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: Tapetum is a cell layer adjacent to the anther locule. There are two main tapetal types, designated as the secretory tapetum and the amoeboid tapetum, in the angiosperms (Pacini et al., 1985; Murgia et al., 1991). The secretory tapetum shows cytoplasm and nucleus shrinkage, loss of the cell wall, and reduction in the size of mitochondria (Pacini et al., 1985; Parish and Li, 2010). Tapetal cells transform into the secretory type at the post-meiotic stage to secrete enzymes for the release of microspores from the tetrad and to provide nutrients for pollen development (Pacini et al., 1985; Piffanelli et al., 1998). Once the transition of tapetum is blocked in anther development in Arabidopsis, the tapetal cells become enlarged and abnormally vacuolated, which is followed by aberrant microspore separation and abnormal pollen-wall development (Zhang et al., 2007; Zhu et al., 2008). In Arabidopsis, TDF1, AMS, and MYB103 are involved in this process. AMS encodes a basic helix–loop–helix protein and acts downstream of TDF1 but upstream of MYB103 (Sorensen et al., 2003; Zhang et al., 2007; Zhu et al., 2008). Histological observation revealed that the critical cause of male sterility in the Bnms3 mutant was attributed to a defect in the timely transition of the tapetum to the secretory type, which was similar to that of Attdf1, Atams, and Atmyb103 mutants. Moreover, the anther of B. napus is bigger than that of Arabidopsis, which made it easier to distinguish the secretory type. Real-time PCR analysis demonstrated that the expression levels of BnTDF1, BnAMS, and BnMYB103 were delayed at the post-meiotic stage of anther development in the Bnms3 mutant. These data suggested that BnMs3 shared the same pathway with BnTDF1, BnAMS, and BnMYB103 in tapetal differentiation and might control the transition of the tapetal cells by affecting the expression of BnMYB103 (Fig. 10A,B).