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DC 欄位 | 值 | 語言 |
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dc.contributor.advisor | 葉開溫 | |
dc.contributor.author | Chin-Hui Shen | en |
dc.contributor.author | 沈金輝 | zh_TW |
dc.date.accessioned | 2021-05-20T19:59:49Z | - |
dc.date.available | 2010-03-10 | |
dc.date.available | 2021-05-20T19:59:49Z | - |
dc.date.copyright | 2010-03-10 | |
dc.date.issued | 2010 | |
dc.date.submitted | 2010-02-22 | |
dc.identifier.citation | Agius, F., Gonzalez-Lamothe, R., Caballero, J.L., Munoz-Blanco, J., Botella,
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Pignocchi, C., Kiddle, G., Hernandez, I., Foster, S.J., Asensi, A., Taybi, T., Barnes, J., and Foyer, C.H. (2006). Ascorbate oxidase-dependent changes in the redox state of the apoplast modulate gene transcript accumulation leading to modified hormone signaling and orchestration of defense processes in tobacco. Plant Physiol. 141: 423-435. Pnueli, L., Liang, H., Rozenberg, M., and Mittler, R. (2003). Growth suppression, altered stomatal responses, and augmented induction of heat shock proteins in cytosolic ascorbate peroxidase (Apx1)-deficient Arabidopsis plants. Plant J. 34: 187-203. Ramírez, I., Dorta, F., Espinoza, V., Jiménez, E., Mercado, A., and Peña-Cortés, H. (2006). Effects of foliar and root applications of methanol on the growth of Arabidopsis, tobacco, and tomato plants. J. Plant Growth Regul. 25: 30-44. Ridley, B.L., O'Neill, M.A., and Mohnen, D. (2001). Pectins: structure, biosynthesis, and oligogalacturonide-related signaling. Phytochemistry 57: 929-967. 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Changes of carbohydrate and free amino acid pools in current pseudobulb of Oncidium Gower ramsey during inflorescence development. J. Agri. Assoc. China 4: 476-488. Wheeler, G.L., Jones, M.A., and Smirnoff, N. (1998). The biosynthetic pathway of vitamin C in higher plants. Nature 393: 365-369. Wolucka, B.A., and Van Montagu, M. (2003). GDP-mannose 3',5'-epimerase forms GDP-L-gulose, a putative intermediate for the de novo biosynthesis of vitamin C in plants. J. Biol. Chem. 278: 47483-47490. Wolucka, B.A., Goossens, A., and Inze, D. (2005). Methyl jasmonate stimulates the de novo biosynthesis of vitamin C in plant cell suspensions. J. Exp. Bot. 56: 2527-2538. Yamamoto, A., Bhuiyan, M.N., Waditee, R., Tanaka, Y., Esaka, M., Oba, K., Jagendorf, A.T., and Takabe, T. (2005). Suppressed expression of the apoplastic ascorbate oxidase gene increases salt tolerance in tobacco and Arabidopsis plants. J. Exp. Bot. 56: 1785-1796. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/8693 | - |
dc.description.abstract | 維他命C是植物對抗環境逆境時所需的抗氧化劑,亦是多種植物荷爾蒙生合成酵素的輔助因子(cofactor),近年來的研究發現植物內生性維他命C的含量多寡具有顯著調控植物花期的功能。文心蘭在營養生長階段,假球莖會累積大量的維他命C,但在進入生殖生長階段時,其含量會明顯地下降。利用0.1M維他命C每日施加於文心蘭假球莖基部的花莖芽,結果發現可有效抑制開花相關基因LEAFY的表現量,並明顯延緩花莖生長,因此可知維他命C在文心蘭開花機制中扮演著抑制者的角色。維他命C含量生合成路徑如Smirnoff-Wheeler路徑與半乳醣醛酸路徑(galacturonate pathway)的基因在營養生長階段都有明顯的表現量,然而當進入生殖生長階段時,僅半乳醣醛酸路徑上的基因表現量有明顯的下降,分析其酵素活性亦呈現相同結果。再進一步轉殖半乳醣醛酸路徑中的果膠甲酯酶(pectin methylesterase)於阿拉伯芥中,發現轉殖植物亦會累積維他命C並且延遲開花。由此可知半乳糖醛酸路徑同時調控植物維他命C含量與開花生理。甲醇是半乳糖醛酸路徑的副產物,處理50mM甲醇於文心蘭擬原球體 protocorm like body,PLB )中發現可提高維他命C 相關基因的表現與維他命C含量。利用羥胺(hydroxylamine)與DPI分別抑制代謝甲醇產生過氧化氫的乙醇氧化酶(alcohol oxidase)與NADPH氧化酶,發現原本受甲醇誘導的維他命C相關基因表現量也會受到抑制,因此認為過氧化氫為植物誘導維他命C相關基因表現的重要次級傳遞訊
息者 (secondary messenger)。綜合這些結果可知文心蘭營養生長階段因具有較高活性的果膠甲酯酶可產生甲醇並進一步產生過氧化氫而誘導維他命C相關基因表現,此訊息傳遞之生理意義在於緩和該階段因為生長而累積的氧化逆境。而當植物細胞壁的果膠含量因為細胞延長而逐漸減少時,果膠甲酯酶活性降低,進而降低維他命C含量而促進開花基因的表現量,而使植物進入生殖生長階段。本論文主要在解析維他命C在文心蘭開花生理中扮演負調控的角色,而果膠甲酯酶則 為調控文心蘭不同生長階段維他命C含量重要的基因 | zh_TW |
dc.description.abstract | We investigated the alteration in L-ascorbate (AsA, reduced form) content and the expression pattern of its related genes during the phase transition in Oncidium orchid. During the vegetative growth, a high hydrogen peroxide (H2O2) level was associated with a high content of the reduced form of AsA. At the bolting period, the AsA content and H2O2 level were greatly reduced in parallel with increased expression of OgLEAFY, the gene encoding a key transcription factor integrating different flowering-inducing pathways. This observation suggests that the reduced AsA content, resulted from the consumption of H2O2, is a prerequisite for mediating the phase transition in Oncidium. A survey of the AsA biosynthetic pathway revealed that the gene expressions and enzymatic activities of the relevant genes in the galacturonate (GalUA) pathway were markedly decreased at the bolting period, as compared with at the vegetative stage. However, the genes involved in the Smirnoff-Wheeler pathway retained a similar expression level in the two growth stages. Moreover, MeOH produced form the demethylation of pectin by pectin methylesterase (OgPME) also could trigger the synthesis of H2O2 and was effective in enhancing the expression of AsA-biosynthetic genes. It suggested that OgPME of the GalUA pathway was the pivotal gene in regulating AsA biosynthesis during the
bolting period. Further elucidation by overexpressing OgPME in Arabidopsis demonstrated a considerable increase in AsA content, as well as a resulting delayed-flowering phenotype. We proposed a model in controlling the phase transition by ascorbate homeostasis which is associated with the functional role of PME in Oncidium. | en |
dc.description.provenance | Made available in DSpace on 2021-05-20T19:59:49Z (GMT). No. of bitstreams: 1 ntu-99-F92b42021-1.pdf: 4948954 bytes, checksum: f09118ce3e6baf20edbf2a8156e04042 (MD5) Previous issue date: 2010 | en |
dc.description.tableofcontents | Table of Contents
Chinese Abstract………………………………………………………..07 English Abstract………………………………………………………..08 1. Chapter 1 Ascorbate Biosynthesis and Phase Transition in Oncidium Decreased L-ascorbate content mediating bolting is mainly regulated by the galacturonate pathway in Oncidium ~ Plant Cell & Physiology, 2009, 50(5): 935-946 1.1 Introduction 1.1.1 Ascorbate level influences the flowering time………………………... 10 1.1.2 The biosynthesis of ascorbate in planta………………………............. 11 1.1.3 The current work on the regulatory mechanism of flowering time in Oncidium…………………................................... 12 1.1.4 The delineation of the development stages of Oncidium……………... 13 1.2 Results 1.2.1 The marked decrease in AsA content and H2O2 level in the pseudobulb during the bolting period……………………..14 1.2.2 Reduction of AsA content during the bolting period is due to the down- regulation of the genes of the GalUA pathway but not the Smirnoff-Wheeler pathway…………….. 15 1.2.3 Pectin accumulation and high PME activity show the availability of the GalUA pathway to carry out AsA biosynthesis at the vegetative stage in Oncidium……………….. 17 1.2.4 Overexpressing OgPME in Arabidopsis demonstrates the elevated AsA level delaying flowering time and a significant effect of the GalUA pathway on flowering………………17 1.3 Discussion……………….......................................................................18 2. Chapter II The signal network of ascorbate homeostasis associated with the growth and development in Oncidium. ~ Plant Signal & Behavior, 2010 5: 1-3 2.1 Introduction 2.1.1 The role of reactive oxygen species in plants………………………… 24 2.1.2 The proposed ascorbate homeostasis in pseudobulb cell of Oncidium orchid in three developmental stages…………………… 26 2.2 Discussion………………...................................................................... 28 2 3. Chapter IIi Ascorbate Homeostasis Mediated by Hydrogen Peroxide Secondary Messenger, but Closely Associated with Pectin Metabolism. Hydrogen peroxide mediates the expression of ascorbate-related genes in response to methanol stimulation in Oncidium ~ Journal of Plant Physiology (in press) 3.1 Introduction 3.1.1 The potential influence of methanol on the growth of Oncidium…………………………………………….31 3.1.2 The extended function of methanol in plant………………………….. 31 3.2 Results 3.2.1 Exogenous application of methanol stimulates AsA biosynthesis in Oncidium PLB cultures…………………………. 33 3.2.2 Characterization of AsA induction by mehtnaol stimulation………….33 3.2.3 Methanol enhances AsA levels by up-regulating AsA-biosynthesis and defense genes………………………………….34 3.2.4 Hydrogen peroxide production in Oncidium PLB cultures through the activation of alcohol oxidase and NADPH oxidase under MeOH stimulation………………36 3.2.5 The up-regulation of AsA-related genes stimulated by MeOH is through H2O2 signal transduction…………… 37 3.3 Discussion………………...................................................................... 37 4. CONCLUSION………………..................................................................... 40 5. Future prospect………………....................................................... 41 6 Materials and Methods 6.1 Plant materials………………................................................................ 42 6.2 Treatment of Oncidium orchids with AsA and H2O2.............................42 6.3 Ascorbate measurement………………................................................. 43 6.4 Ascorbate staining……………….......................................................... 44 6.5 Hydrogen peroxide measurement……………….................................. 45 6.6 Hydrogen peroxide staining………………........................................... 46 6.7 Pectin measurement………………....................................................... 47 6.8 Pectin staining………………................................................................ 48 6.9 Total protein extraction……………….................................................. 49 6.10 Quantitative analysis of protein concentration 6.10.1 Bio-Rad Protein Assay Kit………………............................................. 50 6.10.2 Lowry Assay……………….................................................................. 50 3 6.11 Pectin methylesterase activity measurement......................................... 51 6.12 Polygalacturonase activity measurement.............................................. 52 6.13 Galacturonate reductase activity measurement………………………. 53 6.14 Galacturonate oxidase activity measurement………………………… 54 6.15 GDP-mannose pyrophosphorylase activity measurement……………. 55 6.16 L-Galactono-1,4-lactone dehydrogenase activity measurement……… 56 6.17 Galactose dehydrogenase activity measurement................................... 57 6.18 Ascorbate oxidase activity analysis....................................................... 58 6.19 Ascorbate peroxidase activity analysis.................................................. 60 6.20 Monodehydroascorbate reductase activity assay…………………….. 60 6.21 Superoxidase dismutase (SOD) activity measurement………………. 61 6.22 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) ......................................................................................... 62 6.23 PME activity in native acidic gel electrophoresis…………………….. 64 6.24 RNA extraction...................................................................................... 65 6.25 Rapid amplification of cDNA end (RACE)…………………………... 66 6.26 RT PCR for gene expression.................................................................. 70 6.27 Construction of functional plasmid for overexpressing the interested genes in Arabidopsis………………….. 71 6.28 Preparation of E.coli (DH5α) competence cell……………………….. 73 6.29 Preparation of A. tumefaciens (GV3101) competence cell…………… 74 6.30 Agrobacterium infiltration..................................................................... 75 6.31 Selection of the transgenic Arabidopsis overexpressing interested gene……………………………………….. 76 7. References........................................................................................... 77 4 LIST OF TABLES Table 1 H2O2 amount and pectin concentration of PLBs incubated with various treatments……………………………..……... 82 Table 2 The sequence of primers for RT-PCR.................................................... 83 5 List of Figures Fig. 1 Ascorbic acid content and H2O2 level in different developmental stages of Oncidium pseudobulb..................................... 84 Fig. 2 The expression pattern of OgLEAFY at different developmental stages and under artificial treatments of chemicals........................................................................................... 85 Fig. 3 Expression pattern of AsA biosynthetic genes during vegetative stage and bolting period in Oncidium………………86 Fig. 4 Enzymatic activities of AsA biosynthetic genes during vegetative stage and bolting period in Oncidium………………87 Fig. 5 Pectin content and composition in pseudobulb varied with pectin methylesterase activity…………………………….88 Fig. 6 Overexpression of pectin methylesterase in Arabidopsis……………... 89 Fig. 7 AsA content and AsA redox state in transgenic Arabidopsis overexpressing OgPME…………………………………. 90 Fig. 8 Delayed flowering phenotype of transgenic Arabidopsis overexpressing OgPME……………………………………………….. 91 Fig. 9 Schematic representation of ascorbate homeostasis in pseudobulb cell of Oncidium orchid in three developmental stages…………………………………………………. 92 Fig. 10 An Oncidium cultivar with long vegetative phase (LV) is caused by the higher endogenous ascorbate concentration……………………………………………….. 93 Fig. 11 Effect of methanol (MeOH) doses on ascorbate (AsA) level in Oncidium protocorm-like body (PLB) cultures……………… 94 Fig. 12 The PLB culture markedly varied with MeOH dosage………………. 95 Fig. 13 The AsA level in Oncidium PLB cultures incubated with various compounds for 30 h……………………………………... 96 Fig. 14 The redox state in Oncidium PLB cultures incubated with various compounds for 30 h……………………………………... 97 Fig. 15 Expression of AsA-related genes on treatment of Oncidium PLB cultures with various compounds……………………..98 Fig. 16 Activity assays of AsA-related enzymes under 50 mM MeOH treatments……………………………………………...99 Fig. 17 The effects of hydroxylamine (inhibitor of alcohol oxidase) and diphenyleneiodonium chloride (DPI; inhibitor of NADPH oxidase) on H2O2 production in Oncidium PLB cultures…………….. 100 Fig. 18 Expression of AsA-related genes on treatment with 6 H2O2-producing inhibitors. ................................................................... 101 Fig. 19 The proposed model of the H2O2-signaling network under MeOH stimulation in Oncidium PLB cultures………………… 102 LIST OF Appendixes Appendix 1 The hypothetical effects of low levels of ascorbic acid in regulating flowering time. ................................................. 103 Appendix 2 Four potential branch pathways operating in plants…………….. 104 Appendix 3 Carbohydrate metabolic pathway in the pseudobulb of Oncidium during the flowering process…………. 105 Appendix 4 The production and functional roles of methanol in plant cell….. 106 Appendix 5 Abbreviation.................................................................................. 107 | |
dc.language.iso | zh-TW | |
dc.title | 維他命C調控文心蘭南茜品系開花誘導之研究 | zh_TW |
dc.title | The ascorbate level mediates the
floral initiation of Oncidium Gower ramsey | en |
dc.type | Thesis | |
dc.date.schoolyear | 98-1 | |
dc.description.degree | 博士 | |
dc.contributor.oralexamcommittee | 陳文輝,楊長賢,高景輝,方煒,張耀乾,林讚標 | |
dc.subject.keyword | 文心蘭,開花調控,維他命C, | zh_TW |
dc.subject.keyword | Oncidium,Regulation of flowering time,Ascorbate, | en |
dc.relation.page | 108 | |
dc.rights.note | 同意授權(全球公開) | |
dc.date.accepted | 2010-02-22 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 植物科學研究所 | zh_TW |
顯示於系所單位: | 植物科學研究所 |
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