BnMs3 is required for tapetal differentiation and degradation, microspore separation, and pollen-wall biosynthesis in Brassica napus.
Bottom Line: A total of 76 down-regulated unigenes in the Bnms3 mutant, several of which are associated with tapetum development, callose degeneration, and pollen development, were isolated by suppression subtractive hybridization combined with a macroarray analysis.Moreover, BnMs3 takes part in pollen-wall formation by affecting the expression of a series of genes involved in biosynthesis and transport of sporopollenin precursors.All of the above results suggest that BnMs3 participates in tapetum development, microspore release, and pollen-wall formation in B. napus.
Affiliation: Huazhong Agricultural University, Wuhan, China.
7365AB, a recessive genetic male sterility system, is controlled by BnMs3 in Brassica napus, which encodes a Tic40 protein required for tapetum development. However, the role of BnMs3 in rapeseed anther development is still largely unclear. In this research, cytological analysis revealed that anther development of a Bnms3 mutant has defects in the transition of the tapetum to the secretory type, callose degradation, and pollen-wall formation. A total of 76 down-regulated unigenes in the Bnms3 mutant, several of which are associated with tapetum development, callose degeneration, and pollen development, were isolated by suppression subtractive hybridization combined with a macroarray analysis. Reverse genetics was applied by means of Arabidopsis insertional mutant lines to characterize the function of these unigenes and revealed that MSR02 is only required for transport of sporopollenin precursors through the plasma membrane of the tapetum. The real-time PCR data have further verified that BnMs3 plays a primary role in tapetal differentiation by affecting the expression of a few key transcription factors, participates in tapetal degradation by modulating the expression of cysteine protease genes, and influences microspore separation by manipulating the expression of BnA6 and BnMSR66 related to callose degradation and of BnQRT1 and BnQRT3 required for the primary cell-wall degradation of the pollen mother cell. Moreover, BnMs3 takes part in pollen-wall formation by affecting the expression of a series of genes involved in biosynthesis and transport of sporopollenin precursors. All of the above results suggest that BnMs3 participates in tapetum development, microspore release, and pollen-wall formation in B. napus.
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Mentions: In the Bnms3 mutant, callose dissolution was blocked, but there were still seldom microspores without pollen exine released from the tetrad according to our research. Lipid molecules are a main component of pollen wall. To explore the role of BnMs3 in biosynthesis of lipid molecules, three unigenes obtained from the SSH library and eight ESTs acquired based on the corresponding information from Arabidopsis which participated in this process were analysed by using real-time PCR. According to the different stages at which their differential gene expression initiated, the expression patterns of them could be divided into three clusters. Cluster 1 included BnNEF1 and BnFLP1 which didn’t show any expression changes at the three detected stages between the two materials (Fig. 7A, cluster 1). Cluster 2 was consisted of BnCYP703A2, BnACOS5, BnCYP704B1, BnCHS2 (At4g34850), and BnMS2, showing the initially down-regulated expression in the Bnms3 mutant at the post-meiotic stage (Fig. 7A, cluster 2). The three unigenes (BnMSR20, BnMSR23, and BnMSR73) and BnCHS1 (At4g00040) were attached to cluster 3, which exhibited the down-regulated expression in the Bnms3 mutant initiated at the uninuclear microspore stage (Fig. 7A, cluster 3). Among these differentially expressed genes, BnCYP703A2, BnACOS5, BnCYP704B1, BnMS2, and the three unigenes were relevant to biosynthesis of fatty acids, while BnCHS1 and BnCHS2, which encode chalcone synthase, were involved in biosynthesis of both fatty acids and phenols in Arabidopsis (Dobritsa et al., 2010). These results disclosed that BnMs3 was associated with the biosynthetic pathways of fatty acids and phenolic compounds, which were required for exine development.