Establishment And Extension Of The Molecular Mechanism Of Physiological Male Sterility In Wheat (Triticum Aestivum L.)

Wheat(Triticum aestivum L.) is a valuable agricultural crop and an important food source for humans in our country. A continual increase in wheat consumption has led to a demand for greater wheat yields to guarantee national food security. Heterosis breeding in wheat had significant impacts on grain yield, improved cultivars and led to better adaptation to adverse environments. A number of approaches have been proposed to avoid self-pollination for the commercial production of hybrid wheat seeds, which include genetic male sterility(GMS), cytoplasmic male sterility(CMS), photo-thermo-sensitive male sterility(PTMS) and chemical hybridizing agents(CHAs). Of these, CHA-induced male sterility can provide rapid, flexible and high performance seed-producing female parents for F1 hybrid production; they simultaneously avoid fluctuations of genotype and environmental factors in maintaining male-sterility and/or male-fertility restoration. Specifically, CHA-induced male sterility removes the reproductive isolation necessary in other approaches, the conversion and maintenance of an androsterile line(line A) and the incorporation of factors of fertility restoration in male progenitors, thus facilitating a ‘two-line’ approach to the production of hybrid seed. Thus, the chemical hybridizing agent SQ-1(CHA-SQ-1) offers a new approach to the application of heterosis. Normal fertile male wheat can be made sterile after the spraying of appropriate doses of CHA-SQ-1 at a specific developmental period. The proportion of male sterile wheat can reach 95%–100%, and the outcrossing rate can exceed85%. Today, CHA-SQ-1 has now been used to produce hybrid seeds on a large scale in China.However, the mechanism by which CHA-SQ-1 causes male sterility in wheat is still unknown. With this objective in mind, physiological male-sterile Xinong 1376(PHYMS-XN1376) plants and it maintainer line(MF-XN1376) were used. Morphological,biochemical and physiological responses of wheat flag leaves were detected in this study.This was followed by the application of two-dimensional electrophoresis(2-DE) in combination with tandem mass spectrometry(MS/MS) analysis to compare the different expression patterns of flag leaf membrane proteomes, floret mitochondrial proteomes, and pollen grain proteomes. And also the biochemical characteristics of nuclear DNA fragmentation was detected using terminal deoxynucleotidyl transferase-mediated dUTP nickend-labelling(TUNEL) assay in situ and DAPI staining. The follow most important results were obtained:1. To investigate effects of different treatment times(2h, 4h, 6h, 10 h, and24h) of CHA-SQ-1 used, morphological, biochemical and physiological responses of wheat flag leaves were detected in this study. CHA induced programmed cell death(PCD) as shown in terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling(TUNEL) and DNA laddering analysis. CHA-SQ-1 was rapidly absorbed by wheat flag leaves at 2 h after treatment, and 100% male sterility was induced from 6 h. In the early phase, the CHA-SQ-1triggered organelle alteration and the cells PCD which also broke the balance of the oxidation/antioxidation in wheat leaves. The oxidative stress assays showed that lipid peroxidation was strongly activated and photosynthesis was obviously inhibited in SQ-1-induced leaves. However, CHA contents in wheat leaves gradually reduced along with the time CHA-SQ-1 applied. Young flags returned to an oxidative/antioxidative balance and ultimately developed into mature green leaves.2. We compared their quantitative proteomic profiles in the wheat flag leaves of the control, 2 h and 6 h after CHA-SQ-1 treatment. Our results indicated that a total of 150 spots exhibited at least 1.5-fold(p <0.05) difference in abundance between the control and CHA-SQ-1 treatment, and 149 were successfully identified by MS/MS. Based on the metabolic and functional features of wheat flag leaves, all identified proteins were classified into 12 functional categories, including photosynthesis, ATP synthesis and ion transport,protein folding and assembly, unclassified proteins, protein biosynthesis, cell rescue and defense, redox homeostasis, carbohydrate metabolism, protein degradation, signal transduction, protein transport, and chlorophyll biosynthesis proteins involved in multiple biological processes. Bioinformatic analysis indicated that four biological processes(photosynthesis, ATP production, response to stress, and proteins metabolism) were interrupted after CHA-SQ-1 treatment in flag leaves.3. The cytology of CHA-SQ-1-treated plant anthers at various developmental stages was studied by light microscopy, scanning and transmission electron microscopy, in situ terminal deoxynucleotidyl transferasemediated dUTP nick end-labelling(TUNEL) assay and DAPI staining. The results indicated that the SQ-1-treated plants underwent premature tapetal programmed cell death(PCD), which was initiated at the early-uninucleate stage of microspore development and continued until the tapetal cells were completely degraded; the process of microspore development was then blocked. Microspores with low-viability(fluorescein diacetate staining) were aborted. The study suggests that premature tapetal PCD is the main cause of pollen abortion. Furthermore, it determines the starting period and a keyfactor in CHA-SQ-1-induced male sterility at the cell level.4. This study set out to probe the mechanism of physiological male-sterility(PHYMS)induced by the chemical hybridizing agent(CHA)-SQ-1, and cytoplasmic male-sterility(CMS) of wheat at the proteomic level. A total of 71 differentially expressed mitochondrial proteins were found to be involved in pollen abortion and further identified by MALDI-TOF/TOF MS. These proteins were implicated in different cellular responses and metabolic processes with obvious functional tendencies toward the tricarboxylic acid cycle,the mitochondrial electron transport chain, protein synthesis and degradation, oxidation stress,cell division cycle and epigenetics. Interactions between identified proteins were demonstrated by bioinformatics analysis, enabling a more complete insight into biological pathways involved in anther abortion and pollen defect. Accordingly, we first propose a mitochondria-mediated male sterility protein network in wheat; this network was further confirmed by physiological data, RT-PCR and TUNEL assay. Our results provide intriguing insights into the metabolic pathway of anther abortion induced by CHA-SQ-1 and also give useful clues to identify the crucial proteins of PHYMS and CMS in wheat.