H₂O₂作为信号分子参与基因表达调控和植物生理与发育过程。在本论文中，我在拟南芥中发现并详细研究了H₂O₂的一个新功能--诱导黑暗中的形态建成。光呼吸是植物内源H₂O₂的主要来源，因此我构建了GOX基因过量表达转基因植株，并在黑暗中模拟了光呼吸H₂O₂产生机制，显著诱导了幼苗的黑暗形态建成。细胞周期报告株系的研究表明，在黑暗中细胞周期停滞在G2/M过渡点，而H₂O₂的处理能帮助细胞通过G2/M时期，从而在黑暗中生长出顶端分生组织。这些结果提示H₂O₂可能参与细胞周期调控。 / 然后，我研究了拟南芥中H₂O₂的产生和分布模式。H₂O₂主要集中分布于维管组织、根冠、叶毛和子叶末端。GOX1和GOX2基因恰恰在H₂O₂分布区域较高表达。在gox1-4和gox2-1突变体中，H₂O₂水平显著下降，表明GOX1和GOX2是拟南芥中主要的H₂O₂产生基因。GOX1和GOX2在发芽早期就较高表达，并伴随有H₂O₂的积累。时序基因表达数据表明在发芽过程中GOX酶的底物并非产生于光呼吸，而是脂肪代谢。因此，我提出了两阶段H₂O₂产生机制模型，即：发芽过程中脂肪代谢提供底物，而生长过程中光呼吸提供底物。 / 最后，我探索了H₂O₂诱导黑暗形态建成过程中的信号通路。利用基因芯片分析和生物信息学方法从H₂O₂和光诱导基因的启动子序列中预测了GGGCC（R-box）调控元件。该元件集中分布于-50到-250bp区域，并且在小鼠和人的基因组的相同区域中也大量分布。EMSA实验验证了该调控元件是H₂O₂可诱导的。随后从其结合的核因子中分离鉴定了2个snoRNP蛋白：fibrillarin 2和NOP56，同时还克隆了数个C/D家族snoRNA序列。这些结果表明，在拟南芥中存在着一个参与H₂O₂信号通路的snoRNP/ R-box调控机制。 / For years H₂O₂ has been known as signal molecule involved in gene expression regulation. which affect particular physiological and developmental processes. In this study, I identified and characterized a new function of H₂O₂ in Arabidopsis development. Specifically, I found that H₂O₂ can induce seedling establishment in the dark. Nitrogen and sucrose have substantial influence on the effect of H₂O₂ induced seedling establishment. Because photorespiration is a major source of H₂O₂ production, when photorespiratory H₂O₂ producing pathway was mimicked in glycolate oxidase (GOX) over-expression lines, and significantly increased seedling establishment in the dark was observed. Using reporter lines, I found that the cell cycle is largely blocked at G2/M transition checkpoint in the dark, and H₂O₂ treatment helps the cells pass through the G2/M transition and finally establish shoot apical meristem. These results provide clues on the role of H₂O₂ roles in cell cycle regulation. / Then I characterized the H₂O₂ production and distribution pattern in Arabidopsis. H₂O₂ is accumulated at high level in root vasculature, root cap, veins and distal end in cotyledons, as well as leaf trichomes. The expression sites of GOX1 and GOX2 are in accordance with the places where H₂O₂ is distributed. Both peroxisomal and cytosol H₂O₂ share the same distribution pattern, indicating that the peroxisomal H₂O₂ might be the major source of H₂O₂ in Arabidopsis. In Arabidopsis gox1-4 and gox2-1 mutants, the H₂O₂ levels are both significantly reduced in roots and shoots, the sites where GOX1 and GOX2 are highly expressed, suggesting that GOX1 and GOX2 are likely the major H₂O₂ producing enzymes in Arabidopsis plants. Both GOX1 and GOX2 genes show early expression during germination, along with the H₂O₂ accumulation. Temporal expression data suggest the GOX substrates are not derived from photorespiration, rather from glyoxylate cycle that consumes lipid metabolites to provide carbon sources during germination. A working model is proposed to explain the possible H₂O₂ production mechanism. / Finally, I explored the signaling pathway involving H₂O₂ induced seedling establishment. A GGGCC motif (R-box) was identified from the upstream sequences of H₂O₂ and light induced genes in Arabidopsis. This motif is highly enriched within the -50 to -250 bp region of the induced genes, and it is also specifically distributed in the same region in mouse and human genome. EMSA experiments revealed that several nuclear factors (NF) are bound to this motif, and the binding activities alter under the H₂O₂ treatment. Two C/D family snoRNP proteins, fibrillarin 2 and NOP56 were identified from the R-box binding NFs. Several C/D family snoRNA, including R63, U24a and Z15, were also cloned from R-box binding NFs. These data suggest a snoRNP/ R-box regulation pathway may associate with H₂O₂ signaling in Arabidopsis. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Cheng, Han. / "December 2012." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 101-114). / Abstract also in Chinese. / Thesis/Assessment Committee --- p.ii / Statement: --- p.iii / Abstract --- p.iv / 摘要 --- p.vi / Acknowledgements --- p.vii / Table of Contents --- p.viii / List of Tables --- p.x / Lists of Figures --- p.xi / List of Abbreviations --- p.xii / Chapter 1 --- p.1 / General Introduction --- p.1 / Chapter 1.1 --- H₂O₂ metabolism and homeostasis in plants --- p.2 / Chapter 1.2 --- H₂O₂ Signaling pathway --- p.4 / Chapter 1.3 --- H₂O₂ roles on Plant Growth and Development --- p.6 / Chapter 1.4 --- Project Objectives --- p.7 / Chapter 2 --- p.9 / H₂O₂ Promote Post-germinative Establishment by Accelerating G2/M Transition --- p.9 / Chapter 2.1 --- Introduction --- p.10 / Chapter 2.2 --- Materials and Methods --- p.10 / Chapter 2.2.1 --- Establishment experiment in dark conditions --- p.10 / Chapter 2.2.2 --- GUS staining --- p.10 / Chapter 3.2.4 --- Constructs and generation of transgenic plants --- p.11 / Chapter 2.2.5 --- Ammonia content measurement --- p.12 / Chapter 2.2.6 --- Glycolate oxidase activity measurement --- p.12 / Chapter 2.3 --- Results --- p.12 / Chapter 2.3.1 --- H₂O₂ facilitated Post-germinative establishment in Arabidopsis --- p.12 / Chapter 2.2.2 --- Sucrose and nitrogen source are involved in the establishment --- p.13 / Chapter 2.3.3 --- Overexpression of glycolate oxidase in Arabidopsis --- p.14 / Chapter 2.3.4 --- The H₂O₂ facilitate the establishment by promoting the G2/M transition in the shoot meristem --- p.15 / Chapter 2.4 --- Discussion --- p.16 / Chapter 3 --- p.24 / Glycolate Oxidases mediate H₂O₂ distribution in Arabidopsis --- p.24 / Chapter 3.1 --- Introduction --- p.25 / Chapter 3.2 --- Materials and Methods --- p.26 / Chapter 3.2.1 --- Plant Material and growth conditions --- p.26 / Chapter 3.2.2 --- Phenotype analyses --- p.27 / Chapter 3.2.3 --- Constructs and generation of transgenic plants --- p.27 / Chapter 3.2.4 --- Transient expression --- p.29 / Chapter 3.2.5 --- Confocal microscopy, GUS staining, and stereomicroscopy --- p.29 / Chapter 3.2.6 --- PI, HPF and H2DCFDA staining --- p.30 / Chapter 3.2.7 --- RNA extraction, RT-PCR and quantitative RT-PCR --- p.30 / Chapter 3.2.8 --- Mutants Genotyping --- p.31 / Chapter 3.2.9 --- Expression Data Analysis --- p.32 / Chapter 3.3 --- Results --- p.32 / Chapter 3.3.1 --- Characterization of glycolate oxidase family genes --- p.32 / Chapter 3.3.2 --- Tissue-specific expression of GOX family genes --- p.34 / Chapter 3.3.3 --- H₂O₂ distribution pattern is in accord with GOX1 and GOX2 expression in root and leaf --- p.36 / Chapter 3.3.4 --- The H₂O₂ distribution pattern in the cytosol is similar to that in the peroxisome --- p.37 / Chapter 3.3.5 --- Gox1 and gox2 mutants show decreased H₂O₂ distribution in root and leaf --- p.38 / Chapter 3.3.7 --- A working model for H₂O₂ patterning in Arabidopsis --- p.39 / Chapter 3.3.6 --- Gox1 and gox2 mutants show delayed vegetative-to-reproductive transition --- p.41 / Chapter 3.4 --- Discussions --- p.42 / Chapter 3.4.1 --- H₂O₂ production in plant --- p.42 / Chapter 3.4.2 --- Glycolate can be derived from lipid breakdown during germination --- p.43 / Chapter 3.4.3 --- GOX functions in developmental phases transition regulation --- p.44 / Chapter 4 --- p.63 / The Identification of a New H₂O₂ signaling pathway in Arabidopsis --- p.63 / Chapter 4.1 --- Introduction --- p.64 / Chapter 4.2 --- Materials and Methods --- p.65 / Chapter 4.2.1 --- Plant materials and culture conditions --- p.65 / Chapter 4.2.2 --- Microarray Experiment and Data Analysis --- p.65 / Chapter 4.2.3 --- Crude Nuclear Protein Extraction --- p.66 / Chapter 4.2.4 --- Gel Mobility Shift Assay --- p.67 / Chapter 4.2.5 --- Purification and Identification of R-box Binding NF proteins --- p.68 / Chapter 4.2.6 --- Antibodies --- p.69 / Chapter 4.2.7 --- Western blotting --- p.69 / Chapter 4.2.8 --- Depletion tests --- p.69 / Chapter 4.2.9 --- Shift-Western assay --- p.70 / Chapter 4.2.10 --- Cloning and Detection of snoRNAs --- p.70 / Chapter 4.2.11 --- Bioinformatic Analysis --- p.71 / Chapter 4.3 --- RESULTS --- p.72 / Chapter 4.3.1 --- Transcriptome profiling revealed that H₂O₂ responsive genes also respond to light --- p.72 / Chapter 4.3.2 --- Identification of a putative novel motif from the promoters of co-upregulated genes --- p.73 / Chapter 4.3.3 --- Verification of the GGGCC Motif by Gel Mobility Shift Assay --- p.74 / Chapter 4.3.4 --- R-box is widely found In eukaryotic gene promoters --- p.75 / Chapter 4.3.5 --- R-box is ROS responsive in higher eukaryotic species --- p.76 / Chapter 4.3.6 --- NOP56 and fibrillarin 2 proteins were identified from NF+ samples --- p.77 / Chapter 4.3.7 --- Molecular cloning and detection of box C/D family snoRNA from NF+ samples --- p.79 / Chapter 4.4 --- Discussion --- p.80 / Chapter 5 --- p.99 / Conclusions and Perspectives --- p.99 / Chapter 5.1 --- Conclusions --- p.100 / Chapter 5.2 --- Perspectives --- p.101 / References --- p.103
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_328427 |
| Date | January 2013 |
| Contributors | Cheng, Han, Chinese University of Hong Kong Graduate School. Division of Life Sciences. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | electronic resource, electronic resource, remote, 1 online resource (xiii, 114 leaves) : ill. (some col.) |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
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