Jatropha curcas, a non-edible oilseed, is becoming popular as a source of biodiesel. However, there are some limitations to using J. curcas such as the presence of carcinogenic substances, unpredictable yield, asynchronous flowering and plant height. Understanding the genetic control of these characteristics will help to find solutions. To accumulate genetic information for J. curcas plant breeding programmes, the genetic and phenotypic characterisation of J. curcas seeds obtained from different geographical locations was undertaken (Chapter 2). The results showed a narrow genetic diversity among accessions but apparent phenetic diversity. The screens employed ranged from phenotypic characterizations of seeds to molecular, biochemical, physiological and genotyping assessments. Seed dimensions and weight measurements provided data on seed density and kernel weight, both important parameters for assessing oilseed quality. Biochemical analysis provided data on oil properties relevant to biodiesel e.g. total oil content, oleic/linoleic acid ratio, saponification number, cetane number and gross energy value. Expression analysis of genes (acc, sad, fad, and dgat) essential for the quantity and quality of seed/oil were targeted as molecular screens. Understanding the molecular mechanism of lipid cycling in J. curcas seeds will guide future improvements in oil yield. However, this is still an area of active research because little information exists for this in J. curcas. With regards to oil storage capacity of seeds, no information previously existed on the biology of oil bodies in Jatropha curcas. In this study, oil bodies from J. curcas were studied at the gene transcript and protein levels (Chapter 3). Three Jatropha oleosin genes were isolated. Sequence analysis showed that all three oleosin genes contained the proline knot conserved domain. The upstream regulatory element of one Jatropha oleosin gene was also isolated. In silico analysis of this upstream sequence indicated the presence of a number of seed specific regulatory motifs. Transcript analysis for the three oleosin genes revealed tissue-specific differences. Immunoassay by western blot analysis using antibodies against Arabidopsis oleosin and the J. curcas specific oleosin revealed a diversity of oleosins. Proteomic analysis of oil bodies revealed additional minor proteins including caleosin, steroleosin, aquaporin and curcin. Information from these studies is essential for varietal improvement of Jatropha and/or developing oleosin gene fusion technology for expression of novel compounds. In this study 623 nucleotides upstream of the JcOleosin3 gene have been isolated and characterized in transgenic Arabidopsis plant using β-glucuronidase (GUS) as the reporter gene (Chapter 4). Result showed that the JcOleosin3 promoter directs iii expression of the β-glucuronidase gene in seed and pollen but not in leaves, root, stem or flower. In addition, in transgenic Arabidopsis, the JcOleosin3 promoter drives GUS activity at the early seedling stage; this was expected based on earlier reports of expression of Jatropha oleosins. To understand the molecular basis of seed germination, proteomic studies were performed on resting and germinating seeds of J. curcas. Such studies using 2-DE and MALDI-TOF MS revealed the presence of patatin-like lipases in the germinating seeds but not in the resting seeds (Chapter 5). These proteins showed sequence similarity and conserved domains as found in the patatin lipases from Solanum tuberosum and Arabidopsis thaliana. Patatin lipases play a significant role in the mobilization and lipolysis of TAGs during the germination of oil seeds. Further, there is little knowledge on the triacylglycerides (TAGs) lipid cycling in J. curcas., Bioinformatic data-mining confirmed the TAGs cycling in J. curcas to include the expected stages of synthesis, storage and lipolysis of fatty acids. Based on the data-mining results for the presence of various genes for TAG storage, synthesis and utilization, a scheme is presented for TAG cycling in J. curcas. The scheme presented in this study provides essential information required for oil manipulation in J. curcas.

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The phosphatase AtPTPKISI is involved in the control of starch metabolism in Arabidopsis thaliana leaves at night. The SEX4 (Starch Excess 4) mutants, lacking this predicted phosphatase, have strongly reduced rates of starch metabolism. It is shown that this chloroplastic protein can bind to glucans through a carbohydrate binding domain (CBM) located within its previously predicted kinase interaction sequence (KIS), while another novel KIS containing protein (AKINβy) shows no such interaction. Further analysis of the CBM identifies conserved residues vital for carbohydrate binding and common to CBMs, as well as sugar tongs, not present in similar CBMs or the GBD/KIS domain of the previously studies AMPKβ, but found within the binding domain of the PTPKIS family proteins. While PTPKIS 1 shows activity to generic phosphatase substrates, it is unable to dephosphorylate either phosphotyrosine or phosphothreonine containing peptides. It does however show phosphatase activity towards phosphorylated starch and amylopectin, comparable to that of the mammalian protein laforin. Remarkably, the most closely related protein to PTPKIS 1 outside the plant kingdom is laforin, required for the metabolism of the mammalian storage carbohydrate glycogen and implicated in a severe form of epilepsy (Lafora disease) in humans, through the formation of insoluble starch like polyglucans (lafora bodies). In addition to PTPKIS 1, PTPKIS 2 (At3g015180) is identified, a predicted phosphatase, with a domain structure homologous to that of PTPKIS 1, termed. The PTPKIS2-SALK (PTPKIS2 knockout) mutant, lacking this predicted phosphatase, has a reduced rate of starch metabolism. It is shown that this mutant causes a phenotype similar to SEX4, but less extreme. It is further shown that this protein can bind to glucans through a carbohydrate binding domain (CBM), but unlike PTPKIS 1 shows no activity towards any phosphate substrates. PTPKIS2 does however modulate the activity of PTPKIS 1, causing a 4-fold increase in the activity of PTPKIS 1 against phosphorylated starch, when both enzymes are present in equimolar concentrations. Finally, a hypothesis is proposed as to the roles of PTPKIS 1 and PTPKIS 2 in starch metabolism, and the similarity of function seen in the mammalian protein Laforin.

Sources of carbon for the developing fruits Of Vicia faba L. variety triple white were investigated. Attached leaves and pods were allowed to photosynthesis in [(^14)C]-labelled carbon dioxide. Leaves, pods and seeds were extracted separately with trichloroacetic acid. Amino acid and radioactive analyses were carried out on both the trichloroacetic acid soluble (non-protein) and insoluble (protein) fractions. Quantitative analysis of 90 min leaf photosynthesis indicated that a proportion of new photosynthate is rapidly exported from the leaf. Retention in the leaf of some labelled carbon was demonstrated during a 16 hour chase period. Evidence for protein synthesis from newly formed photosynthate is presented, together with evidence for the rapid turnover of such protein. Labelled amino acids and sugars were shown to be present in the petiole of a leaf photosynthesising in [(^14)C] – CO(_2). Leaves near the plant base contributed carbon to xylem sap amino acids. The bloom node leaf was shown to export preformed sugars and amino acids to the developing (20-35 day old) pod and seeds. Some translocate from the leaf entered the seed directly, but more than half was metabolised in the pod before being re-exported to the seeds. Pod photosynthesis was shown to involve labelling patterns like those of leaf photosynthesis, but to provide the seed with a different, and partly complementary, set of amino acids. Seeds were shown to have a carbon source during the night when transpiration and photosynthesis are negligible. The likelihood of stem tissue functioning as this carbon source is discussed. Bleeding sap from decapitated plants was analysed, and its relationship to xylem sap discussed. Sap contribution to the developing seeds is considered. Quantitative estimates are made of the carbon contribution from leaves and pods to seeds, and these are compared with the results of other workers. Seeds are shown to be capable of amino acid synthesis and interconversion, particularly of compounds readily synthesised from respiratory intermediates. Protein synthesis in leaves, pods and seeds is demonstrated, and the nature of the seed protein is discussed.

Seeds of Vicia faba (L) become fully mature in about 130 days from the fertilization of the ovule. After an initial phase of cell division, storage protein is synthesised in the cotyledons. During the stage of storage protein synthesis, rRNA synthesis occurs concomitant with synthesis of ER, suggesting that storage protein synthesis is mediated on membrane bound ribosomes by mRNA(s). Analysis of the nucleic acids during cotyledon development did not lead to the identification of any non-ribosomal RM species. A fourth rRNA was identified, which had a molecular weight of 52,000 daltons and was hydrogen bonded to the 25S rRNA species. Attempts to identify rRMA precursor molecules in cotyledons and roots were unsuccessful, though in the latter the bacterial precursor molecule to the 163 rRNA was identified. Isolated polyribosomes, from cotyledons at various stages of development, had similar profiles and their constituent nucleic acids were degraded, indicating that they were cleavage products of larger in vivo units. The rRNA cleavage products arising during polyribosome isolation were characterised by their molecular weights. The properties of diethylpyrocarbonate as a nuclease inhibitor were reinvestigated and it was found to be incapable of effectively inhibiting high concentrations of nucleases. The extraction of polyribosomes in the presence of diethylpyrocarbonate effectively protected the polyribosome against HNase during the later stages of the extraction procedure, but not during the initial stages resulting in marked rRNA cleavage and the formation of ribosomal subunits. The use of antllegumin as a method in the identification of a mRNA is discussed.

Traditionally, reactive oxygen species (ROS) have been regarded as toxic by-products of aerobic metabolism. However, in recent years it has become apparent that plants actively produce ROS as signalling molecules. ROS are able to mediate adaptive responses to various environmental stresses as well as processes such as stomatal closure and development. Downstream signalling events that are modulated by ROS include calcium mobilisation, protein phosphorylation and gene expression. This study investigated signalling proteins acting downstream of ROS, in order to understand how ROS are perceived and transduced to elicit such a wide range of responses. To establish a molecular profile provoked by ROS, a microarray experiment of Arabidopsis plants exposed to exogenous H(_2)O(_2) was analysed. Of the 895 differentially expressed transcripts, a substantial proportion had predicted functions in cell rescue and defence, including heat shock, disease resistance and antioxidant genes. Genes encoding candidate H(_2)O(_2) signalling components were identified from this microarray experiment and their H(_2)O(_2) - induced expression was verified by northern RNA-blot analysis. Two transcription factors of the ethylene response factor (ERF) family (AtERFS [At5g47230]) and AtERF6 [At4g17490])and an ankyrin protein kinase (APK [At4g18950]) were selected for further study. Northern blot analysis and comparison with publicly available transcriptome data sets demonstrated that the expression of these three genes was induced by various stress treatments, such as UV-B irradiation, cold and elicitor challenge. To unravel the potential in vivo function of these proteins, loss- and gain-of-function lines were generated and analysed. No abnormal plant phenotypes were observed during development or in response to the stress and hormone treatments tested. A high level of functional redundancy may exist between AtERFS and AtERF6. Microarray analyses were performed on the over-expression lines. Genes that were differentially regulated in APK over-expressor lines gave no indication of its function. However, the microarray analyses revealed that AtERFS and AtERF6 have roles in the plant pathogen defence response, since their over-expression induced defence gene expression. Analysis of cis elements in the promoters of the ERF-differentially regulated genes revealed that both transcription factors displayed GCC box binding activity. However, the GCC box was not over-represented in the promoters of H202-differentially regulated genes, which suggests that this element has a ROS independent regulation.

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