UV-B radiation is an integral component of natural sunlight reaching the Earth’s surface. Although being a potentially harmful and damaging agent, UV-B is a key environmental signal for plants initiating diverse responses that affect their metabolism, development and viability. The majority of these responses involve the differential regulation of gene expression and all require accurate perception of the effective light quality by a photoreceptor. The recent identification of UV RESISTANCE LOCUS8 (UVR8) as a UV B photoreceptor has been an important milestone in plant UV B research (Rizzini et al., 2011; Christie et al., 2012; Wu et al., 2012). The aim of this study was to investigate how the structure of the UVR8 protein determines its function in the UV-B response in Arabidopsis.

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The function of chloroplast ribosomes was investigated by analysing the products of in vitro protein synthesis by isolated pea chloroplasts. Since previous attempts at identifying newly synthesised proteins in isolated chloroplasts had either been unsuccessful or inconclusive, careful attention was paid both to the choice of chloroplast preparation and to the analytical techniques used to identify labelled proteins. A rapid method of chloroplast isolation was used which gave a chloroplast preparation which contained about 50% intact chloroplasts and showed high rates of amino acid incorporation into protein. It was felt that the proteins synthesised by these chloroplasts would accurately reflect the nature and pattern of protein synthesis which occurs in chloroplasts in vivo. High rates of incorporation would also aid identification of newly-synthesised proteins. Amino acid incorporation was shown to be sensitive to selective inhibitors of 70S-type ribosomes, but was not affected by inhibitors of 80S-type ribosomes. In addition, incorporation was shown to by insensitive to ribonuclease, suggesting that protein synthesis was taking place in intact chloroplasts. When the labelled chloroplasts were fractionated by differential centrifugation, approximately 25% of this incorporation was present in a 150 000 x g chloroplast supernatant fraction. Further analysis was confined to this supernatant fraction since only released, and therefore completed, polypeptides should be present in this fraction, thus aiding identification. The 150 000x g supernatant fraction was analysed on polyacrylamide gels in the presence and absence of a denaturant, sodium dodecyl sulphate, and by gel chromatography on Sephadex G100 in a sodium dodecyl sulphate-containing buffer. Only one polypeptide was found to be labelled by all these procedures. This polypeptide was identified as the large subunit of Fraction 1 protein, a major protein constituent of the chloroplast. Identity of the in vitro product present in the soluble phase of the chloroplast with the large subunit of Fraction I protein was established by comparing a two-dimensional tryptic peptide map of its [S35] methionine-labelled peptides with a tryptic peptide map of the large subunit of Fraction I protein labelled in vivo with [S35] methionine. It may therefore be concluded that only one of the many proteins present in the soluble phase of the chloroplast, namely the large subunit of Fraction 1 protein, is synthesised on chloroplast ribosomes.

Stomata account for much of the 70% of global water usage associated with agriculture, and have a profound impact on the water and carbon cycles of the world. Anion channels at the plasma membrane of the guard cell are thought to comprise a major pathway for anion efflux essential for driving stomatal closure. The activity of these channels is therefore tightly linked to abscisic acid (ABA)-dependent stomatal movements. Both the inorganic anion Cl- and the organic acid anion malate (Mal) are transported during stomatal movements. The metabolism of organic acids, primarily Mal plays an especially important role in these processes. However, little is known of the regulation of anion channel current (ICl) or its connection with cytosolic Mal and its immediate metabolite oxaloacetate (OAA). Work described here focuses on the relation of ICl and its connections with metabolism and signals controlling other transport at the guard cell plasma membrane. Thus the impact of Mal, OAA and of acetate in Vicia guard cells was examined, and results shown that all three organic acids affect ICl with different characteristics. Most prominent, the suppression of ICl by OAA within the physiological range in vivo indicates a capacity for OAA to co-ordinate organic acid metabolism with ICl through a direct effect of organic acid pool size. In a second set of studies, the ABA pathway that elevates cytosolic free Ca2+ ([Ca2+]i) in order to activate ICl was explored. These studies build on the discovery of PYR/PRL ABA receptors. Previous analysis of the pyr1/pyl1/pyl2/pyl4 mutant suggested that [Ca2+]i increases are suppressed. However, direct evidence had not been forthcoming. Thus a combination of voltage clamp and fluorescent ratio analysis with the Ca2+-sensitive dye Fura2 was used to show that the activity of Ca2+ channels (ICa) at the plasma membrane in the pyr1/pyl1/pyl2/pyl4 mutant is not activated in ABA, with the effect that [Ca2+]i increases were suppressed. Further studies showed that the normal action of ABA in promoting reactive oxygen species (ROS) was impaired, although adding H2O2 was sufficient to activate the ICa and trigger stomatal closure in the mutant. These results offer direct evidence that the PYR/PYL receptor proteins contribute to the activation by ABA of ICa through ROS, thus affecting [Ca2+]i and its regulation of osmotic solute flux for stomatal closure. Finally, the connection between ICl and the K+ channel currents in the slac1 mutant of Arabidopsis was studied. These studies employed systems dynamic modelling to explain the paradoxical suppression of the inward-rectifying K+ channel current, IK,in and slowing of stomatal opening, by mutation that eliminated the SLAC1 anion channel. Experimental results confirmed the model predictions that the abnormal cytosolic pH (pHi) and [Ca2+]i suppresses IK,in in the slac1 mutant, through measuring pHi and [Ca2+]i in vivo, and experimental manipulation of pHi and [Ca2+]i is sufficient to recover IK,in and stomatal opening. These data uncover a previously unrecognised signaling network that minimises the effects of the slac1 mutant on transpiration, and they underscore the importance of H+-coupled anion transport for pHi homeostasis.

Morphological, cell cycle and molecular events were studied during floral evocation in the shoot terminal meristem of the short-day plant Pharbitis nil. The haploid genome size of P.nil was 3.7 X 10e8 bp. Morphological and cell doubling time observations demonstrated that the apex widened and flattened follOwing an inductive dark treatment, the cell doubling time decreased in the peripheral zone and increased in the central zone. An inductive treatment comprising 48 h darkness given to 4 d old plants produced. 100~ flowering at the shoot terminal meristem. An inhibitory treatment was developed using 5 min RL breaks during the 48 h dark period. This treatment prevented flowering at the shoot terminal apex and discriminated between events essenttat for flowering and changes resulting from shifts from light to darkness and vice versa. It was shown using shading experiments that the shoot terminal apex may be directly involved in the reception of red-light. Maximal differences in the proportion of cells in G2/G 1 (G2/G 1 ratio) were recorded at the light/dark transition (i.e. at the end of the 48 h dark period). Apices were sampled at this time to construct floral and vegetative cDNA libraries. Less growth occurred in the central zone of the shoot apex. For example, the mitotic index of the central zone was consistently lower than that of the peripheral zone in both vegetative and induced apices. Unusual PLM curves were generated which meant that the component phases of the cell cycle were determined from microdensitometric data. The duration of the cell cycle in the peripheral zone was two-fold longer in vegetative apices compared to floral apices. However, the proportion of cycling cells (the growth fraction) was similar in the peripheral zones of both vegetative and floral apices. Following exposure to RL the duration of G2 in the peripheral zone increased five-fold. mRNA was separately extracted from 40 floral and vegetative shoot apices. The mRNA was copied into cDNA and amplified using the polymerase chain reaction. The amplified cDNA was then cloned into two A phage cDNA libraries (floral and vegetative). These libraries will form the foundation of future work on the molecular biology of floral evocation.

A fundamental event in any intracellular trafficking pathway is the fusion of a cargo vesicle with its target membrane. In eukaryotes, this membrane fusion event requires members of the SNARE family (soluble N-ethylmaleimide-sensitive factor adaptor protein receptors). SNAREs are characterised by the presence of a C-terminal transmembrane domain, a cytosolic N-terminal regulatory motif, and a cytoplamically oriented SNARE motif essential for the fusion event. Three SNAREs (t-SNAREs) present on the target membrane and one (v-SNARE) on the transport vesicle specifically interact, thus bringing membranes into close proximity and driving the fusion between them. In the present research project, I studied protein transport routes to the vacuole within the plant secretory pathway and aimed to shed light on the role of SNAREs in the biogenesis and filling of of protein storage vacuoles (PSVs) in maturing seeds of the model plant Arabidopsis thaliana. This thesis deals with the preliminary study of the t-SNARE BET11, a potential candidate for a role in the protein transport to PSVs in seeds. The expression patterns and the hypothetical function of the Arabidopsis thaliana BET11 were analysed by fluorescent protein fusions and confocal laser scanning microscopy, GUS assays and biochemical approaches. Transient expression in Nicotiana tabacum demonstrated that, fused to fluorescent proteins, BET11 localises at the trans-Golgi-Network (TGN), a compartment with a central role in both biosynthetic and endocytic protein transport. This led to the more extended analysis of BET11 in the model plant to ascertain its localisation in the secretory pathway. In addition, a bet11 knockout was identified and characterised, and its phenotypes analysed. Also an Arabidopsis line expressing a reporter protein bearing the characterised C-terminal vacuolar sorting signal (ctVSS) (sp-RFP-AFVY) in the bet11 background was generated. Within the knockout background, the vacuolar reporter was secreted, especially in vegetative tissues, and surprisingly not in seeds. The missorting phenotype could be complemented by ectopic expression of BET11.

At the late stages of seed maturation, Arabidopsis thaliana seeds become filled with storage lipids. This source of energy is then used to support seed germination and seedling establishment. Although the metabolic pathways for synthesis and mobilisation of storage oil are well known, the regulation of these pathways remains more unclear. The aim of this project was to study the mechanisms of regulation of storage oil metabolism at the transcriptional level by identifying the transcriptional regulators and their target binding sequences in the promoters of genes involved in oil biosynthesis and breakdown in Arabidopsis seeds. A reverse genetic screen allowed the identification a transcription factor belonging to the bZIP family that is required for normal seed fatty acid composition. Seeds of the bzip67 mutant contain more linoleic (18:2) and less oleic (18:1) and linolenic (18:3) acid in their oil indicating that the desaturation process is compromised. Real-time PCR suggests that bZIP67 is a positive regulator of FATTY ACID DESATURASE 3 and a repressor of REDUCED OLEATE DESATUATION 1. Both genes are involved in fatty acid desaturation. Data from over-expression of bZIP67 also indicates that other partners are likely to be required to promote FAD3 gene expression. A different approach was taken to study the regulators of oil breakdown. Promoter analysis study of MALATE SYNTHASE (MLS) was carried out. MLS is a marker gene for the glyoxylate cycle, which allows the conversion of oil to sugar upon seed germination. Characterisation of T-DNA mutants disrupted in the MLS promoter suggests that the information required to drive MLS expression falls in the first ~140 bp of the promoter. The results show that this region is conserved in different Brassicales species and contains a number of conserved putative elements possibly required for MLS post-germinative expression. Different promoter-reporter constructs lacking each of the conserved elements were tested with a transient assay system using biolistics and the results indicate that putative ABI5, R1MYB, ABI4 and DOF binding elements are required for full MLS expression. Transcription factors from these families are therefore strong candidates as regulators of MLS expression.

Senescence is a highly regulated developmental process in plants in which nutrients are remobilised from organs which are no longer required or are stressed so that they may be used by organs which are only just developing. Whilst much is known about the causes of and the resulting outcomes of this process, very little is known about the genetic machinery which link them. Some genes have been identified as having a regulatory role in the senescence process, but many of these have been determined by a forward genetics approach whereby mutants are randomly screened for phenotypical e↵ects. A much better approach, where possible, is the reverse genetics approach whereby mutants are sought for testing as they are suspected to demonstrate a phenotypical e↵ect. It was the purpose of this study to find novel ways of identifying those genes which may be highly regulatory of the senescence process and to determine how they are able to lead from several di↵erent known causes of senescence through to the senescence response itself. It was hypothesised that, using measurements of the expression levels of a very large number of genes throughout the senescence process, theoretical models of regulation between those genes could be determined and that these theoretical models would allow specific interactions to be identified and explained using biological validation techniques. A large microarray experiment, performed prior to the start of this project, measured the expression of over 30,000 genes in the Arabidopsis thaliana genome over a period of 21 days during natural senescence. By cleaning this data and fitting an ANOVA driven model to the resulting intensity measurements, it has been possible to separate e↵ects leading to observed expression changes. The levels of each of these e↵ects were tested by F-tests and this has allowed the identification of 8,878 genes which are significantly di↵erentially expressed during senescence. By first using theoretical models to find genes amongst the set of 8,878 which demonstrate highly robust regulatory behaviour on other genes in the set, 118 genes were able to be isolated for further study. A senescence phenotype screen was developed to assess reduced-expression mutants of many of those 118 genes and 8 were shown to have a significantly altered timing of senescence when compared with wild-type plants. The surrounding networks of each of those 8 genes were formed by applying theoretical regulatory network modelling in another novel manner similar to a Metropolis-Hastings approach which identified a set of 75 genes providing a testable regulatory network model. The resulting network model has been tested biologically to establish the accuracy of the predictions. Whilst many of the predictions were not confirmed, a vast network has been identified surrounding two of the highly regulatory genes indicating a junction of two separate pathways leading to the senescence response and providing a network structure which could be used in another round of theory and validation. Additionally, these results introduce new interesting questions about how the senescence network may have evolved to respond to so many inputs.

Transcriptional regulation controlling pathogen-responsive gene expression in Arabidopsis is believed to underlie the plant defence response, which confers partial immunity of Arabidopsis to infection by Botrytis cinerea. In this thesis networks of transcriptional regulation mediating the defence response are studied in various ways. First transcriptional regulation was predicted for all genes differentially expressed during B. cinerea infection by development of a novel clustering approach, Temporal Clustering by Affinity Propagation (TCAP). This approach finds groups of genes whose expression profile time series have strong time-delayed correlation, a measure that is demonstrated to be more predictive of transcriptional regulation than conventionally used similarity measures. TCAP predicts the known regulation of GI by LHY, and co-clusters ORA59 and some of its downstream targets. Predicted novel regulators of pathogen-responsive gene expression were then studied in a reverse genetics screen, which discovered several novel but weakly altered susceptibility phenotypes. Comparison of predicted targets to known targets was complicated by the sparsity of mutant versus wildtype gene expression experiments performed during B. cinerea infections in the literature. To explore the context-dependence of transcriptional regulation, evidence of transcriptional regulation in different contexts was collected. This was compiled to generate a qualitative model of transcriptional regulation during the defence response. This model was validated and extended by experimental analysis of transcription factor-promoter binding in Yeast and transcriptional activation in planta. Comparative transcriptomics showed that downstream genes of some of these regulators | TGA3, ARF2, ERF1 and ANAC072 | are over-represented in the list of genes differentially expressed during B. cinerea infection, which is consistent with these targets being regulated by them during B. cinerea infection. Finally this qualitative model was used as prior information and was used along with gene expression time series to infer quantitative models of the gene regulatory network mediating the defence response. Some known regulation was predicted, and additionally ANAC055 was predicted to be a central regulator of pathogenresponsive gene expression.

Our understanding of the evolutionary dynamics of selection for herbicide resistance is limited by the time and space required to conduct meaningful selection experiments in higher plants. This constrains the study of the dynamics of resistance evolution predominantly to mathematical models. The primary goal of this thesis was to overcome these limitations, and to study the evolutionary phenomena underpinning several management strategies. To do so, a series of experimental evolution studies were conducted using Chlamydomonas reinhardtii, a single-­‐cell green chlorophyte susceptible to a range of commercial herbicides. In particular, this thesis explored the impact of herbicide sequences, rotations and mixtures, as well the impact of herbicide dose, on evolution of resistance. Applying herbicides in sequence allowed the study of the impact of environmental perturbation on the dynamics of resistance and the associated fitness costs, finding more rapid selection for resistance to a second and third mode of action in some populations. Cycling between herbicides creates conditions of temporal environmental heterogeneity, the outcomes of which are not easily predictable as resistance was slowed down in some cycling regimes, while in others it accelerated the evolution of resistance or gave rise to cross-­‐resistance. Herbicide mixtures are a management strategy relying on increases in environmental complexity to provide better control of resistance. The results presented show that mixtures were effective at slowing the evolution of resistance when all mixture components were used at fully effective doses, while low doses of mixtures accelerated resistance evolution and led to more cross-­‐resistance. Finally, modifications of the applied herbicide dose allowed the study of local adaptation along an environmental gradient, where the differences in outcomes based on the specific herbicides used were again evident. Overall, the work presented here uses applied scenarios to study the underlying evolutionary phenomena, in order to feed back into the applied thinking.