| During the life cycle of higher plants, there exists the alternation between the diploid sporophytic generation and the haploid gametophytic generation. In angiosperm, the development of male and female gametophyte is critical to the plant fertility. Anther and ovule development processes play a key role inthe development of male and female gametophyte since they construct a proper and necessary developmental microenvironment with nutrient supply and positional developmental cues. Thus,investigations on the molecular and genetic mechanisms controlling these processes become an essential step for the understanding regulatory mechanism of plant fertility. Therefore, we used tobacco and Arabidopsis as plant material to investigate the molecular mechanism of male and female gametophyte development concerning its relation to developmental microenvironment. The main results are as follows:1. Based onprevious study in our lab, two specifically expressed genes were found in subtractive library of egg and zygote in tobacoo. Bio informatics analysis indicates that they belong to the WOX family, therefore, theywere named NtWOX9A and NtWOX9B respectively. NtWOX9A and NtWOX9B encode transcription factors containing the Homeobox, which consistsof a60-amino acid helix-turn-helix structure and can bind toDNA or RNA directly via the second helix. Proteins encoding HB domain has have been found exclusively in eucaryote. In addition, NtWOX9A and NtWOX9B also have another conserved domain in C-terminal belong to the subfamily.2. The results of Real time RT-PCR and fusion protein fluorescence observations showed that NtWOX9A and NtWOX9B had the similar expression pattern. They are expressed in the stem apex, anther, ovary and style continuously and specifically, but not in mature vegetative organs. NtWOX9A and NtWOX9B are expressed highly in the young anther, ovary and style, and decreased later as the structures become matured. This result indicates that NtWOX9A and NtWOX9B are most likely to be significant in the progress of sexual reproduction in tobacco, especially in the development of anther, ovary and style.The result of subcellular location of NtWOX9A protein showed that it was only detected in the nucleus, which is consistent to the usual location of a transcription factor.3. NtWOX9A RNAi and overexpression(OX) vectors were constructed and transformed into Nicotiana tabacum cv. Petite Havana SRI plants. Reciprocal cross experiments were performed between the transgenic lines and wild-type (WT), and all F1ovaries produced fewer seeds than WT. This result demonstrates NtWOX9A play an important role in development of both male and female gametophytes. Interestingly, fertility was reduced severely when transgenic lines acted as female parent, indicating its paternal and maternal allele may have different biological effects on male and female gametophyte development.4. We investigated the role of NtWOX9A in pollen development. According to FDA staining and germination assay in vitro, both the pollen vitality and germination ratewere reduced in transgenic lines.The statistics analysis of the pollen density exhibited a significant reduction in the number of pollen, this suggests the pollen abortion may occur in the early developmental stage. Mononuclear pollen in the transgenic lines showed obvious abnormal according the morphological analysis. Result of DAPI staining suggests NtWOX9A does not affect the cell cycle. In the histological sections of RNAi or OX anthors, the tapetal cells were obviously abnormal at the tetrad stage. The results showed proliferation of tapetal in OX lines, but earlier degradation of tapetal in RNAi lines. Subsequently the tapetum irregularly degraded at the mononuclear microspore stage. In OX lines, the tapetum became thicker and some residues of tapetal cells retained until the connective cell degenerated. But in RNAi lines, the tapetum showed higher degradation than the wild type. Callose deposition in ovules development was observed using aniline blue staining. Callose was thicker in the RNAi lines than WT type, but thinner in OX lines. We surmise that tapetum regulated callose deposition, which leading to pollen fertility severely. 5. In addition, we also investigated the effect of NtWOX9A during ovary development. The ovules of transgenic lines and WT were cleared in Methyl salicylate or Hoyer solution. Results showed that ovule primordia initiation and megaspore mother cell(MMC) were normal in RNAi transgenic lines. But in OX transgenic lines, more than half of MMC could not elongate, which showed smaller nucleus and high vacuolization. All transgenic lines showed variety of abnormal structures during subsequent development, including that MMC could not initiate meiosis, or functional megaspore selection and megaspore mitosis was defeated. This result indicates that NtWOX9A could in fluence archesporial cell fate and its devolopment.6. Then we observed the callose deposition in both transgenic lines using aniline blue staining. Part of callose distribution polarity in RNAi transgenic ovules were disturbed. Some ovules showed normal callose distribution polarity, but callose wall appeared much thicker than that of WT. Callose distribution in OX transgenic lines was also abnormal. The polar deposition was disturbedand callose wall became thinner and discontinuous. This result indicates NtWOX9A may influenced MMC development through the regulation of callose dynamic accumulation.7. On the basis of previous working in our laboratory, we also identified a gene exine formation defect (EFD)in Nicotiana tobaccum egg cell-zygote subtractive library, which encods a methyltransferase. Its homolog in Arabidopsis was then functionally analyzed. EFD mutation leads to defective exine and anomalously expression of genes relevant to the pollen primexine and exine development.8. EFD is predominately expressed in the tapetum at the tetrad stage. Via Aniline blue staining, the callose deposition of efdtetrads was irregular and the callose wall was much thinner than that of WT. Meanwhile, genes related to sporopollenin synthesis and transport, such as AT1G01280, AT3G07540, were irregularly expressed in the efd mutant. In vitro DNA methyltransferase activity assay revealed that Ac?, a restriction enzyme sensitive to methylation, couldn't digest the fragment of thepromoters of genes,which was treated by EFD. This result demonstrated that EFD regulatsthe callose deposition in during male gametophyte generation and the exine formation depends on the methyltransferase function.In conclusion, NtWOX9A and EFD were sifted from the Nicotiana tobaccum egg cell-zygote subtractive library, and proved to be participated in the plant sexual reproduction. Utilizing conventional biologic methods as bioinformatics, RT-PCR and expression pattern analysis, we confirmed that NtWOX9A regulated the early pollen development and the functional megaspore selection via effecting tapetum development and callose wall formation respectively. EFD was involved in regulating callose deposition and exine formation, and therefore, controls microsporegenesis and male gametophyte fertility. The above-mentioned results reveal that male and female gametophyte development depends on the proper development of anther and nucellus tissue, which offers a necessary microenvironment. In the micro environment callose directional deposition is a critical factor in the determination of archesporial cell fateand regulation of gametophyte generation, and thus, regulating male and female fertility. |
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