| Plant sexual reproduction is not only a major approach of plant propagation, but also one of the foundations for evolution and adaptation to the environment. Anther is the male reproductive organs of plants which is the place for pollen development. Therefore, the study of anther and pollen development has important theoretical significance. As the male-sterile varieties are valuable resources that greatly facilitate the production of hybrids via cross-pollination, in-depth study of anther and pollen development has important application value. In flowering plants, anther development is a very complex process which consists of two sequential phases: microsporogenesis and microgametogenesis. In Arabidopsis microsporogenesis, stamen primordia gradually formed the primarily morphologic structure of anther and filament through cell differentiation and fate decision. In microgametogenesis, pollen matured with two asymmetric mitotic divisions to form the multi-nulcei male gametophyte. Pollen development depends on the diploid sporophytic cell layer, especially the tapetal cells. The tapetum, which arises from secondary parietal cells, surrounds developing reproductive cells. It plays an important role in pollen development by contributing to microspore release, nutrition, pollen-wall synthesis and sporopollenin deposition. With the pollen development, it initiates the programmed cell death (PCD) process. With the completion of mitosis of pollen, tapetal cells undergo lysis and provide the materials for pollen coat. So far, in Arabidopsis, several leucine-rich repeat receptor-like protein kinases were reported for cell fate determination of tapetum. Besides, several transcription factors were also identified which involved in regulating tapetum development and function. However, for the complexity of anther development, it should be there are many key factors not characterized, and the relationship of these reported factors is still not clear. Based on these reasons, we use forward genetics to identify novel key genes which important of anther development, and illustrate the function of them.In this study, male sterile mutant ms188 was generated by ethyl-methane sulfonate (EMS) mutagenesis. A map-based cloning approach was used, and ms188 was mapped to a 95.8-kb region on chromosome 5 between molecular marker MDA7 and K24C1. Sequence analysis revealed that ms188-1 had a pre-mature stop codon in the R2R3 region of AtMYB103 which belongs to MYB transcription factor family. Allelism tests and genetic complementation indicated that AtMYB103 corresponded to MS188. The observations semi-thin section of wild-type and ms188 anthers revealed that mutant tapetal cell walls remained intact in late stages, suggested that the sceretory function of tapetum was altered. Most of the mutant microspores underwent degradation during late phases. Aniline blue staining showed the degradation of callose wall was altered, resulted in separation of tetrad. The scanning and transmission electron microscopy observation showed the surviving microspores lacked exine in ms188 locules. Semi-quantitive and quantitive RT-PCR analysis indicated that the callase-related gene A6 and the exine formation gene MS2 were obviously downregulated in mutant anthers. These results implicate that AtMYB103 plays an important role in tapetum development, callose dissolution and exine formation in A. thaliana anthers.We also characterized the detail expression pattern of AtMYB103. it was specifically expressed in tapetum and microsporocyte, indicated that it regulated tapetum development and secretory function. In addition, disrupted development and secretory function of tapetal cells in ms188 mutant were analyzed by TEM. Furthermore, the aberrant exine formation of ms188 is due to the aborted tapetal functions rather than the primexine formation. In order to illustrate the regulatory mechanism of AtMYB103, we performed global expression profiling analysis of wild-type and ms188 mutant. A total of 821 genes (728 downregulated and 93 upregulated) in ms188 flower buds were identified. Based on 'Electronic Fluorescent Pictograph'(e-fp) Browser dataset, we assigned these differentially expressed genes to nine groups as diverse stages of anther development, such as bud expressed, late anther expressed, pollen expressed, and so on. Selected differentially expressed genes for quantitative RT-PCR analysis showed the microarray experiments are reliable. Functional classification of microarray data showed that loss-of-function of AtMYB103 impairs several key metabolic and signaling pathways throughout anther development: expression of many late-stage-expressed kinases is affected, suggested that AtMYB103 might act as a regulator in signal transduction of anther development at the late stage; several pectinase and callase related genes were suppressed, indicated that they probable involved in degradation of tapetal cell wall and callose wall; many genes involved in lipid synthesis and transport were also significantly downregulated, resulted in absent of exine of ms188 pollen. However, TUNEL assay illustrated the PCD process of tapetum was not regulated by AtMYB103. In addition, we applied RNA in situ hybridization to characterize several novel regulators of microsporogenesis which identified in the microarray experiments. These results indicated that AtMYB103 acts as a key regulator for several pathways during Arabidopsis anther development.To elucidate the transcriptional regulatory pathway in anther development, we analyze the mutants of reported transcription factors through cytological observation. Besides, we preliminarily identify the relationship of several key regulators in anther development though RT-PCR strategy. Further double mutant strategy and in situ analysis confirm the DYT1-TDF1-AMS-AtMYB103-MS1 transcriptional regulatory pathway in anther development.Summary, the results indicated that AtMYB103 acts as a key transcription factor of anther transcriptional regulatory pathway, and affects several pathways to regulate tapetum development and pollen maturation. Investigation of these mechanisms deepenes our understanding of the roles of tapetal cells in pollen development.
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