Submitted in fulfillment for the Degree of Doctor of Technology: Biotechnology, Durban University of Technology, 2010. / Ceratatheca triloba (Bernh.) E. Mey. Ex Hook. f. is one of four species that is common to the summer rainfall areas in South Africa, especially the grasslands. It is used in traditional medicine to treat stomach cramps, nausea, fever and diarrhea. Like many other plants used in the traditional medicine system, these uses are not justified through scientific investigations. This study was undertaken to characterize the functionality of the main bioactive compounds from Ceratatheca triloba. This was achieved by isolating and identifying predominant chemicals from the non polar extracts using conventional chromatography techniques. Once identified the crude extracts and identified compounds were tested for their antimicrobial, anti-oxidant activity, anti-inflammatory activity and anticancer activity. This was followed by investigating the safety of the crude extracts and the purified compounds by the Brine shrimp lethality assay, and its toxicity to HepG2 cells and the Salmonella mutagenecity test. For large scale production, we set up a protocol to produce 9, 10 anthracenedione in a cell suspension culture system. Following the complete chemical profile of the roots, stems, flowers and leaves the predominant compounds were isolated, characterized and identified by UV-Vis, IR, EI-LCMS and NMR (COSY, HMQC, HMBC and DEPT). Three anthraquinone derivatives and one steroid, 9, 10 anthracenedione, 1-hydroxy-4-methylanthraquinone, 5, 8-dimethoxy-2, 3, 10, 10a-tetrahydro-1H-phenanthrene-4, 9-dione and androst-5-ene-3, 17, 19-triol were determine by analysis of spectral data (UV, 1H NMR, 13C NMR and EI-LC-MS) 9, 10 anthracenedione and 1 hydroxy-4-methylanthraquinone showed antibacterial activity against S.aureus, M. luteus, B cureus and E. coli. Due to the synergistic effect of the individual compounds, the crude extract exhibited good potency (>500) against S.aureus and M. luteus, medium potency against E. coli. and S. typhimurium (<100) and very low potency against B cureus (<10). Although a similar trend was observed for 9, 10 anthracenedione and 1 hydroxy-4-methylanthraquinone unlike the crude extract. A very low potency against S.aureus for 9, 10 anthracenedione and a high potency for 1 hydroxy-4-methylanthraquinone. Thus 9, 10 anthracenedione is an effective drug against E. coli and S. typhimurium and 1 hydroxy-4-methylanthraquinone is effective against S.aureus and M. luteus. The crude root extracts and 9, 10 anthracenedione, 1 hydroxy-4-methylanthraquinone, 8-dimethoxy-2, 3, 10, 10a-tetrahydro-1H-phenanthrene-4 showed a ± 50% reduction of the free radicals. No anti-inflammatory activity was observed. The purified extracts showed moderate toxicity against HepG2 cells at high concentrations and no toxicity was observed against brine shimp larvae. No mutagenecity was observed with the crude extracts using the Ames test. All purified and crude extracts showed potent inhibition of the human topoisomerase II enzyme. In conclusion, although this study does not indicate any relationship to its traditional usage it provides valuable information that paves a way for commercial exploitation of C. triloba. 9, 10 anthracenedione and 1 hydroxy-4-methylanthraquinone can be used as antibacterial agents. Their antioxidative potential can be exploited for anti-cancer as in many cancers reactive oxygen species are implicated in the aetiology of these cancers. Furthermore, in this study 9, 10 anthracenedione was produced from both callus cultures and cell suspension cultures. This compound demonstrates potent anti-topoisomerase II activity which is vital to cancer treatment. Thus, the synergistic effect of 9, 10 anthracenedione and 1 hydroxy-4-methylanthraquinone as antibacterial, anti-oxidative and anti-cancer compounds demonstrate the importance of C. triloba. / Centre for Research Capacity Development ; National Research Foundation

Submitted in complete fulfillment for the Degree of Master of Technology: Biotechnology, Durban University of Technology, 2012. / Many secondary metabolites that have been extracted from medicinal plants have been used as source of clinical drugs. However, the concentration of the active metabolites in plants is generally low. An attractive alternative for producing these important secondary metabolites is via plant tissue culture technology. More particularly, the genetic transformation of a plant tissue by Agrobaterium rhizogenes has been employed for producing high yields of secondary metabolites. In a previous study, three structurally similar anthraquinones: 9,10-Anthracenedione, 1-Hydroxy-4-methylanthraquinone and 5,8-Dimethoxy-2,3,10,10a-tetrahydro-1H,4aH-phenanthrene-4,9-dione, and one steroid; Androst-5-ene-3, 17, 19-triol were isolated from the root extracts of C. triloba. The anthraquinones have shown to exhibit the anticancer mechanism which involves the inhibition of the activity of the human topoisomerase II enzyme that transforms supercoiled DNA to linear DNA. However, these anthraquinones were found in very low concentrations. Therefore, in this study we used plant cell and tissue culture systems (cell suspension, shoot and hairy root cultures) of C. triloba to increase the production of anthraquinones. Since the establishment of C. triloba in vitro plant systems required a source sterile explants, a protocol that involved the use of NaCIO was optimized for the sterilization and subsequent germination of C. triloba seeds which were micro-propagated into shoot cultures. These cultures provided a source explants for the induction of callus and hairy root cultures. The biomass of these plant cell and tissue cultures were subsequently bulked up for the extraction for anthraquinones and the yields were compared followed by fractionation and identification of the major compounds. The bioactivity of the fractions was evaluated by testing their cytotoxicity on cancer cells and anti-topoisomerase activity. The sterilization protocol that provided sterile seeds was found to be a solution of 30% NaCIO at an exposure time of 10 minutes. From the sterilized seeds shoot cultures were established on MS medium. The leaf explants of the shoot cultures were then used to induce callus cultures which subsequently were transferred to liquid medium whereby the total biomass of suspension cultures increased from 4 g to 134.18 g (wet weight). Also hairy roots cultures were established from stem explants with a low cell density inoculum of A. rhizogenes at a transformation efficiency of 73%. The growth of these hairy roots was slow in hormone free medium. This was overcomed with the use NAA and IAA which increased the xvii biomass from 1.03 g in the control culture (without hormone) to 23.91 g and 46.13 g respectively. An evaluation of the anthraquinones in the field root and hairy root, cell suspension and shoot culture extracts was carried out by using their Thin Layer Chromatography profiles and the High Performance Liquid Chromatography profiles as well as the standards, 9,10-Anthracenedione and 1-Hydroxy-4-methylanthaquinone. TLC analysis showed that the RF values of the fractions CT01 and CT02 matched the RF values of anthraquinones standards while HPLC analysis revealed that hairy root cultures supplemented with IAA (125.03 μg.mg-1) or NAA (98.25 μg. mg-1) produced a higher concentration of anthraquinones than the control culture (without hormone) (13.33 μg.mg-1), the field roots (33.51 μg. mg-1) and the shoot (3.23 μg.mg-1) and cell suspension cultures (13.17 μg.mg-1). Due to co-elution of the compounds in HPLC analysis, six fractions were isolated by Preparative Thin Layer Chromatography from the hairy root extract (obtained from the culture supplemented with NAA) and were coded as CT01, CT02, CT03, CT04, CT05 and CT06. The compounds in these fractions were identified by Electron Ionization-Liquid chromatography-Mass Spectroscopy and it was found that the hairy roots produced one acridone derivative; 5-Methoxy-2-nitro-10H-acridin-9-one, one naphthoquinone derivative; 2H-Naphto[2,3-b]pyran-5,10-dione,3,4-dihydro-2,2-dimethyl- and seven anthracenedione derivatives. These were: i) 5,8-Dimethoxy-2,3,10,10a-tetrahydro-1H,4aH-phenanthrene-4,9-dione, ii) 9,10-Anthracenedione, 2-methyl-, iii) 1-Hydroxy-4-methylanthraquinone, iv) 9,10-Anthracenedione, 2-ethyl-, v) 1,5-Diaminoanthraquinone, vi) Phenanthrene, 3,6-dimethoxy-9-methyl-, vii) 9,10-Anthracenedione, 1,4-dimethyl-. Fractions CT01 (5,8-Dimethoxy-2,3,10,10a-tetrahydro-1H,4aH-phenanthrene-4,9-dione, 9,10-Anthracenedione, 2-methyl- and 1-Hydroxy-4-methylanthraquinone) and CT02 (9,10- Anthracenedione, 2-ethyl-) were cytotoxic to the DU-145 cancer cell line at concentrations of 125 μg.mg-1 to 1000 μg.mg-1. These fractions also showed anti-topoisomerase activity as they inhibited the conversion of supercoiled DNA into linear DNA. In conclusion this is the first study that describes the transformation of C. triloba by A. rhizogenes mediated transformation and compares the production of anthraquinones in C. triloba hairy roots to the field roots, shoot and cell suspension cultures. This study has xviii indicated that hairy root cultures is a high-yielding production system for anthraquinones (5,8-Dimethoxy-2,3,10,10a-tetrahydro-1H,4aH-phenanthrene-4,9-dione, 1-Hydroxy-4-methylanthraquinone, 9,10-Anthracenedione, 2-methyl- and 9,10- Anthracenedione, 2-ethyl-) which could have the potential to be used in cancer therapy. In addition the discovery of C. triloba hairy roots having the biosynthetic capacity to synthesize five valuable anthraquinone derivatives that are not found the field roots has also been revealed. / National Research Foundation.

Thesis (MSc (Physics))--University of Stellenbosch, 2007. / Imaging and spectroscopy of laser induced chlorophyll fluorescence (LICF) are emerging as useful tools in plant physiology and agriculture since these methods allow an early detection of plant stress and transformation of plant tissue, before visual symptoms appear. Chlorophyll fluorescence is governed by photosynthetic efficiency and it depends on the plant species and physiological state. In addition, the laser induced fluorescence of chlorophyll molecules in the red and far red spectral range is also used to study basic processes and phenomena in photo-excited molecules. In the work reported here experimental setups used for laser induced chlorophyll fluorescence imaging and spectroscopy techniques were developed to investigate chlorophyll fluorescence under constant illumination and also to detect green-fluorescent protein (GFP) by looking at the chlorophyll fluorescence spectrum and image. He-Ne (wavelength 632 nm), tunable argon ion (wavelength 455 nm), and excimer (wavelength 308 nm) lasers were used as excitation sources. An Ocean Optics spectrometer was used to record the spectrum of the chlorophyll fluorescence and the variation of the chlorophyll fluorescence spectrum with time. The chlorophyll fluorescence spectrum of tobacco leaves expressing GFP was compared to that of control leaves. A charge-coupled device (CCD) camera was used to image the fluorescence from GFP expressing and control tobacco leaves to investigate the effect of GFP genes on chlorophyll fluorescence in relation to the state of the plant material. The spectral analysis technique and image processing procedures were elaborated in order to obtain better information on chlorophyll fluorescence. The results of this work show that the experimental setups and analytical procedures that were devised and used are suitable for laser induced chlorophyll fluorescence analysis. Fluorescence bleaching could be obtained from the time variation of the fluorescence spectrum, and plant expressing GFP can be distinguished from control plants by differences in the laser induced chlorophyll fluorescence.

Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Plant secondary compounds have been exploited as medicines, flavourants, incense and are widely used for different culinary purposes. Efficacy of herbal remedies is determined by the phytochemical profile which is dependent on the species, locality and growth conditions. Salvia stenophylla (Burch. ex Benth.) is a local aromatic sage growing in almost all South African biomes, with the volatile compound accumulation varying depending on season, locality and genotype. Salvia stenophylla extracts have proven anti-inflammatory, anti-plasmodial, anti-oxidant and anti-anxiety properties due to the presence of essential oils, flavonoids and caffeic acid derived phytochemicals. As a result, S. stenophylla extracts are increasingly being used for the formulation of pharmaceutical and cosmeceutical products. However, these industries largely depend on the wild populations for raw materials, and increased commercialization of sage-based natural products and medicine exerts harvesting pressure. This might reduce the sustainability of herbal medicines since there is no formal cultivation of S. stenophylla in South Africa. Medicinal plant biotechnology provides options for propagation and manipulation of herbal plant to increase synthesis of secondary compounds. The aims of this study were to develop tissue culture propagation system so as to provide an alternative to wild-harvesting, to improve essential oil quality and accumulation in S. stenophylla by applying third generation biotechnological tools to alter terpene biosynthesis via heterologous gene expression aided by Agrobacterium-gene transfer and to charaterize the resultant metabolite profiles using thin layer chromatography, gas chromatography mass spectrometry and liquid chromatography mass spectrometry. Initially, seeds were germinated in-vitro on one-tenth Murashige and Skoog medium (MS) (1962). Seeds were decontaminated by washing them in 3.5% (w/v) hypochloride and were either scarified using 70% (v/v) sulphuric acid, placed on media with smoke solution or both. Both scarification and smoke supplementation induced germination, but seeds subjected to both treatments, as a combination, showed very poor germination. Seedlings produced were used to establish an efficient tissue culture system for S. stenophylla. Shoot tips, nodal and basal explants were placed on MS media with different plant growth regulator (PGR) combinations and concentrations. The best plantlet regeneration and shoot elongation were observed on plants on medium supplemented with 5.7 μM indole acetic acid (IAA) and 8.9 μM N-6-benzyladenine (BA), producing 4-6 shoots per explant with 6.67 cm mean length. Treatment with 4.5 μM 2,4 dichlorophenoxyacetic acid (2,4 D) did not produce shoots but had the highest biomass production, which was all in the form of callus. The resultant shoots from the micropropation system had similar metabolites as wild type plant and also they had some compounds that were not detected in their wild type counterparts. The effects of nitrogen, potassium, water stress, and phytohormones on metabolite accumulation were also studied in a S. stenophylla microplant system. Plant growth regulator free half strength MS medium encouraged the most prolific growth. Increasing potassium and nitrogen concentrations correlated to the accumulation of (-)-α-bisabolol whilst changes in other major compounds like ß-bisabolene, α-muurolene, α-patchoulene, and D-limonene were insignificant. To investigate the effects of water stress on metabolite accumulation and profile, water stress was induced using sorbitol and polyethyl glycol. Reduced water availability only negatively affected rooting in-vitro whilst the chemical profile was not affected. The study also focused on terpene production in S. stenophylla by over-expressing the heterologous geranyl diphosphate synthase gene (AgGPPS2) from Abies grandis, using Agrobacterium tumefaciens (EHA105). The AgGPPS2 gene was ligated to a pCAMBIA1301 vector which was cloned in EHA105 and this in turn was used to transform S. stenophylla. Resultant transgenic plantlets exhibited normal growth characteristics but showed variation in the metabolite accumulation. GC-MS analysis showed a 6% increase in (-)-α-bisabolol accumulation whilst 3-δ-carene, α-pinene and camphor were lowered. Solvent extracts were analysed using LC-MS and these had rosmarinic acid in greater abundance in transgenic than in the wild type plants. However, the chemical profiles of the wild type and transgenic plants showed some similarities suggesting AgGPPS2 expression only alters the abundance of some secondary compounds, whilst the overall integrity of the metabolome is maintained. Unlike most metabolite engineering studies elsewhere, this is the first attempt a local South African sage has been genetically engineered to enhance terpene biosynthesis. This is crucial in an economy that is increasing becoming plant based for pharmaceutical, industrial and food needs. Further studies are required to elucidate the efficacy of transgenic and in-vitro plant-derived extracts. / AFRIKAANSE OPSOMMING: Plant sekondêre produkte word gebruik as medisinale-, geur-en reukmiddels en word ook as algemene bestandeel gebruik in kookkuns. Die doeltreffendheid van kruie middels word bepaal deur die fitochemiese profiel. Hierdie profiel is afhanklik van die spesie, ligging en groei kondisies. Salvia stenophylla (Burch. ex Benth.) is ‘n plaaslike aromatiese salie wat voorkom in byna al die biome van Suid-Afrika , waar die versameling van vlugtige verbindings varieer op grond van die seisoen, ligging en genotipe. Salvia stenophylla ekstraksies is bewys om anti-inflamatories, antiplasmodiaal, anti-oksidant en anti-angs eienskappe te besit as gevolg van die noodsaaklike olies, flavonoïede en afgeleide kaffieksuur fitochemikalieë wat die plant besit. As gevolg word S. stenophylla ekstraksies meer en meer aangewend in die ontwikkeling van farmaseutiese en kosmetiese produkte. Hierdie industrieë benodig rou plant material vanuit die natuurlike populasies wat druk op die biome sit as gevolg van die verhoogde kommersialisering van salie-gebaseerde natuurlike produkte en medisyne. Gevolglik is die volhoubaarheid van medisyne afkomstig vanaf kruie soos salie in bedwang omdat geen formele kultivasie van S. stenophylla in Suid-Afrika al in plek gestel is nie. Medisinale plant biotegnologie bied moontlikhede vir kultiveering, voortplanting en manipuleering van kruie om sodoende die produksie van sekondêre verbindings in kruie te verhoog. Die doel van hierdie studie was om ‘n weefselkultuur voortplantingssisteem te ontwikkel om sodoende ‘n alternatief te bied vir wild oesting. Die studie het gefokus op die verbetering van noodsaaklike olie kwaliteit en akkumulasie in S. stenophylla deur die gebruik van derde generasie biotegnologiese gereedskap om terpeen biosintese te modifiseer. Hierdie is bereik deur weefselkultuur en heterologiese geen uitdrukking wat aangehelp is deur Agrobacterium geen oordrag. Die gevolglike metaboliet profiele was gekarakteriseer deur gebruik te maak van dun laag chromatogragie, gas chromatografie massa spektrometrie en vloeistof chromatografie massa spektrometrie. Aanvanklik is die sade ontkiem in-vitro op een-tiende Murashige en Skoog medium (MS) (1962). Dekontaminasie van die sade is gedoen deur die sade te was in 3.5% (w/v) hipochloried waarna die sade óf geskarifideer is deur te was met 70% (v/v) swawelsuur óf op medium geplaas is wat ‘n rook oplossing bevat óf albei. Beide skarifikasie en rook aanvulling het ontkieming aangehelp, maar sade wat aan albei behandelings blootgestel is, het baie swak ontkieming getoon. Saailinge was gebruik om ‘n effektiewe weefselkultuursisteem vir S. stenophylla te produseer. Stingel punte, nodale en basale eksplante was op MS medium geplaas met verskillende kombinasies en konsentrasies van verskeie plant groei reguleerders (PGR). Die beste plant regenerasie en stingel verlenging was geobserveer met plante op medium aangevul met 5.7 μM indool asynsuur (IAS) en 8.9 μM N-6-bensieladenien (BA), waar dit 4 tot 6 stingels per plant met ‘n gemiddelde lengte van 6.67 cm gegroei het. Behandeling met 2,4 Dichlorophenoksiasynsuur (2,4 D) het nie stingels produseer nie, maar het die hoogste biomassa produksie getoon in die vorm van kallus. Die gevolglike stingels van die mikrovoortplantingsisteem het soortgelyke metaboliete as die wilde tipe plante, maar verbindings wat nie in die wilde tipe plante voorkom nie was ook ontdek. Die effek van stikstof, kalium, water stres en fitohormone op metaboliet akkumulasie was ook bestudeer in S. stenophylla mikroplantsisteem. Plant groei reguleerder vrye half sterkte MS medium het die produktiefste groei aangemoedig. Verhoging van kalium en stikstof konsentrasies het gekorreleer met die versameling van (-)-α-bisabolol en terwyl veranderinge in ander belangrike verbindings soos ß-bisaboleen, α-muuroleen, α -patchouleen en D-limoneen onbeduidend was. Om die effek van water stres op metaboliet- akkumulasie en -profiel te ondersoek was water stres geinduseer deur sorbitol en poli-etielglikol te gebruik. Die verlaging van water beskikbaarheid het slegs ‘n negatiewe invloed op in-vitro wortelvorming gehad terwyl die chemiese profiel onveranderd gebly het. Die studie het ook gefokus op die terpeen produksie in S. stenophylla deur die ooruitdrukking van die heterologiese geranieldifosfaatsintase geen (AgGPPS2) vanaf Abies grandis met behulp van Agrobacterium tumefaciens (EHA105). Die AgGPPS2 geen was geligeer in ‘n pCAMVIA1301 vektor wat geklooneer was in EHA105 en dit op sy beurt was getransformeer in S. stenophylla. Die gevolglike transgeniese plante het normale groei ten toongestel, maar het variase in metaboliet akkumulasie getoon. GC-MS analises het gewys dat ‘n 6% toename in (-)-α-bisabolol versameling terwyl 3-δ-kareen, α-pineen en kamfor verlaag het. Opgelosde ekstraksies was geanaliseer deur VC-MS en het gewys dat roosmariensuur in groter hoeveelhede in transgeniese plante as wilde tipe plante akkumuleer. Nogtans het die chemiese profiele van die twee genotipes sommige ooreenkomste getoon, wat voorstel dat AgGPPS2 uitdrukking slegs sekere sekondêre verbindings menigte male vermeerder terwyl die algehele integriteit van die metaboloom gehoue bly. In teenstelling met die meederheid studies oor metaboliet ingenieuring wat elders plaasvind, is hierdie die eerste poging om ‘n plaalike Suid-Afrikaanse salie genieties te ingenieur met die oog om die terpeen biosintese te verbeter. Hiedie is krities in ‘n ekonomie wat voortdurend vorder om plant gebaseerde produkte te maak vir farmaseuties, industrieël en voedsel behoeftes. Verdere studies is nodig om helderheid te gee oor die effektiewiteit van transgeniese en in-vitro plant afkomstige ekstraksies.

Thesis (MTech (Agriculture))--Cape Peninsula University Of Technology, 2016. / Bambara groundnut (Vigna subterranea), a dicotyledonous plant is a legume which has a potential to contribute to food security and nutrition. Protoplasts are naked plant cells lacking cell walls. Viable protoplasts are potentially totipotent. Therefore, when given the correct stimuli, each protoplast is capable, theoretically, of regenerating a new wall and undergoing repeated mitotic division to produce daughter cells from which fertile plants may be regenerated through the tissue culture process. Protoplast systems are valuable and versatile cell based systems that are useful in observing cellular processes and activities. In this study, the isolation of protoplast from the leaves of Bambara groundnut plant was extensively optimised. The factors affecting protoplast isolation considered in this study were ages of plant material, mannitol concentration, combinations and concentrations of enzymes and duration of incubation. Effects of ages of Bambara groundnut plant (4, 6, 8, 10 weeks), molarities of mannitol (0.4 M, 0.5 M. 0.6 M and 0.7 M), concentration and combination of enzymes (1%, 2% and 4% cellulase, 0.5% and 1% macerozyme and, 0.5% and 1% pectinase) at different incubation duration (4, 18, 24, 42 hours) were investigated. Overall, it can be deduced from this study that the optimal protoplast yield (4.6 ± 0.14×105ml-1/gFW) and viability (86.5 ± 2.12%) were achieved by digesting the leaves of four week old Bambara groundnut plant with 2% cellulase and 0.5 % macerozyme with 0.5M mannitol for 18 hours. Freshly isolated protoplasts were then cultured at different densities of 1 × 104 - 2 ×106 protoplasts/ml using MS in three different culture (Liquid, agar and agarose bead) methods. First cell division was observed only in liquid medium. With several attempts, no division was achieved in the agar and agarose bead methods, division also did not progress in the liquid medium and hence, plant regeneration from Bambara groundnut protoplasts could not be achieved in this study. Consequently, a further study is underway to compare the proteomic profiles of freshly isolated protoplasts and cultured protoplasts in order to gain insights into the expression of proteins that could perhaps be contributing to the difficulty in regenerating Bambara groundnut plant through protoplast technology. The present study is novel because it is the first study to optimise the various factors that could affect protoplast isolation from the leaves of Bambara groundnut and thus developed an efficient protocol for protoplasts isolation from leaves of Bambara groundnut for cell manipulation studies.

Chiao Ying Ann. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 127-140). / Abstracts in English and Chinese. / Thesis Committee --- p.I / Statement --- p.II / Abstract --- p.III / 摘要 --- p.V / Acknowledgements --- p.VII / Abbreviations --- p.IX / Abbreviation of chemicals --- p.XI / Table of Contents --- p.XII / List of figures and tables --- p.XVIII / Chapter Chapter 1. --- Literature review / Chapter 1.1 --- Significances of manipulation of nitrogen sink-source relationship --- p.1 / Chapter 1.2 --- Nitrogen sink-source relationship in plants --- p.2 / Chapter 1.3 --- Aspartate family amino acid metabolism --- p.5 / Chapter 1.3.1 --- Asparagine metabolism --- p.9 / Chapter 1.3.1.1 --- "Asparagine synthetase (AS, EC 6.3.5.4)" --- p.9 / Chapter 1.3.1.2 --- "Asparaginase (ANS, EC 3.5.1.1)" --- p.10 / Chapter 1.3.2 --- Metabolism of aspartate-derived essential amino acids --- p.10 / Chapter 1.3.2.1 --- "Aspartate kinase (AK, EC 2.7.2.4)" --- p.10 / Chapter 1.3.2.2 --- "Homoserine dehydrogenase (HSD, EC 1.1.1.3)" --- p.12 / Chapter 1.3.2.3 --- "Dihydrodipicolinate synthase (DHPS, EC 4.2.1.52)" --- p.13 / Chapter 1.3.2.4 --- "Lysine a-ketoglutarate reductase (LKR, EC 1.5.1.7)" --- p.14 / Chapter 1.3.2.5 --- "Threonine synthase (TS, EC 4.2.3.1)" --- p.15 / Chapter 1.3.2.6 --- Cystathionine γ-synthase (CGS，EC 2.5.1.48) --- p.16 / Chapter 1.3.2.7 --- Threonine deaminase (TD，EC 4.3.1.19) --- p.17 / Chapter 1.4 --- Previous attempts to manipulate seed protein quantity and quality --- p.18 / Chapter 1.4.1 --- Enhancement of amino acids transported from source to sink --- p.18 / Chapter 1.4.2 --- Redirection of metabolic pathways to increase target amino acids --- p.19 / Chapter 1.4.2.1 --- Production of aspartate by Aspartate Aminotransferase (AAT) --- p.24 / Chapter 1.4.2.2 --- Deregulation of AK to increase the common substrate for all essential aspartate family amino acids --- p.25 / Chapter 1.4.2.3 --- Inhibition of TS and enhancement of CGS to increase Met biosynthesis --- p.25 / Chapter 1.4.2.3.1 --- Inhibition of TS --- p.26 / Chapter 1.4.2.3.2 --- Enhancement of CGS --- p.26 / Chapter 1.4.2.4 --- Deregulation of DHPS and reduction of lysine catabolism to increase lysine content --- p.27 / Chapter 1.4.2.4.1 --- Deregulation of DHPS --- p.28 / Chapter 1.4.2.4.2 --- Reduction of Lys catabolism --- p.29 / Chapter 1.4.2.3.3 --- Deregulation of DHPS and reduction of LKR --- p.29 / Chapter 1.4.3 --- Expression of seed storage proteins to entrap the free amino acids --- p.30 / Chapter 1.5 --- Expression of multiple transgenes in plants --- p.34 / Chapter 1.5.1 --- Significance of multiple genes manipulation in seed quality improvement --- p.34 / Chapter 1.5.2 --- Difficulties in introduction of multiple genes into plant genomes --- p.34 / Chapter 1.5.3 --- Recent advances in introduction of multiple genes into plant genome --- p.35 / Chapter 1.6 --- Global nitrogen regulators in plants --- p.36 / Chapter 1.6.1 --- Global regulation of nitrogen metabolism --- p.36 / Chapter 1.6.2 --- General amino acid control by GCN system --- p.38 / Chapter 1.6.3 --- General amino acid control in plants --- p.39 / Chapter 1.6.4 --- GCN system in plants --- p.41 / Chapter 1.7 --- Hypothesis and specific objectives of this study --- p.42 / Chapter Chapter 2 --- Materials and methods --- p.46 / Chapter 2.1 --- Materials --- p.46 / Chapter 2.1.1 --- "Vectors, bacterial strains and plants" --- p.46 / Chapter 2.1.2 --- Chemicals and reagents used --- p.49 / Chapter 2.1.3 --- "Buffer, solution, gel and medium" --- p.49 / Chapter 2.1.4 --- Commercial kits used --- p.49 / Chapter 2.1.5 --- Equipments and facilities used --- p.49 / Chapter 2.2 --- Methods --- p.50 / Chapter 2.2.1 --- Molecular techniques --- p.50 / Chapter 2.2.1.1 --- DNA gel electrophoresis --- p.59 / Chapter 2.2.1.2 --- PCR technique --- p.50 / Chapter 2.2.1.3 --- Restriction digestion --- p.50 / Chapter 2.2.1.4 --- Ligation (for sticky-end ligation) --- p.51 / Chapter 2.2.1.5 --- DNA purification --- p.51 / Chapter 2.2.1.6 --- DNA sequencing --- p.51 / Chapter 2.2.1.7 --- Transformation of competent E. coli cells --- p.52 / Chapter 2.2.1.8 --- Preparation of plasmid from bacterial cells --- p.53 / Chapter 2.2.1.9 --- Transformation of competent Agrobacterium tumefaciens cells --- p.53 / Chapter 2.2.1.10 --- DNA extraction from plant tissue (Small-scale) --- p.54 / Chapter 2.2.1.11 --- RNA extraction from plant tissue --- p.55 / Chapter 2.2.2 --- Growth conditions of A. thaliana --- p.55 / Chapter 2.2.2.1 --- Surface sterilization of A. thaliana seeds --- p.55 / Chapter 2.2.2.2 --- Growing A. thaliana --- p.55 / Chapter 2.2.3 --- Characterization of transgenic A. thaliana with altered sink-source relationship --- p.57 / Chapter 2.2.3.1. --- Determination of amino acid contents in seeds --- p.57 / Chapter 2.2.3.2. --- Expression study of developing siliques of transgenic lines --- p.58 / Chapter 2.2.3.2.1 --- Tagging siliques of different developmental stages --- p.58 / Chapter 2.2.3.2.2 --- Extraction of silique RNA --- p.58 / Chapter 2.2.3.2.3 --- cDNA synthesis --- p.58 / Chapter 2.2.3.2.4 --- Real-time PCR --- p.59 / Chapter 2.2.4 --- Characterization of transgenic A. thaliana overexpressing GCN2 --- p.60 / Chapter 2.2.4.1 --- Gene expression study of vegetative tissues by real-time PCR --- p.60 / Chapter 2.2.4.2 --- Gene expression study of developing siliques by real-time PCR --- p.61 / Chapter 2.2.5 --- Making transgenic A. thaliana --- p.61 / Chapter 2.2.5.1 --- Cloning of multigene construct --- p.61 / Chapter 2.2.5.1.1 --- Subcloning of target genes into donor vectors --- p.61 / Chapter 2.2.5.1.1.1 --- Cloning of LRP into donor vector VS --- p.61 / Chapter 2.2.5.1.1.2 --- Cloning of dapA into donor vector SV --- p.64 / Chapter 2.2.5.1.1.3 --- Cloning of ansB into donor vector VS --- p.67 / Chapter 2.2.5.1.1.4 --- Cloning of antisense LKR fragment into donor vector SV --- p.70 / Chapter 2.2.5.1.2 --- Preparation of phosphorylated linkers --- p.73 / Chapter 2.2.5.1.3 --- Introduction of target genes to acceptor vector --- p.73 / Chapter 2.2.5.2 --- Agrobacterium-mediated transformation of A. thaliana via Vacuum infiltration --- p.78 / Chapter 2.2.5.3 --- Screening of transformants --- p.79 / Chapter Chapter 3. --- Results --- p.80 / Chapter 3.1 --- Characterization of transgenic lines with altered sink-source relationship --- p.80 / Chapter 3.1.1 --- Amino acid analysis of mature seeds of transgenic lines --- p.80 / Chapter 3.1.1.1 --- Aspartate family amino acids levels remain steady in seeds of transgenic plants --- p.83 / Chapter 3.1.1.2 --- Increase in seed Met content in Met-rich protein expressing transgenic plants --- p.85 / Chapter 3.1.1.3 --- Increase in seed Lys content in phas-dapA/phas-LRP transgenic plants --- p.87 / Chapter 3.1.2 --- Gene expression study of transgenic line --- p.89 / Chapter 3.1.2.1 --- Down-regulation of akthr1 and akthr2 in transgenic plants with altered N sink-source relationship --- p.89 / Chapter 3.1.2.2 --- Down regulation of GCN2 in transgenic plants with altered N sink-source relationship --- p.90 / Chapter 3.1.2.4 --- Expression study of other genes in aspartate family pathway --- p.90 / Chapter 3.2 --- Characterization of GCN2 overexpressing line --- p.93 / Chapter 3.2.1 --- Gene expression study of seedlings of GCN2 overexpressing plants --- p.93 / Chapter 3.2.1.1 --- Increased GCN2 expression by azaserine treatment --- p.93 / Chapter 3.2.1.2 --- Increased akthrl and akthr2 expression in GCN2 overexpressing plants --- p.96 / Chapter 3.2.1.3 --- Expression study of other genes in aspartate family pathway --- p.96 / Chapter 3.2.2 --- Gene expression study of GCN2 overexpressing plants during seed development --- p.98 / Chapter 3.3 --- Construction of transgenic plants by multigene assembly system --- p.100 / Chapter 3.3.1 --- Successful construction of recombinant plasmid carrying four target genes --- p.100 / Chapter 3.3.2 --- Transformation of A. thaliana with multigene vector --- p.103 / Chapter Chapter 4 --- Discussion --- p.104 / Chapter 4.1 --- Characterization of transgenic plants with altered sink-source relationship of aspartate family amino acid metabolism --- p.104 / Chapter 4.1.1 --- Total content of aspartate family amino acids remains steady in transgenic lines --- p.105 / Chapter 4.1.2 --- Methionine content increases in phas-PN2S and phas-MetL transgenic plants --- p.106 / Chapter 4.1.3 --- Relative lysine content increases in phas-dapA/phas-LRP transgenic plants --- p.107 / Chapter 4.1.4 --- Coordinated regulation of gene expressions of akthrl and akthr2 with GCN2 expression in transgenic plants with altered sink-source relationship --- p.109 / Chapter 4.2 --- GCN system in plants --- p.110 / Chapter 4.2.1 --- Transcriptional regulation of GCN2 in A. thaliana --- p.110 / Chapter 4.2.2 --- Regulation of amino acid biosynthesis by GCN system --- p.111 / Chapter 4.2.2.1 --- Regulation of akthrl and akthr2 by GCN2 --- p.111 / Chapter 4.2.2.2 --- GCN4 homolog in plants? --- p.112 / Chapter 4.2.2.3 --- Regulation of amino acid metabolism by GCN system --- p.113 / Chapter 4.3 --- Generation of transgenic plants with a combination of altered sink- source relationship --- p.114 / Chapter Chapter 5. --- Conclusion and Future Prospective --- p.116 / Appendix I: The major chemicals and reagents used in this research --- p.118 / "Appendix II: Major buffers, solutions and mediums used in this research" --- p.120 / Appendix III: Commercial kits used in this research --- p.125 / Appendix IV: Major equipment and facilities used in this research --- p.126 / References --- p.127

Expanding upon our understanding of plant defense is critical, particularly with the perilous threats of climate change and overpopulation to our food security, health and well-being. In this study, we focused on plant defense using two distinct approaches. First, we performed a proteomic analysis of plant exosome-like nanoparticles in order to elucidate their defense related protein cargo. Secondly, we used a wheat antimicrobial protein, puroindoline, as a fusion partner for the expression of recombinant proteins in rice endosperm. Plant exosome-like nanoparticles (ELP) were isolated from fresh tomato and subjected to mass spectrometry (MS) analysis. The ELPs were compared to fresh pressed tomato juice, and the proteins that were significantly upregulated in the ELPs were analyzed for their defensive properties. Bioinformatic analysis identified 30 proteins upregulated in the ELPs, with a majority of these being involved in plant defense. Puroindoline is a protein found in soft wheat varieties. A unique feature of this protein is the presence of a tryptophan-rich domain, which causes it to localize and tether onto starch granule surfaces; a property we are seeking to exploit for recombinant protein isolation. We hypothesized that when expressed in a pin-null crop, such as rice, puroindoline along with its fusion partner will localize and adhere to starch granule surfaces. PIN fusions were expressed in rice, and their subcellular localization was determined by immunolocalization. It was observed that PIN localizes to rice starch ii granules in vitro and in planta, and retains its starch granule binding abilities as a fusion partner. To identify other possible starch granule binding fusion partners, an anhydrous cleavage method was developed that can scan dry biological materials for associated proteins, in this case the starch granule surface. Incubation of our cleavage reagent with isolated rice starch granules yielded several cleavage products as determined through SDS-PAGE. These cleavage products were compared with previous proteomic data of trypsin digested rice starch granules.

Organophosphate hydrolase (OPH, EC 3.1.8.1) is a bacterial enzyme with a broad spectrum of potential substrates that include organophosphorus pesticides, herbicides, and chemical warfare agents. OPH has been expressed successfully in bacterial, fungal, and insect cell culture systems; however, none of these systems produces amounts of enzyme suitable for applications outside of the research laboratory. Therefore, a transgenic Zea mays L. (maize) system was developed to express OPH as an alternate to the current OPH expression systems. The bacterial gene encoding the OPH protein was optimized for transcriptional and translational expression in maize. The optimized gene was inserted into the maize genome under the control of embryo specific, endosperm specific, and constitutive plant promoters. Select transformants were analyzed for the expression of OPH. Expression was observed in the seeds of plants transformed with each of the three constructs with the highest expression observed with the embryo specific and constitutive promoter constructs. The highest OPH expressing lines of transgenic maize had expression levels higher than those reported for the E. coli expression system. OPH was purified from transgenic maize seed and analyzed for posttranslational modification and kinetic properties. OPH was observed to undergo a glycosylation event when expressed in maize that yielded at least two forms of OPH homogolous dimer. The glycosylated form of OPH bound tightly to the Concanavalin A sepharose and remained active after months of storage at room temperature. OPH activity was checked against a number of organophosphate herbicides. Enzymatic activity was observed against the herbicide Amiprophos-methyl and kinetic properties were measured. Enzymatic activity was also tested against the organophosphate Haloxon. Transgenic maize callus, leaf, and seed tissue could be screened for the presence of the optimized opd gene by enzymatic activity. Comparison of the growth of transgenic and control callus on media containing organophosphates showed that the transgenic callus was resistant to the herbicidal effects of haloxon. Transgenic plants expressing OPH were also resistant to the herbicide bensulide when compared to control plants. This indicates that OPH can be used as a screenable marker in plant systems and may be a potential scorable marker system as well.

Since the first signs of life on planet earth, organisms have had to adapt to the daily changes between light and dark, and high and low temperatures. This has led to the evolution of an endogenous time keeper, known as the circadian clock. This biological timing system helps the organism to synchronize developmental and metabolic events to the most favorable time of the day. Such a mechanism is of considerable value to plants, since they in contrast to animals cannot change location when the environment becomes unfavorable. Thus is the ability to predict coming events of central importance in a plants life. This thesis is a study of the molecular machinery behind the clockwork in the small weed plant Arabidopsis thaliana as well as its close relative perennial; the woody species Populus. We have characterized a novel component of the circadian clock, EARLY BIRD (EBI). EBI is involved in transcriptional and translational regulation, via interaction with the known post-translational clock regulator ZEITLUPE (ZTL). In Populus, we describe the role of the circadian clock and its components with respect to entry and exit of dormancy and show that gene expression of the Populus LATE ELONATED HYPOCOTYL (LHY) genes are crucial importance for freezing tolerance and thereby survival at high latitudes. Furthermore, the input to the Populus clock is mediated via the phytochrome A (phyA) photoreceptor. / Liv på jorden har alltid behövt anpassa sig till de dagliga växlingarna mellan främst ljus och mörker. Detta har lett till evolutionen av en intern, biologisk klocka, känd som den circadianska klockan, efter latinets ”circa diem”, som betyder ”ungefär en dag”. Denna inre klocka hjälper organismer att styra biologiska processer till den tid på dygnet som är mest gynnsam för deras utveckling och överlevnad. Denna mekanism är av stort värde för växter, eftersom de inte kan söka skydd på mera lämpliga platser om de blir utsatta för olika former av stress. Det gör att förmågan att förutse kommande händelser är av yttersta vikt för växter. Denna avhandling är en studie av det molekylära nätverk som styr denna biologiska klocka i den lilla örtplantan Arabidopsis thaliana (backtrav), och den besläktade träd-arten Populus (hybrid-asp). Vi har karaktäriserat en ny komponent i den circadianska klockan i Arabidopsis, EARLY BIRD (EBI). EBI är involverad i transkriptionell och translationell reglering av klockan, via interaktion med den kända post-translationella klock-regulatorn ZEITLUPE (ZTL). I Populus har vi beskrivit den interna klockan och dess roll i processer som invintring, vinterdvala och återstart av tillväxt. LATE ELONATED HYPOCOTYL (LHY) generna i Populus är avgörande för förvärv av köld-tolerans och således överlevnad på högre latituder. Dessutom har vi visat att signaler till den circadianska klockan i Populus är medierade via fotoreceptorn phytochrome A (phyA).

The major objectives of this study were to analyze the spatial and temporal distribution of starch branching enzyme (SBE) activity in wheat (Triticum aestivum L. cv. Fielder), to clone and characterize Sbe cDNA and to analyze the expression of genes encoding the SBE isoforms. An assay based on SBE-mediated unprimed synthesis of á-1,4 glucan by phosphorylase a was standardized to detect SBE activity in the protein extracts of wheat tissues. Analysis of SBE activity in the various parts of a wheat plant revealed highest activity in developing kernels. Within a kernel, the embryo axis exhibited higher SBE activity than either the endosperm or scutellum. During kernel development, SBE activity was highest in kernels 10 days post-anthesis (DPA). As the kernels matured beyond 10 DPA SBE activity decreased. Kernel dry weight, starch and amylose concentration increased rapidly until 20 DPA whereas the moisture concentration decreased during this period. Quantitative image analysis of starch granules suggested two distinct granule types (type A and type B that varied in size and shape) were present in kernels after 15 DPA. Image analysis of starch granules isolated at the different stages of kernel development suggested that granules formed immediately after anthesis developed as type A granules while the type B granules were initiated at 12 to 15 DPA. The reverse transcriptase-polymerase chain reaction (RT-PCR) was used to isolate partial cDNA that corresponded to Sbe1 and Sbe2 transcripts expressed in wheat kernels. Northern blot analysis, using the isolated Sbe1 and Sbe2 cDNA detected two transcripts of 4.9 and 2.8 kb in kernels and a single transcript of 2.8 kb in leaves and roots. The 2.8 kb Sbe transcript detected in kernels corresponded in size to that of Sbe transcripts reported from other plants. However, the 4.9 kb transcript detected in wheat kernels seems to represent a unique Sbe transcript. During kernel development, Sbe1 and Sbe2 genes were differentially expressed. Northern blot analysis also revealed that 4.9 and 2.8 kb Sbe1 transcripts were expressed in kernels of three hexaploid wheat cultivars, two tetraploid wheat cultivars and in a barley cultivar. A cDNA library constructed from 12 day old kernels was screened to isolate full length Sbe1 and Sbe2 cDNA clones. The longest clone isolated using the Sbe1 cDNA probe contained a truncated cDNA that represented the 4.9 kb transcript expressed in wheat kernels. RT-PCR analysis suggested that the 4.9 kb transcript was formed as a result of duplication of Sbe1 mRNA sequences. A full-length cDNA (2970 bp) that corresponded to the 2.8 kb Sbe2 transcript expressed in wheat kernels was also isolated. The translated region of the cDNA predicted a 823 amino acid primary product with a molecular mass of 91.4 kDa. The isolated cDNA clones were able to restore BE activity in BE deficient E. coli indicating that the isolated cDNA coded for a functional BE. Southern blot analysis suggested that the Sbe1 and Sbe2 transcripts were encoded by more than one gene.