3%nucleotide sequence identity and65.2%amino acid sequence identity with each other. Real-time RT-PCR suggested that BcMF8and BcMF18were pollen-expressed, whose transcripts were both first detected at the uninucleate stage and maintained throughout to the mature pollen stage. However, the RNA level of BcMF8was higher than BcMF18in the same tissue or organization. In addition, BcMF8transcript was also detected in the pollen tube of the pollinated pistil. The functional interruption of BcMF8and BcMF18by antisense RNA technology resulted in aborted pollen with higher frequencies. Although the aborted pollen in the antisense BcMF8and BcMF18transgenic plants mix the pheynotype of antisense BcMF8pollen and antisense BcMF18pollen, but no more severe. Partial were slipper-shaped and bilaterally sunken pollen with abnormal intine development and increased aperture formation, with some other were collapsed with no cellulose deposition and intine formation. The inhibition of either BcMF8or BcMF18could not affect the tapetum development, but an early degradation of tapetum would happen when both of them were interrupted, suggesting that BcMF8and BcMF18, are partially functionally redundant, required for tapetum PCD.(4) BcMF23a and BcMF23b, two putative pectin methylesterase genes, were both expressed in pollen during the late stages of pollen development. Full length cDNA and DNA of two homologous genes of PME gene At5g07410, BcMF23a and BcMF23b, were obtained by homologous cloning. They shared86%nucleotide sequence identity and89.3%amino acid sequence identity with each other. Structure and the deduced amino acid analysis indicated that BcMF23a and BcMF23b possessed the features of PMEs. Results of RT-PCR, in situ hybridization and analysis of promoter activity suggested that BcMF23a and BcMF23b were pollen-expressed, whose transcripts were first detected at the binucleate pollen and maintained throughout to the mature pollen grains. These finding suggested that BcMF23a and BcMF23b may function in pollen development. In addition to the pollen, BcMF23b promoter drove an extra gene expression in the valve margins, abscission layer at the base of the first true leaves, taproot and lateral roots in seedlings. Prokaryotic expression and western blot indicated that both of the putative BcMF23a and BcMF23b proteins were approximately40kDa and soluble, which exhibited intracellular and extracellular localization revealed by transient expression analysis in subcellular localization. PME activity assay suggested that only BcMF23a had PME activity, while BcMF23b not.