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Ricinoleic acid (12-hydroxyoleic acid) has a wide range of industrial uses. Its current source is the castor plant (Ricinus communis ) which contains up to 90% ricinoleic acid in its seed storage lipid. R. communis has significant limitations as an agricultural source of ricinoleic acid; it produces potent allergens, requires hand harvesting and only grows effectively in limited climatic zones. A solution to these limitations is to identify the components of the seed storage lipid biosynthetic pathway and transfer them to an agronomic host such as oil seed rape. The developing seed ER is the ma jor compartment of storage oil biosynthesis, whereas during germination these storage compounds are broken down to support the germinating seedling. In this study, a quantitative gel-based proteomic approach has been used to identify the proteins elevated in the developing seed ER compared to the germinating seed ER. On identification of the protein components of storage lipid biosynthesis in the developing seed, their influence on oil quality will be assessed. The use of yeast may be useful in this regard as the influence of transformed gene products on oil production can be measured within days of transformation. A protocol for the analysis of lipid production, including triricinolein production, in a yeast model has been established. Developing ER preparations were made from seed material harvested between 25 to 30 days after flowering; a stage where lipid biosynthesis is at its maximum. ER samples prepared from 3-day germinated seed were used in a differential screen to identify a subset of proteins elevated in the developing seed. Four independent replicates of developing and germinating ER were prepared and analysed using 2-Dimensional Difference In-Gel Electrophoresis (2D DIGE). Spots elevated by ?10%, with the criteria that they were present in all four gels with a student t-test value of p <0.02, were deemed significant and selected for picking and mass spectrometry analysis. Protein sequence data and peptide mass fingerprints were generated and used to search a complete R. communis protein database. Prior to the 2D DIGE analysis, all stages of the proteomic methodology were validated and if necessary optimised for the R. communis samples, from sample preparation through fluorescent labelling, isoelectric focussing, reproducibility of the analytical gels to the ability to identify and effectively pick spots from high loading preparative gels. 91 protein spots were identified as significantly elevated in the developing preparations and 15 spots as significantly elevated in the germinating preparations. On analysis with mass spectrometry a total of 54 developing spots and 10 germinating spots gave confident identities. The ma jority of the developing spots identified were protein chaperones, folding proteins, and storage proteins. No components of lipid biosynthesis were identified in the 2D DIGE analysis, likely due to their membrane bound nature and the loss of these proteins due to poor solubility in 2D electrophoresis buffers or their precipitation during isoelectric focussing. The oleaginous yeast Yarrowia lipolytica was identified as a suitable candidate for in-vivo assays of the effect of R. communis lipid biosynthesis gene products on oil composition. This yeast can utilise hydrophobic substrates for growth including ricinoleic acid, makes significant amounts of storage oil, has a complete genome sequence available and mature genetic tools facilitating its transformation. A protocol for its growth on hydrophobic substrates, lipid extraction and analysis of triricinolein production has been established. 2D DIGE provides a statistically rigourous method for identifying and quantifying elevated proteins in differential screens of plant seed ER. For the identification of the components of lipid biosynthesis in the developing ER, attention now turns to the membranes. Gel-free mass spectrometry based approaches provide the best chance of identifying these proteins and will complement the proteomic analysis presented here.

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Potassium (K+) is an essential nutrient required by plants in high concentration and its uptake is mediated by a number of different K+ channels and K+ transporters including the KT/KUP/HAK transporter family. Phylogenetic analysis of the KT/KUP/HAK transporters in Arabidopsis and rice reveals that the proteins are arranged in four distinct clusters (I-IV). Members of cluster I and II are suggested to act as high- and low-affinity transporters respectively, while transporters in cluster III and IV have been less studied. Elaeis guineensis (oil palm) is a palm oil producing crop. The palm oil is a rich source of vitamins, carotenoids, iron and antioxidant activity and is now the largest source of edible oil. Despite the scientific and economic importance of the oil palm, Elaeis guineensis jacq has been relatively unstudied compared to other important crops such as rice, tomato, and barley. The aim of this study was to clone and characterise KT/KUP/HAK transporters from the oil palm. By a combination of database searching, PCR and 5’ and 3’ RACE, three genes encoding KT/KUP/HAK transporters from the oil palm were identified and named EgKUP3, EgKUP8 and EgKUP11. Phylogenetic analysis of the obtained sequences together with other KT/KUP/HAK transporters from various plant species suggested that they belong to this transporter superfamily and grouped in either cluster II (EgKUP3 and EgKUP8) or cluster III (EgKUP11). The hydrophobicity and topology analysis by TMHMM predicted a cytoplasmic N-terminus and an extracellular C-terminus, whereas EgKUP8 was predicted to have extracellular N- and C- termini. EgKUP11 was predicted to feature cytoplasmic N- and C- termini. TMHMM predicts 13, 10 and 14 transmembrane domain for EgKUP3, 8, and 11 respectively. Transcriptomic analysis was conducted on EgKUPs seedlings at 7-, 14-, and 21-day after treatment with a range of different K+-concentrations; 0.2 mM, 12.3 mM and 21.3 mM. The results suggested EgKUP8 expression is significantly upregulated in root tissues under depleted K+ conditions (0.2 mM) at 14 and 21 day. The expression of EgKUP3 and EgKUP11 on the other hand was not sensitive to changes in external K+ concentration. Functional complementation using Escherichia coli knockout strain defective in K+ uptake systems revealed that all EgKUPs complemented growth at 50 mM K+ concentration while EgKUP8 was also able to complement growth at 5 mM K+ when tested at pH 7.5. In contrast none of the EgKUPs were able to grow at pH 5.5 under all external K+ concentrations tested. Observations from gene expression and functional complementation studies may indicate the importance of EgKUP8 in K+ sufficient environment. Next, the EgKUPs were expressed as C-terminal GFP fusions in S. cerevisiae to establish the pre-crystallisation information needed for structural characterisation. The whole-cell florescent counts of each fusion proteins were used to calculate the approximate amount of protein expression. Although all the fusion proteins expressed between 1 – 1.3 mg/L (1 mg/L is a set threshold for protein expression), it proved impossible to extract the monodispersed transporters into our test detergents, including DDM and DM, as assessed by fluorescent size exclusion chromatography (FSEC). Cellular localisation studies indicated the EgKUPs do not localise to the plasma membrane plasma as expected but rather to the endoplasmic reticulum or cytoplasm, likely as a result of misfolding and faulty processing of the proteins in the S. cerevisiae system. Attemps to optimise the protein expression were performed by producing N- or/and C-terminally truncated proteins, but to no success. I also conducted experiments searching for novel solubilizing agents to improve general membrane protein stability in solution. However, this experiment has been conducted on a different protein, the xanthine/uric acid transporter (UapA) from Aspergillus nidulans, due to lack of success in obtaining a soluble and stable EgKUPs outside the lipid bilayer environment. The UapA which was, exchanged into Maltose Neopentyl Glycol 34 (MNG-34) amphiphile retained a greater amount of folded protein (53.2%) compared to protein exchanged into the conventional detergent, DDM (40%) after 130 min of heating at 40°C. This observation was confirmed by hFSEC assay in which MNG-34 was found to confer the greatest stability to UapA at 50°C.

Willow (Salix spp.) is a rich source of naturally occurring bioactive compounds. This study focussed specifically on exploring the condensed tannins (CT) in the National Willow Collection, which is a unique germplasm collection held at Rothamsted Research. Willow leaves were analysed by two different tannin methods, thiolysis and acid-butanol-acetone, in order to evaluate their suitability for assessing inter-accession variation and to identify accessions with a potential medicinal use. This is the first report of the tannin contents and compositions of a willow germplasm collection. Large variations were found for tannin contents, i.e. 2.8 to 19.5 g CT/100 g dry weight, and also for composition; mean degrees of polymerisation (mDP) ranged from 4.9 to 12.0, the procyanidin (PC)/prodelphinidin (PD) ratios from 19.1/80.9 to 52.0/48.0 and the cis/trans flavan-3-ol ratios from 13.0/87.0 to 52.1/47.9. Catechin was the major terminal unit and gallocatechin the major extension unit, which clearly distinguishes willow tannins from most other plant tannins. Interestingly, some accessions also had leaves with relatively unusual CT types, i.e. high mDP-values and high molar pcercentages of trans-flavan-3-ols. Cluster analysis succeeded in allocating several accessions into distinct groups that coincided with Salix breeding populations. This study has now laid the foundation for identifying the Salix tannin genome in the future. Given the time-comsuming nature of two tannin analysis methods. This study also evaluated the potential of Near-Infrared Spectroscopy (NIRS), which a much faster method, for predicting CT contents and compositions in these willow leaves. The results confirmed that NIRS is an excellent tool for estimating CT contents, PC/PD and c/s/frans-flavan-3-ol ratios in willow leaves. This finding could be of interest to plant breeders in developing new willow accessions. The final part of the thesis evaluated the antiparasitic potential of willow leaves against an intestinal parasite. CT with contrasting compositions were purified to test their efficacy against Haemonchus contortus, which is a major gastrointestinal parasite of livestock. As expected PD- were more active than PC-tannins. In addition, this study demonstrated for the first time that the PC activity could be synergistically enhanced by some Salix flavonoids.

In many regions of the world, the crop, oilseed rape (Brassica napus), is giving rise to populations of volunteer weeds in fields and feral plants outside fields, both of which can retain crop genes and hybridize with compatible wild relatives. Feral oilseed rape has received global attention as a means by which genetically modified (GM) traits may persist in the environment. There are still major uncertainties, however, over the long term environmental and economic consequences of its persistence and invasiveness, particularly in relation to GM coexistence and environmental risk assessment. This thesis presents a demographic study of feral oilseed rape over an 11 year period from 1993 to 2004 within a 500 km2 area of Tayside (Scotland). The number of feral oilseed rape populations increased almost five-fold during a period when the number of fields and total area cropped with oilseed rape decreased. Ferals did not usually remain at the same location for more than one or two years, and did not spread by gradual movement out from the sites of initial colonization. They persisted and spread in the region by occurring at different places each year, most likely through long range dispersal. Transport corridors hosted higher densities than farmland, in which ferals were more prevalent in areas having a high density of oilseed rape crops. The insect communities associated with feral oilseed rape and a related ruderal plant charlock were compared to gauge the potential ecological impact of ferality. Ferals did not appear to compete with charlock but provided an additional host for those invertebrate species already living on charlock. They also had the potential to function as a bridge for crop pests between growing seasons. At current levels of feral oilseed rape there are unlikely to be any issues related to coexistence (i.e. ferals will bring a negligible contaminant to crops), but feral oilseed rape can persist and flower outside the range of cropped oilseed rape plants. It has become part of the native weed and wildflower community, but to date has had no major ecological impact. The long term demographic changes in feral oilseed rape that were found in the 11 year study could not have been predicted from the initial early years when there were few populations or from prior estimates of risk carried out at small spatial scales. A long term approach is therefore needed at realistic scales for successful ecological risk assessment. The Tayside study could provide a baseline and model for assessing the ecological impact of new GM traits such as cold tolerance or insect resistance.

Quinidinone was formed diastereoselectively from quinine and reacted with a variety of nucleophiles to produce a series of 9-substituted quinidine analogues. The configuration at the C9 position was determined to be (S) by X-ray crystallography, which supports the proposed chelation control mechanism of nucleophilic addition to quinidinone. Hydrogenation of the C10-C11 double bonds of the new compounds also allowed access to a series of 9-substituted-10,11-dihydroquinidine derivatives. 8-Fluoroquinidinone and 8-fluoroquininone were prepared from quinidinone, and both were reacted independently with nucleophiles to produce a series of 9-(R)-substituted-8-(S)-fluoro-epi-quinidines and 9-(S)-substituted-8-(R)-fluoro-epi-quinines, the configurations of which were confirmed by X-ray crystallography, supporting a Felkin-Ahn type mechanism for nucleophilic addition to 8-fluoroquinidinone and 8-fluoroquininone. A small series of 9-(R)-substituted-8-(S)-fluoro-10,11-dihydro-epiquinidines were also synthesised by hydrogenation of the C10-C11 double bonds in the corresponding 9-(R)-substituted-8-(S)-fluoro-epi-quinidine derivatives. All of the novel Cinchona alkaloid derivatives were screened as enantioselective electrophilic fluorinating reagents in the asymmetric fluorination of ethyl-1-indanone-2-carboxylate. Moderate enantioselectivities were achieved; the best, 64% enantiomeric excess, was obtained with 9-phenyl-8-fluoro-10,11-dihydro-epi-quinidine and was a significant improvement on the 30% enantiomeric excess obtained using quinidine. Excellent enantioselectivities were obtained when the novel Cinchona derivatives were screened as chiral aminoalcohol ligands in the enantioselective addition of diethylzinc to benzaldehyde. The best enantiomeric excess achieved was 92%, using 9-(3,5-bis-trifluoromethylphenyl)-10,11-dihydroquinidine. Preliminary screening of antimalarial activity has revealed that five of the novel Cinchona alkaloid derivatives possess antimalarial activity against the Plasmodium falciparum parasite. The active Cinchona alkaloid derivatives all contained either a fluorine atom at the C8 position or a trifluoromethyl group on the aromatic ring of the 9-aryl quinidine or 9-aryl-10,11-dihydroquinidine derivatives.

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