蛋白質分泌及胞吐作用是指蛋白質在內質網(ER)中合成後前往質膜(PM) ， 隨後， 被分泌到達細胞外的過程。 而細胞分泌路徑是指蛋白質途經數個含有膜包被的細胞器後運送到细胞之外。 這些細胞器，包括內質網， 高爾基體， 反式高爾基網絡(TGN) 及質膜。 分泌蛋白分泌出细胞之外後， 在细胞外基質中進行其功能。 / 為達到這項研究的目的，我們結合了細胞、分子和生物化學上的方法， 來對蛋白質運輸路徑及參與蛋白質分泌的細胞器進行研究。首先，通過MALDI-MS/MS對煙草懸浮BY-2細胞中的原態分泌性蛋白質進行分析。第二，把已識別的分泌蛋白包括陽離子過氧化物酶同工酶40K（40K）和N1過氧化物酶（N1），透過轉基因細胞的GFP融合表達方式、及應用特異性抗體於免疫螢光和膠體金免疫電鏡上的測定來對其特性作進一步分析。 第三，總合以上的研究，煙草懸浮BY-2細胞的典型蛋白質分泌路徑次序為質網 - 高爾基體 - 反式高爾基網絡 - 質膜。 / Protein secretion or exocytosis is the process by which proteins synthesized in the endoplasmic reticulum (ER) travel to the plasma membrane (PM) for their subsequent secretion outside of the cell. The secretory pathway responsible for protein secretion contains several membrane-bounded organelles such as the ER, Golgi apparatus, trans-Golgi Network (TGN), and PM. The secreted proteins move outside of the cell and perform their functions in the extracellular matrix. / The general objective of this study was to examine the transport pathways and organelles involved in protein secretion in plant cells using a combination of cellular, molecular and biochemical approaches. First, major native secreted proteins in suspension cultures of tobacco BY-2 culture cells were identified via MALDI-MS/MS analysis. Second, the identified secreted proteins, cationic peroxidase Isozyme 40K (40K) and peroxidase N1 (N1), were further characterized by examining the GFP fusion expression of transgenic cell lines and by generating specific antibodies in immunofluorescent and immunogold electron microscope (EM) studies. Third, throughout all of these studies, a typical ER-Golgi-TGN-PM pathway was mapped for protein secretion in tobacco BY-2 cells. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Lam, Chun Kok. / "December 2012." / Thesis (M.Phil.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 79-84). / Abstracts also in Chinese. / Thesis /Assessment Committee --- p.i / Statement --- p.i / Abstract --- p.ii / 摘要 --- p.iv / Acknowledgements --- p.v / List of Abbreviations --- p.xiii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1. --- Secreted protein --- p.1 / Chapter 1.2. --- Secretory Pathway --- p.1 / Chapter 1.3. --- Protein secretion --- p.2 / Chapter 1.4. --- Plant Peroxidases --- p.3 / Chapter 1.5. --- Project Objective --- p.4 / Chapter 1.6. --- Significance --- p.4 / Chapter Chapter 2 --- Materials and Methods --- p.6 / Chapter 2.1. --- Mass spectrometry analysis --- p.6 / Chapter 2.2. --- Generation of 40K/N1-GFP construct --- p.7 / Chapter 2.2.1. --- For transient expression --- p.7 / Chapter 2.2.2. --- For stable expressing constructs --- p.7 / Chapter 2.3. --- Transient expression of 40K/N1-GFP --- p.7 / Chapter 2.4. --- Generation of transgenic cell lines --- p.8 / Chapter 2.5. --- Fluorescence microscopic screening --- p.9 / Chapter 2.6. --- Generation and characterization of antibodies specific for 40K/N1 peroxidase --- p.9 / Chapter 2.7. --- Confocal immunofluorescence studies --- p.10 / Chapter 2.8. --- (TIRF) Total internal reflection fluorescence microscopy --- p.11 / Chapter 2.9. --- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis --- p.11 / Chapter 2.10. --- Drug Treatment --- p.12 / Chapter 2.10.1. --- Dexamethasone (dex) --- p.12 / Chapter 2.10.2. --- Brefeldin A (BFA)/Concanamycin A (ConcA) --- p.12 / Chapter 2.11. --- Salt treatment (plasmolysis) --- p.13 / Chapter 2.12. --- EM (electron microscopy) study --- p.13 / Chapter Chapter 3 --- Results --- p.14 / Chapter 3.1. --- Protein secretion from tobacco BY2 cells --- p.14 / Chapter 3.2. --- Western blot analysis --- p.15 / Chapter 3.3. --- Protein expression in tobacco plant tissues --- p.15 / Chapter 3.4. --- EM labeling on the wild type BY-2 cells --- p.16 / Chapter 3.5. --- Localization in the tobacco root tip apoplast --- p.17 / Chapter 3.6. --- 40K/N1 peroxidase transient/stable cell line expression --- p.18 / Chapter 3.7. --- Time course study of 40K/N1 peroxidase-GFP cell line expression after induction --- p.18 / Chapter 3.8. --- Plasmolysis (salt treatment analysis) --- p.20 / Chapter 3.9. --- Brefeldin A (BFA) and concanamycin A (ConcA): Trafficking through the Golgi and TGN --- p.20 / Chapter 3.10. --- Examining exocytosis by total internal reflectance fluorescence (TIRF) --- p.22 / Chapter 3.11. --- Immunolabeling study --- p.23 / Chapter 3.12. --- EM study on transgenic 40K & N1 peroxidase-GFP cell lines --- p.24 / Chapter Chapter 4 --- Discussion --- p.26 / Chapter 4.1. --- Trafficking from the ER to the extracellular matrix --- p.26 / Chapter 4.2. --- Secretion through PM by exocytosis --- p.28 / Chapter 4.3. --- Time required for the secretory pathway --- p.29 / Chapter 4.4. --- Similarities of 40K and N1 --- p.30 / Chapter 4.5. --- Future perspectives --- p.30 / References --- p.79

Endocytosis is essential for eukaryotic cells. However, relatively little is known about the endocytic pathway and its molecular machinery in plant cells. In this research, a highly conserved membrane protein called secretory carrier membrane protein 1 (SCAMP) from rice (Oryza sativa) (OsSCAMP1) was employed as a tool to study the plant endocytosis. Toward this goal, I have generated polyclonal antibodies specific to SCAMP and transgenic tobacco BY-2 cell lines expressing yellow fluorescence protein (YFP)-SCAMP fusion. Confocal microscopy study showed that SCAMP localized to both plasma membrane (PM) and motile organelles. Further drug treatment and uptake studies demonstrated that these organelles are early endosomes distinct from Golgi and prevacuolar compartment (PVC), because they colocalized with the endosomal marker FM4-64. Immunogold electron microscopy study with SCAMP antibodies has identified the early endosome (EE) as a vesicular tubular membrane organelle, which resembles the structure of trans-Golgi network (TGN). These results indicate that the secretory and endocytic pathways are merged at the TGN which may serve as the sorting station for both pathways. / Since brefeldin A (BFA) induced both TGN and Golgi to form similar aggregates or BFA compartments in tobacco BY-2 cells, studies were also performed to sort out these BFA-induced compartments. Here I have demonstrated that the BFA-induced compartments derived from Golgi and TGN are physically distinct where the TGN aggregates were usually found to be surrounded by the Golgi aggregates in the same cells in both confocal immunofluorescent and immunogold EM studies. Furthermore, the internalized endosomal marker FM4-64 was found to colocalize with the TGN-derived BFA compartments but separated from the Golgi aggregates, whereas the endocytosis inhibitor tyrphostin A23 prevented TGN but not Golgi from forming BFA compartments. Therefore, the secretory Golgi organelle is functinally distinct from the endocytic TGN/EE in their responses to BFA treatment in plant cells. / The possible roles of SCAMPs in cytokinesis were also investigated. In transgenic tobacco BY-2 cells expressing the TGN/EE marker SCAMP-YFP, SCAMPs were found to be concentrated in the developing cell plate together with the internalized endosomal marker FM4-64 under confocal microscopy and this was further confirmed by immunogold electron microscopy studies with SCAMP antibodies. These results have demonstrated that SCAMPs, TGN and endocytosis are all involved in the cell plate formation during cytokinesis in plant cells. / Lam, Sheung Kwan. / Adviser: Jiang Liwen. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3266. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 176-191). / 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.

Optical cell manipulation allows precise and non-invasive exploration of mammalian cell function and physiology for medical applications. Plants, however, represent a vital component of the Earth's ecosystem and the knowledge gained from using optical tools to study plant cells can help to understand and manipulate useful agricultural and ecological traits. This thesis explores the potential of several biophotonic techniques in plant cells and tissue. Laser-mediated introduction of nucleic acids and other membrane impermeable molecules into mammalian cells is an important biophotonic technique. Optical injection presents a tool to deliver dyes and drugs for diagnostics and therapy of single cells in a sterile and interactive manner. Using femtosecond laser pulses increases the tunability of multiphoton effects and confines the damage volume, providing sub-cellular precision and high viability. Extending current femtosecond photoporation knowledge to plant cells could have sociological and environmental benefits, but presents different challenges to mammalian cells. The effects of varying optical and biological parameters on optical injection of a model plant cell line were investigated. A reconfigurable optical system was designed to allow easy switching between different spatial modes and pulse durations. Varying the medium osmolarity and optoinjectant size and type affected optoinjection efficacy, allowing optimisation of optical delivery of relevant biomolecules into plant cells. Advanced optical microscopy techniques that allow imaging beyond the diffraction limit have transformed biological studies. An ultimate goal is to merge several biophotonic techniques, creating a plant cell workstation. A step towards this was demonstrated by incorporating a fibre-based optical trap into a commercial super-resolution microscope for manipulation of cells and organelles under super-resolution. As proof-of-concept, the system was used to optically induce and quantify an immunosynapse. The capacity of the super-resolution microscope to resolve structure in plant organelles in aberrating plant tissue was critically evaluated.

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The current state of global biodiversity is one of sustained and increasing decline especially in developing countries such as South Africa, where, medicinal plants face a particular threat due the herbal medicine trade, and because in situ conservation measures have not stemmed the exploitation of these plants (Chapter 1). Furthermore, seed storage, which offers an efficient ex situ conservation technique, cannot presently be applied to many medicinal plants, either because these species produce short-lived, recalcitrant seeds, or the post-shedding behaviour of the seeds is altogether unknown. This study investigated three medicinal plant species indigenous to Kenya and South Africa: Trichilia dregeana and T. emetica, of which no population inventories exist and no wild populations were encountered locally during the course of this study; and Warburgia salutaris, one of the most highly-utilised medicinal plants in Africa, and which is currently endangered and virtually extinct in the wild in some countries such as South Africa. Aspects of post-shedding seed physiology (Chapter 2) and the responses of the germplasm of the three species to cryopreservation (Chapter 3) were studied using viability and ultrastructural assessment, with the aim of establishing methods for both short-term and the long-term preservation, via appropriate seed storage and cryopreservation, respectively. The effect of cryopreservation on genetic fidelity, a crucial aspect of germplasm conservation, was assessed by polymerase chain reaction (PCR) based random amplified polymorphic DNA (RAPDs), using W. salutaris as a case-study (Chapter 4). The seeds of all three species were found to exhibit non-orthodox behaviour. On relatively slow-drying, seeds of T. dregeana and T. emetica lost viability and ultrastructural integrity at axis water contents of 0.55 g g-l (achieved over 6 d) and 0.42 g g-l (after 3 d) respectively, while flash-drying of embryonic axes facilitated their tolerance of water contents as low as 0.16 g g-l (T. dregeana, flash-dried for 4 h) and 0.26 (T. emetica, flash-dried for 90 min). Seeds of W. salutaris were relatively more tolerant to desiccation, remaining viable at axis water contents below 0.1 g g-l when desiccated for 6 d in activated silica gel. However, excised embryonic axes flash-dried to similar water contents over 90 min lost viability and were ultrastructurally damaged beyond functionality. In terms of storability of the seeds, those of T. dregeana could be stored in the fully hydrated state for at least 5 months, provided that the quality was high and microbial contamination was curtailed at onset of storage, while those T. emetica remained in hydrated storage for about 60 d, before all seeds germinated in storage. Seeds of W salutaris, even though relatively tolerant to desiccation, were not practically storable at reduced water content, losing viability within 49 d when stored at an axis water content of 0.1 g g-l. The seeds of all three species were sensitive to chilling, suffering extensive subcellular derangement, accompanied by loss of viability, when stored at 6 °C. Thus, T. dregeana and T. emetica are typically recalcitrant, while those of W. salutaris are suggested to fit within the intermediate category of seed behaviour. For either recalcitrant or intermediate seeds, the only feasible method of long-term germpalsm preservation may be cryopreservation. Subsequent studies established that whole seeds of W. salutaris could be successfully cryopreserved following dehydration in activated silica gel. However, whole seeds of T. dregeana and T. emetica were unsuitable for cryopreservation, and excised embryonic axes were utilised. For these, in vitro germination methods, as well as cryoprotection, dehydration, freezing and thawing protocols were established. Post-thaw survival of the axes of both species was shown to depend on cryoprotection, rapid dehydration and cooling (freezing) in cryovials. Embryonic axes of T. dregeana regenerated only as callus after cryopreservation, while those of T. emetica generated into apparently normal plantlets. Thawing/rehydration in a 1:1 solution of 1 µM CaC12.2H2O and 1 mM MgC12.6H2O increased the percentage of axes surviving freezing, and that of T. emetica axes developing shoots. The effect of the extent of seed/axis development on onward growth after cryopreservation was apparent for seeds of W. salutaris and excised axes of T. emetica. The seeds of W. salutaris surviving after cryopreservation germinated into seedlings which appeared similar to those from non-cryopreserved seeds, both morphologically and in terms of growth rate. Analysis using PCR-RAPDs revealed that there were no differences in both nucleotide diversity or divergence, among populations of seedlings from seeds which had been sown fresh, or those which had either been dehydrated only, or dehydrated and cryopreserved. Thus, neither dehydration alone, nor dehydration followed by cryopreservation, was associated with any discernible genomic change. The above results are reported and discussed in detail in Chapters 2 to 4, and recommendations and future prospects outlined in Chapter 5. / Thesis (Ph.D.)-University of Natal, Durban, 2002.

The zygotic germplasm of plant species producing desiccation-sensitive seeds can be conserved in the long-term only by cryopreservation. Usually the embryonic axis is excised from the cotyledons and is used as the explant for cryopreservation as it is small and provides a large surface area:volume ratio. However the shoot of the axis of most species studied does not develop after excision, with the result that survival after cryopreservation is often recorded as callus production or simply explant enlargement and/or greening. Thus, besides explant size, factors such as in vitro regeneration techniques, physical injury induced upon excision and developmental status of the seed could compromise the success of cryopreservation. This study investigated the effect of the factors mentioned above, with particular attention to the developmental status of the seeds on explant in vitro development (section 3.1), response to dehydration (section 3.2) and cryopreservation of the desiccation-sensitive embryonic axes (section 3.3) of two species: Trichilia dregeana, T. emetica and embryos of a third, Strychnos gerrardii. For all three species, investigations were conducted on the embryonic axes/embryos excised from mature seeds immediately after fruit harvesting and from mature seeds stored under hydrated conditions for different periods (in order to achieve different degrees of development). In addition, preliminary studies were carried out on axes of T. dregeana to assess whether generation of reactive oxygen species (ROS) occurs in response to wounding upon axis excision (section 3.4). Excised embryonic axes of T. dregeana and T. emetica did not develop shoots in vitro unless the explants included attached cotyledonary segments. Following the development associated with short-term storage, however, the excised axes could develop shoots after complete cotyledon excision. The embryos from the (endospermous) seeds of S. gerrardii which included the paper-thin cotyledons, developed normally in vitro, with percentage germination increasing with seed storage time. For all three species, in vitro axis germination was promoted when activated charcoal was included in the germination medium, regardless of the developmental stage of the seeds. When dehydrated to approximately 0.3 g H2O g-1 dry mass (g g-1), embryonic axes from all three species failed to develop shoots even though a minimum of 50% produced roots in all cases. Hence, shoot production was shown to be more sensitive to desiccation than was root production. Furthermore, the sensitivity of the shoot apical meristem to desiccation was not ameliorated with seed storage for T. dregeana and T. emetica, but did decrease for S. gerrardii when seeds were stored for 6 – 8 weeks. The application of certain cryoprotectants did facilitate production of shoots after dehydration by a few axes of both Trichilia spp. For T. dregeana explants, combination of glycerol and sucrose allowed for 10% of the axes to retain the ability for shoot production after dehydration while for T. emetica explants, the combination of DMSO and glycerol (10 - 20% shoot production after dehydration) was best. The efficacy of the cryoprotectants was not influenced by storage period. The provision of cryoprotectants still needs to be tested for S. gerrardii. Survival of subsequent cryopreservation of T. dregeana and S. gerrardii explants was best achieved with rapid cooling in nitrogen slush, with the cooling procedure for T. emetica explants still to be optimized. The highest post-cryopreservation survival of T. dregeana axes was achieved when seeds had been stored for three months, while the seed storage period did not affect post-thaw survival of the axes of T. emetica or S. gerrardii. A small proportion of S. gerrardii explants only, produced shoots after cryopreservation, whereas the surviving embryonic axes of T. dregeana and T. emetica regenerated only as non-embryogenic callus. Although callus production is less desirable than successful seedling establishment, it has the potential for micropropagation if embryogenicity can be induced. Ultrastructural examination of the shoot apical meristem of T. dregeana after a 3-d recovery period, following excision, revealed considerable cellular derangement, although damage of individual organelles could not be resolved microscopically. Preliminary studies on T. dregeana involving a colorimetric assay using epinephrine, confirmed the generation of ROS in response to wounding associated with axis excision. Reactive oxygen species generated appeared to persist over prolonged periods rather than occurring only as a single oxidative burst. Hence, ROS production at the wound site could be the primary factor contributing to lack of shoot development. Axes immersed in the anti-oxidant, ascorbic acid (AsA) immediately after excision, showed lower ROS production and 10% shoot development when cultured in vitro, indicating that the oxidative burst coincident with, and after excision might be counteracted if immediate ROS production can be adequately quenched. Future investigations should aim to identify the specific ROS associated with wounding and optimize an anti-oxidant treatment(s) that will facilitate shoot development. Thus, the successful cryopreservation of the germplasm of the species tested, and others producing recalcitrant seeds, depends on a spectrum of species-specific factors, some still to be elucidated. / Thesis (M.Sc.)-University of KwaZulu-Natal, Westville, 2007.

Recalcitrant seeds are those that are shed at high water contents, are actively metabolic throughout development, when they are, and remain, desiccation-sensitive, and may also be chilling sensitive. These properties preclude their conventional storage. Because recalcitrant seeds lose viability rapidly (within a few days to several months depending on the species) the long-term storage of their germplasm is achievable only by cryopreservation [i.e. storage at very low temperatures, generally in or over, liquid nitrogen at -196°C or -150°C, respectively. Generally the seeds are far too large to be cryostored, thus explants - most conveniently, excised zygotic embryonic axes - are used. As the axes of recalcitrant seeds are highly hydrated, specific pre-treatments prior to freezing have to be applied in order to avoid lethal ice crystal formation. During the course of this study, cryopreservation protocols were developed for excised zygotic embryonic axes of two different species (Quercus robur L. and Ekebergia capensis Sparrm.). Surface-sterilisation regimes were tested for axes of both species, with the use of a 1% sodium hypochlorite solution containing a wetting agent, emerging as the best. For both species, the vigour and viability of axes, assessed by in vitro germination performance, was tested after the implementation of four different rates of desiccation (achieved by a laminar-airflow; silica-gel-; flash- and fast flash-drying). The most rapid dehydration rate (fast flash-drying) facilitated the best germination rates (vigour) for both Q. robur and E. capensis axes after 240 and 20 min, when water contents were reduced to 0.37 ±0.04 and 0.39 ±0.06 g g-1 (dmb), respectively. Consequently, fast flash-drying was used in combination with three different freezing rates (slow, intermediate and ultra-rapid cooling). While axis viability was lost after slow or intermediate cooling, good survival was obtained for each species after ultra-rapid cooling. In addition to the optimisation of culture conditions, desiccation and freezing rates, the efficacy of different thawing media (distilled water, mannitol, sucrose, full-strength MS medium supplemented with sucrose and a 1 µM calcium/1 mM magnesium solution) was also assessed. The only thawing medium that ensured normal seedling production was the Ca2+Mg2+ solution, in which electrolyte leakage was significantly curtailed. In addition to vigour and viability assessment the responses of the embryos to the various manipulations were monitored by light microscopy and/or transmission electron microscopy. The results of the various manipulations are discussed in terms of the stresses imposed on the excised axes, by each of the procedures. For axes of Q. robu, the outcome of the presently developed successful procedure and two unsuccessful protocols from the published literature are compared and contrasted. It is concluded that while in vitro germination media need to be assessed on a species basis, use of the mildest effective surface-sterilant, in conjunction with the most rapid means to achieve dehydration and cooling/freezing, are likely to underlie generally successful cryopreservation. Additionally, thawing parameters have emerged as being critically important. / Thesis (M.Sc.)-University of Natal, Durban, 2000.

The ultrastructure and physiology of the secretory glands of Dionaea muscipula Ellis (The Venus's Flytrap) have been examined in order to investigate the mechanism of obtaining nutrients practised by this plant. The dynamic changes in the ultrastructure following stimulation have been examined. Particularly prominent features are a decrease in the size of the main cell vacuole and an increase in smaller vacuoles. Important changes are also observed in the distribution of ribosomes within the secretory cells. By the use of high resolution histochemical, cytochemical, and autoradiographic methods, it is shown that the secretory hydrolases are probably stored in a sub-compartment of the vacuole, the smooth endoplasmic reticulum and the cell walls. It is shown by quantification of the cell and by autoradiography that the dictyosomes do not appear to be involved in the discharge of secretion, but rather that this probably occurs both by direct fusion of the endoplasmic reticulum with the plasmalemma and by the migration of vesicles derived from the endoplasmic reticulum directly to the cell periphery. Evidence is presented to show that, in addition to being released from a site of storage, some of the protein discharged is synthesized de novo during the cycle. This is suggested by the observed formation of numerous polysomes associated with the endoplasmic reticulum in the secretory cells and confirmed by radiolabelling techniques. A preliminary analysis of the enzymology of the secretion is presented and it is tentatively suggested that there are several peptide hydrolase activities present, possibly including a serine proteinase and a carboxypeptidase. It is shown that the secretion can hydrolyse chitin and has peroxidase activity. By following the absorption of chloride ions, it is shown that the plasmodesmata are important in the uptake of these ions. The overall architecture of the gland is considered in relation to the possible pathway of absorption of digestive products and it is suggested that the plasmo-desmata are probably the primary route for the movement of nutrients into the glands. A model is put forward, based on these observations, for the way in which the gland conducts a bi-directional flow of material. It is hypothesized that, while absorption takes place via the symplast, secretory proteins are discharged directly into the apoplast and flushed from there by hydrostatic pressure generated by the inner secretory cells. Suggestions are made for how this might be achieved.

Thesis (Ph. D.)--Ohio University, March, 2004. / Includes bibliographical references (leaves 158-181).