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published_or_final_version / Botany / Doctoral / Doctor of Philosophy

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published_or_final_version / Botany / Master / Master of Science

published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

published_or_final_version / Botany / Master / Master of Philosophy

By immumolabeling and electronmicroscopy, the subcellular localization of GmPAP3 has been proved to be mainly localized in mitochondria, a primary site for ROS production. Ectopic expression of GmPAP3 in transgenic tobacco BY-2 cells mimicked the protective effects exhibited by the antioxidant ascorbic acid by: (1) increase the percentage of cells with active mitochondria; (2) reduce the percentage of dead cells; and (3) lower the accumulation of ROS under NaCl and osmotic stress treatments. However, when ectopically express a truncated GmPAP3 with the mitochondria transit peptide removed, such protective effect was not observed. This provides evidences on the significance of mitochondria localization to the physiological function of GmPAP3. In addition, when GmPAP3 transgenic Arabidopsis thaliana seedlings were subjected to NaCl, osmotic stress, and oxidative stress treatments, the growth performance of the transgenic lines was significantly better than the wild type. To summarize, these studies has demonstrate that the mitochondrial localized GmPAP3 may play a role in stress tolerance by enhancing ROS scavenging. / Mitochondrion is one of the major sites for the production of reactive oxygen species (ROS). Abiotic stresses such as salinity and osmotic stress can cause oxidative damage to organelle membranes due to excess accumulation of ROS. The inducibility of GmPAP3 gene expression by salinity and oxidative stresses and the putative mitochondrial localization of GmPAP3 prompt us to further investigate the possible physiological roles of GmPAP3 under abiotic stress-induced oxidative stress. / My Ph.D. study has been focused on the detailed functional analysis of the GmPAP3 gene. The objectives of my research include: (i) to verified the subcellular localization of GmPAP3; (ii) to investigate the physiological functions of GmPAP3 under NaC1 and osmotic stress in both cellular level and in planta level. and (iii) to examine the significance of mitochondria] localization of GmPAP3 in relationship to its protective roles. / Purple acid phosphatases (PAPs) represent a diverse group of acid phosphatases in animals and plants. While the mammalian PAPs were found to be related to Reactive Oxygen Species (ROS) evolution in important physiological functions, the roles of plant PAPs remain largely unknown. / Recently, we have isolated a novel PAP-like gene (GmPAP3) from soybean that is induced by NaC1 and oxidative stresses. Subcellular localization prediction programs suggested that GmPAP3 may be a novel PAP that localized in mitochondria. Most other PAPs are extracellularly located and membrane localization of PAPs was only verified in a few cases. / by Li, Wing Yen Francisca. / "December 2009." / Adviser: Lam Hon-Ming. / Source: Dissertation Abstracts International, Volume: 72-01, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 123-134). / 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 Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Collection of the author's previous publications. Includes bibliographies.

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

D.Phil. (Biochemistry) / This thesis concerns a study of the effect of isonitrosoacetophenone on plant metabolism. Three different systems were investigated; cultured tobacco and sorghum cells as well as Arabidopsis thaliana plants, and a metabolomic approach was followed. Unlike most scientific studies, metabolomics is a discipline which is not driven by a specific hypothesis, but rather by the obtained data to add scientific insights to the topic under investigation. As such, the current study lacks a definite overarching hypothesis, but specific objectives were outlined and answered in each experimental chapter. This thesis is therefore presented as a compilation of nine chapters in which experimental/research work is described in Chapter 3- 8. It is important to note that each chapter is presented in accordance with the guidelines for the respective journal in which the corresponding manuscript was published or submitted to.