GmSAL1 enhances vacuolar sodium ion compartmentalization and ROS scavenging in a calcium dependent manner.

Koo, Siu Chung Nicolas.Thesis submitted in: November 2007.Thesis (M.Phil.)--Chinese University of Hong Kong, 2008.Includes bibliographical references (leaves 78-86).Abstracts in English and Chinese.Thesis committee --- p.iStatement --- p.iiAbstract --- p.iiiChinese Abstract --- p.vAcknowledgements --- p.viAbbreviations --- p.viiiTable of contents --- p.xiList of figures --- p.xvList of tables --- p.xviiChapter 1. --- General IntroductionChapter 1.1 --- General introduction to salt tolerance in plant --- p.1Chapter 1.1.1 --- Adverse effecst of high salinity in plant cellsChapter 1.1.1.1 --- Ion toxicity --- p.1Chapter 1.1.1.2 --- Disturbed osmotic homeostasis --- p.2Chapter 1.1.1.3 --- Oxidative stress --- p.3Chapter 1.1.2 --- Major salt tolerance strategy in plantChapter 1.1.2.1 --- Maintenance of ion homeostasis --- p.4Chapter 1.1.2.2 --- Maintaining osmotic homeostasis --- p.4Chapter 1.1.2.3 --- Detoxification of Reactive oxygen species --- p.4Chapter 1.2 --- Cytosolic Calcium signal in plantChapter 1.2.1 --- General introduction of calcium in plant --- p.6Chapter 1.2.2 --- Calcium transport in plant cell --- p.7Chapter 1.2.3 --- Cytosolic calcium signals in plant under abiotic stress --- p.9Chapter 1.2.4 --- Responding to cytosolic calcium signals --- p.12Chapter 1.3 --- Calcium mediated ion homeostasis in plant under salt stressChapter 1.3.1 --- General introduction on Calcium dependent ion channels in plant --- p.13Chapter 1.3.2 --- SOS family cascade in Arabidopsis --- p.13Chapter 1.4 --- The interaction between cytosolic calcium and reactive oxygen species in plants --- p.14Chapter 1.5 --- "Calcium signaling mediated by Inositol 1,4,5 triphosphate in plant" --- p.15Chapter 1.6 --- Study on HAL2 and its homolog in plant --- p.18Chapter 1.7 --- Previous studies on GmSAL1 in Prof. Lam's lab --- p.20Chapter 1.8 --- Hypothesis and significant of this project --- p.21Chapter 2 --- Materials and MethodsChapter 2.1 --- MaterialsChapter 2.1.1 --- "Plants, bacterial strains and vectors" --- p.23Chapter 2.1.2 --- Chemicals and Regents --- p.25Chapter 2.1.3 --- Commercial kits --- p.26Chapter 2.1.4 --- Primers and Adaptors --- p.27Chapter 2.1.5 --- Equipments and facilities used --- p.27Chapter 2.1.6 --- "Buffer, solution, gel and medium" --- p.27Chapter 2.1.7 --- Software --- p.28Chapter 2.2 --- MethodsChapter 2.2.1 --- Molecular TechniquesChapter 2.2.1.1 --- Bacterial cultures for recombinant DNA and plant transformation --- p.29Chapter 2.2.1.2 --- Recombinant DNA techniques --- p.29Chapter 2.2.1.3 --- Preparation and transformation of Agrobacterium competent cells --- p.30Chapter 2.2.1.4 --- Gel electrophoresis --- p.31Chapter 2.2.1.5 --- DNA and RNA extractions --- p.32Chapter 2.2.1.6 --- Generation of single-stranded DIG-labeled PCR probes --- p.34Chapter 2.2.1.7 --- Testing the concentration of DIG-labeled probes --- p.36Chapter 2.2.1.8 --- Northern blot analysis --- p.36Chapter 2.2.1.9 --- PCR techniques --- p.37Chapter 2.2.1.10 --- Sequencing --- p.38Chapter 2.2.2 --- Plant cell culture and transformationChapter 2.2.2.1 --- Arabidopsis thaliana --- p.39Chapter 2.2.2.2 --- Nicotiana tabacum L. cv. Bright Yellow 2 (BY-2) cells --- p.39Chapter 2.2.3 --- Growth and treatment conditions for plantsChapter 2.2.3.1 --- Growth and salt treatment condition of soybean samples for gene expression studies of GmSAL1 --- p.40Chapter 2.2.3.2 --- Root assay of GmSAL1l transgenic Arabidopsis thaliana --- p.41Chapter 2.2.4 --- "Cell viability, ROS detection and confocal microscopy"Chapter 2.2.4.1 --- Cell viability assay --- p.42Chapter 2.2.4.2 --- Detection of intracellular contents of Na+ --- p.42Chapter 2.2.4.3 --- Detection of Reactive oxygen species (ROS) --- p.42Chapter 2.2.4.4 --- Confocal microscopy --- p.43Chapter 2.2.4.5 --- Images processing and analysis --- p.43Chapter 2.2.5 --- Statistical analysis --- p.44Chapter 3 --- ResultsChapter 3.1 --- GmSAL1 sequence analysis --- p.45Chapter 3.2 --- Expression of GmSAL1 was induced by NaCl stress --- p.49Chapter 3.3 --- Construction of GmSAL1 transgenic tobacco BY-2 cell line --- p.50Chapter 3.4 --- Ectopic expression of GmSAL1 alleviates NaCl stress in transgenic tobacco BY-2 cells --- p.52Chapter 3.5 --- GmSAL1 enhances vacuolar sodium compartmentalization in transgenic tobacco BY-2 cell under NaCl treatment --- p.55Chapter 3.6 --- GmSAL1 helps maintain cell turgidity in transgenic tobacco BY-2 cell under NaCl treatment --- p.58Chapter 3.7 --- GmSAL1 enhances ROS scavenging in transgenic tobacco BY-2 cell under NaCl treatment --- p.61Chapter 3.8 --- Effect of expressing GmSAL1 in Arabidopsis thaliana under salt stress --- p.64Chapter 4 --- Discussion --- p.66Chapter 4.1 --- Sequence analysis and enzyme activity of GmSAL1 --- p.68Chapter 4.2 --- Gene expression profile of GmSAL1 --- p.70Chapter 4.3 --- Functional analysis of GmSAL1 in transgenic tobacco BY-2 cellsChapter 4.3.1 --- GmSAL1 protects transgenic BY-2 cells under salt treatment --- p.71Chapter 4.3.2 --- GmSAL1 regulates Na+ compartmentalization and ROS scavenging in transgenic BY-2 cells under NaCl treatment in a calcium dependent manner --- p.72Chapter 4.4 --- Functional tests of GmSAL1 transgenic A. thaliana --- p.75Chapter 5 --- Conclusion and perspective --- p.76References --- p.78"Appendix I: Substrate specificity and Km, Kcat values of GmSAL1 protein" --- p.87Appendix II: Restriction and modifying enzymes --- p.89Appendix II: Chemicals --- p.90Appendix III: Commercial kits --- p.94Appendix IV: Equipments and facilities used --- p.95"Appendix V: Buffer, solution, gel and medium formulation" --- p.9