鹽鹼化是世界上最主要非生物脅迫之一，它主要是由於土壤中的鹽（氯化鈉）過量積累所導致的, 不僅影響植物的生長而且也影響農作物的產量。大豆是世界上最重要的經濟類豆科植物之一，由於其種子內含有豐富的營養物質例如蛋白質，油，糖分和纖維，所以它為我們提供了極為重要和大量的油脂和蛋白。在鹽脅迫下大豆的產量會有明顯的降低。由於以上這些情況，我們希望搞清楚大豆對於鹽脅迫的反應機制。首先是通過蛋白質組學弄清楚鹽脅迫的生理過程和大豆如何耐受鹽脅迫的。一般來說，蛋白質組學包括了鑑定蛋白類的化合物和測量在生物系統中的蛋白含量的學科。近期，質譜的發展提供了一個去研究細胞內蛋白質的動態變化十分有用的平台。定量蛋白質組學的發展對於我們系統性的了解蛋白質的功能的分子是十分重要的並且預期會提供給我們各種生物過程和系統的分子機制的見解.其次，表觀遺傳性特別是組蛋白修飾。因為組蛋白修飾通過重新編排染色體的結構和組成參與了許多重要的生物過程並且這些翻譯後修飾對於植物的耐受鹽脅迫也發揮著十分重要的作用。因此我們希望了解組蛋白修飾是如何參與這一過程的。 / Salinity stress, which is caused by the accumulation of excessive amount of salts in the soil, is one of the most severe abiotic stresses that constraint not only crop plant growth but also crop productivity. Soybean (Glycine max) is one of the most important economical legume crops in the world because of its richness of nutritional compositions including protein, oil, sugar and fiber in the seed. Soybean yield of sensitive cultivars is decreased dramatically under salt stress. Because of this, we tried to figure out the mechanism of how soybean response to salinity stress. Firstly, Proteomics--to elucidate the affected physiological process in the salinity stress and the way to tolerant the stress. In general, proteomics involves the identification of protein components and the measurement of protein abundance in biological systems. Recent mass spectrometry (MS)-based technology developments have provided useful platforms for the study of quantitative changes in protein components within the cell. Quantitative proteomics is important for the system-based understanding of the molecular function of each protein component and expected to provide insights into molecular mechanisms of various biological processes and systems. Secondly, Epigenetics--particularly histone modifications. Because histone modifications play important roles in many fundamental biological processes by rearranging the structure and composition of chromatin and PTMs have more roles in response salinity stress. So we want to understand how PTMs involve in this process from epigenetics. / Detailed summary in vernacular field only. / Peng, Xu. / "October 2012." / Thesis (M.Phil.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 44-54). / Abstracts also in Chinese. / Thesis committee --- p.i / Declaration --- p.ii / Abstract (in English) --- p.iii / Abstract (in Chinese) --- p.iv / Acknowledgements --- p.v / Table of contents --- p.vi / General abbreviations --- p.vii / Chapter Chapter 1: --- Introduction --- p.1 / Chapter 1.1 --- Soybean --- p.1 / Chapter 1.2 --- Salinity stress and plants’ response to salinity stress --- p.1 / Chapter 1.3 --- Mass Spectrometry base Proteomics --- p.3 / Chapter 1.3.1 --- Introduction to proteomics --- p.3 / Chapter 1.3.2 --- Proteomic studies in plants --- p.5 / Chapter 1.4 --- Introduction to Epigenetics --- p.6 / Chapter 1.5 --- Present studies --- p.8 / Chapter Chapter 2 --- Proteomic studies in soybean --- p.9 / Chapter 2.1 --- Materials and methods --- p.9 / Chapter 2.1.1 --- Plant materials and stress treatment --- p.9 / Chapter 2.1.2 --- Protein extraction and nuclei extraction and histone isolation --- p.10 / Chapter 2.1.3 --- Protein Preparation for Mass Spectrometry --- p.11 / Chapter 2.1.4 --- Analysis of Protein using nanoLC-MS/MS and Data analysis --- p.12 / Chapter 2.2 --- Results and discussions --- p.13 / Chapter 2.2.1 --- One thousand two hundred seventeen proteins were identified by LC MS/MS-based proteomics technique --- p.13 / Chapter 2.2.2 --- Functional analyses of identified proteins --- p.22 / Chapter 2.2.3 --- One hundred sixty-three proteins are changed under salinity stress as identified by LC MS/MS-based proteomics technique --- p.26 / Chapter 2.2.4 --- Important stress relate proteins were identified by LC MS/MS-based proteomics technique --- p.30 / Chapter 2.2.5 --- Histone PTMs in soybean under salinity stress --- p.34 / Chapter Chapter 3 --- Conclusions and perspectives --- p.35

大豆的豐富營養和經濟價值使它成為重要的農產品。但是，土壤鹽漬化影響著大豆的產量。這個問題在沿岸地方特別嚴重。若要改善大豆的耐鹽能力，必先增加對大豆耐鹽機理的了解。 / 本實驗室從大豆中發現了一個受鹽脅迫誘導表達的基因GmSAL1。以往透過體外酶反應分析法， GmSAL1蛋白被介定為一個能作用於1,4,5-三磷酸肌醇 (IP₃) 的肌醇磷酸-5-磷酸酶。這有別於在擬南芥中的SAL1同源蛋白AtSAL1, AtSAL1是一個肌醇磷酸-1-磷酸酶。由於IP₃是信號傳導途徑中的重要分子，本課題對與IP₃信號及耐鹽性相關的GmSAL1蛋白功能進行研究。 / 本課題旨在：（一）　研究GmSAL1對於細胞質內IP₃的累積的體內作用； （二） 研究 GmSAL1 在脫落酸 (ABA)　的信號傳導中的可能角色； （三） 研究 GmSAL1 在鹽脅迫下的保護作用。 / 本研究利用體內報告系統證明了 GmSAL1 對於減少細胞質內IP₃的累積的作用。這種影響IP₃水平的功能減弱了由 ABA 信號所引起的氣孔關閉和種子萌發抑制。利用 GmSAL1 轉基因煙草細胞 (BY-2)　和擬南芥，證明 GmSAL1 在鹽脅迫下起著短暫的保護作用。GmSAL1在鹽脅迫下的保護功能可能是由於蒸騰作用的局部恢復和細胞鈉離子區室化的作用。 / 本研究展示了肌醇信號，ABA信號和鹽脅迫反應三者之間的關係。這是在以前的研究中未被清楚闡釋的。 / Soybean is nutritionally and economically important. However, high soil salinity, particularly in coastal regions, impedes the production of soybean. Understanding the salt tolerance mechanism is the first step towards the enhancement of salt tolerance of soybean. / Our laboratory identified a salt-responsive gene from soybean namely GmSAL1. Previous in vitro enzyme assay suggested that the GmSAL1 protein is an inositol 5’-phopsphatase acting on inositol 1,4,5-trisphosphate (IP₃), which is different from the enzymatic activity reported for the SAL1 homologue (AtSAL1) in Arabidopsis thaliana (A. thaliana) which is an inositol 1-phopsphatase. Since IP₃ is an important molecule involved in signal transduction pathways, this project is to explore the in vivo functions of GmSAL1 in relation to IP3 signaling and salinity tolerance. / The specific objectives of this research are (1) to study the in vivo role of GmSAL1 on cytosolic IP₃ accumulation; (2) to study the possible involvement of GmSAL1 in ABA signaling; (3) to study the protective roles of GmSAL1 under salt stress. / In this project, the function of GmSAL1 to reduce cytosolic IP₃ was demonstrated using an in vivo reporter system. This activity on IP₃ levels reduced the sensitivity of stomatal closure and seed germination inhibition mediated by ABA signals. A transient protection effect against the ionic effect under salt stress by GmSAL1 was shown by gain-of-function tests in transgenic BY-2 cells and transgenic A. thaliana. The protective effect conferred by GmSAL1 may be due to a partial resuming of transpiration through reduction of ABA signals and compartmentalization of Na⁺ into vacuoles. / The study of GmSAL1 in this research demonstrated the link among inositol signaling, ABA signaling, and salinity response which was not well addressed in previous reports. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Ku, Yee Shan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 97-104). / Abstracts also in Chinese. / Statement --- p.i / Abstract --- p.ii / 摘要 --- p.iv / Acknowledgements --- p.v / General Abbreviations --- p.vii / Abbreviations of Chemicals --- p.ix / Table of Contents --- p.xi / List of Figures --- p.xviii / List of Tables --- p.xxi / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- General introduction to salinity and agriculture --- p.1 / Chapter 1.1.1 --- Adverse effects of salinity on plants --- p.2 / Chapter 1.1.1.1 --- Osmotic stress --- p.2 / Chapter 1.1.1.2 --- Ionic stress --- p.3 / Chapter 1.1.1.3 --- Separation of ionic effect from osmotic effect --- p.3 / Chapter 1.1.1.4 --- Oxidative stress --- p.4 / Chapter 1.1.2 --- Major physiological responses of plants to achieve salt tolerance --- p.5 / Chapter 1.1.2.1 --- Maintenance of cellular ion homeostasis --- p.5 / Chapter 1.1.2.2 --- Balance between Na⁺ and K⁺ influx --- p.5 / Chapter 1.1.2.3 --- Efflux of Na⁺ from cell --- p.9 / Chapter 1.1.2.4 --- Enhanced compartmentalization of Na⁺ and Cl⁻ in vacuole --- p.11 / Chapter 1.1.2.5 --- Enhanced vacuolar ion compartmentalization --- p.13 / Chapter 1.1.2.6 --- Biosynthesis of osmolytes --- p.13 / Chapter 1.2 --- Signal transduction under salt stress --- p.14 / Chapter 1.2.1 --- General introduction to signal transduction under salt stress --- p.14 / Chapter 1.2.2 --- ABA signaling under salinity --- p.15 / Chapter 1.2.2.1 --- General introduction to ABA signaling --- p.15 / Chapter 1.2.2.2 --- IP₃ and ABA signaling --- p.16 / Chapter 1.2.2.3 --- Introduction to inositol phosphate --- p.16 / Chapter 1.2.2.4 --- Phosphatidylinositol-3-monophosphate --- p.18 / Chapter 1.2.2.5 --- Phosphatidylinositol-4-monophosphate --- p.18 / Chapter 1.2.2.6 --- Phosphatidylinositol-5-monophosphate --- p.18 / Chapter 1.2.2.7 --- Phosphatidylinositol (3,5) bisphosphate --- p.19 / Chapter 1.2.2.8 --- Phosphatidylinositol (4,5) bisphosphate --- p.19 / Chapter 1.2.2.9 --- Inositol (1,4,5) trisphosphate (IP₃) --- p.19 / Chapter 1.2.2.10 --- Inositol metabolism under salt stress --- p.19 / Chapter 1.2.2.11 --- The involvement of IP₃ in ABA signaling --- p.20 / Chapter 1.2.3 --- General introduction to Ca²⁺ signaling --- p.22 / Chapter 1.2.4 --- Ca²⁺ channels --- p.23 / Chapter 1.2.4.1 --- Ligand-gated Ca²⁺ channels --- p.23 / Chapter 1.2.4.1.1 --- IP₃ gated Ca²⁺ channels --- p.23 / Chapter 1.2.4.1.2 --- Cyclic nucleotide gated channels (CNGCs) --- p.24 / Chapter 1.2.4.1.3 --- Glutamate receptor homologs (GLRs) --- p.24 / Chapter 1.2.4.2 --- Voltage-gated Ca2⁺ channels --- p.25 / Chapter 1.2.4.2.1 --- Two-pore channels (TPCs) --- p.25 / Chapter 1.2.4.2.2 --- Mechanosensitive Ca2²⁺permeable channels (MSCCs) --- p.25 / Chapter 1.2.4.2.3 --- Ca²⁺ and ABA signaling --- p.26 / Chapter 1.2.5 --- ABA, IP₃ and Ca²⁺ signaling --- p.26 / Chapter 1.2.5.1 --- Ca²⁺ signaling under salt stress --- p.30 / Chapter 1.2.5.2 --- Ca²⁺ signal mediated cellular responses --- p.30 / Chapter 1.3 --- Introduction to inositol phosphatases --- p.30 / Chapter 1.3.1 --- Previous studies on inositol phosphatases in plant --- p.33 / Chapter 1.3.1.1 --- Inositol polyphosphate 1-phosphatase --- p.33 / Chapter 1.3.1.2 --- Inositol polyphosphate 5-phosphatase --- p.34 / Chapter 1.4 --- Previous research on GmSAL1 in Prof. Hon-Ming Lam’s lab --- p.37 / Chapter 1.5 --- Objective and Significance of this project --- p.38 / Chapter Chapter 2 --- Materials and methods --- p.39 / Chapter 2.1 --- Materials --- p.39 / Chapter 2.1.1 --- Plants, bacterial strains and vectors --- p.39 / Chapter 2.1.2 --- Chemicals and enzymes --- p.40 / Chapter 2.1.3 --- Buffer, medium and solution --- p.41 / Chapter 2.1.4 --- Primers --- p.41 / Chapter 2.1.5 --- Equipments and facilities --- p.44 / Chapter 2.1.6 --- Software --- p.44 / Chapter 2.2 --- Methods --- p.44 / Chapter 2.2.1 --- Measurement of osmolarity --- p.44 / Chapter 2.2.2 --- Plant growth and treatment conditions --- p.45 / Chapter 2.2.2.1 --- NaCl, PEG and ABA treatment on soybean plant --- p.45 / Chapter 2.2.3 --- Artificial crossing of A. thaliana --- p.46 / Chapter 2.2.3.1 --- Screening of double homozygous transgenic A. thaliana lines --- p.46 / Chapter 2.2.4 --- Transformation of tobacco BY-2 cells --- p.47 / Chapter 2.3 --- Molecular techniques --- p.48 / Chapter 2.3.1 --- DNA extraction --- p.48 / Chapter 2.3.2 --- PCR --- p.48 / Chapter 2.3.2.1 --- Screening of transgenes --- p.48 / Chapter 2.3.2.2 --- Synthesis of DIG-labeled DNA probe for northern blot analysis --- p.49 / Chapter 2.3.3 --- DNA gel electrophoresis --- p.49 / Chapter 2.3.4 --- RNA extraction --- p.50 / Chapter 2.3.5 --- Northern blot analysis --- p.51 / Chapter 2.3.5.1 --- RNA treatment --- p.51 / Chapter 2.3.5.2 --- Electrophoresis --- p.51 / Chapter 2.3.5.3 --- RNA blotting --- p.51 / Chapter 2.3.5.4 --- GmSAL1 mRNA detection --- p.52 / Chapter 2.4 --- Cell viability assay --- p.52 / Chapter 2.5 --- Na⁺ compartmentalization assay --- p.53 / Chapter 2.6 --- ABA sensitivity assays --- p.53 / Chapter 2.6.1 --- Seed germination assay --- p.53 / Chapter 2.6.2 --- Stomatal opening assay --- p.54 / Chapter Chapter 3 --- Results --- p.55 / Chapter 3.1 --- Differential response of GmSAL1 expression level to NaCl and PEG treatment --- p.55 / Chapter 3.2 --- The expression of GmSAL1 in host plant is responsive to ABA --- p.59 / Chapter 3.3 --- Effect of GmSAL1 on cytosolic IP₃ level in vivo --- p.62 / Chapter 3.4 --- Overexpression of GmSAL1 down-regulates in planta IP₃ level in guard cell --- p.62 / Chapter 3.5 --- Ectopic expression of GmSAL1 in A. thaliana alters stomatal aperture in the presence of ABA in a Ca²⁺ dependent manner --- p.67 / Chapter 3.6 --- Ectopic expression of GmSAL1 in A. thaliana reduces the ABA inhibitory effect on seed germination --- p.71 / Chapter 3.7 --- Overexpression of GmSAL1 transiently protects A. thaliana against ionic effect under salinity --- p.76 / Chapter 3.8 --- Overexpression of GmSAL1 enhances the survival of tobacco BY-2 cells under salt treatment but not near iso-osmotic PEG treatment --- p.80 / Chapter 3.9 --- Overexpression of GmSAL1 confers enhanced vacuolar compartmentalization of Na⁺ in NaCl treated BY-2 cells --- p.85 / Chapter Chapter 4 --- Discussion --- p.90 / Chapter 4.1 --- GmSAL1 as a novel inositol 5-phosphatase --- p.90 / Chapter 4.2 --- The effect of GmSAL1 expression on ABA signaling --- p.91 / Chapter 4.3 --- Involvement of GmSAL1 in tolerance toward ionic effect under salt stress --- p.92 / Chapter 4.4 --- The protective function of GmSAL1 under salinity --- p.93 / Chapter Chapter 5 --- Conclusion --- p.96 / References --- p.97 / Chapter Appendix I --- Chemicals --- p.105 / Chapter Appendix II --- Formulations of buffer, medium and solution --- p.107 / Chapter Appendix III --- Equipments and facilities --- p.110 / Chapter Appendix IV --- Osmolarity of solutions --- p.111 / Chapter Appendix V --- Result of biological repeat of northern blot analysis of GmSAL1 in soybean leaf under NaCl --- p.113 / Chapter Appendix VI --- Result of biological repeat of northern blot analysis of GmSAL1 in soybean root under NaCl --- p.114 / Chapter Appendix VII --- Result of biological repeat of northern blot analysis of GmSAL1 in soybean leaves under 100μM ABA treatment for 0hr, 0.5hr, 1hr, 2hr and 4hr --- p.115 / Chapter Appendix VIII --- Result of biological repeat experiment on the survival rate of tobacco BY-2 cell in 150mM NaCl supplemented MS medium --- p.116 / Chapter Appendix IX --- Result of biological repeat experiment on the survival rate of tobacco BY-2 cell in 13.3% PEG-6000 supplemented MS medium --- p.117

The radiotracer methodology was combined with the Hoagland solution culture of growing soybean in a greenhouse to investigate the absorptions of phosphorus (P), calcium (Ca), and sodium (Na) as a function of salinity. Salt stress was varied by using zero to 120 mM NaCl. The research was initiated because of a need to increase soybean production in the saline soils of the semi-arid regions of the world. Although P absorption increased with time at each concentration of NaCl, increasing its concentrations ([NaCl]) to 120 mM reduced P uptake considerably. The addition of inorganic P (Pi) to the salt medium improved P absorption significantly (P < 0.0001) in stem, petiole, and roots. Polynomial regressions showed the relationship between 22Na activity and [NaCl] for leaves and petiole to be cubic (R2 = 1) while in the stem a quadratic relationship prevailed. A maximum of P and Na absorption was observed at 40 mM NaCl. The relationship between 32P activity and increasing [NaCl] was linear for the roots (a positive slope) and the stem (a negative slope). 45Ca and 32P dual labelling part of the experiments failed to produce results because an unexpectedly high degree of tissue quenching which prevented from obtaining the minimum counting requirements for separation. Shoot fresh and dry weights decreased linearly with increasing [NaCl] as did the root fresh and dry weights. Leaf chlorophyll content during the last week of the final harvest showed a linear relationship with time. Chlorophyll increased with time linearly when the growth medium contained zero and 40 mM NaCl; whereas a negative slope was obtained for 80 and 120 mM NaCl. It seems that P fertilization of the soil could ameliorate the salt effect. 22 Na uptake results indicated that there is a mechanism for exclusion of Na from soybean plant parts.

No description available.

我們在大豆的耐鹽候選基因中進行了篩選和調查，確定了一個穀胱甘肽-S -轉移酶（Glutathion-S-transferase ）基因（ GmGSTL1 ）具抗鹽特性，其表達量也跟隨鹽處理上調。系統發育分析表明，GmGSTL1 屬於LAMBDA 類，文獻對這類蛋白功能的記載甚少。我們在異源系統，包括煙草BY- 2 細胞和擬南芥，測試其保護細胞/植物對鹽脅迫的功能。結果表示GmGSTL1 轉基因細胞的活性氧積累比對照顯著降低，存活率也有所改善。同樣，轉基因擬南芥在鹽處理壓力下的症狀也得以緩解。為了進一步剖析GmGSTL1 的保護機制，我們在大豆葉片中提取多元酚，並發現兩個候選黃酮（槲皮素，山奈酚）與GmGSTL1 起相互作用。槲皮素的外源性應用同樣可以緩解細胞/植物在鹽脅迫下的症狀，表示槲皮素在功能上與GmGSTL1 相約。 / In a survey of candidate genes located in the salinity tolerance locus of soybean, we identified a putative glutathione-S-transferase (GST) gene (GmGSTL1) which was up-regulated in response to salt treatment. Phylogenetic analyses revealed that this putative GST belongs to the Lambda class, a plant-specific group with unknown functions. We expressed GmGSTL1 in heterologous systems, including tobacco BY-2 cells and Arabidopsis thaliana, to test its ability to protect cell/plant against salinity stress. Compare to the wild type control, we observed a marked reduction of ROS accumulation in transgenic cells under salt treatment, and their survival rate was also improved. Similarly, expression of GmGST1 in transgenic A. thaliana also alleviated stress symptoms under salt treatment. To further address the possible protective mechanisms of GmGSTL1, we identified two candidate flavonoid interactants (quercetin and kaemferol) of the GmGSTL1 protein from soybean leaf extract. Exogenous application of quercetin could reduce salinity-induced ROS accumulation in BY-2 cells and leaf chlorosis in A. thaliana. / Detailed summary in vernacular field only. / Chan, Ching. / Thesis (Ph.D.) Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 80-104). / Abstracts also in Chinese.

Wong, Tak Hong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 96-104). / Abstracts in English and Chinese. / Thesis Committee --- p.i / Statement --- p.ii / Abstract --- p.iii / Chinese Abstract --- p.v / Acknowledgements --- p.vii / Abbreviation --- p.ix / Table of Content --- p.xi / List of figures --- p.xiv / List of tables --- p.xv / Chapter 1. --- Introduction --- p.1 / Chapter 1.1 --- Problem of soil salinization and sodification: reducing crop productivity --- p.1 / Chapter 1.2 --- Effects of high salinity on plant growth --- p.2 / Chapter 1.2.1 --- Ion toxicity --- p.2 / Chapter 1.2.2 --- Osmotic stress --- p.3 / Chapter 1.2.3 --- Oxidative stress --- p.3 / Chapter 1.3 --- Overview of salt tolerance mechanisms in plant --- p.4 / Chapter 1.3.1 --- Maintenance of ion homeostasis --- p.4 / Chapter 1.3.2 --- Maintaining osmotic homeostasis --- p.5 / Chapter 1.3.3 --- Detoxification of Reactive oxygen species --- p.5 / Chapter 1.4 --- The important role of CI- in plant salt stress tolerance research --- p.6 / Chapter 1.5 --- Introduction to chloride channel (CLC) family --- p.7 / Chapter 1.6 --- E. coli CLC-ecl: The first CLC member found to function as antiporter --- p.8 / Chapter 1.7 --- Yeast GEF1: eukaryotic model for early plant CLC complementation studies --- p.9 / Chapter 1.8 --- Mammalian CLC family: 4 channels and 5 antiporters --- p.10 / Chapter 1.8.1 --- CLC-4 and -5: First eukaryotic CLC member found to be function as antiporter --- p.13 / Chapter 1.8.2 --- CLC-7 function as antiporter and regulate lysosomal acidification --- p.13 / Chapter 1.8.3 --- "CLC-6 select nitrate over chloride, unlike other mammalian CLC members" --- p.14 / Chapter 1.9 --- Introduction to Plant CLC members --- p.14 / Chapter 1.10 --- Tobacco CLC-Ntl co-localized with mitochondrial markers in plant and may cause current on Xenopus oocytes membrane --- p.15 / Chapter 1.11 --- Rice CLCs may involved in salt tolerenace and growth regulation --- p.16 / Chapter 1.12 --- Arabidopsis CLC members are extensively studied --- p.18 / Chapter 1.12.1 --- AtCLCa regulates nitrate accumulation --- p.20 / Chapter 1.12.2 --- "AtCLCb, a nitrate/proton antiporter with unclear physiological role" --- p.22 / Chapter 1.12.3 --- "AtCLCc selective chloride over nitrate, involved in salt tolerance" --- p.23 / Chapter 1.12.4 --- AtCLCd and AtCLCf both localized on Golgi network --- p.25 / Chapter 1.12.5 --- AtCLCe may regulate ionic strength of chloroplast thylakoid membrane --- p.26 / Chapter 1.13 --- Previous work in Prof. Lam's laboratory --- p.26 / Chapter 1.14 --- "Reason, Hypothesis, Objective and long term significance" --- p.28 / Chapter 2. --- Materials and Methods --- p.30 / Chapter 2.1 --- Materials --- p.30 / Chapter 2.1.1 --- "Bacterial strains, animals, plants and plasmid vectors" --- p.30 / Chapter 2.1.2 --- Chemicals and Enzymes --- p.33 / Chapter 2.1.3 --- Commercial kits --- p.33 / Chapter 2.1.4 --- Primers --- p.35 / Chapter 2.1.5 --- Equipments and facilities used --- p.36 / Chapter 2.1.6 --- "Buffer, solution, gel and medium" --- p.36 / Chapter 2.1.7 --- Software --- p.36 / Chapter 2.2 --- Methods --- p.37 / Chapter 2.2.1 --- Growth and treatment of soybean seedling --- p.37 / Chapter 2.2.2 --- RNA extraction from root tissue --- p.37 / Chapter 2.2.3 --- RNA denaturing gel electrophoresis --- p.39 / Chapter 2.2.4 --- Generation and testing of single-stranded DIG-labeled PCR probes --- p.39 / Chapter 2.2.5 --- Northern blot analysis --- p.41 / Chapter 2.2.6 --- Transformation of V7/GmCLCl electro-competent Agrobacterium tumefaciens --- p.42 / Chapter 2.2.7 --- PCR screening of transformed Agrobacterium tumefaciens colonies --- p.43 / Chapter 2.2.8 --- DNA gel electrophoresis --- p.43 / Chapter 2.2.9 --- Agrobacterium-mediated transformation of tobacco BY-2 cells --- p.44 / Chapter 2.2.10 --- Verifying the expression of GmCLCl in transgenic tobacco BY-2 cells --- p.45 / Chapter 2.2.11 --- Salt treatment of tobacco BY-2 cells and cell viability assay --- p.46 / Chapter 2.2.12 --- Subcloning of GmCLCl cDNA into pgh21 vector --- p.47 / Chapter 2.2.13 --- In vitro synthesis of GmCLCl cRNA --- p.51 / Chapter 2.2.14 --- Obtaining oocyte from Xenopus laevis ovaries --- p.52 / Chapter 2.2.15 --- Microinjection of GmCLCl cRNA into Xenopus oocyte and oocyte incubation --- p.53 / Chapter 2.2.16 --- Two electrode voltage clamp of Xenopus oocytes --- p.54 / Chapter 3. --- Results --- p.56 / Chapter 3.1 --- Phylogenetic analysis of GmCLCl --- p.56 / Chapter 3.2 --- Expression of GmCLCl in root was induced by NaCl and alkaline condition --- p.60 / Chapter 3.3 --- Construction of GmCLCl transgenic tobacco BY-2 cell line --- p.62 / Chapter 3.4 --- GmCLCl improve NaCl stress tolerance of transgenic tobacco BY-2 cells in a pH dependent manner --- p.67 / Chapter 3.5 --- Subcloning of GmCLCl into pgh21 --- p.70 / Chapter 3.6 --- GmCLCl cRNA synthesis by in vitro transcription --- p.72 / Chapter 3.7 --- Two electrode voltage clamp (TEVC) of GmCLCl cRNA injected Xenopus oocytes --- p.75 / Chapter 4. --- Discussion --- p.81 / Chapter 4.1 --- Implications from phylogenetic and sequence analysis on the function of GmCLCl --- p.81 / Chapter 4.2 --- Electrophysiological characterization of GmCLC 1 by Xenopus oocytes --- p.82 / Chapter 4.3 --- Some plant CLCs contributed in salt tolerance response --- p.84 / Chapter 4.4 --- Relationship between pH and physiological function of plant CLCs --- p.85 / Chapter 5. --- Conclusion and Perspectives --- p.88 / Chapter 6. --- Appendices --- p.90 / Chapter Appendix I: --- Major Chemicals and reagents used in this research --- p.90 / Chapter Appendix II: --- Enzymes used in this research --- p.92 / Chapter Appendix III: --- Major equipment and facilities used in this research --- p.93 / Chapter Appendix IV: --- "Buffer, solution, gel and medium formulation" --- p.94 / Chapter 7. --- References --- p.96

Histone modifications and histone variants are of importance in many biological processes. Whether they play some roles in regulating soybean salinity stress response is unknown. Previously, no study of histone modifications and histone variants in soybean were reported. In this study, I elucidated that in soybean leaves, mono-, di- and tri-methylation at Lysine (K) 4, 27 and 36, and acetylation at Lysine 14, 18 and 23 were present in histone H3. Moreover, H3K27 methylation and H3K36 methylation usually excluded each other. Although H3K79 methylation was not reported in Arabidopsis, they were detected in soybean. In soybean histone H4, Lysine 8 and 12 were acetylated. In addition, the variants of histone H3 and H4 and their modifications were also determined. The variants of histone H3 were different at positions of A31F41S87S90 (histone variant H3.1) and T31Y41H87 L90 (histone variant H3.2), respectively. Lysine 4 and 36 methylation were only detected in histone H3.2, suggesting that histone variant H3.2 might associate with actively transcribing genes. The two variants of histone H4 (H4.1 and H4.2) were different at amino acid 60. Moreover, I also found that the abundance of most of the histone modifications and histone variants did not change under the salinity stress except that H3K79 methylation would be up-regulated by the salinity stress. / In a parallel study, a PHD (plant homeodomain) finger domain containing protein, GmPHD1, was able to decipher the 'code' underlying H3K4 methylation. GmPHD1 was ubiquitously expressed in soybean and its expression increased upon salinity stress. GmPHD1 could bind to histone H3K4 methylation, with the preference to H3K4 dimethylation. It could then recruit several proteins, which were GmGNAT1, GmElongin A, and GmISWI. The interaction between GmPHD1 and GmGNAT1 was regulated by the self-acetylation of GmGNAT1. GmGNAT1 could also acetylate histone H3; GmElongin A was a transcription elongation factor; and GmISWI was a chromatin remodeling protein. Our data also indicated that the GmPHD1 located at the promoter of several soybean salt stress inducible genes. Therefore, the GmPHD1 recruited proteins to remodel the chromatin structure and facilitate the transcription of those salt stress inducible genes. Moreover, GmGNAT1 exhibited the preference to acetylate histone H3K14, therefore representing a kind of histone crosstalk between H3K4 methylation and H3K14 acetylation. / Proteomics studies with 2-DE revealed that salt treatment may affect soybean photosynthesis and chloroplast formation. Comparison between the proteomic profiles of salt tolerant soybean variety (wild type) and salt sensitive soybean variety (cultivated, Union) indicated that protein levels in the detoxification and defense pathway as well as energy metabolism were higher in the wild type soybean, while the process of protein metabolism was less active. In addition, proteomic profiles of the cultivated soybean roots at different developmental stages were also compared to identify proteins related to soybean development. The expression of proteins which play critical roles in detoxification and defense pathways were higher at the seedling stage, especially the proteins which regulated the formation of ROS. / Soybean is an important economic crop and its production can be severely affected by salinity stress. At present, the soybean response to salinity stress is not clear. In my studies, I tried to understand this process from the perspective of proteomics and epigenetics, especially histone modifications. / Wu, Tao. / Advisers: Njai Sai Ming; Lam Hon Ming. / Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 126-151). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Cheng, Chun Chiu. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 164-183). / Abstracts in English and Chinese. / Thesis Committee --- p.i / Statement --- p.ii / Abstract --- p.iii / 摘要 --- p.v / Acknowledgements --- p.vi / General Abbreviations --- p.viii / Abbreviations of Chemicals --- p.xi / List of Figures --- p.xv / List of Tables --- p.xvii / Table of Contents --- p.xix / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Salt stress in plants --- p.1 / Chapter 1.2 --- Overview of the molecular basis of salt tolerance in plants --- p.2 / Chapter 1.2.1 --- Stress perception --- p.3 / Chapter 1.2.2 --- Signal transduction --- p.3 / Chapter 1.2.2.1 --- Protein phosphatases --- p.4 / Chapter 1.2.2.2 --- The SOS pathway for ion homeostasis --- p.4 / Chapter 1.2.3 --- DNA and RNA helicases in post-transcriptional control --- p.6 / Chapter 1.2.4 --- ROS scavengers --- p.7 / Chapter 1.2.5 --- Proteases and proteinase inhibitors --- p.8 / Chapter 1.2.6 --- Heat shock proteins (Hsps) --- p.9 / Chapter 1.2.7 --- Highlights on DnaJ/Hsp40 --- p.9 / Chapter 1.3 --- Review on functional genomics of salt stress responses in plants --- p.11 / Chapter 1.3.1 --- Genomics on model organisms --- p.12 / Chapter 1.3.2 --- Transcriptomics for identifying salt stress responsive genes --- p.12 / Chapter 1.3.2.1 --- Multiple stress transcriptome analysis --- p.13 / Chapter 1.3.2.2 --- Genome-wide transcriptome analysis on molecular crosstalk --- p.14 / Chapter 1.3.2.3 --- Tissue specific transcriptome analysis --- p.16 / Chapter 1.3.2.4 --- Comparative transcriptome analysis --- p.17 / Chapter 1.3.2.5 --- Transcriptome analysis of soybean --- p.24 / Chapter 1.3.3 --- Proteomics in plant salt stress studies --- p.26 / Chapter 1.3.4 --- Beyond the transcriptome and proteome --- p.27 / Chapter 1.4 --- Significance of using soybean germplasms for identifying salt stress responsive genes --- p.28 / Chapter 1.5 --- Objectives --- p.29 / Chapter Chapter 2 --- Materials and Methods --- p.30 / Chapter 2.1 --- Materials --- p.30 / Chapter 2.1.1 --- "Plants, bacterial strains，and vectors" --- p.30 / Chapter 2.1.2 --- Enzymes and major chemicals --- p.33 / Chapter 2.1.3 --- Primers --- p.34 / Chapter 2.1.4 --- Commercial kits --- p.34 / Chapter 2.1.5 --- Equipment and facilities --- p.34 / Chapter 2.1.6 --- "Buffer, solution, gel and medium" --- p.34 / Chapter 2.2 --- Methods --- p.35 / Chapter 2.2.1 --- cDNA microarray analysis --- p.35 / Chapter 2.2.1.1 --- Construction of cDNA subtraction libraries --- p.35 / Chapter 2.2.1.2 --- Assembly of cDNA microarray --- p.36 / Chapter 2.2.1.3 --- External control RNA synthesis --- p.39 / Chapter 2.2.1.4 --- Probe labelling and hybridization --- p.40 / Chapter 2.2.1.5 --- Hybridization signal collection --- p.41 / Chapter 2.2.1.6 --- Image analysis --- p.41 / Chapter 2.2.1.7 --- Data analysis --- p.42 / Chapter 2.2.1.8 --- Selection of salt responsive genes using fold difference in expression --- p.45 / Chapter 2.2.1.9 --- DNA sequencing --- p.46 / Chapter 2.2.1.10 --- Real-time PCR analysis --- p.47 / Chapter 2.2.2 --- Growth conditions and treatments of plants --- p.48 / Chapter 2.2.2.1 --- Soybean for microarray hybridization and real-time PCR --- p.48 / Chapter 2.2.2.2 --- Soybean for the study of GmDNJ1 expression under ABA treatment --- p.48 / Chapter 2.2.2.3 --- Wild-type and transgenic Arabidopsis for functional analysis --- p.49 / Chapter 2.2.2.4 --- Wild-type and transgenic rice for functional analysis --- p.49 / Chapter 2.2.3 --- "DNA, RNA, and protein extraction" --- p.50 / Chapter 2.2.3.1 --- Plasmid DNA extraction from E. coli cells --- p.50 / Chapter 2.2.3.2 --- RNA extraction from plant tissues --- p.51 / Chapter 2.2.3.3 --- Soluble protein extraction from plant tissues --- p.51 / Chapter 2.2.4 --- Blot analysis --- p.51 / Chapter 2.2.4.1 --- Northern blot analysis --- p.52 / Chapter 2.2.4.2 --- Western blot analysis --- p.53 / Chapter 2.2.5 --- Subcloning of GmDNJ1 into pGEX-4T-1 --- p.53 / Chapter 2.2.5.1 --- "Restriction digestion, DNA purification and ligation" --- p.53 / Chapter 2.2.5.2 --- Transformation of competent Escherichia coli (DH5a and BL21) --- p.54 / Chapter 2.2.6 --- Luciferase refolding assay --- p.54 / Chapter 2.2.6.1 --- Culture of E. coli strain BL21 (DE3) --- p.54 / Chapter 2.2.6.2 --- Cell lysis --- p.55 / Chapter 2.2.6.3 --- Purification of the GST-GmDNJ1 fusion protein --- p.55 / Chapter 2.2.6.4 --- Quantitation of protein --- p.55 / Chapter 2.2.6.5 --- Luciferase refolding assay --- p.56 / Chapter Chapter 3 --- Results --- p.57 / Chapter 3.1 --- Overview of cDNA microarray analysis --- p.57 / Chapter 3.2 --- Identification of salt responsive genes in subtraction libraries concerning two contrasting soybean germplasms --- p.61 / Chapter 3.3 --- Data processing before selection of salt stress responsive genes --- p.75 / Chapter 3.3.1 --- M-A plots --- p.75 / Chapter 3.3.2 --- Boxplots --- p.76 / Chapter 3.3.3 --- Scatterplots --- p.76 / Chapter 3.4 --- Selection of salt responsive genes using fold difference in expression --- p.77 / Chapter 3.4.1 --- Selection of genes with differential expression between tolerant and sensitive germplasms --- p.77 / Chapter 3.4.2 --- Selection of genes with differential expression between cultivated and wild germplasms --- p.89 / Chapter 3.4.3 --- Data validation by real-time PCR analysis --- p.91 / Chapter 3.5 --- Selection of salt responsive genes using statistical tools --- p.95 / Chapter 3.5.1 --- Quantitative trait analysis for salt responsive genes --- p.95 / Chapter 3.5.2 --- Identification of salt stress correlation genes --- p.100 / Chapter 3.5.3 --- Cluster analyses --- p.104 / Chapter 3.5.3.1 --- Clustering genes --- p.104 / Chapter 3.5.3.2 --- Clustering samples --- p.108 / Chapter 3.5.4 --- Data validation by real-time PCR analysis --- p.111 / Chapter 3.6 --- Summary of cDNA microarray analysis --- p.112 / Chapter 3.7 --- Studies on GmDNJ1 --- p.120 / Chapter 3.7.1 --- Sequence analysis of GmDNJ1 --- p.120 / Chapter 3.7.2 --- GmDNJ1 was induced by salt stress and ABA treatment in soybean (Glycine max) --- p.127 / Chapter 3.7.3 --- Expressing GmDNJ1 in transgenic Arabidopsis (Arabidopsis thaliana) enhances the tolerance to salt stress and dehydration stress --- p.129 / Chapter 3.7.4 --- Expressing GmDNJ1 in transgenic rice (Oryza sativa) enhances the tolerance to salt stress and dehydration stress --- p.135 / Chapter 3.7.5 --- The GmDNJ1 protein can replace DnaJ in the in vitro luciferase refolding assay --- p.141 / Chapter Chapter 4 --- Discussion --- p.145 / Chapter 4.1 --- Overview of expression profiling of the 20 soybean germplasms --- p.145 / Chapter 4.2 --- Identification of salt responsive genes from subtraction libraries --- p.146 / Chapter 4.3 --- Normalization of data from microarray experiments --- p.148 / Chapter 4.4 --- The fold difference analysis --- p.149 / Chapter 4.4.1 --- Response to stress --- p.149 / Chapter 4.4.2 --- Gene expression --- p.150 / Chapter 4.4.3 --- Molecular function --- p.150 / Chapter 4.4.4 --- Metabolic activity --- p.151 / Chapter 4.4.5 --- Cellular component --- p.152 / Chapter 4.4.6 --- Genes with 2.5-fold difference in expression between cultivated and wild germplasms --- p.153 / Chapter 4.5 --- Selection of salt responsive genes using statistical tools --- p.153 / Chapter 4.5.1 --- Quantitative trait analysis --- p.153 / Chapter 4.5.2 --- Cluster analyses --- p.154 / Chapter 4.6 --- Studies on GmDNJ1 --- p.157 / Chapter 4.6.1 --- GmDNJ1 is a good candidate for gene studies --- p.157 / Chapter 4.6.2 --- Sequence analysis of GmDNJ1 suggested it to be a DnaJ/Hsp40 homologue in soybean --- p.158 / Chapter 4.6.3 --- GmDNJ1 was induced by salt stress and ABA treatment --- p.158 / Chapter 4.6.4 --- GmDNJ1 has a higher expression in salt tolerant soybean germplasms over sensitive ones --- p.159 / Chapter 4.6.5 --- Ectopic expression of GmDNJ1 enhanced the tolerance to salt stress and dehydration stress in transgenic Arabidopsis --- p.159 / Chapter 4.6.6 --- Ectopic expression of GmDNJ1 enhanced the tolerance to salt stress and dehydration stress in transgenic rice --- p.160 / Chapter 4.6.7 --- Luciferase activity assay showed that GmDNJ 1 functioned as a DnaJ/Hsp40 in vitro --- p.161 / Chapter Chapter 5 --- Conclusion --- p.162 / References --- p.164 / Appendix I - Enzymes and major chemicals --- p.184 / Appendix II - Primers --- p.188 / Appendix III - Major commercial kits --- p.192 / Appendix IV - Major equipment and facilities --- p.193 / "Appendix V - Formulation of buffer, solution, gel, and medium" --- p.194 / Appendix VI - Plots in microarray experiments --- p.198 / Appendix VII - Clones with differential expression (>2.5-fold or >1.8-fold) between germplasms --- p.208 / Appendix VIII - Salt responsive genes revealed by quantitative trait analysis --- p.216 / Appendix IX - Supplementary data in real-time PCR analysis --- p.221 / Appendix X - Supplementary data in functional analyses --- p.233