| In hybrid crop breeding, hybrid vigor or heterosis is a common phenomenon, whichcrossing different inbred lines produces F1hybrids that usually have higher yields than theparents. In wheat (Triticum aestivum L.), production of sufficient quantities of hybrid seeds isnot so successful because it is a self-pollination crop. Chemical hybridizing agents (CHA) hasbeen shown to induce male sterility in wheat so that hybrid seeds of any parental combinationcan be produced. However, little is known about wheat male sterility-responsive proteins orthe molecular mechanisms of male sterility induced by CHA. In wheat, male reproductiveprocesses take place in the anther, which contains diploid sporogenous cells that experiencemeiosis to form haploid microspores and finally develop into pollen grains. Once theelaborate chronological order process becomes disordered, pollen might be aborted, resultingin male sterility. Recently, there has been an increasing application of proteomic approachesto study anther development and pollen reproduction. Many proteins specifically expressed inanthers were detected, such as in rice, Arabidopsis, maize, wolfberry and tomato. For wheat,CHA could provide a favourable system for inducing male sterility without fertilityrestoration. However, the very large size and polyploidy complexity of the wheat genomehave been substantial barriers to genome analysis. Thus，the exploitation of wheat malesterility induced by CHA-SQ-1could be enhanced by proteome analysis of developinganthers.1. Proteomic studies were performed to identify the protein species involved in malesterility induced by chemical hybridizing agents in wheat (CIMS). A total of128differentialspots were determined by using the threshold of significance p <0.05and a≥2-fold increaseor decrease in protein expression. For MALDI-TOF/TOF MS/MS analysis, the differentlyexpressed spots of common up or down-regulated at different stage of anthers were excised.16spots were successfully identified as15individual proteins by searching against NCBInrand uniprot databases, and more than half of the identified protein spots were involved incarbon metabolism and cell wall related proteins and oxidative-redox stress proteins.Oxidative-redox stress proteins up-regulated in CIMS anthers include putative glutathioneS-transferase GSTF1, superoxide dismutase and putative In2.1protein, which were activated in antioxidant systems to scavenge ROS. Meanwhile, the ROS level was significantly higherthan NW anthers, which indicated the abortion of microspores was possible happened in theprocess of chronic oxidative stress by the abnormal increased ROS in CIMS plant. Moreover,β-1,3-glucanase and vacuolar invertase were thought to directly related to the occurrence ofmale sterility. Photosynthesis-related protein, ABC transporter, transcriptional repressor andreversibly glycosylated polypeptide were also identified in this experiment. In combinationwith phenotypic analysis and tissue section analysis, the identified proteins from2D-PAGEindicate regulating patterns of CIMS pollen development is a complex network. The presentstudy provided important clues for understanding the mechanism of male sterility induced byCHA in wheat.2. Protein polyubiquitination is a significant regulator of diverse physiological functions,including sexual reproduction, in plants. CHA-SQ-1has been shown to induce male sterilityin wheat through inhibition of pollen development. This mechanism by which CHA inducesmale sterility in wheat is unclear. In this study, differential proteomic analysis ofpolyubiquitinated proteins associated with wheat male sterility was investigated. Wheat plantsof the same genetic background were treated with or without CHA. Ubiquitinated proteinswere then extracted and enriched for proteomic analysis. Differentially expressedpolyubiquitinated proteins in trinuclear stage anther were identified by nanospray liquidchromatography/tandem mass spectrometry. A total of127and131differentially expressedpolyubiquitinated proteins, including heat shock protein70, ATPase subunit,glycosyltransferase, ubiquitin related enzyme and20S proteasome subunit, were successfullyidentified by searching against wheat protein database and NCBInr database, respectively.Most of these proteins are related to photosynthesis, carbohydrate and energy metabolism,and multiple metabolic processes. These findings show that alteration of polyubiquitinatedproteins is assoicated with male sterility in wheat.3. In previous studies, we first isolated one different protein β-1,3-glucanase andUGPase by using two-dimensional electrophoresis and MALDI-TOF-TOF-MS technologiesfrom normal wheat and CHA induced male sterility wheat. In this experiment, β-1,3-glucanase activity and the expression of callose deposition related gene UGPase weredeterminate in the normal wheat, CIMS and genetic male sterility (GS) wheat. The resultsshowed that β-1,3-glucanase activity is significantly lower than NW in the stage ofmeiophase to mononuclear stage in CIMS and GS, but significantly higher in the stage ofbinuclear and trinuclear. The level of β-1,3-glucanase activity was a roughly trend of“high-low-high-low-low” in the three wheat anthers. From the stage of meiosis to tetrad, β-1,3-glucanase activities had a sharply decrease which created the condition of callose deposition. And then, the activities were sharply increase which indicated the callose needed todegradation. In the stage of callose synthesis and degradation, β-1,3-glucanase activity hadsignificantly different between the fertility line and sterility line, but had no different betweenCIMS and GS. β-1,3-glucanase activity had significantly different between the fertility lineand sterility line in the stage of callose synthesis and degradation, but had no differencebetween CIMS and GS. The UGPase gene of callose deposition required was highlyexpressed in the meiophase and had a sharply decrease in the tetrad stage. However, theexpression of UGPase gene had significantly different between the fertility line and sterilityline. And then, the expression remained the same amount from mononuclear stage totrinuclear stage. In CIMS and GS anthers, UGPase gene is also expressed highly in meiosisstage, but significantly lower than expression of normal anther (p <0.01). In the tetrad period,expression level was significantly decreased, and no significant differences were detected inmononuclear stage. But UGPase gene expression of binuclear and trinuclear of CIMS was amarked increased in relative to normal trinuclear stage anther (p <0.01). The expression ofUGPase had maintained stable in the binuclear and trinuclear of GS. The level of UGPasegene expression was a roughly trend of “high-low” in the three wheat anthers from meiosis totetrad These data indicated that β-1,3-glucanase activity and the expression of UGPase geneplay important role in the male sterility of wheat. Consequently, the PMCs might be began todegenerate at the early meiosis stage, the difference of UGPase gene expression and β-1,3-glucanase activity eventually resulting in complete pollen collapse. In addition, the criticalperiod of anthers abortive might be the meiosis stage to the tetrad stage rather than wepreviously thought the mononuclear period.4. The combination of using SDS-PAGE and western blot methods, a preliminaryresearch of the ubiquitination of mitochondrial proteins for physiologically male sterilityinduced with CHA-SQ-1and corresponding normal lines of complete fertility. Theubiquitination of80kD protein was not obvious in the NW plant, and the ubiquitin level ofbinuclear was higher than mononuclear and trinuclear florets. In CIMS florets, the level ofubiquitination presented the obvious down-regulate trend with the development of the floretsstage. When the anther development to the trinuclear stage, the ubiquitination of80kD proteinwas down to the limit. In mononuclear stage of CIMS wheat florets, the ubiquitin levels aresignificantly higher than NW. Development to the stage of binuclear anthers, the florets of theprotein ubiquitin level was lower than NW. When the anther development to trinuclear stage,the ubiquitin of CIMS florets were almost undetectable level, while the NW florets still have ahigh level at this period. These results were indicated that the process of ubiquitination of themitochondrial proteins from florets may be abnormal because of the spraying of CHA-SQ-1. It is also likely to affect the normal metabolism of mitochondria, which lead to respirationdisorders, decrease the production of ATP, namely energy metabolism abnormity. Finalabortive pollen might be happened because of the energy in short supply and anther starchaccumulation was blocking.5. Separation histone of different processing materials at different periods by13%SDS-PAGE gel electrophoresis, subsequently, the results of western blot found that theubiquitination of histone H3, H2A and H2B were happened, but the histone H2Bubiquitination was high level. In NW plants, the ubiquitin level of histone H2B atmononuclear period was significantly higher than the last period. In sterile florets, highestperformance of histone H2B ubiquitination was happened at the anther stage of binuclear.Compared to ubiquitin level of H2B in NW and CIMS, mononuclear stage performance hassignificant differences. The ubiquitin level of histone H3was much lower than H2B, but alsoshowed higher than sterile flowers at mononuclear stage in NW florets. There is no differentin the following two periods between NW and sterile florets. The expression level of RAD6gene which has the function of catalytic H2B ubiquitination was consistent with the changetrend of H2B ubiquitination in the three periods. These results showed that the ubiquitinationlevel of histone H2B in male sterile wheat was seemed to be significantly less than that infertile ones, meanwhile, the expression of the RAD6gene in sterility wheats was apparentlysuppressed. It can be seen that there is a close relationship between the ubiquitination of thehistones on chromosomes and the physiological male sterility induced by the CHA-SQ-1inwheat. |
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