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Molecular characterization of genes essential for early development of germ cells in rice Molecular characterization of genes essential for early development of germ cells in rice 上田, 弥生ウエダ, ヤヨイ UEDA, Yayoi In flowering plants, after the transition from vegetative growth to reproductive development, primordial germ cell initials differentiate from somatic cells in flowers. Several genes that control the early stages of germ-cell development have been identified, however, little is known about the molecular mechanism determining the germ cell fate during sexual reproduction in plants. The MEL1 (MEIOSIS ARRESIED ATLEPTOTENE1) is the only plant ARGONAUTE (AGO) gene specifically expressing at the primordial germ cells. AGO proteins are known to be a key player in gene-silencing pathways guided by small RNAs. PlWl-domain containing subfamily has important roles in maintaining germ stem cells in animal. In plant, however, there is no AGO gene which belonging to PIWI subfamily. Therefore, a specific AGO gene system in plant germ cell development would be suggested. 　In this study, I focused on the characterization gene expressed at early stages of germ cells development specifically in relation to the MEL1 function. MEL1 is well-characterized ARGONAUTE family that has essential functions in germ cell development. To date the rice MEL1 is the only gene available for investigating a genetic system conducting plant germ cell initiation and maintenance. A large portion of the genes identified specific in very early stages in germ cell development had unknown function However, comparison of gene expression profiles between wild-type and mell-1 mutant down- or up-regulated inmell-1 mutant revealed many possible causal genes for mell-1 dysfunction, i.e. most of which are not characterized previously. Of these genes, the OsSPLl4, one of SQUAMOSA(SQUA) promoter-binding-like(SPL) family genes, encoding a putative transcription factor, revealed to up-regulated in the mell-l mutant in both Affymetrix (Fold change=4.37) and Agilent (=3.01) microarray. Sequence and experimental analyses indicated that eleven OsSPL genes including OsSPL14 were putative targets of OsmiR156, suggesting a possibility that the OsSPL14 should be directly targeted by the rice MEL1 AGO with miR156 microRNA as a guide molecule. Moreover, the volume of miR156 RNA detected in the co-IP fraction with the anti-MEL1antibody 3.2-fold more than in that with the pre-immune serum. The detection level of miRl56 depended on that of the MEL1 protein, as the miR156 co-IP with the MEL1 immune complex protein, but few or not with precipitates recovered using mell-1 mutant or preimmune serum. These data strongly suggest that the MEL1forms a complex with the miR156 microRNA in vivo. These results strongly suggested that the miR156microRNA was one of good candidates of guide RNA molecules of the MEL1 AGO. 　Analysis performed in each section and results obtained from the analysis are summarized as follows. 　In the Section 1, the whole transcriptome profiles of plant reproductive process, including early stages that are difficult to be dissected in Arabidopsis, was obtained by using the Affymetrix rice genome array analysis and provided as a dataset of rice reproductive expression atlas. In addition, using the atlas data, the gene expression patterns of several genes that the are highly expressed in early development stage were investigated. A large part of genes that are expressed in early reproductive development remains uncharacterized because most of pre-meiotic stage specific genes were not categorized to any functions by GO analysis. However, the expression patterns of other meiosis-related genes were well corresponded previous reports. The specific genes that were found in this study may have the crucial function at early germ cells development stages. 　In the section 2, to confirm the spatial and temporal expression of the MEL1 gene, mRNA in situ hybridization was performed on the anther sections with the MSP1 gene as cellular marker of young anther tissues. Expression of the MEL1 was earlier than that of the MSP1, which clearly indicates that the MEL1mRNA expression start at the archesporial initials. The MEL1 expression is not required for the germ-cell initiation, but for the maintenance of germ cells. In the Section 2, in situ hybridization of the OsNOZZLE(OsNZZ), a putative rice ortholog of Arabidopsis SPOLOCYTELESS|NOZZLE was also performed. Different from the MEL1 and the, MSP1,the OsNZZ mRNA was expressed both in developing anther wall layers and sporogenous cells similar that of Arabidopsis SPL/NZZ, but it was not detected in archesporial cells. Although their function is not the same as Arabidopsis SPL/NZZ, OsNZZ may have function in early germ cell development. 　In the Section 3, the microarray at 1-cm young panicle, several genes responsive for environmental stresses and/or hormone-responsive genes up-regulated. It suggests that absence of the MEL1 may cause stressful condition, thus many stress-response genes and ethylene signaling-related gene are induced. TheMEL1 may inhibits stress responses in germ cells to accomplish precise germ cell division and meiosis. In addition, some transposable element-like transcripts were also up-regulated in mel-1 mutant. It was demonstrated that Drosophila piwi mutations impact retrotransposon mobility. MEL1 may also suppress the activity of transposable elements during rice germ-cell development. On the other hand, many genes related to cell structure and cycle were down-regulated in the mell-1 mutant. These results may indicated that inmell-1 mutant, the failure of pre-meiotic mitosis of sporogenous cells in the mell-1 mutant anther (Nonomura et al. 2007) was caused by down-regulation of these genes. At pre-meiotic S/G2 stage of young anthers, few genes were affected by the mell-1 mutation. Most of up- and down-regulation of genes in the later stages of pre-meiotic germ-cell development might be secondary effects of mell-1 mutation. 　Finally, the Section 4 suggested a possibility that the rice MEL1 AGO directly regulated the OsSPL14gene with the plant specific microRNA, miR156 as described above. 　Results obtained in this thesis will provide new and useful information to understand the gene functions in initiation of germ cells development and maintenance of germ cell identity in rice. application/pdf 総研大甲第1249号 eng thesis https://ir.soken.ac.jp/records/1442 博士（理学） 2009-03-24 総合研究大学院大学 https://ir.soken.ac.jp/record/1442/files/甲1249_要旨.pdf application/pdf 305.9 kB 2016-02-17 https://ir.soken.ac.jp/record/1442/files/甲1249_本文.pdf application/pdf 13.9 MB 2016-02-17