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The plant hormone gibberellin (GA) promotes several processes during Arabidopsis reproductive development, including the transition to flowering, floral organ growth and fertility. GA functions during stamen development to promote degradation of the tapetum cell layer through programmed cell death (PCD) and in post-anthesis pollen development. Bioactive GA is synthesised through a multi-step pathway, in which the last two biosynthetic steps are expressed as conserved multigene families. One of these, the GA 20-oxidases (GA20ox) consists of five paralogues in Arabidopsis, though physiological functions have only been ascribed to two (AtGA20ox1 and -2). Through a reverse genetics approach, this project demonstrates that AtGA20ox1, -2 and -3 account for almost all GA20ox activity in Arabidopsis, with very little evidence of any functions for AtGA20ox4 or -5. Unlike AtGA20ox1, -2, -3 and -4, AtGA20ox5 possesses only partial GA20ox activity, performing the first two out of three sequential catalytic conversions in vitro. Partial functional redundancy occurs between AtGA20ox1, -2 and -3 across Arabidopsis development, although AtGA20ox1 and -2 dominate. Mapping of floral AtGA20ox expression through qPCR and the creation of transgenic GUS reporter lines found that the relationship between these three paralogues is complex, and not explicable through the simple hypothesis of co-expression in the same tissues. During anther development, the reported expression of AtGA20ox1, -2, -3 and -4 is mainly restricted to the tapetum cell layer, and loss of AtGA20ox1, -2 and -3 results in an anther developmental arrest in which the tapetum does not degrade. This project demonstrates that stamen development is dependent on an optimum level of GA, with GA-deficiency restricting filament elongation to prevent pollination and GA-overdose negatively affecting anther development. DELLA repression of GA signalling is necessary for successful pollen development, with two of the five DELLA paralogues, RGA and GAI, critical to this process in the Columbia ecotype.

Notably, the hormone regulates the formation and emergence of lateral roots (LRs). In Arabidopsis thaliana the initiation of lateral root primordia (LRP) is induced by auxin and takes place deep within the parental root. Also, the emergence of the LRP through the overlying tissues is regulated by auxin. It has been shown that the gene LAX3 is expressed in cortical and epidermal directly overlying a LRP. External auxin induces LAX3's expression in all cortical and epidermal cells suggesting that it actsas the activating signal. There are two objectives in this study: the first one is to understand how the expression of LAX3 is regulated and the second one is to identify and characterise novel inhibitors of the induction of LAX3. It has been shown that mutations in ARF7 and ARF19 or IAA14/SLR are sufficient to block LAX3 auxin induction. Using classical genetics approaches, it is shown that ARF7 and ARF19 actually regulate LAX3 positively and negatively, respectively. Furthermore, a canonical Auxin Response Element present in the promoter of LAX3 is shown to negatively regulate its expression. Using transcriptomics datasets, a regulatory network is proposed and several putative candidates have been selected. In order to obtain alternative approaches to dissect the induction of LAX3, a suite of 13 inhibitors (representing 8 distinct classes of compounds) have identified. The major and most promising class has been investigated and shown to interfere most probably with the E2 conjugating enzymes. A model and preliminary results with some of the other inhibitors identified are proposed.

It has long been known that fossil fuels are a finite source of energy. With this in mind research has turned to the development of renewable energy sources. One solution is the conversion of biomass to useable energy sources. These resources are located in the cell walls of currently available agronomic crops in the form of complex biopolymers, lignocelluloses, which are highly recalcitrant. In the following thesis I explore the novel mechanism of Impacting cell wall composition using mutants involved in trafficking to the cell wall. The following work shows that Rab GTPasemutants impact on cell wall deposition, with specific sub-clades impacting particular cell wall polymers. I then go on to show these mutants have significant effect on recalcitrance and thus increase saccharification of the biomass, without impacting on agronomic properties. Finally I go on to show the same impact on cell wall composition in a presumed orthogolous Rab in tomato. These findings all have significant Implications in the fields of Intracellular trafficking, cell wall biology and bioenergy.

Breeding plants that produce equivalent growth with reduced water input (improved water use efficiency (WUE)) is necessary for sustained future crop production. Water deficit induces redistribution and synthesis of the phytohormone abscisic acid (ABA) thereby restricting transpiration. ABA is synthesised via cleavage of oxygenated carotenoids (xanthophylls). This work involved two approaches and three key ratelimiting enzymes in the ABA biosynthesis pathway: 9-cis-epoxycarotenoid dioxygenase (NCED) β-carotene hydroxylase (BCH); and phytoene synthase (PSY). Increasing ABA production under optimal conditions by overexpressing SlNCED1 has been shown to reduce stomatal conductance and improve WUE. The first approach, part of a larger programme exploring the allelic variation of NCED1 in wild tomato species, describes the introgression of S. galapagense and S. neorickii NCED1 alleles into the cultivated tomato background S. lycopersicum cv. Ailsa Craig. Plants homozygous for SgNCED1, SnNCED1 and SlNCED1 alleles were compared for water use gravimetrically. It was concluded that SgNCED1 and SnNCED1 did not improve WUE. The second approach involved creating a transgenic rootstock that might provide sufficient extra root-sourced ABA under non-stressed conditions to improve WUE of a non-transgenic scion. Root tissues contain less carotenoids than photosynthetic tissues, which may limit increases in root ABA biosynthesis. To increase precursor flux through the pathway, transgenic lines simultaneously over-expressing SlPSY1, SlBCH2, and SlNCED1 were created by crossing two double transgenic lines. Isolated roots of a selected triple line (H-22-8-8) accumulated increased concentrations of carotenoids, ABA and ABA catabolites. Gravimetric water use trials revealed that WT/H-22-8-8 (scion/rootstock) grafts frequently showed improved; TEp, δ13C, exudate flow rate and ABA concentration. H-22-8-8 rootstock conferred a consistent, modest, daily water saving; significant in the absence of water stress. This indicates that the triple rootstock is capable of restricting stomatal opening and water use, without reducing biomass production.

Apples are a major dietary source of polyphenols in the Western diet and contain procyanidins, hydroxycinnamic acids, flavanols, dihydrochalcones and flavonols. Despite their abundance and familiarity very little research into their metabolism has been performed; research is required to elucidate the metabolic products of these polyphenols and characterise their absorption and excretion pathways. A human intervention study was designed specifically to investigate the absorption, metabolism, excretion and biokinetcs of apple polyphenols. Male volunteers (n = 9) consumed a supermarket apple juice substituted with water as the control phase, and the same apple juice substituted with a high polyphenol cider apple extract as the test phase. Blood samples were taken over 0-24 h and urine samples were collected at 0-4 h, 4-8 h and 8-24 h. A rapid, validated and novel single LC/ES/IMS/MS method was developed and validated for the analysis of a wide range of polyphenols and their metabolites in these urine and plasma samples (after sample preparation). Apple polyphenolrelated metabolites were identified using LC/MS/MS and MS2; nine urinary metabolites and seven plasma metabolites were identified, mostly for the first time after apple consumption. Data on the excretion, bioavailability and biokinetics of these metabolites, including products of the colonic micro flora, were obtained. In urine, the major apple-related polyphenolic metabolites identified were dihydroxyphenyl valerolactone sulfate and 5- (3', 4'- dihydroxyphenyl) -y- valerolactone glucuronide; both colonic bacterial metabolites which appear at their maximum concentrations 4-8 h post apple ingestion. Minor metabolites included (-) epicatechin sulfate and glucuronide conjugates. In plasma, 3, 4-dihydroxyphenylacetic acid, 5- (3',4'-dihydroxyphenyl) -y- valerolactone glucuronide and dihydroxyphenyl valero lactone sulfate predominate; Tmax values of 5-6 h were observed. Minor plasma metabolites included phloretin (Cmax291 ± 175 nM) and p-coumaric acid (Cmax 634 ± 225 nM). In conclusion, the project has identified apple-related polyphenol metabolites in human urine and plasma; many for the first time after apple consumption. Important biokinetic parameters have also been reported for these metabolites.

Oilseeds store energy as triacylglycerides during periods of dormancy in preparation for germination and the early stages of development. The triacylglyceride is stored in discrete organelles termed oil bodies. Oil bodies are formed during the synthesis of neutral lipids within the bilayer of cellular endoplasmic reticulum (ER); as lipid is synthesised it forms droplets of oil that swell distending the ER membrane and at a critical diameter separate from the ER by vesiculation forming independent organelles. These organelles are structurally stabilised by a phospholipid monolayer originating from the ER and the addition of highly amphiphilic oleosin proteins. Oil bodies have been shown previously to be extremely stable organelles that can be easily extracted and purified from oilseeds; our aim was to develop an understanding of the physical and chemical properties of sunflower oil bodies ex-vivo prior to their subsequent use in commercial products. Several novel findings were elucidated through this work: oil body phytochemical composition, their physical and oxidative stability and their ability to store and deliver flavour compounds. It was hypothesised that tocopherol is tightly associated with sunflower oil bodies. This was tested by recovering oil bodies from sunflower seed and washing them to remove extraneous proteins and associated phenolic compounds. Tocopherol remained with washed oil bodies (392 mg tocopherol.kg-1 oil body oil) and this population of tocopherol represented 38% of the total seed tocopherol. It was hypothesised that this high tocopherol concentration and its intrinsic association to oil body structures would contribute to an increased level of oxidative stability. Sunflower seed lipids were significantly more resistant to thermally induced oxidation when stabilised in oil body suspensions compared to sunflower oil emulsions stabilised by a range of commercial emulsifiers (sodium dodecyl sulfate, polyoxyethylenesorbitan monolaurate (tween 20) and dodecyltrimethylammonium bromide). Oxidative stability was assessed through lipid hydroperoxide concentration and the concentration of headspace hexanal. Maximum lipid hydroperoxide concentration in surfactant stabilised emulsions after 8 days at 45oC ranged between 26 and 333 mmol lipid hydroperoxide.kg-1 oil whereas lipid hydroperoxide concentrations in oil body suspensions did not exceed 12 mmol lipid hydroperoxide.kg-1 oil. In addition there was no development in oxidative rancidity over the 8 day storage trial of oil bodies stored at 5oC. The composition of phospholipids in a range of oil body preparations was assessed; purified oil bodies contained principally phosphatidylcholine (91%) and a smaller fraction of phosphatidylethanolamine (9%). Less purified preparations contained other phospholipid species; the presence of which was explained by contamination with either non-intrinsic cellular phospholipids or phospholipase D that catalysed the breakdown of phospholipids to phosphatidic acid. Mechanisms and the extent of oil body physical stability were assessed using charge analysis and resistance of oil body preparations to changes in temperature and pH. Oil bodies are stabilised by a combination of steric hindrance and electrostatic repulsion provided by the surface proteins and phospholipids. Oil bodies had a zeta potential of -30mV at neutral pH and the surface charge was pH dependant with an apparent isoelectric point of between pH3 and pH6 was calculated from electrophoretic mobility, streaming potential and creaming stability measurements. Purified oil bodies were physically stable to thermal stresses up to 45oC for 2 days, although less purified preparations lost structural integrity at temperatures above 25oC. When assessed for their ability to delivery flavour molecules, oil bodies had comparable bulk phase properties to artificial emulsions stabilised by tween 20. Oil bodies did show a greater rate of flavour delivery during headspace dilution, when compared with the model artificial emulsions, suggesting commercial benefits may be gained through the incorporation of oil bodies into commercial emulsions. The key findings of this work are that oil bodies are extremely stable organelles that are resistant to thermal stress and physical processing. When lipid is stored within oil bodies it has greater resistance to the onset of lipid oxidation which may be explained by the intrinsic association of phospholipids, proteins and phytochemicals (vitamin E).

Besides photosynthesis, plastids are responsible for starch storage, fatty acid biosynthesis and nitrate metabolism. Our understanding of plastids can be improved with observation by microscopy, but this has been hampered by the invisibility of many plastid types. By targeting green fluorescent protein (GFP) to the plastid in transgenic plants, the visualisation of plastids has become routinely possible. Using GFP, motile, tubular protrusions can be observed to emanate from the plastid envelope into the surrounding cytoplasm. These structures, called stromules, vary considerably in frequency and length between different plastid types, but their function is poorly understood. During tomato fruit ripening, chloroplasts in the pericarp cells differentiate into chromoplasts. As chlorophyll degrades and carotenoids accumulate, plastid and stromule morphology change dramatically. Stromules become significantly more abundant upon chromoplast differentiation, but only in one cell type where plastids are large and sparsely distributed within the cell. Ectopic chloroplast components inhibit stromule formation, whereas preventing chloroplast development leads to increased numbers of stromules. Together, these findings imply that stromule function is closely related to the differentiation status, and thus role, of the plastid in question. In tobacco seedlings, stromules in hypocotyl epidermal cells become longer as plastids become more widely distributed within the cell, implying a plastid density-dependent regulation of stromules. Co-expression of fluorescent proteins targeted to plastids, mitochondria and peroxisomes revealed a close spatio-temporal relationship between stromules and other organelles. Stromule and plastid fusion could not be induced under conditions which promote substantial fusion of mitochondria. Data are presented suggesting that organelles may be able to pass between cells, and an experiment was designed to test this possibility in the C4 photosynthetic cells of maize. Inhibitor studies have shown that stromule and plastid movement is dependent on the actin cytoskeleton and the ATPase activity of myosin. An Arabidopsis gene, CHUP1, is responsible for chloroplast relocation in response to light intensity and encodes a chloroplast-localised actin-binding protein. To assess whether this protein is involved in stromule movement, CHUP1 was down-regulated with RNAi. Whilst plants with reduced CHUP1 expression exhibited a chup1 mutant phenotype, no significant effect on stromules was discovered. It was thus concluded that chloroplast relocation and stromule formation are two independent processes that employ different actin-dependent mechanisms. It is proposed that stromules act primarily to increase the plastid surface area in response to a number of developmental and environmental factors.

Seed germination is a key process in world agriculture. For this reason, the capacity of a seed to germinate with minimum input from farmers is highly desirable, keeping the production costs as low as possible. The physiological mechanism of germination is well known, involving the rupture of the endosperm and testa envelope by the expanding embryo, but the molecular and biomechanical changes underlying this process are poorly understood. In order to answer the question of how the plant developmentally regulates changes in cell wall stiffness associated with germination, an innovative molecular biomechanics approach was developed. It combines biophysical, engineering and molecular biology approaches. A comparative approach was taken using the related species Arabidopsis thaliana and Lepidium sativum, the former due to the wealth of genomic resources, and the latter due to larger size and ease of use in biomechanics experiments. Environmental scanning electron microscopy imaging revealed that the endosperm structure is intact after protrusion of the radicle, confirming the fact that rupture occurs between individual cells. Germination is a process that requires targeted cell separation or/ and cell wall remodeling. For this reason, following the predictions of the gene network SeedNet, containing the endosperm-specific sub-network cluster 19 (Bassel et al., 2011), endosperm specific genes were studied. In order to localize their expression, promoter::GUS constructs were used for the genes DELTA-VPE, SCPL51 and DOF2.1, and different mutant alleles of four transcription factors from cluster 19 (athb23, bhlh-115, bee2 and dof2.1) were screened to identify changes in germination behaviour. DELTA-VPE and SCPL51 were proven to be endosperm specific and ABA insensitive. DELTA-VPE was GA insensitive and SCPL51 expression required GA. Also, DELTA-VPE expression could be observed after 15 minutes of imbibition in the whole endosperm, while SCPL51 showed a temporal expression requiring 18 hours of imbibition before being observed in all endosperm cells. The analysed T-DNA lines showed an epistatic relationship between ATHB23 and DOF 2.1 and a decreased sensitivity to stress factors like osmotic and salt stresses, than the wild type. Using nanoindentation, a differentiation between different regions of endosperm was attempted, but the methodology was not sensitive enough. However, different elastic modulus values for imbibed and dry Lepidium seeds were registered. To image the internal changes in seed structure during germination, micro-CT was used, estimating values for endosperm thickness from dry state to germinated one. This work enforces the knowledge of the molecular biology and biomechanical properties of the endosperm.