M.Sc. / Plants have the ability to continuously respond to various stimuli which alter their physiology, morphology and development. These stimuli may be abiotic or biotic and range from essential to toxic in their effects. One of these stimuli is a steroid from fungal membranes, ergosterol (C28H44O), which does not occur in plants. Ergosterol acts as a pathogen-associated molecular pattern molecule and triggers defence mechanisms in plants, characterised by highly regulated and interrelated events that include the elicitation of the oxidative burst and expression of a number of defencerelated genes. However, the ergosterol-induced global cellular reprogramming of the host has not been fully investigated in all aspects. No metabolomic study has previously been conducted to elucidate, for instance, the effect of ergosterol on plant metabolism. A clear and broader understanding of the molecular mechanisms involved in plant : ergosterol interactions is of paramount importance, for it would open up possibilities of developing novel, more effective and sustainable strategies to control or eradicate fungal diseases in plants. In plants, the metabolome is a compilation of all primary and secondary metabolites. The latter are the final recipients of genetic information, and their levels can influence gene expression and protein stability. Metabolite patterns reveal the actual cellular dynamic environment. Hence, qualitative and quantitative measurements of extra- and intracellular metabolites yield insights into the cellular processes that control the biochemical phenotype of the cell, tissue or whole organism. Metabolomics, the most recent of the ‘omics’ approaches, is the holistic analysis of metabolites present within a biological system under specific physiological conditions. In the present study a metabolomic approach was used to elucidate and analyse changes in the metabolism of tobacco (Nicotiana tabacum) cells following ergosterol treatment. Special attention is given to sesquiterpenoids since the antimicrobial compounds (phytoalexins) isolated from plants within the Solanaceae are mostly bicyclic sesquiterpenoids. Suspension of tobacco cells were treated with different concentrations (0 - 1000 nM) of ergosterol and incubated for different time periods (0 - 24 h). A viability assay, based on the ability of viable cells to reduce 2,3,5- triphenyltetrazolium chloride (TTC), was used to determine whether cell death occurred due to ergosterol treatment. No loss of cell viability was observed over the concentration range and time periods used in this study, indicating that the observed responses were due to the treatment alone and possible secondary responses due to cell death could be excluded. Intracellular metabolites were extracted with two methods: a selective dispersive liquid-liquid micro extraction and a general methanol extraction. Chromatographic techniques (TLC/HPTLC, GC-FID, GC-MS, GC×GC-TOF-MS, UPLC-MS) and 1H NMR spectroscopy were used for quantitative and qualitative analyses. Multivariate data analyses (PCA and OPLS-DA models) were used to extract interpretable information from the multidimensional data generated from the aforementioned techniques.

The optimal defense hypothesis predicts that the allocation of plant defenses across plant organs is proportional to the importance of a given organ to plant fitness. Despite this, much less work has been devoted to the study of reproductive defenses in plants relative to vegetative structures like leaves. This study examines the apparancy hypothesis and the resource availability hypothesis using a phylogenetic comparative approach within the genus Cornus . During the 2016 growing season, plants of 25 species of Cornus were tracked for flower and fruit phenology as well as sampled for floral and fruit tissue in a common garden experiment at the Arnold Arboretum of Harvard University. This tissue was used to quantify floral and fruit defensive chemistry (e.g. tannin activity, total phenolics, total flavonoids, titratable acidity), and fruit palatability traits (e.g. water, sugar, lipid, and protein content), and the color of reproductive structure using reflectance spectroscopy. Native habitat environmental data was obtained using digitized herbarium records and publicly available environmental data layers. Trait-trait and trait-environment relationships were assessed with phylogenetic generalized least squares regression. The evolution of later flowering phenology was correlated with increased floral phenolics and tannins, and the evolution of increased fruiting duration was correlated with increased fruit tannins, both supporting the apparancy hypothesis. Additionally, the evolution of higher fruit sugar content was correlated with higher fruit tannins, and a strong evolutionary trade-off between the production of tannins and the production of flavonoids was observed. With respect to habitat, floral and fruit flavonoids and tannins were consistently lower in species native to warmer environments, while fruit phenolics and was higher in drier environments, which may support the resource availability hypothesis.

No description available.

Ph.D. / Current models regarding plant : pathogen interactions assume that recognition of pathogen-associated molecular pattern (PAMP) molecules can occur through pattern recognition receptors (PRRs) on the surface of plant cells. Lipopolysaccharides (LPS) embedded in the cell wall of Gram-negative bacteria can trigger defence responses or prime the plant in order to respond more rapidly, following perception of bacterial pathogens. Limited data has been reported on signal transduction and the nature of the LPS receptors in plants since no receptors have been identified yet. Parallels have been shown to exist between self-incompatibility and pathogen recognition with regard to self / non-self recognition. The two processes were reviewed and conceptual and mechanistic links between microbial recognition and self-incompatibility were discussed herein. The role of S-domain receptor-like kinases (RLKs) in defence mechanisms has previously not been widely recognized or explored. It was reasoned that S-domain RLKs could be utilized to function as resistance (R) genes or as pattern recognition receptors in perception of PAMPs of a non-protein nature. It has been found that genes encoding receptors may be up-regulated in response to perception of its ligand. A putative receptor-like kinase was previously reported to be induced by LPS. This 153 bp differentially expressed transcript, HAP3-15 (GenBank accession number DR109311), might be an expressed sequence tag (EST) for a gene encoding a receptor for LPS. The experimental characterisation of this EST was reported herein. Gene-walking, reverse transcriptase polymerase chain reaction (RT-PCR), rapid amplification of cDNA ends (RACE), cloning, sequencing and bio-informatic analyses were used to identify the full gene. These results revealed that it encoded a receptor-like protein kinase with an extracellular S-domain recognition motif. The 2842 bp genomic sequence obtained, showed that the sequence had a defined promoter region and six major domains. The first five domains were encoded by the first exon. These domains included a B-lectin / agglutinin domain, an S-locus glycoprotein domain, an EGF-like repeat, a PAN domain, a transmembrane region and part of the 6th domain. The 6th domain was a kinase domain consisting of eleven sub-domains interspersed by three introns. The gene was therefore designated as the N. tabacum S-domain Receptor-like kinase (NS-RLK).

Au Yeung, Wan Kin. / "August 2011." / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 89-103). / Abstracts in English and Chinese. / Thesis committee --- p.i / Statement --- p.ii / Abstract --- p.iii / Acknowledgements --- p.v / General abbreviations --- p.vi / Abbreviations of chemicals --- p.vii / List of figures --- p.ix / List of figures in Appendix VI --- p.xii / List of tables --- p.xiv / Table of Contents --- p.xv / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- The significance of studying rice disease resistance --- p.1 / Chapter 1.1.1 --- Economic importance of rice --- p.1 / Chapter 1.1.2 --- Diseases caused by pathogens virulent to rice --- p.1 / Chapter 1.1.2.1 --- Bacterial leaf blight diseases --- p.1 / Chapter 1.1.2.2 --- Fungal blast diseases --- p.2 / Chapter 1.1.3 --- Approach to enhance resistance of crops towards pathogens --- p.2 / Chapter 1.2 --- Literature review on plant immunity system --- p.3 / Chapter 1.2.1 --- Pathogen associated molecular patterns (PAMP) and PAMP -triggered immunity (PTI) --- p.4 / Chapter 1.2.2 --- Pathogen effectors and effector-triggered immunity (ETI) --- p.5 / Chapter 1.2.3 --- Roles of phytohormones in plant defense responses --- p.6 / Chapter 1.2.4 --- G protein signaling and plant defense responses --- p.9 / Chapter 1.3 --- Literature review on FK506 binding proteins (FKBPs) --- p.10 / Chapter 1.4 --- Background information of this study - origin of the clone chosen for study in this project --- p.11 / Chapter 1.5 --- Hypothesis and Objectives --- p.12 / Chapter Chapter 2 --- Materials and Methods --- p.13 / Chapter 2.1 --- Materials --- p.13 / Chapter 2.1.1 --- "Plants, bacterial strains and vectors" --- p.13 / Chapter 2.1.2 --- Chemicals and Regents --- p.18 / Chapter 2.1.3 --- Commercial kits --- p.18 / Chapter 2.1.4 --- Primers and Adaptors --- p.19 / Chapter 2.1.5 --- Equipments and facilities used --- p.23 / Chapter 2.1.6 --- "Buffer, solution, gel and medium" --- p.23 / Chapter 2.2 --- Methods --- p.24 / Chapter 2.2.1. --- Bacterial and yeast cultures --- p.24 / Chapter 2.2.2 --- Plant growth conditions and treatments --- p.25 / Chapter 2.2.2.1 --- Surface sterilization of J. thaliana seeds --- p.25 / Chapter 2.2.2.2 --- Environmental conditions of A. thaliana for germination of seeds and growing of seedlings --- p.26 / Chapter 2.2.2.3 --- Environmental conditions of A. thaliana for growing of plants --- p.26 / Chapter 2.2.2.4 --- Pathogen inoculation test of A. thaliana with Pst DC3000 --- p.27 / Chapter 2.2.3 --- Cloning and subcloning of OsFKBP 12 and OsUCCl --- p.27 / Chapter 2.2.3.1 --- Sub-cloning of OsFKBP12 to pGEX-4T-l and pMAL-c2 --- p.27 / Chapter 2.2.3.2 --- Cloning of OsUCCl to pGEX-4T-l --- p.29 / Chapter 2.2.4 --- "DNA, RNA and protein extractions" --- p.29 / Chapter 2.2.4.1 --- Plasmid extraction from bacterial cells --- p.29 / Chapter 2.2.4.2 --- Genomic DNA extraction from plant through CTAB method --- p.29 / Chapter 2.2.4.3 --- RNA extraction from plant tissues --- p.30 / Chapter 2.2.4.4 --- Protein extraction from plant tissues --- p.31 / Chapter 2.2.4.5 --- Fusion protein extraction from E. coli --- p.31 / Chapter 2.2.5 --- Western blot analyses --- p.32 / Chapter 2.2.5.1 --- Western blot analysis of GST tag and MBP tag fusion proteins --- p.32 / Chapter 2.2.5.2 --- Western blot analysis native OsYchFl proteins --- p.33 / Chapter 2.2.6 --- Real-time PCR study --- p.33 / Chapter 2.2.6.1 --- cDNA synthesis --- p.33 / Chapter 2.2.6.2 --- Real-time PCR --- p.34 / Chapter 2.2.7 --- Yeast two hybrid --- p.35 / Chapter 2.2.7.1 --- Screening of OsFKBP 12 interaction protein partners by yeast mating --- p.35 / Chapter 2.2.7.2 --- Identification of positive interacting protein partners by extracting DNA plasmid from yeast --- p.35 / Chapter 2.2.7.3 --- Re-transformation of pGBKTl-OsFKBP 12 with their interacting partner clones into yeast (AH 109) by co-transformation --- p.36 / Chapter 2.2.8 --- In vitro pull down assay of OsFKBP 12 with their putative protein interacting partner --- p.36 / Chapter 2.2.8.1 --- In vitro pull down of native OsYchFl by MBP-His-OsFKBP12 --- p.36 / Chapter 2.2.8.2 --- In vitro pull down of GST-AtYchF 1 by MBP-His-OsFKBP12 --- p.37 / Chapter 2.2.8.3 --- In vitro pull down of MBP-His-OsFKBP12 by GST-OsUCCl --- p.37 / Chapter 2.2.8.4 --- In vitro pull down of MBP-His-OsFKBP12 by GST-OsYchFl G domain --- p.38 / Chapter 2.2.9 --- GTPase assay ofOsYchF with OsFKBP12 --- p.38 / Chapter 2.3.0 --- Phylogenetic analysis and sequence alignment --- p.39 / Chapter Chapter 3 --- Results --- p.40 / Chapter 3.1 --- Identification of OsFKBP 12 encoding a FKBP (FK506 binding protein)-domain containing protein in Oryza sativa (rice) --- p.40 / Chapter 3.2 --- OsFKBP12 was down-regulated in the pathogen-inoculated Xal4 rice line CBB14 --- p.47 / Chapter 3.3 --- Ecotpic expression of OsFKBP 12 repressed the expression of defense marker genes in transgenic A. thaliana --- p.50 / Chapter 3.4 --- Expressing OsFKBP 12 in transgenic A. thaliana enhanced the susceptibility to the bacterial pathogen Pst DC3000 --- p.54 / Chapter 3.5 --- OsFKBP 12 protein interacted with a putative defense-related G-protein and a copper binding protein --- p.57 / Chapter 3.6 --- "OsFKBP 12 protein interacted with the G domain of defense-related G protein, OsYchFl" --- p.69 / Chapter 3.7 --- OsFKBP 12 protein enhanced the in vitro phosphate release of OsYchFl --- p.72 / Chapter Chapter 4 --- Discussion --- p.74 / Chapter 4.1 --- The identification and characterization of OsFKBP 12 --- p.74 / Chapter 4.2 --- Expression pattern of OsFKBP 12 upon biotic stress in bacterial blight resistant near isogenic line (NIL) --- p.75 / Chapter 4.3 --- OsFKBP 12 repressed the expression of SA-regulated defense marker genes when ectopically expressed in A. thaliana --- p.75 / Chapter 4.4 --- Ectopic expression of OsFKBP 12 enhanced susceptibility towards Pst DC3000 in transgenic A. thaliana --- p.76 / Chapter 4.5 --- The interacting partners of OsFKBP 12 in relation to plant defense response --- p.78 / Chapter 4.6 --- The specific biochemical interaction of OsFKBP 12 with OsYchFl --- p.80 / Chapter 4.7 --- Future perspectives --- p.85 / Chapter Chapter 5 --- Conclusion --- p.87 / References --- p.89 / Appendix --- p.104

Lam Sze Kwan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 172-200). / Abstracts in English and Chinese. / Acknowledgements --- p.ii / Table of contents --- p.ii / Abstract --- p.xii / 撮要 --- p.xv / List of Abbreviations --- p.xvi / List of Tables --- p.xvii / List of Figures --- p.xix / TABLE OF CONTENTS / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1. --- Overview of Chitinases --- p.3 / Chapter 1.1.1. --- Classification of Chitinases --- p.7 / Chapter 1.1.1.1. --- Family 19 Chitinases --- p.7 / Chapter 1.1.1.1.1. --- Class I Chitinases --- p.9 / Chapter 1.1.1.1.2. --- Class II Chitinases --- p.10 / Chapter 1.1.1.1.3. --- Class IV Chitinases --- p.10 / Chapter 1.1.1.1.4. --- Class V Chitinases --- p.11 / Chapter 1.1.1.1.5. --- Class VI Chitinases --- p.11 / Chapter 1.1.1.2. --- Family 18 Chitinases --- p.12 / Chapter 1.1.1.2.1. --- PR-8/Class III Chitinases --- p.12 / Chapter 1.1.1.2.2. --- PR-11 Chitinases --- p.15 / Chapter 1.1.1.3. --- The PR-4 Family --- p.16 / Chapter 1.1.2. --- Catalytic Mechanism of Chitinases --- p.19 / Chapter 1.1.2.1. --- Catalytic Mechanism of Family 18 Chitinases --- p.20 / Chapter 1.1.2.2. --- Catalytic Mechanism of Family 19 Chitinases --- p.21 / Chapter 1.1.3. --- Biological Properties of Chitinases --- p.22 / Chapter 1.1.3.1. --- Antifungal Activity of Chitinases in vitro --- p.22 / Chapter 1.1.3.2. --- Antifungal Activity of Chitinases in vivo --- p.23 / Chapter 1.1.3.3. --- Other Functions --- p.23 / Chapter 1.2. --- Overview of Ribonucleases --- p.25 / Chapter 1.2.1. --- Classification of Ribonucleases --- p.26 / Chapter 1.2.1.1. --- RNase T1 Family --- p.26 / Chapter 1.2.1.1.1. --- Action Mechanism of RNase T1 Family --- p.32 / Chapter 1.2.1.2. --- RNase T2 Family --- p.34 / Chapter 1.2.1.2.1. --- Action Mechanism of RNase T2 Family --- p.36 / Chapter 1.2.2. --- Biological Activities of Plant Ribonucleases --- p.38 / Chapter 1.2.2.1. --- Phosphate Remobilization --- p.38 / Chapter 1.2.2.2. --- Senescence --- p.39 / Chapter 1.2.2.3. --- Programmed Cell Death --- p.40 / Chapter 1.2.2.4. --- Plant Defense --- p.41 / Chapter 1.2.2.5. --- RNA Processing and Decay --- p.43 / Chapter 1.2.2.6. --- Antitumor Activities --- p.43 / Chapter 1.3. --- Overview of plant ribosome-inactivating proteins (RIPs) --- p.45 / Chapter 1.3.1. --- General properties of RIPs --- p.46 / Chapter 1.3.1.1. --- Classification of RIPs --- p.46 / Chapter 1.3.2. --- Activities of Ribosome-inactivating Proteins --- p.52 / Chapter 1.3.2.1. --- RNA N-glycosidase activity --- p.52 / Chapter 1.3.2.2. --- Protein synthesis inhibitory activity --- p.58 / Chapter 1.3.2.3. --- Abortifacient activity --- p.59 / Chapter 1.3.2.4. --- Immunosuppressive activity --- p.60 / Chapter 1.3.2.5. --- Antiviral activity --- p.61 / Chapter 1.3.3. --- Roles of RIPs in plants --- p.63 / Chapter 1.3.3.1. --- Defensive role of RIPs in plants --- p.63 / Chapter 1.3.3.2. --- Role of RIPs in stress adaptation in plants --- p.66 / Chapter 1.3.4. --- Possible application of RIPs --- p.67 / Chapter 1.3.4.1. --- Use of RIPs in therapies --- p.67 / Chapter 1.3.4.1.1. --- Antiviral agents --- p.67 / Chapter 1.3.4.1.2. --- Immunotoxins --- p.68 / Chapter 1.3.4.1.3. --- Anti-HIV drugs --- p.69 / Chapter 1.3.4.2. --- Use of RIPs in agriculture --- p.71 / Chapter 1.4. --- Overview of the PR-5 Family: Thaumatin-Like Proteins (TLPs) --- p.72 / Chapter 1.4.1. --- Occurrence of Thaumatin-Like Proteins --- p.76 / Chapter 1.4.2. --- Biological properties of TLPs --- p.77 / Chapter 1.4.2.1. --- Antifungal Activity --- p.77 / Chapter 1.4.2.2. --- TLPs as Anti-Freeze Protein --- p.78 / Chapter 1.4.3. --- Biotechnological Application ´ؤ Transgenic Plants --- p.79 / Chapter Chapter 2 --- Materials and Methods --- p.81 / Chapter 2.1. --- Materials --- p.81 / Chapter 2.2. --- Preparation of Crude Extract --- p.82 / Chapter 2.3. --- Purification --- p.83 / Chapter 2.4. --- Chromatography --- p.84 / Chapter 2.4.1. --- CM-Cellulose Chromatography --- p.84 / Chapter 2.4.2. --- Mono S® HR 5/5 and Mono Q® HR 5/5 --- p.85 / Chapter 2.4.3. --- Affi-gel Blue gel --- p.86 / Chapter 2.4.4. --- Superdex75 --- p.87 / Chapter 2.5. --- Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.88 / Chapter 2.6. --- Protein Concentration Determination --- p.89 / Chapter 2.7. --- Preparation of Rabbit Reticulocyte Lysate --- p.90 / Chapter 2.8. --- Determination of N-terminal Amino Acid Sequence --- p.91 / Chapter 2.9. --- Biological Activity Assays --- p.92 / Chapter 2.9.1. --- Assay for Antifungal Activity --- p.92 / Chapter 2.9.2. --- Assay for Cell-Free Translation Inhibitory Activity --- p.93 / Chapter 2.9.3. --- Assay of Cytotoxic Activity on Cancer Cell Lines --- p.94 / Chapter 2.9.4. --- Assay for HIV-1 Reverse Transcriptase (RT) Inhibitory Activity --- p.95 / Chapter 2.9.5. --- Assay of Mitogenic Activity --- p.97 / Chapter 2.9.6. --- Assay for N-Glycosidase Activity --- p.98 / Chapter 2.9.6.1. --- RNA Extraction --- p.98 / Chapter 2.9.6.2. --- Aniline Treatment --- p.99 / Chapter 2.9.6.3. --- Formaldehyde Gel Electrophoresis --- p.99 / Chapter 2.9.7. --- Assay of Ribonuclease Activity --- p.100 / Chapter 2.9.7.1. --- Assay for Yeast tRNA --- p.100 / Chapter 2.9.7.2. --- Activity toward Polyhomoribonucleotides --- p.100 / Chapter Chapter 3 --- Purification and Characterization of Pathogenesis-Related Proteins from their Respective Sources --- p.101 / Chapter 3.1. --- Purification and Characterization of Chitinase and Ribonuclease from the Roots of Panax notoginseng --- p.102 / Chapter 3.1.1. --- Introduction --- p.102 / Chapter 3.1.2. --- Results --- p.104 / Chapter 3.1.3. --- Purification --- p.107 / Chapter 3.1.3.1. --- Cation-Exchange Chromatography on CM-Cellulose --- p.108 / Chapter 3.1.3.2. --- Affinity Chromatography on Affi-gel Blue gel --- p.111 / Chapter 3.1.3.3. --- Cation-Exchange Chromatography on Mono S Column --- p.114 / Chapter 3.1.3.4. --- Gel Filtration on Superdex 75 Column --- p.115 / Chapter 3.1.4. --- Characterization of Chitinase --- p.117 / Chapter 3.1.4.1. --- N-terminal Amino Acid Sequence --- p.117 / Chapter 3.1.4.2. --- Assay for Antifungal Activity --- p.118 / Chapter 3.1.4.3. --- Assay for Cell-Free Translation-inhibitory Activity --- p.120 / Chapter 3.1.4.4. --- Assay for HIV-1 Reverse Transcriptase Inhibitory Activity --- p.120 / Chapter 3.1.5. --- Characterization of Ribonuclease --- p.121 / Chapter 3.1.5.1. --- N-terminal Amino Acid Sequence --- p.121 / Chapter 3.1.5.2. --- Assay for Ribonuclease Activity --- p.122 / Chapter 3.1.5.3. --- Assay for Cell-Free Translation-inhibitory Activity --- p.125 / Chapter 3.1.5.4. --- Assay for Antifungal Activity --- p.125 / Chapter 3.1.5.5. --- Assay for Antiproliferative Activity --- p.126 / Chapter 3.1.6. --- Discussion --- p.127 / Chapter 3.2. --- Purification and Characterization of Ribosome-Inactivating Protein and Antifungal Protein from the mushroom Lyophyllum shimeji --- p.131 / Chapter 3.2.1. --- Introduction --- p.131 / Chapter 3.2.2. --- Results --- p.132 / Chapter 3.2.3. --- Purification --- p.134 / Chapter 3.2.3.1. --- Cation-Exchange Chromatography on CM-Cellulose --- p.135 / Chapter 3.2.3.2. --- Affinity Chromatography on Affi-gel Blue Gel --- p.137 / Chapter 3.2.3.3. --- Cation-Exchange Chromatography on Mono S --- p.140 / Chapter 3.2.4. --- Characterization of Ribosome-Inactivating Protein and Antifungal Protein from Lyophyllum shimeji --- p.142 / Chapter 3.2.4.1. --- N-terminal Amino Acid Sequence --- p.142 / Chapter 3.2.4.2. --- Assay for Antifungal Activity --- p.144 / Chapter 3.2.4.3. --- Assay for N-glycosidase Activity --- p.147 / Chapter 3.2.4.4. --- Assay for Mitogenic Activity --- p.147 / Chapter 3.2.4.5. --- Assay for HIV-1 Reverse Transcriptase Inhibitory Activity --- p.148 / Chapter 3.2.5. --- Discussion --- p.150 / Chapter 3.3. --- Purification and Characterization of Ribosome-inactivating Protein from the Hypsizigus marmoreus --- p.153 / Chapter 3.3.1. --- Introduction --- p.153 / Chapter 3.3.2. --- Result --- p.154 / Chapter 3.3.3. --- Purification --- p.155 / Chapter 3.3.3.1. --- Cation-Exchange Chromatography on CM-Cellulose --- p.156 / Chapter 3.3.3.2. --- Affinity-Chromatography on Affi-gel Blue Gel --- p.158 / Chapter 3.3.3.3. --- Anion-Exchange Chromatography on Mono Q Column --- p.160 / Chapter 3.3.4. --- Characterization of Ribosome-inactivating Protein from Hypsizigus marmoreus --- p.162 / Chapter 3.3.4.1. --- N-terminal Amino Acid Sequence --- p.162 / Chapter 3.3.4.2. --- Assay for Cell-Free Translation-Inhibiting Activity --- p.163 / Chapter 3.3.4.3. --- Assay for Antifungal Activity --- p.164 / Chapter 3.3.4.4. --- Assay for N-glycosidase Activity --- p.166 / Chapter 3.3.4.5. --- Assay for HIV-1 Reverse Transcriptase Inhibitory Activity --- p.166 / Chapter 3.3.4.6. --- Assay for mitogenic Activity --- p.167 / Chapter 3.3.4.7. --- Assay for Antiproliferative Activity --- p.167 / Chapter 3.3.5. --- Discussion --- p.159 / Chapter Chapter 4 --- General Discussion --- p.170 / References --- p.172

植物合成多種發病機理相關蛋白以對抗病原體的侵襲。植物發病機理相關蛋白包括：核糖核酸酶；抗真菌蛋白；凝集素；胰蛋白酶抑制因子等。這些發病機理相關蛋白具有抗病毒，抗細菌，抗真菌，免疫調節及抗腫瘤等活性。從六種植物中提純了七個發病機理相關蛋白，包括三個凝集素，一個核糖核酸酶，兩個種抗真菌蛋白及一個胰蛋白酶抑制因子。 / 從西洋參須中提純了新的核糖核酸酶。核糖核酸酶分子量為26kDa，具有特异N末端氨基酸序列。此核糖核酸酶在 pH5 及 60℃ 條件下活性最高。它能抑制腫瘤細胞分裂及抑制人類後天免疫力缺乏症候群病毒逆轉錄酶活性。 / 從粉色菜豆及日本大花豆中提純了兩種凝集素。它們由兩個分子量為32kDa的亞基構成雙倍體。他們的活性穩定于0-60℃及3-12 pH。粉色菜豆凝集素的特异性糖基為木糖，日本大花豆凝集素的特异性糖基為半乳糖。從太子參中提純的凝集素分子量為33kDa，其活性穩定于0-60℃及2-5 pH。 這三種凝集素都具有抑制腫瘤細胞分裂及抑制人類後天免疫力缺乏症候群病毒逆轉錄酶活性。 / 提純的胰蛋白酶抑制因子分子量為21kDa。具有高耐熱及耐酸鹼性并表現出抑制腫瘤細胞分裂及抑制人類後天免疫力缺乏症候群病毒逆轉錄酶活性。從豇豆中提純的抗真菌肽分子量為5447Da，具有類防御素N末端氨基酸序列。 / Plants produce a diversity of proteins with antipathogenic activities. These proteins comprise among others, (i) ribonucleases, (ii) antifungal proteins, (iii) lectins and (iv) trypsin inhibitor with antiviral, antifungal and anticancer activities. The aim of this project was to isolate antipathogenic plant proteins including a ribonuclease from American ginseng branch roots, a trypsin inhibitor from rambutan seeds, defensin-like antifungal peptides from borlotti beans and king pole beans, and lectins from borlotti beans, Japanese large pinto beans and Pseudostellaria heterophylla. / The isolated 26-kDa ginseng branch root ribonuclease was monomeric with a novel N-terminal amino acid sequence. It exhibited maximal robonucleolytic activity toward yeast tRNA at pH 5 and 60℃. It inhibited proliferation of MCF7 human breast cancer cells and HepG2 human hepatoma cells. It also inhibited the activity of HIV-1 reverse transcriptase. / Both borlotti bean lectin and Japanese large pinto bean lectin were dimeric with a subunit molecular mass of 32-kDa. They were stable from 0℃ to 60℃ and from pH 3 to pH 12. Borlotti bean lectin was xylose-specific whereas Japanse large pinto bean lectin was galactose-specific. The 33-kDa Pseudostellaria heterophylla lectin could not be inhibited by the simple sugars tested. It was stable from 0℃ to 60℃ and from pH 2 to 5. All three isolated lectins suppressed proliferation of MCF7 and HepG2 cells and inhibited HIV-1 reverse transcriptase. / The isolated 21-kDa rambutan trypsin inhibitor has relatively high pH stability and thermostability, and exhibited HIV-1 reverse transcriptase inhibitory activity and antiproliferative activity toward a variety of tumor cells. The isolated 5447-Da king pole bean defensin-like peptide inhibited fungal growth. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Zhao, Yuan. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 202-222). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / 論文摘要 --- p.iv / Declaration --- p.v / Abbreviations --- p.vi / Table of Contents --- p.vii / List of Tables --- p.x / List of Figures --- p.xii / Chapter Chapter 1 --- Overview of Plant Defense-related Protein --- p.1 / Chapter 1.1 --- Overview of Lectins and hemagglutinins --- p.4 / Chapter 1.1.1 --- History and definition of lectins and hemagglutinins --- p.4 / Chapter 1.1.2 --- Occurrence and distribution of plant lectins --- p.6 / Chapter 1.1.3 --- Classification of lectins --- p.7 / Chapter 1.1.3.1 --- Classification of lectins on the basis of overall structure of lectin subunits --- p.7 / Chapter 1.1.3.2 --- Classification of lectins based on binding specificty to carbohydrates --- p.11 / Chapter 1.1.3.3 --- Classification of lectins according to families --- p.12 / Chapter 1.1.3.3.1 --- Legume lectins --- p.12 / Chapter 1.1.3.3.2 --- Monocot mannose-binding lectins --- p.13 / Chapter 1.1.3.3.3 --- Other lectins --- p.14 / Chapter 1.1.4 --- Defensive role of plant lectins --- p.15 / Chapter 1.1.5 --- Applications of plant lectins --- p.18 / Chapter 1.1.5.1 --- The antibacterial activity --- p.18 / Chapter 1.1.5.2 --- Anti-insect activity --- p.19 / Chapter 1.1.5.3 --- Antifungal activity --- p.21 / Chapter 1.1.5.4 --- The antiviral activity --- p.22 / Chapter 1.1.5.5 --- Lectin affinity chromatography --- p.23 / Chapter 1.1.5.6 --- Lectin microarray --- p.23 / Chapter 1.2 --- Overview of Ribonucleases --- p.26 / Chapter 1.2.1 --- History and definition of Ribonucleases --- p.26 / Chapter 1.2.2 --- Classification of Ribonucleases --- p.27 / Chapter 1.2.2.1 --- T1 Ribonucleases family --- p.27 / Chapter 1.2.2.2 --- RNase T2 family --- p.28 / Chapter 1.2.3 --- Biological activities of plant ribonucleases --- p.28 / Chapter 1.2.3.1 --- Phosphate remobilization --- p.28 / Chapter 1.2.3.2 --- Senescence --- p.29 / Chapter 1.2.3.3 --- Programmed cell death --- p.30 / Chapter 1.2.3.4 --- Plant defense --- p.31 / Chapter 1.2.3.5 --- RNA processing and decay --- p.32 / Chapter 1.2.3.6 --- Antitumor activities --- p.33 / Chapter 1.3 --- Other plant pathogen-related proteins --- p.34 / Chapter 1.3.1 --- Overview of chitinase --- p.34 / Chapter 1.3.1.1 --- Classification of chitinases --- p.35 / Chapter 1.3.1.2 --- Biological properties of chitinases --- p.38 / Chapter 1.3.2 --- Overview of plant ribosome-inactivating proteins (RIPs) --- p.41 / Chapter 1.3.2.1 --- Classification of RIPs --- p.42 / Chapter 1.3.2.2 --- Roles of RIPs in plants --- p.44 / Chapter 1.3.2.3 --- Possible application of RIPs --- p.46 / Chapter 1.3.3 --- Overview of thaumatin-like proteins (TLPs) --- p.50 / Chapter 1.3.3.1 --- Occurrence of TLPs --- p.51 / Chapter 1.3.3.2 --- Biological properties of TLPs --- p.52 / Chapter 1.4 --- Aim of this study --- p.54 / Chapter Chapter 2 --- Isolation of a lectin and an antifungal protein from Phaseolus vulgaris cv. Borlotti beans / Chapter 2.1 --- Introduction --- p.55 / Chapter 2.2 --- Materials and Methods --- p.55 / Chapter 2.3 --- Results --- p.64 / Chapter 2.4 --- Discussion --- p.79 / Chapter Chapter 3 --- Isolation of a lectin from Pinto beans (Phaseolus vulgaris pinto bean) / Chapter 3.1 --- Introduction --- p.82 / Chapter 3.2 --- Materials and Methods --- p.83 / Chapter 3.3 --- Results --- p.87 / Chapter 3.4 --- Discussion --- p.103 / Chapter Chapter 4 --- Isolation of a lectin from Pseudostellaria hetorophylla roots / Chapter 4.1 --- Introduction --- p.105 / Chapter 4.2 --- Materials and Methods --- p.107 / Chapter 4.3 --- Results --- p.110 / Chapter 4.4 --- Discussion --- p.122 / Chapter Chapter 5 --- Isolation of a ribonuclease from branch roots of American ginseng (Panax quinquefolium) / Chapter 5.1 --- Introduction --- p.124 / Chapter 5.2 --- Materials and Methods --- p.126 / Chapter 5.3 --- Results --- p.129 / Chapter 5.4 --- Discussion --- p.142 / Chapter Chapter 6 --- Isolation of a trypsin inhibitor in rambutan (Nephelium lappaceum L) seeds / Chapter 6.1 --- Introduction --- p.144 / Chapter 6.2 --- Materials and Methods --- p.147 / Chapter 6.3 --- Results --- p.152 / Chapter 6.4 --- Discussion --- p.163 / Chapter Chapter 7 --- Isoation of a defensin-like antifungal peptide from Phaseolus vulgaris cv. 'King Pole Bean' / Chapter 7.1 --- Introduction --- p.168 / Chapter 7.2 --- Materials and Methods --- p.170 / Chapter 7.3 --- Results --- p.173 / Chapter 7.4 --- Discussion --- p.181 / Chapter Chapter 8 --- Overall discussion --- p.183 / References --- p.186

In this thesis, I combined field, greenhouse and common-garden experiments to examine the ecological and evolutionary consequences of plant-herbivore interactions and the genetic architecture of fitness-related traits in the insect-pollinated, self-incompatible, perennial herb Arabidopsis lyrata. More specifically, I examined (1) whether damage to leaves and inflorescences affects plant fitness non-additively, (2) whether trichome production is associated with a cost in terms of reduced tolerance to leaf and inflorescence damage, (3) whether young plant resistance to a specialist insect herbivore varies among populations, and (4) whether the evolution of flowering time, floral display and rosette size is constrained by lack of genetic variation or by genetic correlations among traits. A two-year field experiment in a Swedish population showed that damage to rosette leaves and to inflorescences can affect both current and future plant performance of A. lyrata, and that effects on some fitness components are non-additive. A two-year field experiment in another Swedish population indicated that trichome-producing plants are not less tolerant than glabrous plants to leaf and inflorescence damage. In a greenhouse experiment, acceptability of young plants (5-6 weeks old) to ovipositing females and damage received by Plutella xylostella larvae varied considerably among twelve A. lyrata populations. Both oviposition and leaf damage were positively correlated with rosette size, but trichome density in the trichome-producing morph was apparently too low at this developmental stage to influence resistance to P. xylostella. In a common-garden experiment, flowering time, floral display and rosette size varied among four Scandinavian A. lyrata populations, and displayed significant additive genetic variation in some populations. Yet, strong genetic correlations between flowering start and number of flowers, and between petal length and petal width suggest that these traits may not evolve independently. Taken together, the results indicate the need to consider possible long-term and non-additive effects of herbivore damage to different plant parts, that there is no trade-off between trichome production and tolerance to herbivory, that the importance of morphological defenses against herbivory may change through plant ontogeny, and that considerable genetic variation for traits such as flowering time and floral display can be maintained in natural plant populations.

Because organisms have limited resources to allocate to multiple life history traits, the Optimal Defense Theory (ODT) and the Growth-Differentiation Balance Hypothesis (GDBH) were developed by terrestrial plant ecologists to predict intraindividual defense allocation based on the cost of defense and these life history trade-offs. However, these theories have garnered equivocal experimental support over the years and are rarely experimentally extended from predictions of plant physiology to the palatability of the tissues an herbivore experiences. We therefore examined tissue palatability, nutritional value, and defense mechanisms in multiple Dictyotalean seaweeds in two Caribbean locations, using two herbivores. Relative palatability of tissues varied greatly with algal species, grazer species, and location. Because older bases were not consistently defended, GDBH did not predict relative palatability. We could not reject ODT without intensive measures of tissue fitness value and herbivore risk, and this theory was therefore not useful in making broad predictions of tissue palatability. In testing the physiological predictions of these theories, we found the young, growing apices of these seaweeds to be generally more nutritionally valuable than the old, anchoring bases and found organic-rich apices to be more chemically deterrent, thus supporting ODT. However, the combined chemical, nutritional, and structural traits of these algae all influenced herbivore choice. As a result, these patterns of apical value and chemical defense reflected palatability of live tissues for only one of five algal species, which rendered ODT and GDBH poor predictors of relative palatability for most algae.

Thesis (M. Sc.)--University of Stellenbosch, 2002. / Includes bibliographical references. Also available via the Internet.