Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/90092
標題: Characterization and Functional Analysis of a RING-Type E3 Protein Ligase in Regulating Ovule Development in Arabidopsis thaliana
阿拉伯芥中參與胚珠發育之一個RING-type E3蛋白質接合酶之功能性分析
作者: 林哲逸
Jhe-Yi Lin
關鍵字: 泛素
接合酶
胚珠發育
ubiquitin
E3
RING finger
ovule development
引用: 劉祥欽(2003). Molecular cloning and functional analysis of E3 RING finger genes from Arabidopsis thaliana. 國立中興大學生物科技研究所碩士論文。 廖珮君(2004). Characterization and functional analysis of genes regulating cell division and differentiation in Arabidopsis.國立中興大學生物科技研究所碩士論文。 彭妍筑(2009). Characterization and functional analysis of AtRING3 and AtRING4 genes in regulating stamen development in Arabidopsis thaliana. 國立中興大學生物科技研究所碩士論文。 許巍瀚(2012). Functional analysis of genes regulating cell division and gametophyte development in Arabidopsis. 國立中興大學生物科技研究所博士論文。 Alexander M.P. (1969). Differential staining of aborted and nonaborted pollen. Stain Technology 44, 117-122. Azevedo C., Santos-Rosa M.J. and Shirasu K. (2001). The U-box protein family in plants. Trends Plant Sci. 6, 354-358. Bates P.W. and Vierstra, R.D. (1999). UPL1 and 2, two 405 kDa ubiquitin-protein ligases from Arabidopsis thaliana related to the HECT-domain protein family. Plant J. 20, 183-195. Callis J., Carpenter T., Sun C.W., Vierstrat R.D. (1995). Structure and evolution of genes encoding polyubiquitin and ubiquitin-like proteins in Arabidqpsis thaliana ecotype Columbia. Genetics 139, 921-39. Chattopadhyay S., Ang L.H., Puente P., Deng X.W. and Wei, N. (1998). Arabidopsis bZIP protein HY5 directly interacts with light-responsive promoters in mediating light control of gene expression. Plant Cell 10, 673-683. Deshaies R.J. (1999). SCF and cullin/RING H2-based ubiquitin ligases. Annu. Rev. Cell Dev. Biol. 15, 435-467 Disch S., Anastasiou E., Sharma V.K., Laux T., Fletcher J.C. and Lenhard M. (2006). The E3 ubiquitin ligase BIG BROTHER controls Arabidopsis organ size in a dosage-dependent manner. Curr. Biol. 16, 272–279. Downes B.P., Stupar R.M., Gingerich D.J. and Vierstra R.D. (2003). The HECT ubiquitin-protein ligase (UPL) family in Arabidopsis: UPL3 has a specific role in trichome development. Plant J. 35, 729-742. Eudes A., Pollet B., Sibout R., Do C.T., Seguin A., Lapierre C., Jouanin L. (2006). Evidence for a role of AtCAD 1 in ligniWcation of elongating stems of Arabidopsis thaliana. Planta 225, 23–39. Fu H., Reis N., Lee Y., Glickman M.H. and Vierstra R.D. (2001). Subunit interaction maps for the regulatory particle of the 26S proteasome and the COP9 signalosome. EMBO J. 20, 7096-7107. Gagne J.M., Downes B.P., Shiu S.H., Durski A.M. and Vierstra R.D. (2002). The F-box subunit of the SCF E3 complex is encoded by a diverse superfamily of genes in Arabidopsis. Proc. Natl. Acad. Sci. U.S.A. 99, 11519-11524. Gingerich D.J., Gagne J.M., Salter D.W., Hellmann H., Estelle M. and Vierstra R.D. (2005). Cullin 3A and B assemble with members of the Broad complex/Tramtrack/Bric-a-brac (BTB) protein family to form essential uqiquitin-protein ligases (E3s) in Arabidopsis. J. Biol. Chem. 280, 18810-18820. Glickman M.H. (2000). Getting in and out of the proteasome. Semin. Cell Dev. Biol. 11, 149-158. Greve K., Cour T.L., Jensen M.K., Poulsen F.M. and Skriver K. (2003). Interactions between plant RING-H2 and plant-specific NAC (NAM/ATAF1/2/CUC2) proteins : RING-H2 molecular specificity and cellular localization. Biochem. J. 371, 97-108. Hardtke C.S., Okamoto H., Stoop-Myer C. and Deng X.W. (2002). Biochemical evidence for ubiquitin ligase activity of the Arabidopsis COP1 interacting protein 8 (CIP8). Plant J. 30, 385-394. Hatfield P.M., Gosink M.M., Carpenter T.B., Vierstra R.D. (1997). The ubiquitin-activating enzyme (E1) gene family in Arabidopsis thaliana. Plant J. 11, 213-26. Hellmann H., Hobbie L., Chapman A., Dharmasiri S., Dharmasiri N., del Pozo C., Reinhardt D. and Estelle M. (2003). Arabidopsis AXR6 encodes CUL1 implicating SCF E3 ligases in auxin regulation of embryogenesis. EMBO J. 22, 3314–3325. Hershko A. and Ciechanover A. (1998). The ubiquitin system. Annu. Rev. Biochem. 67, 425-479 Höfgen R. and Willmitzer, L. (1988). Storage of competent cells for Agroacterium transformation. Nucl. Acids Res. 16, 9877. Ishiguro S., Kawai-Oda A., Ueda J., Nishida I. and Okada K. (2001). The DEFECTIVE IN ANTHER DEHISCENCE1 gene encodes a novel phospholipase a1 catalyzing the initial step of jasmonic acid biosynthesis, which synchronizes pollen maturation, anther dehiscence, and flower opening in Arabidopsis .Plant Cell. 13, 2191–2209. Jackson P.K., Eldridge A.G., Freed E., Furstenthal L., Hsu J.Y., Kaiser B.K. and Reimann J. D. R. (2000). The lore of the RINGs : substrate recognition and catalysis by ubiquitin ligases. Trends Cell Biol. 10, 429-439. Jefferson R.A., Kavanagh T.A. and Bevan M.W. (1987). GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plant. EMBO J. 6, 3901-3907. Kraft E., Stone S.L., Ma L., Su N., Gao Y., Lau O.S., Deng X.W. and Callis J. (2005). Genome analysis and functional characterization of the E2 and RING-Type E3 ligase ubiquitination enzymes of Arabidopsis. Plant Physiol. 139, 1597-1611. Lam Y.A., Lawson T.G., Velayutham M., Zweier J.L. and Pickart C.M. (2002). A proteasomal ATPase subunit recognizes the polyubiquitin degradation signal. Nature 416, 763-767. Lee J.H., Terzaghi W., Gusmaroli G., Charron J.B., Yoon H.J., Chen H., He Y.J., Xiong Y. and Deng X.W. (2008). Characterization of Arabidopsis and rice DWD proteins and their roles as substrate receptors for CUL4-RING E3 ubiquitin ligases. Plant Cell 20, 152-167. Lovering R., Hanson I.M., Borden K.L., Martin S., O'Reilly N.J., Evan G.I., Rahman D., Pappin D.J., Trowsdale J. and Freemont P.S. (1993). Identification and preliminary characterization of a protein motif related to the zinc finger. Proc. Natl. Acad. Sci. U.S.A. 90, 2112-2116. Moon J., Parry G. and Estelle M. (2004). The ubiquitin-proteasome pathway and plant development. Plant Cell 16, 3181-95. Oyama T., Shimura Y. and Okada K. (1997). The Arabidopsis HY5 gene encodes a bZIP protein that regulates stimulus-induced development of root and hypocotyl. Genes Dev. 11, 2983-2995. Peters J.M. (2002). The anaphase-promoting complex: proteolysis in mitosis and beyond. Mol. cell 9, 931-943. Peng Y.J., Shih C.F., Yang J.Y., Tan. C.M., Hsu W.H., Huang Y.P., Liao P.C., Yang C.H. (2013) A RING-type E3 ligase controls anther dehiscence by activating the jasmonate biosynthetic pathway gene DEFECTIVE IN ANTHER DEHISCENCE1 in Arabidopsis. Plant J. 74, 310-327. Pickart C.M. (2001). Mechanisms underlying ubiquitination. Annu. Rev. Biochem.70, 503-33. Salinas-Mondragon R.E., Garcidueñas-Piña C. and Guzm´an P. (1999). Early elicitor induction in members of a novel multigene family coding fornhighly related RING-H2 proteins in Arabidopsis thaliana. Plant Mol. Biol. 40, 579–590. Serrano M. and Guzmán P. (2004). Isolation and gene expression analysis of Arabidopsis thaliana mutants with constitutive expression of ATL2, an early elicitor-response RING-H2 zinc-finger gene. Genetics 167, 919-929. Stone S.L., Hauksdóttir H., Troy A., Herschleb J., Kraft E. and Callis J. (2005). Functional analysis of the RING-type ubiquitin ligase family of Arabidopsis. Plant Physiol. 137, 13-30. Stone S.L., Williams L.A., Farmer L.M., Vierstra R.D. and Callisa J. (2006). KEEP ON GOING, a RING E3 ligase essential for Arabidopsis growth and development, is involved in abscisic acid signaling. Plant Cell 18, 3415–3428. Vierstra R.D. (1996). Proteolysis in plants: mechanisms and functions. Plant Mol. Biol. 32, 275-302. Vierstra R.D. (2003). The ubiquitin/26S proteasome pathway, the complex last chapter in the life of many plant proteins. Trends Plant Sci. 8, 135-42. Vierstra R.D. (2009). The ubiquitin–26s proteasome system at the nexus of plant biology. Nature Rev. Mol. Cell Biol. 10, 385-397. Wing S.S. (2003). Deubiquitinating enzymes-the importance of driving in reverse along the ubiquitin–proteasome pathway. Int. J. Biochem. Cell Riol. 35, 590-605. Xie Q., Guo H.S., Dallman G., Fang S., Weissman A.M., Chua N.H. (2002). SINAT5 promotes ubiquitin-related degradation of NAC1 to attenuate auxin signals. Nature 419, 167-70. Xu R. and Li Q.Q. (2003). A RING-H2 zinc-finger protein gene RIE1 is essential for seed development in Arabidopsis. Plant Mol. Biol. 53, 37-50. Zheng N. Schulman B.A., Song L., Miller J.J., Jeffrey P.D., Wang P., Chu C., Koepp D.M., Elledge S.J., Pagano M., Conaway R.C., Conaway J.W., Harper J.W. and Pavletich N.P. (2002). Structure of the Cul1-Rbx1-Skp1-F box Skp2 SCF ubiquitin ligase complex. Nature 416, 703-709.
摘要: 真核生物中其中一種主要的分解蛋白質途徑就是泛素/26S 蛋白酶系統,是一種藉由連接泛素當作標記,形成一種蛋白質降解訊號,在細胞內部此標記的蛋白質得以被辨識,因而控制細胞內蛋白質的生命週期。阿拉伯芥RING finger gene DAF-Like gene 1 (DAFL1) 預測是一個E3 protein ligase,在26S proteosome pathway系統中扮演了重要的角色。在演化樹分析中DAFL1與實驗室前人研究的DAF (DAD1-Activating Factor)基因親緣關係非常接近,但這兩個基因表現的位置卻有明顯的差異。在阿拉伯芥發育早期的花器裡,DAF在雄蕊中表現,而DAFL1專一地表現在雌蕊與胚珠中,推測DAFL1可能與DAF的功能有所不同。異位表現DAFL1 RNAi與antisense的基因轉殖植株可觀察到不孕的性狀,是由於花藥不開裂造成花粉無法正常授粉,異位表現DAFL1-H135A點突變的基因轉殖植株同樣也有此性狀。將野生型阿拉伯芥花粉與不孕植株的雌蕊雜交,能得到正常授粉而延長的果莢,不過果莢中的種子只有野生型的一半左右。另外構築可以專一表現在雌蕊與胚珠的載體,使得基因轉殖植株可以專一地表現DAFL1-H135A點突變蛋白質,其花藥可以正常開裂,果莢中的種子數量同樣只有大約野生型的一半左右。利用酵母菌雙雜交系統,藉由營養篩選方法從阿拉伯芥的花苞cDNA library中去找出能與DAFL1有交互作用的蛋白質,經過高嚴謹度篩選後得到了數個可能是DAFL1的候選受質。其中CAD1經由啟動子活性分析實驗得知其表現位置在胚珠,與DAFL1啟動子表現的部位有重疊,異位表現CAD1的轉基因植株也觀察到種子數目減半的性狀。CAD1已被證實為合成木質素重要成分的關鍵酵素,並參與果莢木質化的過程,與DAFL1同樣表現在雌蕊非木質化的部位。因此推測在雌蕊發育早期的階段,胚珠藉由DAFL1降解CAD1避免木質素過早合成。
In eukaryotes, the ubiquitin/26S proteasome system is one of the major mechanisms that act on the protein degradation. Proteins are tagged with ubiquitin as the signals to be degraded. In the cells, the ubiquitin-tagged proteins can be recognized and to regulate the life cycle of proteins. It was proposed that RING finger gene DAF-Like gene 1 (DAFL1) encodes an E3 protein ligase that plays a key role in 26S proteasome pathway in Arabidopsis thaliana. Based on phylogenetic analysis, DAFL1was closely related to DAF (DAD1-Activating Factor); however, the spatial specificities of DAFL1 and DAF gene expression patterns in Arabidopsis thaliana were different. In the early age of floral organs of Arabidopsis thaliana, DAFL1 specifically accumulated in the pistil and the ovule whereas DAF was expressed in stamen. It was suggested that DAFL1 and DAF might behave divergently in regulating plant development. Transgenic Arabidopsis plants that ectopically expressing DAFL1 RNAi and antisense caused indehiscent anthers and resulted in the unreleased pollen from the anthers. Similarly, ectopic expression of DAFL1-H135A caused indehiscent anthers in transgenic Arabidopsis plants. Pollens of wild-type Arabidopsis were crossed to the stigmas of sterile transgenic plants. At 7 days after pollination, the siliques of sterile transgenic plants developed and elongated. However, the amount of seeds of the transgenic plants is about a half of that of the wild-type Arabidopsis. Furthermore, transgenic plants that specifically expression of DAFL1-H135A in the pistil and the ovule produced dehiscent anthers, but the amount of seeds of the transgenic plants is about a half of that of the wild-type Arabidopsis. To discover the proteins that might interact with DAFL1, yeast-two hybrid system was performed to screen putative ones from flower buds cDNA library of Arabidopsis. One of the candidate genes, CAD1, was applied in the promoter assay. The result indicated that the gene expression patterns of CAD1 and DAFL1 was the same in ovule. In addition, transgenic plants that ectopic expressing CAD1 produced the amount of seeds is about a half of that of the wild-type Arabidopsis. CAD1 is a key enzyme in the process of lignin synthesis and has been thought to be involved in the lignification of siliques. Interestingly, DAFL1 and CAD1 also highly accumulated in the non-lignification site such as in the carpel. Our results revealed that DAFL1 degrades CAD1 and inhibits lignin biosynthesis at the early stage of pistil development.
URI: http://hdl.handle.net/11455/90092
文章公開時間: 2018-02-04
Appears in Collections:生物科技學研究所

文件中的檔案:

取得全文請前往華藝線上圖書館



Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.