Primarily Study On Molecular Mechanism Of Male Sterility Induced By Chemical Hybridization Agent Monosulfuron Ester Sodium In Rapeseed(Brassica Napus L.)

Rapeseed(Brassica napus L.) exhibits significant heterosis and the chemical hybridization agent(CHA) induced male sterility has become an important tool for rapeseed heterosis utilization. A number of CHAs have been developed from sulfonylurea herbicids, however, the molecular mechanism of which is largely unknown. Previously, our lab found a CHA(Monosulfuron Ester Sodium, MES) can induce male sterility in rapeseed at very low concentration. In order to get some clues to uncover the molecular mechanism of CHA-MES inducing rapeseed male sterility, we carried out a series of researches in this study. Firstly, the ultrastructure of the meiocyte and tapetum cells from fertile and MES induced male sterile rapeseed anthers was observed and compared; secondly, using Brassica Oligo Microarray, the genome-wide gene expression profile during normal anther development was detected; thirdly, the comparative transcriptome analysis was also performed between fertile and MES induced male sterile anthers, a series of lipid and carbohydrate metabolism related genes were selected as candidates for male sterility; finally, we selected 42 genes for experimental verification using virus induced gene silencing(VIGS) system in Arabidopsis. The main results were as follows:1. The ultrastructure observation was performed between fertile and CHA-MES induced sterile anthers. At pollen mother cell(PMC) stage, several layers of anther cells were formed in the fertile anthers, in which the tapetum cells and PMCs exhibiting highly cell divisive; while the meiocytes became abnormal plasmolysis in MES induced sterile anthers. At tetrad stage, the fertile microspores packed in the callose initiate to form primary pollen exitine and the tapetum cells became secretory with many endoplasmic reticulum(ER) structures dispersed in the cytoplasm; while, the meiosis process could not perform normally in sterile anthers, and the meiocytes just separated from each other and further plasmolysis. At uninuclear microspore stage, a big vacuole and abundant cellular organelles formed in fertile microspores, and the cell wall of tapetum celles degraded and cytoplasm fused, two types of plastids(elaioplasts and tapetosomes) appeared, which were transmitted to complete the pollen extine; while, in MES induced sterile anthers, a small part of cells might successfully go through meiosis and microspores seemingly produced, however, the cytoplasm of microspores almost empty, some could not form complete pollen wall, and the tapetum cells exhibited several types of defects, such as no degradation of the cell wall or cytoplasm, abnormal elaioplasts and tapetosomes. Until the mature pollen grain stage, in fertile anthers, many granule storage and ER structures were filling in two or three nucleate pollen grain; while, the sterile pollen grains were thorough male sterily with no cytoplasm in, and the tapetum cell residues formed solidified bulks or still showed the visible cell wall. Furthermore, the chloroplasts were also abnormal in exodermis and endodermis cells at uninuclear microspore stage. Overall, the normal anther development process involved with many types of cells and organelles, however, the CHA-MES treatment destroyed the cellular structures of meiocytes, tapetum cells and anther wall cells, especially in the structure of the plastids.2. The dynamic transcriptome analysis of leaves and anthers in normal fertile rapeseed indicated the gene expression alteration during anther development process. Six pairs of student’s T-test comparisons were performed between three anther tissues and leaves, and 11,678 differentially expressed transcripts(DETs) were obtained. The distribution of the DETs indicated that a small part of genes expression was altered at early anther development stage in contrary with large number of genes regulation at late stage. Using the K means method, 11,678 DETs were clustered into 15 dominant patterns(DPs) according to their expression levels, each DP containing 89 to 4,319 DETs. Furthermore, DP1-DP5 containing 705 DETs, exhibited high expression level during early anther development stages(SBs and An-MBs), in contrary with DP6-DP12 showing very high expression levels at late stages(AnLBs), which contained 1,517 DETs. The GO enrichment analysis of genes in each DP revealed that the endomembrane system related genes were enriched at both early and late anther development stages(DP1, DP5, DP6, DP8, DP9). In addition, at early stages(DP1-DP5), the pollen extine formation and glycerol metabolism related genes were enriched; while, at late stages, large amounts of pectin metabolism or cell wall related genes were enriched. The distribution of carbohydrate and lipid metabolism related genes indicated that lipid metabolism related genes were dominant in early stages anthers while carbohydrate metabolism related genes were enriched at late stage.3. The comparative transcriptome analysis was also performed between fertile and CHAMES induced sterile anthers. Through the vertical and horizontal comparisons, 1,501 DETs were selected as genes which were related to anther development and the expression was altered by MES. In which, 64% of the DETs were down-regulated in male sterile rapeseed plants, 36% of the DETs were up-regulated. Furthermore, there were small number of genes(77 and 67) differentially expressed in leaves(Ls) and small buds(SBs), respectively. However, a large number of differentially expressed genes(161 and 1,238) were detected in anthers from middle buds(An-MBs) and large buds(An-LBs). The subcellular localization analysis revealed that most of the DETs were localized in plastids/chloroplasts, mitochondria or nucleus. The gene function categories and enrichment analysis results indicated that MES affected the carbohydrate and lipid metabolism related genes expression during anther development. The metabolism pathway analysis using Map Man also confirmed the results. At last, we extracted all the lipid and carbohydrate metabolism related genes which expression altered in Ls, SBs and An-MBs, which might be tightly related with MES inducing male sterility. The carbohydrate contents in leaves and flower buds were also detected and they were indeed altered.4. It is reported that the basic action mode of sulfonylurea herbicides was to target the acetolactate synthetase(ALS) in plants, however, it is not clear when they are used as CHA. In this study, using the Arabidopsis sulfonylurea resistant mutant CS204 as reference, we confirmed that ALS was also the target of CHA-MES. Furthermore, the male sterility plants were also obtained using TRV based gene silencing(TRV-VIGS) of ALS, and more serious developmental defects were observed when the m RNA level of ALS was severely silenced. This phenomenon was similar to CHA-MES effects on rapeseed plants, that is, inducing male sterility at low concentration and repressing development at high concentration. In addition, according to the results of comparative transcriptome analysis, we selected 42 differentially expressed genes as candidates which might be directly related to plant fertility. Using TRVVIGS system, 2 genes were quickly verified.Taken together, we proposed a primary mechanism of CHA-MES inducing rapeseed male sterility: MES firstly inhibited the ALS activity in rapeseed anther, then lipid and carbohydrate metabolism processes were disordered during anther development, resulting in abnormal structure of plastids and finally male sterility. Furthermore, because of the high requirement level of lipid and carbohydrate metabolism in anthers, the anther tissue is more sensitive to MES than other tissues.