The biosynthesis of ABA from R-[2-¹⁴C]-MVA was demonstrated in Persea americana cv. Fuerte mesocarp and in mature seeds of Hordeum vulgare cv. Dyan and cv. Himalaya. Radioactivity from R-[2-¹⁴-C]-MVA was also incorporated into the 1',4'-trans ABA diol in Persea americana mesocarp and a possible role for this metabolite as a precursor of ABA in plants is discussed. The biosynthesis of ABA from MVA could not be demonstrated in either turgid and waterstressed Hordeum vulgare cv. Dyan, Pisum sativum cv. Black-eyed Susan and Phaseolus vulgaris cv. Top-crop or in immature seeds of Pisum sativum and Phaseolus vulgaris. (R,S,)-[2-¹⁴C]-ABA was catabolised to PA, DPA and aqueous conjugates in leaves and mature seeds of Hordeum vulgare cv. Dyan, seedlings and immature seeds of Pisum sativum and Phaseolus vulgaris and in mesocarp from ripening fruits of Persea americana. PA and DPA were identified by either microchemical methods and/or capillary GC-MS. 7'-Hydroxy ABA was characterised as a novel ABA catabolite in light-grown and etiolated leaves of Hordeum vulgare by capillary GC-MS. Circular dichroism analysis revealed that it was derived predominantly from the (R)-enantiomer of ABA. This catabolite was absent in similar studies using the dicotyledons Pisum sativum and Phaseolus vulgaris. Refeeding studies with [¹⁴C]-PA, [C]-DPA and [¹⁴C]-7'-hydroxy ABA were used to confirm the metabolic interrelationship between ABA and its catabolites in both vegetative and non-vegetative tissues from monocotyledonous and dicotyledonous species. The methyl ester of (R,S,)-ABA was hydrolysed efficiently by light-grown leaves of Hordeum vulgare. Older, vegetative tissues catabolised (R,S,)-ABA more efficiently than their younger counterparts. In contrast, small, immature seeds of Pisum sativum catabolised (R,S,)-ABA more effectively than larger, immature seeds of this species. Light did not appear to influence ABA biosynthesis but markedly enhanced ABA catabolism. Light stimulated the overall rate of ABA catabolism in both vegetative and non-vegetative tissue. Water stress reduced ABA catabolism in Hordeum vulgare leaves but had little effect on this process in Phaseolus vulgaris seedlings. Pretreatment of tissues with (R,S,)-ABA retarded the catabolism of (R,S,)-[2-¹⁴C]-ABA, negating ABA-induced conversion to PA. Cycloheximide inhibited ABA biosynthesis and catabolism but did not affect ABA conjugation. Chloramphenicol and lincomycin had little or no effect on ABA metabolism suggesting that the enzymes involved were labile and cytoplasmic in origin. Ancymidol and cycocel inhibited ABA biosynthesis while AM01618 stimulated this process. The cytokinins, benzyladenine, kinetin, isopentenyl adenine and zeatin also inhibited ABA biosynthesis. These results are discussed in relation to the possible involvement of carotenoids in ABA biosynthesis. AM01618, ancymidol andcycocel did not significantly influence the conversion of ABA to PA and DPA while cytokinins appeared to enhance this process only in vegetative tissue. The information derived from these studies was then used in attempts to develop a cell-free system from higher plants capable of metabolising ABA. A cell-free system prepared from imbibed Hordeum vulgare cv. Dyan embryos biosynthesized and catabolised ABA. This is the first demonstration of a cell-free system from non-vegetative tissue capable of metabolising ABA and could prove useful for elucidating its biosynthetic route. This cell-free system generated the terpenyl pyrophosphates IPP, FPP and GGPP from MVA. ABA was produced from both MVA and IPP in the presence of 0₂ and NADPH. The biosynthesis of ABA was stimulated by the addition of the squalene 2,3-oxide cyclase and kaurene synthetase inhibitor, AM01618 and a "cold-pool trap" of (R,S,)-ABA. Ancymidol, cycocel and cytokinins reduced incorporation of label from MVA into ABA. Similar cell-free preparations, in the absence of AM01618, converted (R,S,)-[2-¹⁴-C]-ABA into PA, 7'-hydroxy ABA and water-soluble conjugates. Although the methyl ester of (R,S,)-ABA was efficiently hydrolysed in this cell-free system no DPA was generated. The possible involvement of mixed function oxidase activity and soluble oxidases is discussed in relation to ABA metabolism. While cell-free preparations from Persea americana cv. Fuerte mesocarp and immature seeds of Pisum sativum and Phaseolus vulgaris were unable to synthesize ABA from MVA, these tissue homogenates converted ABA into more polar acidic products. PA and DPA were identified as products of ABA catabolism in extracts from immature seeds of Phaseolus vulgaris and the l',4'-cis diol of ABA in extracts from Pisum sativum immature seeds

Zea mays plants kept at ambient (ca 375ppm) and elevated CO₂ (ca 650 to 700ppm) were used to examine the possibility of a symplasmic loading, unloading and transport pathway in dark-adapted and illuminated (200μmolm⁻²sec⁻¹ ) sink and source leaves. 5,6-carboxyfluorescein diacetate was introduced into the mesophyll cells and symplasmic transfer observed 3h after application. In sink and source leaves exposed to ambient CO₂ and illuminated at 200 molm-2sec-1, the fluorescence front was observed approximately 3cm from the point of application, while in dark-adapted plants, the fluorescence front was observed approximately 1cm from the point of application. Under elevated CO₂ conditions the fluorescence front in illuminated plants appeared to transport faster moving approximately 5cm from the point of application, and in dark-adapted plants, only 3cm from the point of application. Based on the increase in 5,6-CF accumulation under elevated CO₂ conditions, the present study suggests that there was an increase in capacity for assimilate loading and transport under elevated CO₂ conditions. In source leaves, 5,6-CFDA was taken up into the mesophyll cells, loaded symplasmically and transported basipetally. In sink leaves 5,6- CFDA was taken up from basal mesophyll and after symplasmic loading, was transported acropetally where it was offloaded into the younger immature sink region. Transport in the sieve tubes was confirmed by using aniline blue, which was applied 3h after 5,6-CF transport. Aniline blue coupled with 5,6-CF transport studies showed that the sieve tubes of both cross and longitudinal veins are involved in symplasmic unloading, loading and transport processes in sink and source leaves. Apoplasmic uptake of 5,6-CFDA by cut leaves showed that after apoplasmic transport via the transpiration stream, 5,6-CFDA was offioaded to the xylem parenchyma where it was metabolically cleaved , releasing fluorescent 5,6-CF into the xylem parenchyma. Transverse sections cut after 3h of uptake were observed after 120 and 180 min suggesting that a retrieval of solutes occurs from the xylem to the xylem parenchyma, bundle sheath, phloem parenchyma and to the th in-walled sieve tubes. It was not possible to determine if the thick-walled sieve tubes were involved or if they took up 5,6-CF. Given the available data on loading and offioading of assimilates in sink and source leaves respectively, this study demonstrated that a slow symplasmic pathway exists from the mesophyll to the phloem, and that offloading from the phloem in sink leaves can occur via a symplasmic route.

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Protoplasts were isolated from tobacco and barley leaves in sucrose or mannitol using commercially available cellulases and macerozymes. Barley growth and protoplast isolation and purification conditions were optimized so that protoplasts were obtained in high yields free of unwanted debris and organelles. A technique for processing barley and tobacco protoplasts for examination by scanning electron microscopy was developed in which protoplasts seem to have maintained their structural integrity. Barley and tobacco protoplasts took up 3H-B. subtilis DNA, 125I-B. subtilis DNA or 125I-M. luteus DNA as a linear function of time (0-6 hr) and DNA concentration (0-200 μg/ml). Up to 16 pg of exogenous DNA was taken up per protoplast of which approximately one half became nuclear associated. Protoplasts were viable after the uptake as shown by standard staining and culturing techniques. Approximately 20% of the DNA taken up after typical 4 hr uptake reactions was of average gene size (5-10 x 105 daltons), and therefore of potential significance to host gene expression.

Landolphia kirkii is scrambling shrub forming an integral part of the flora along the coastal areas of north-eastern South Africa. The non-sustainable harvesting of fruit as food source, by monkeys and rural communities and the highly recalcitrant nature of their seeds threatens the continuation of the species. In addition, the ability of the plants to produce high quality rubber makes its long-term conservation highly desirable. Previously, attempts have been made to cryopreserve germplasm of L. kirkii, but no survival had been recorded at cryogenic temperatures of below -140ºC. The present study reports on the effects of rapid dehydration, chemical cryoprotectants and various cooling rates, thawing and imbibition treatments on survival of embryonic axes excised with cotyledons completely removed, as well as with 3 mm portion of each cotyledon attached, from fresh, mature, recalcitrant seeds of L. kirkii. Survival was assessed by the ability for both root and shoot development in in vitro culture, the tetrazolium test and electrolyte leakage readings. At seed shedding, embryonic axes were at the high mean water content of 2.24 g gˉ¹ (dry mass basis). All axes (with and without attached cotyledonary segments) withstood rapid (flash) drying to a water content of c. 0.28 g gˉ¹; however, the use of chemical cryoprotectants, singly or in combination, before flash-drying was lethal. Rapid cooling rates were detrimental to axes flash-dried to 0.28 g gˉ¹, with no explants showing shoot production after exposure to -196ºC and -210ºC. Ultrastructural examination revealed that decompartmentation and loss of cellular integrity were associated with viability loss after rapid cooling to cryogenic temperatures, although lipid bodies retained their morphology regardless of the thawing temperature employed. Furthermore, analysis of the lipid composition within embryos of L. kirkii revealed negligible amounts of capric and lauric acids, suggested to be the medium-chained saturated fatty acids responsible for triacylglycerol crystallisation when lipid-rich seeds are subjected to cryogenic temperatures. Hence, lipid crystallisation was not implicated in cell death following dehydration, exposure to cryogenic temperatures and subsequent thawing and rehydration. Rapid rehydration of embryonic axes of L. kirkii by direct immersion in a calcium-magnesium solution at 25ºC for 30 min (as apposed to slow rehydration on moistened filter paper or with rehydration in water) was associated with highest survival post-dehydration. Cooling at 1ºC minˉ¹ and 2ºC minˉ¹ facilitated survival of 70 and 75% respectively of axes with attached cotyledonary segments at 0.28 g gˉ¹ after exposure to - 70ºC. Viability retention of 40 and 45% were recorded when embryonic axes with attached cotyledonary segments were cooled at 14 and 15ºC minˉ¹ to temperatures below -180ºC. However, no axes excised without attached cotyledonary segments produced shoots after cryogenic exposure. The use of slow cooling rates is promising for cryopreservation of mature axes of L. kirkii, but only when excised with a portion of each cotyledon left attached. / Thesis (M.Sc.)-University of KwaZulu-Natal, Westville, 2011.

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.

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No description available.

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.