Both structural and immunogold EM studies have also been carried to identify the storage PVCs in developing tobacco seeds and mungbean cotyledons. Biochemically, storage PVCs in both developing tobacco seeds and mungbean cotyledons were labeled by VSRAt-1, S2 (globulin-like proteins), BiP and DIP antibodies. Structurally, storage PVCs in developing tobacco seeds, sized about 200 nm diameter, contain wavy limiting membrane with electron-dense core and periphery translucent outer layer with internal vesicles. In contrast, storage PVCs in mungbean cotyledon, sized about 400 nm diameter, contain electron-dense and translucent area located adjacent to each other. / Further drug treatments studies demonstrated that the lytic PVCs/MVBs in tobacco BY-2 cells were distinct from the storage PVCs in seed cells. BFA and wortmannin treatments respectively caused the lytic PVCs in tobacco BY-2 cells to become aggregate and vacuolated, whereas the storage PVCs in seed cells remained unchanged in response to treatments of these drugs. Therefore, the storage PVCs in developing seeds are biochemically distinct from the lytic PVCs in tobacco BY-2 cells. / Plant cells contain both lytic vacuole and protein storage vacuole. Prevacuolar compartments (PVCs) are membrane-bounded organelles mediating protein trafficking between the Golgi apparatus and vacuoles in the plant secretory pathways. Multivesicular bodies (MVBs) have recently identified as the lytic PVCs in tobacco BY-2 cells. However, little is known about the dynamics of the lytic PVCs. In addition, the existence and identity of PVCs for protein storage vacuole (termed storage PVCs in this study) remain unknown. / This thesis research addressed two important biological questions: the dynamics of the lytic PVCs and the identity of the storage PVCs in plant cells. Towards this goal, I have demonstrated that the Golgi apparatus and the lytic PVCs, marked by YFP fusion reporters in transgenic tobacco BY-2 cells, have different sensitivity to brefeldin A (BFA) treatments. BFA at high concentrations (50 to 100 microg/mL) caused both YFP-marked Golgi stacks and lytic PVCs to form aggregates in a dosage-dependent and time-dependent manner. Confocal immunofluorescence and immunogold EM studies with specific organelle antibody markers further demonstrated that BFA-induced aggregates derived from the lytic PVCs were distinct from but physically associated with the Golgi aggregates. Thus, the BFA effects on the secretory organelles have been extended to the lytic PVCs. / Tse, Yu Chung. / "September 2007." / Source: Dissertation Abstracts International, Volume: 69-08, Section: B, page: 4521. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (p. 156-164). / 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, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Lo Sze Wan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 108-115). / Abstracts in English and Chinese. / Thesis committee --- p.ii / Statement --- p.iii / Acknowledgements --- p.iv / Abstract (in English) --- p.vi / Abstract (in Chinese) --- p.viii / Table of content --- p.x / List of tables --- p.xv / List of figures --- p.xvi / List of abbreviations --- p.xix / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- The secretory pathway --- p.2 / Chapter 1.1.1 --- Endoplasmic reticulum --- p.2 / Chapter 1.1.2 --- Golgi complex --- p.3 / Chapter 1.1.3 --- Vacuoles --- p.3 / Chapter 1.1.4 --- Prevacuolar compartment --- p.4 / Chapter 1.2 --- The secretory pathway in plant cells --- p.5 / Chapter 1.2.1 --- The secretory pathway in yeast and mammalian cells --- p.7 / Chapter 1.2.2 --- The lytic pathway in plant cells --- p.8 / Chapter 1.2.3 --- The protein storage vacuole pathway in plant cells --- p.10 / Chapter 1.3 --- Dynamic studies between organelles --- p.12 / Chapter 1.4 --- Objectives of this thesis research --- p.13 / Chapter Chapter 2 --- Development of Transgenic Cell Lines Expressing PVC and Golgi Markers --- p.15 / Chapter 2.1 --- Introduction --- p.16 / Chapter 2.1.1 --- Putative PVC marker --- p.16 / Chapter 2.1.2 --- Golgi marker --- p.17 / Chapter 2.1.3 --- Dynamic studies --- p.18 / Chapter 2.1.4 --- Cell culture study --- p.18 / Chapter 2.2 --- Materials and Methods --- p.21 / Chapter 2.2.1 --- Plant material --- p.21 / Chapter 2.2.2 --- Construction of fusion reporters --- p.22 / Chapter 2.2.2.1 --- Cloning materials --- p.22 / Chapter 2.2.2.2 --- Vector preparation --- p.22 / Chapter 2.2.2.3 --- Cloning of pGFP-BP-80K and pGFP-BP-80H --- p.24 / Chapter 2.2.2.4 --- Cloning of pGFP-α-TIPH --- p.28 / Chapter 2.2.3 --- Transformation of tobacco BY-2 cells --- p.30 / Chapter 2.2.3.1 --- Agrobacterium transformation --- p.30 / Chapter 2.2.3.2 --- BY-2 cell transformation --- p.30 / Chapter 2.2.4 --- Screening of transgenic BY-2 cells --- p.31 / Chapter 2.2.4.1 --- Killing curve study --- p.31 / Chapter 2.2.4.2 --- Antibiotic selection --- p.32 / Chapter 2.2.4.3 --- Fluorescence microscopy screening (For single-construct cell lines) --- p.33 / Chapter 2.2.4.4 --- Confocal laser scanning microscopy (CLSM) screening (For double-construct cell lines) --- p.33 / Chapter 2.2.5 --- Detection of fluorescent protein expression --- p.35 / Chapter 2.2.5.1 --- Confocal imaging --- p.35 / Chapter 2.2.5.2 --- Protein extraction and subcellular fractionation --- p.36 / Chapter 2.2.5.3 --- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) --- p.36 / Chapter 2.2.5.4 --- Western blot analysis --- p.37 / Chapter 2.2.5.5 --- Cell culture study --- p.37 / Chapter 2.3 --- Results --- p.39 / Chapter 2.3.1 --- Hygromycin concentration at 50 mg/L was optimal for selection --- p.39 / Chapter 2.3.2 --- Lower transformation efficiency for double-construct cell lines --- p.40 / Chapter 2.3.3 --- Screening of transgenic cell lines --- p.41 / Chapter 2.3.4 --- Both pGFP-BP-80K and pGFP- a -TIPH expressed as punctate signals in single-construct cell lines --- p.45 / Chapter 2.3.5 --- Weak punctate or diffuse signals were detected from PVC markers in double-construct cell lines --- p.47 / Chapter 2.3.6 --- GFP reporters were successfully transformed into BY-2 cells --- p.51 / Chapter 2.3.7 --- Profiles of fluorescent signals in transgenic cells during cell culture --- p.53 / Chapter 2.4 --- Discussion --- p.59 / Chapter 2.4.1 --- Abnormal cell growth might be due to high selection pressure --- p.59 / Chapter 2.4.2 --- Double-construct cell lines developed were not yet suitable for further study --- p.60 / Chapter 2.4.3 --- Single-construct cell lines expressing putative PVC markers were developed --- p.62 / Chapter 2.4.4 --- 2- to 3-day-old cells were more suitable for subsequent studies --- p.63 / Chapter Chapter 3 --- Characterization of Transgenic Tobacco BY-2 Cell Expressing Reporters for Distinct Prevacuolar Compartments --- p.66 / Chapter 3.1 --- Introduction --- p.67 / Chapter 3.1.1 --- Wortmannin --- p.69 / Chapter 3.1.2 --- Brefeldin A --- p.70 / Chapter 3.1.3 --- FM4-64 --- p.71 / Chapter 3.2 --- Materials and Methods --- p.73 / Chapter 3.2.1 --- Plant material --- p.73 / Chapter 3.2.2 --- Confocal immunofluorescence studies --- p.73 / Chapter 3.2.3 --- Drug treatment studies --- p.74 / Chapter 3.2.3.1 --- Wortmannin treatment --- p.74 / Chapter 3.2.3.2 --- BFA treatment --- p.75 / Chapter 3.2.4 --- FM4-64 uptake study --- p.76 / Chapter 3.3 --- Results --- p.78 / Chapter 3.3.1 --- Organelles marked by GFP- a -TIP CT reporters did not localize at Golgi compartment --- p.78 / Chapter 3.3.2 --- Wortmannin induced GFP- a -TIP marked organelles to vacuolated --- p.81 / Chapter 3.3.3 --- GFP- a -TIP CT reporters partially colocalized with VSRin wortmannin-treated cells --- p.83 / Chapter 3.3.4 --- BFA induced GFP- a -TIP marked organelles to form BFA- induced compartments --- p.88 / Chapter 3.3.5 --- GFP-α -TIP CT reporter colocalized with internalized FM4-64 --- p.91 / Chapter 3.4 --- Discussion --- p.94 / Chapter 3.4.1 --- GFP- α -TIP CT reporter was a putative PVC marker --- p.94 / Chapter 3.4.2 --- GFP- a -TIP marked organelles behaved differently from lytic PVCs --- p.95 / Chapter 3.4.3 --- GFP- a -TIP marked organelles were not lytic PVCs --- p.96 / Chapter 3.4.4 --- FM4-64 uptake study reveals a new PVC marker --- p.98 / Chapter Chapter 4 --- Summary and Future Prospects --- p.100 / Chapter 4.1 --- Summary --- p.101 / Chapter 4.1.1 --- Hypothesis --- p.101 / Chapter 4.1.2 --- Development of transgenic cell lines --- p.102 / Chapter 4.1.3 --- Characterization of organelles marked by GFP- a -TIP CT reporter --- p.103 / Chapter 4.2 --- Future prospects --- p.106 / Reference --- p.108

No description available.

Thesis (Ph. D.)--University of Queensland, 2002. / Includes bibliographical references.

The tonoplast H$ sp+$-ATPase of Beta vulgaris L. was partially purified by Triton X-100 solubilization and Sepharose 4B chromatography resulting in the enrichment of two polypeptides (57 and 67 kDa). Kinetic analysis of ($ alpha$-$ sp{32}$P) BzATP labeling identified the 57 kDa polypeptide as a nucleotide-binding subunit with a possible regulatory function. In addition, ($ sp{14}$C) DCCD-labeling identified a 16 kDa polypeptide as a putative transmembrane proton channel. It is concluded that the tonoplast H$ sp+$-ATPase is a multimer composed of at least three polypeptides. / Anti-57 and anti-67 kDa sera reacted with polypeptides of the corresponding size in bovine chromaffin granules, bovine clathrin-coated vesicles, and yeast vacuolar membranes, suggesting common structural features and common ancestry for endomembrane H$ sp+$-ATPases of different organelles and different phyla. Anti-57 serum was used to isolate a cDNA encoding the corresponding subunit from Arabidopsis. Protein sequence analysis revealed homologies between endomembrane, F$ sb0$F$ sb1$ and archaebacterial ATPases, suggesting that these different classes of ATPases have evolved from a common ancestor.

Thesis (Ph. D.)--Carleton University, 1992. / Also available in electronic format on the Internet.

Cheung Siu Chung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 86-91). / Abstracts in English and Chinese. / Thesis Committee --- p.ii / Statement --- p.iii / Acknowledgements --- p.iv / Abstract --- p.v / 摘要 --- p.vii / Table of Contents --- p.viii / List of Tables --- p.xiii / List of Figures --- p.xiv / Lists of Abbreviations --- p.xvii / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- The plant secretory pathways --- p.2 / Chapter 1.1.1 --- Three different protein sorting pathways to plant vacuoles --- p.3 / Chapter 1.1.2 --- VSD and VSR --- p.6 / Chapter 1.2 --- Prevacuolar compartments --- p.7 / Chapter 1.2.1 --- Lytic PVC --- p.7 / Chapter 1.2.2 --- BP-80 reporter as a lytic PVC marker --- p.8 / Chapter 1.2.3 --- PVC of PSV --- p.9 / Chapter 1.2.4 --- α-TIP CT reporter as a PVC of PSV marker --- p.10 / Chapter 1.3 --- Project objectives --- p.11 / Chapter Chapter 2 --- Development of Transgenic Tobacco BY-2 Cell Lines Expressing Fluorescent Reporters for Golgi and Prevacuolar Compartments / Chapter 2.1 --- Introduction --- p.13 / Chapter 2.2 --- Materials and Methods --- p.15 / Chapter 2.2.1 --- Chemicals --- p.15 / Chapter 2.2.2 --- Oligonucleotides: Primers and Adapters --- p.15 / Chapter 2.2.3 --- Bacterial Strains --- p.17 / Chapter 2.2.4 --- "Preparation of single-reporter constructs (GONST1 -CFP, CFP-BP-80 and CFP-a-TIP CT reporters)" --- p.17 / Chapter 2.2.4.1 --- "Cloning of pGONSTl-CFPK, a Golgi marker" --- p.17 / Chapter 2.2.4.2 --- "Cloning of pCFP-BP-80K, a lytic PVC marker" --- p.20 / Chapter 2.2.4.3 --- "Cloning of pCFP-α-TIP CTK, a putative marker for PVC of PSV" --- p.22 / Chapter 2.2.5 --- "Preparation of double-reporter constructs (CFP-BP-80-GONST1 - YFP, CFP-α-TIP CT-GONST1-YFP, CFP-BP-80-YFP-α-TIP CT and CFP-α-TIP CT-YFP-BP-80 reporters)" --- p.24 / Chapter 2.2.5.1 --- Insertion ofAdapter-XH to pCFP-BP-80K and pCFP-α-TIP CTK --- p.24 / Chapter 2.2.5.2 --- "Cloning of pCFP-BP-80-GONST 1 -YFPK, pCFP-α-TIP CT- GONST 1-YFPK, pCFP-BP-80-YFP-α-TIP CTK and pCFP- α-TIP CT-YFP-BP-80K" --- p.26 / Chapter 2.2.6 --- Agrobacterium electroporation --- p.30 / Chapter 2.2.7 --- Agrobacterium-mediated transformation of tobacco BY-2 cells --- p.30 / Chapter 2.2.8 --- Selection and screening of transformed BY-2 cells --- p.31 / Chapter 2.2.8.1 --- Antibiotic selection --- p.31 / Chapter 2.2.8.2 --- Fluorescence microscopic screening --- p.31 / Chapter 2.2.9 --- Detection of CFP and YFP reporter genes and their expressions --- p.32 / Chapter 2.2.9.1 --- CTAB genomic DNA extraction --- p.32 / Chapter 2.2.9.2 --- PCR test for CFP (and YFP) transgene in genomic DNA --- p.33 / Chapter 2.2.9.3 --- Subcellular fractionation and protein extraction --- p.33 / Chapter 2.2.9.4 --- Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis --- p.34 / Chapter 2.2.9.5 --- Confocal microscopic study --- p.35 / Chapter 2.3 --- Results --- p.36 / Chapter 2.3.1 --- Establishment of kanamycin-resistant BY-2 cells expressing CFP (and YFP) reporters --- p.36 / Chapter 2.3.2 --- Fluorescence microscopic screening of transgenic BY-2 cell lines --- p.37 / Chapter 2.3.3 --- CFP (and YFP) reporter was successfully integrated into transgenic BY-2 cell genome --- p.41 / Chapter 2.3.4 --- CFP (and YFP) reporter was expressed in transgenic BY-2 cell lines --- p.44 / Chapter 2.3.5 --- Punctate CFP (and YFP) signals were detected in transgenic BY-2 cell lines expressing single (or double) reporter --- p.48 / Chapter 2.4 --- Discussion --- p.53 / Chapter 2.4.1 --- "Transgenic BY-2 cell lines expressing single reporter marking Golgi, lytic PVC and putative PVC of PSV have been developed" --- p.53 / Chapter 2.4.2 --- "Golgi, lytic PVC and putative PVC of PSV were separate and distinct organelles" --- p.53 / Chapter 2.4.3 --- Transgenic BY-2 cell lines expressing double reporter were not yet suitable for subsequent study --- p.55 / Chapter Chapter 3 --- Characterization of Transgenic Tobacco BY-2 Cell Lines Expressing Fluorescent Reporters for Prevacuolar Compartments / Chapter 3.1 --- Introduction --- p.58 / Chapter 3.2 --- Materials and Methods --- p.60 / Chapter 3.2.1 --- Confocal immunofluorescence study --- p.60 / Chapter 3.2.2 --- Drug treatment study (for single-reporter transgenic tobacco BY-2 cell line) --- p.62 / Chapter 3.2.2.1 --- Wortmannin treatment --- p.62 / Chapter 3.2.2.1.1 --- Dosage effect --- p.62 / Chapter 3.2.2.1.2 --- Time-course study --- p.62 / Chapter 3.2.2.2 --- Brefeldin A treatment --- p.63 / Chapter 3.2.2.1.1 --- Dosage effect --- p.63 / Chapter 3.2.2.1.2 --- Time-course study --- p.63 / Chapter 3.2.3 --- Drug treatment study (for double-reporter transgenic tobacco BY-2 cell line) --- p.64 / Chapter 3.2.3.1 --- Wortmannin treatment --- p.64 / Chapter 3.2.3.2 --- Brefeldin A treatment --- p.64 / Chapter 3.3 --- Results --- p.65 / Chapter 3.3.1 --- CFP-α-TIP CT reporter-marked compartment was not Golgi apparatus --- p.65 / Chapter 3.3.2 --- Wortmannin induced CFP-α-TIP CT reporter-marked compartment to vacuolate --- p.69 / Chapter 3.3.3 --- BFA induced CFP-α-TIP CT reporter-marked compartment to form aggregates --- p.72 / Chapter 3.3.4 --- Wortmannin and BFA treatment caused lytic PVC to form small vacuole and Golgi to form aggregate respectively in transgenic BY-2 cell lines expressing double-reporter --- p.75 / Chapter 3.4 --- Discussion --- p.77 / Chapter 3.4.1 --- CFP-α-TIP CT reporter-marked compartment was not Golgi apparatus --- p.77 / Chapter 3.4.2 --- CFP-α-TIP CT reporter-marked compartment was not lytic PVC --- p.77 / Chapter 3.4.3 --- Transgenic BY-2 cell lines expressing double reporter could successfully mark two compartments simultaneously in the same cell --- p.78 / Chapter Chapter 4 --- Summary and Future Prospects / Chapter 4.1 --- Summary --- p.80 / Chapter 4.1.1 --- Hypothesis --- p.80 / Chapter 4.1.2 --- Development of transgenic tobacco BY-2 cell lines --- p.81 / Chapter 4.1.3 --- Characterization of α-TIP CT reporter-marked PVC-like compartment --- p.82 / Chapter 4.2 --- Conclusions --- p.84 / Chapter 4.3 --- Future prospects --- p.85 / References --- p.86

Wang, Hao. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 83-93). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Cui, Yong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 73-84). / Abstracts in English and Chinese. / Thesis/Assessment Committee --- p.ii / Statement --- p.iii / Acknowledgements --- p.iv / Abstract --- p.v / 摘要 --- p.vi / Table of Contents --- p.vii / List of Figures --- p.xi / List of Supplemental Tables --- p.xiii / List of Abbreviations --- p.xiii / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- The plant secretory and endocytosis pathways --- p.2 / Chapter 1.2 --- Rab proteins --- p.4 / Chapter 1.2.1 --- Overview of the small GTPases --- p.4 / Chapter 1.2.2 --- Function of Rab proteins in Arabidopsis --- p.6 / Chapter 1.3 --- Prevacuolar compartments --- p.9 / Chapter 1.3.1 --- PVCs in mammalian and yeast cells --- p.9 / Chapter 1.3.2 --- PVCs in plant cells --- p.9 / Chapter 1.4 --- Vacuolar Sorting Receptors --- p.10 / Chapter 1.5 --- Project objectives --- p.10 / Chapter CHAPTER 2 --- Early and Late Prevacuolar Compartments in Arabidopsis thaliana Cells --- p.12 / Chapter 2.1 --- Introduction --- p.13 / Chapter 2.2 --- MATERIALS AND METHODS --- p.19 / Chapter 2.2.1 --- Plasmid Construction --- p.19 / Chapter 2.2.2 --- Plants materials and growth conditions --- p.19 / Chapter 2.2.3 --- Transient Expression of Arabidopsis suspension cultured cells --- p.20 / Chapter 2.2.4 --- Confocal imaging studies --- p.21 / Chapter 2.3 --- RESULTS --- p.23 / Chapter 2.3.1 --- Organelle markers serve as a tool to study biogenesis and turnover of PVCs --- p.23 / Chapter 2.3.2 --- AtRab5 and AtRab7 proteins show distinct but closely associated patterns in the PVC-to-Vacuole pathway --- p.26 / Chapter 2.3.3 --- AtRab5 and AtRab7 proteins localize on the distinct organellein Arabidopsis thaliana protoplasts --- p.32 / Chapter 2.3.4 --- AtRab5 proteins are closely associated with AtRab7 proteins --- p.35 / Chapter 2.3.5 --- ARA7-Q69L proteins recruit a SNARE complex onto the enlarged PVCs --- p.37 / Chapter 2.4 --- Discussion --- p.40 / Chapter 2.4.1 --- PVC dynamics in Arabidopsis cells --- p.40 / Chapter 2.4.2 --- AtVSR and its point mutation form defined different stages of PVCs in Arabidopsis thaliana protoplasts --- p.41 / Chapter 2.4.3 --- AtRab7 proteins localized on the tonoplast and newly defined late PVCs --- p.41 / Chapter CHAPTER 3 --- AtRab7 proteins play a critical role in mediating vacuolar trafficking in Arabidopsis thaliana Cells --- p.43 / Chapter 3.1 --- Introduction --- p.44 / Chapter 3.2 --- MATERIALS AND METHODS --- p.45 / Chapter 3.2.1 --- Plasmid Construction --- p.45 / Chapter 3.2.2 --- Plants materials and growth conditions --- p.45 / Chapter 3.2.3 --- Transient Expression of Arabidopsis suspension cultured cells --- p.45 / Chapter 3.2.4 --- Confocal imaging studies --- p.45 / Chapter 3.2.5 --- Drug treatment --- p.46 / Chapter 3.3 --- RESULTS --- p.48 / Chapter 3.3.1 --- Mutations at GTP-binding motifs and the effector domain affect the subcellular localization of AtRabG3e --- p.48 / Chapter 3.3.2 --- "AtRabG3e-T22N induced vacuolation of YFP-ARA7 marked PVCs, which remains separated from ER, Golgi and TGN but colocalizes with early PVC markers" --- p.51 / Chapter 3.3.3 --- AtRab7-T22N inhibits vacuolar trafficking of cargo proteins --- p.54 / Chapter 3.3.4 --- Wortmannin-induced vacuolation of late PVCs in transgenic plants --- p.57 / Chapter 3.4 --- Discussion --- p.59 / Chapter 3.4.1 --- The proper targeting of AtRab7 proteins --- p.59 / Chapter 3.4.2 --- AtRab5 and AtRab7 proteins are essential for vacuolar protein trafficking --- p.59 / Chapter CHAPTER 4 --- Summary and Future Perspectives --- p.61 / Chapter 4.1 --- Summary --- p.62 / Chapter 4.1.1 --- Localization of AtRab5 and AtRab7 proteins on different populations of PVCs --- p.62 / Chapter 4.1.2 --- Functions of AtRab7 proteins in Arabidopsis cells --- p.63 / Chapter 4.1.3 --- The Rab conversion maturation model --- p.63 / Chapter 4.2 --- Future perspectives --- p.64 / References --- p.73

Liu, Yang. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 99-104). / Abstracts in English and Chinese. / Table of Contents / 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.xi / List of Figures --- p.xii / List of Abbreviations --- p.xiv / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1. --- The plant secretory pathway --- p.2 / Chapter 2. --- Vesicular pathways in plant cells --- p.3 / Chapter 3. --- Prevacuolar Compartments --- p.6 / Chapter 4. --- Vacuolar sorting receptors (VSRs) --- p.7 / Chapter 5. --- BP-80 & Arabidopsis VSR Proteins --- p.8 / Chapter 6. --- Research Objectives --- p.9 / Chapter Chapter 2 --- Development and Expression of GFP-OsVSRs Fusion Reporters in Tobacco BY-2 and Rice Suspension Cultured Cells --- p.11 / Chapter 1. --- Introduction --- p.12 / Chapter 2. --- Materials and methods --- p.14 / Chapter 2.1 --- Construction of GFP-OsVSR chimeric reporters --- p.14 / Chapter 2.2 --- Construction of Golgi Marker and PVC marker --- p.18 / Chapter 2.3 --- Agrobacterium electroporation --- p.27 / Chapter 2.4 --- Transformation of tobacco BY-2 cells --- p.27 / Chapter 2.5 --- Transient expression of GFP-OsVSRs in protoplasts of tobacco BY-2 cells and rice suspension cultured cells --- p.28 / Chapter 2.6 --- Screening of transgenic BY-2 cells expressing GFP-OsVSR reporters --- p.30 / Chapter 2.7 --- Chemicals --- p.33 / Chapter 3. --- Results --- p.34 / Chapter 3.1 --- Study subcellular localization of OsVSR proteins with chimeric GFP-OsVSR reporters --- p.34 / Chapter 3.2 --- Generation of transgenic tobacco BY-2 cell lines expressing GFP-OsVSR reporter constructs --- p.38 / Chapter 3.3 --- Transient expression of GFP-OsVSR reporters in tobacco BY-2 and rice cell protoplasts --- p.40 / Chapter 4. --- Conclusion --- p.42 / Chapter Chapter 3 --- Subcellular Localization of GFP-OsVSR Fusion Reporters in Tobacco BY-2 and Rice Suspension Cultured Cells --- p.43 / Chapter 1. --- Introduction --- p.44 / Chapter 2. --- Materials and methods --- p.45 / Chapter 2.1 --- Confocal immunofluorescence studies --- p.45 / Chapter 2.2 --- Antibodies --- p.46 / Chapter 2.3 --- Wortmannin and BFA drug treatment --- p.46 / Chapter 2.4 --- Electron microscopy of resin-embedded cells --- p.47 / Chapter 2.5 --- Two-dimensional (2-D) gel analysis --- p.47 / Chapter 3 --- Results --- p.49 / Chapter 3.1 --- "Distinct subcellular localizations of GFP-OsVSRl, GFP-OsVSR2 and GFP-OsVSR4 reporters in transgenic BY-2 cell lines" --- p.49 / Chapter 3.2 --- "Subcellular localizations of GFP-OsVSRl, GFP-OsVSR2 and GFP-OsVSR4 in protoplasts of rice suspension cultured cells" --- p.58 / Chapter 3.3 --- Distinct localizations of GFP-OsVSR3 and GFP-OsVSR5 --- p.62 / Chapter 3.4 --- Immunogold EM localization of VSR proteins in rice suspension cultured cells --- p.65 / Chapter 3.5 --- 2-D western blot detection of VSR proteins in various plants --- p.68 / Chapter 4. --- Conclusions --- p.70 / Chapter Chapter 4 --- Summary and Discussion --- p.71 / Chapter 1. --- The significance of this study --- p.72 / Chapter 2. --- The hypothesis in this study --- p.73 / Chapter 3. --- A reporter system to study subcellular localization of OsVSR proteins in both tobacco BY-2 cells and rice suspension cultured cells --- p.75 / Chapter 4. --- Transiently expression of GFP-OsVSR reporters in BY-2 and rice protoplasts ..… --- p.76 / Chapter 5. --- Distinct PVC and Golgi localizations of GFP-OsVSR fusions --- p.77 / Chapter 6. --- Summary and future perspective --- p.78 / Appendix --- p.79 / Characterization of A Novel Rice Protein --- p.79 / Chapter 1. --- Introduction --- p.80 / Chapter 2. --- Materials and methods --- p.83 / Chapter 2.1 --- Antibodies --- p.83 / Chapter 2.2 --- Sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) and western blot analysis of proteins from different plant species --- p.84 / Chapter 2.3 --- Sucrose gradient fractionation with protein F antibody --- p.84 / Chapter 2.4 --- Confocal immunofluorescence studies --- p.85 / Chapter 2.5 --- Affinity purification of Protein F by its antibody --- p.85 / Chapter 3. --- Results --- p.87 / Chapter 3.1 --- Protein F is presented in different plant species --- p.87 / Chapter 3.2 --- Protein F is an integral membrane protein --- p.89 / Chapter 3.3 --- Subcelluar localization of Protein F --- p.91 / Chapter 3.4 --- Affinity purification of Protein F for identification --- p.95 / Chapter 4. --- Summary and future perspectives --- p.97 / Chapter 4.1 --- Summary --- p.97 / Chapter 4.2 --- Future Perspectives --- p.97 / References --- p.99