Differential Proteomic Analysis Between Recessive Genic Male Sterility 7365A And Its Isogenic Line 7365B In Brassica Napus

In Brassica napus, the recessive genetic male sterility system was considered to be an effective pollination control system in heterosis utilization, because its abortion completely, stable sterility, and broad recovery source. Multienzyme associations or metabolon localized to specific subcellular organization are involved in several critical functions in plant metabolism, such as protein processing and degradation, respiratory electron-transport chain, and plant reproduction. To date, few multienzyme complexes involved in male fertility have been examined in B. napus. In this study, 7365 AB, a near isogenic lines(NILs) were chosen as materials. Proteomics combined with biochemistry were used to analyse the molecule mechanism of tapetal lipid synthesis abnormity, and function of sporopollenin metabolon in tapetal cells during the anther development. The results were described as follows: 1. Defective anther cuticle in the muant 7365AWe used scanning electron microscopy(SEM) to investigate the ultrastructure of the anther surface. At stage 12 of the anther development, the anther epidermis cell length and width were decreased in the mutant 7365 A as compared to the wild-type. Furthermore, scanning examination indicated a glossy and smooth anther surface in the mutant as compared to the normal outermost linear shaped of the wild-type, suggesting that the mutant 7365 A was defective in the synthesis of anther cuticle.We used gas chromatography-mass spectrometry(GC-MS) and gas chromatography-flame ionization detection(GC-FID) to analyze the constituents of chloroform-extractable cuticular waxes and aliphatic cutin monomers in the mutant 7365 A and wild-type. The cutin in the wild-type anther was 2.96 μg/mm2, while that of the mutant anther epidermis was only 1.70 μg/mm2, which is a 42.6% reduction compared with the wild-type. Moreover, the total amount of cuticle wax in the mutant was significantly decreased as compared to the wild-type, with a reduction of 60%. Chemical analysis indicated that the synthesis of lipidic compounds in the 7365 A anther cuticle were abnormality. 2. Comparative proteomic analysis in the mutant 7365 A and the wild-type in B. napusTo identify the key lipid metabolism proteins and their involvement in the microsporogenesis in B. napus, we used 2-dimensional electrophoresis(2-DE) to investigate the protein profiles in the buds of mutant 7365 A and wide-type(1–2 mm bud length). Approximately 1000 spots were detected on each 2-DE gel. Statistical analysis identified 103 spots that exhibited at least 1.5-fold differences. Among these 103 spots, 52 unique proteins with significant expression changes were obtained after removing redundant proteins and were analyzed by using matrix-assisted laser desorption ionization(MALDI)-time-of-flight(TOF)/TOF mass spectrometry(MS). To evaluate the accuracy of the 2-DE data, western blot analysis was performed to determine the expression of BnPKSA, BnPKSB, and BnTKPR1 between buds of both the mutant 7365 A and wild-type.Gene ontology(GO) was performed to understand the potential function of these 50 proteins, and to calculate the functional category distribution based on the level 2 biological processes. The annotated proteins were functionally grouped into 16 biological processes, including metabolic process, cellular process, stimulus response, developmental process, and reproductive process, etc. Many proteins involved in metabolic and cellular processes were down-regulated, indicating that the normal development of the anther in the mutant was affected. Proteins involved in the stress response and protein degradation were found to be up-regulated, indicating that the ubiquitin-proteasome system in the mutant was activated to respond to adverse reactions caused by the abnormal male reproduction. 3. Proposed model of a sporopollenin metabolon in tapetal cells of B. napusSporopollenin, the major component of exine is associated with lipid metabolism during the pollen development. In 2-DE analysis showed that BnPKSA, BnPKSB, and BnTKPR1 were significantly down-regulated in the mutant. Their homologous proteins in Arabidopsis were reported to contribute to sporopollenin precursor synthesis. Given the similar spatial-temporal patterns and functionality of these four sporopollenin biosynthetic enzymes, the potential protein-protein interactions may exist among these enzymes. To test this hypothesis, firstly, we used qRT-PCR, western-blot analysis, and GUS analysis to confirm the existence of similar expression patterns in BnPKSA, BnPKSB, BnTKPR1, and BnACOS5. Then In vitro and in vivo analysis showed interaction among BnPKSA, BnPKSB, and BnACOS5, while BnTKPR1 did not. Taken together, we propose that BnPKSA and BnPKSB may compose a heterodimer combined with BnACOS5, constituting a sporopollenin metabolon in tapetal cells that related with male reproductive development in B. napus.