Hyaloperonospora arabidopsidis is an obligate biotrophic oomycete shown to cause downy mildew in Arabidopsis thaliana. The main focus of this project is examining plant stress response and the strategies employed by H. arabidopsidis to infect Arabidopsis and evade plant stress responses. Two regions of the H. arabidopsidis genome containing genes expressed in planta during infection were bioinformatically annotated. The results indicated the genes were involved in regulatory processes associated with the pathogenicity of H. arabidopsidis but not a direct role in pathogenicity. H. arabidopsidis infects its host by secreting effector proteins into the cytoplasm and apoplastic space of the host. The secretome of H. arabidopsidis was analysed to identify classes of cysteine rich apoplastic effectors. This identified 15 candidate elicitin (ELI) and elicitin-like (ELL) sequences, three Kazal-like serine protease inhibitors and four candidates similar to the protein sequences of Ppats 14 and 24, expressed during infection. A second set of aims was to identify potential signalling networks up activated during plant defence responses to infection by H. arabidopsidis using a new model developed by Beal et al (Beal, Falciani et al. 2005) to eventually engineer transcriptional networks. Unfortunately this failed due to problems with the experiment. However, it was still possible to identify signalling networks from a second microarray time course experimental data set centred on signalling networks up regulated in response to the onset of senescence, as they share overlapping signalling pathways. The modelling methodology was used to model the anthocyanin biosynthesis pathway. The model predicted the presence of AtMYB15 as a positive regulator of anthocyanin biosynthesis along with AtMYB90. Research carried out by Nichola Warner (Warner 2008) suggested that AtMYB90 was not essential for anthocyanin biosynthesis during senescence based on by comparing the phenotype of the MYB90 knock out, IM28, with the wild type (WT) Col-0 using a time course microarray. Models of networks of transcriptional regulation of the anthocyanin biosynthesis pathway for IM28 and WT implicate AtMYB29 as a positive regulator of anthocyanin biosynthesis.

This thesis describes the development of a new approach to measuring the growth of plant roots. Work on changing the growth patterns of plants by the introduction of the right materials into their feed as well as the process of genetic manipulation is enhanced by being able to measure the growth of the plants roots in real time. Previous work in doing this has been subject to low reliability due in part to the nature of the problem. Plant root growth rates are of the order of 0.1 μm per second and thus have to be captured under the microscope. The plant surfaces show low contrast and have few predictable features so many methods prove to be inappropriate. Previous work in the measurement made use of the RootFlowRT software that uses a combination of a tensor based method and a correspondence method. However, the results from these methods have a high level of unreliability. The tensor method as applied shows a reliability of less than 10% and work carried out in this thesis shows that the correspondence method on its own cannot reliably predict the growth rates for large areas in any root. The work has introduced the use of Scale Space Optical Flow method to replace the previous tensor method and this has been shown to have a reliability of greater than 30% in almost all cases. The results of this method are then used to refine the search space for the correspondence method and again increase the reliability of the measurements. The validity of the final results using the current method are thus shown to be a great improvement on the previous method. For comparison: Percentage of measurements in the correct direction and size • RootFlowRT 70% • Current method 95% Maximum spread of invalid results • RootFlowRT +/-200% in size and 100% in direction • Current method +/-10% in size or direction

WEE 1 is a key eukaryotic cell cycle regulator. In plants it has a clear role at the DNA damage/ DNA replication checkpoints. I aimed to discover the functional significance of interactions between WEE1 and other cellular proteins in Arabidopsis thaliana and Nicotiana tabacum. First I examined effects of ectopic expression of Arabidopsis WEE1 (Arath;WEE1) in transgenic tobacco and tobacco WEE1 (Nicta;WEE1) in transgenic Arabidopsis. Western blotting using a plant WEE1 antibody showed that expression of Nicta;WEE1 in Arabidopsis caused increases in total WEE1 protein. The response of primary root length, numbers of lateral roots and primordia, and meristem length to zeocin (a DNA damaging agent) and hydroxyurea, (which perturbs DNA replication), resembled the wee1-1 insertional mutant rather than Arath;WEE1 over-expression. Expression of Arath;WEE1 in tobacco resulted in reduced WEE1 protein but also induced similar phenotypic changes as Nicta;WEE1 expression in Arabidopsis under zeocin and HU stress. I concluded that interactions with cellular proteins in the alien species resulted in down-regulation of WEE1 activity. In a yeast 2-hybrid screen Arath;WEE1 interacted with the glutathione-S-transferase protein, GSTF9. To test the functionality of this interaction I analysised the root and cell cycle phase phenotypes of single mutants: wee1-1 and gstf9 and I generated the double mutant wee1-1;gstf9. I demonstrated that both Arath;WEE1 and GSTF9 have roles in the DNA replication and damage checkpoints, but largely act in different genetic pathways. Arath;WEE1 also interacts with GF14ω, a 14-3-3 protein in a yeast 2-hybrid assay. In other eukaryotes this stabilizes WEE1. I confirmed that over-expression of GFF14ω in transgenic Arabidopsis (GFF14ω OEX) results in a very similar root phenotype to over-expression of Arath;WEE1 as predicted from a stabilization of WEE1. However the GFF14ω OEX phenotype was not abolished in a wee1-1 genetics background.indicating that Arath;WEE1is not required for the action of GF14ω.

The ability of plants to respond to environmental cues is crucial for the success of the organism. Light is an important source of energy for the plant as well as providing information for plant growth and development. Plant responses to the electromagnetic spectrum are controlled by a number of different photoreceptors. Phytochromes respond to red and far-red light and are responsible for controlling photomorphogenesis in seedlings (Chen and Chory, 2011). The recently identified UVR8 responds to UV-B light and prevents damage to the plant from these harmful wavelengths (Rizzini et al., 2011). Responses to UVA/blue light are controlled by three different photoreceptors: the cryptochromes that are involved in the circadian clock as well as regulating a number of aspects of photomorphogenesis (Chaves et al., 2011); the Zeitlupe family which control the circadian clock and flowering responses (Demarsy and Fankhauser, 2009) and finally the phototropins which regulate the light-dependent processes that increase photosynthetic efficiency of plants (Christie, 2007, Christie and Murphy, 2013). Within Arabidopsis there are two phototropins, phototropin 1 (phot1) and phototropin 2 (phot2), which share approximately 60% sequence identity at the amino acid level (Christie et al., 2002). The phototropins have also been identified in algae, ferns and mosses as well as higher plants where their mode of action appears to be conserved (Onodera et al., 2005). The phototropins function redundantly to control phototropism, chloroplast movement, leaf flattening and positioning, stomatal opening whilst phot1 alone controls the rapid inhibition of hypocotyl growth upon transfer of dark-grown seedlings to light (Christie, 2007). The phototropins are serine/threonine (Ser/Thr) kinases consisting of two Light, Oxygen and Voltage (LOV) domains in the N terminus and a Ser/Thr kinase at the C terminus (Figure 1). In darkness, the protein associates with the plasma membrane and upon illumination partially internalises to cytosolic strands. Illumination with blue light also results in the the LOV domains forming a covalent linkage with the chromophore, Flavin Mononucleotide (FMN) and the protein undergoes autophosphorylation at a number of serine residues including upstream of the LOV1 domain and between the LOV1 and LOV2 linker region, as well as within the kinase activation loop (Inoue et al., 2008; Sullivan et al., 2008). Whilst the physiological functions of the phototropins from Arabidopsis are well characterised, the function of the membrane association and subsequent internalisation is unknown. Therefore the aims of this project were to identify the mechanism of phot1 association with the membrane and to determine if there are specific regions of membrane interaction within the kinase domain of phot1, since the C-terminus of phot2 including the kinase domain is known to direct localisation to the plasma membrane (Kong et al., 2007). It was therefore of interest to analyse the kinase domain of various phototropins to assess if there are sequences that may direct protein localisation to the plasma membrane. Chapter 3 describes the Lysine Rich Motif (LRM) that was identified and subsequently mutated to assess localisation when transformed in to the phot1-5 phot2-1 double mutant as well as subsequent complementation analysis of the physiological responses controlled by phot1. The analysis suggests that phot1 may interact with the membrane through a lipid interaction that is not mediated by the LRM. The consequences of these results are discussed further in the chapter. Further analysis of the role played by the kinase domain in membrane localisation of phot1 is described in the truncation analysis in Chapter 4. A similar approach to the deletion analysis employed by Kong et al. (2013) was used. A predicted secondary structure was generated to ensure that truncations were performed outside secondary structures that may be important for the protein structure. The insect cell system as well as transient expression in N. benthamaiana provided convenient methods to analyse various truncations of the kinase domain of phot1. The method was also used to analyse the membrane association of phot2. The effect of 1-butanol treatment on Arabidopsis seedlings was also investigated in relation to phot1 associating with the plasma membrane by interaction with lipids such as phosphatdic acid (PA). The work presented in this chapter suggests that there may be more than one region of phot1 that interacts with the membrane and the complications of this are discussed further. In order to investigate the function of internalised phot1 after irradiation, the protein was constitutively targeted to the membrane using a farnesyl tag. A farnesylation sequence targets proteins to the membrane via a lipid modification (Sorek, Bloch and Yalovsky, 2009). The farnesyl-tagged phot1-GFP was transformed into the phot1-5 phot2-1 double mutant and the physiological responses controlled by the phototropin were assessed. The results of targeting phot1-GFP to the membrane are shown in Chapter 6. Constitutive targeting of phot1-GFP to the plasma membrane showed that the soluble protein visualised after blue light illumination is, perhaps surprisingly, dispensable for the physiological responses tested. There may however be a role to play in the fine-tuning of the Arabidopsis response to illumination. Together these studies provide new perceptions in the possible mechanism of membrane attachment of phot1. Finally, investigation of the phototropin from the algae Ostreococcus tauri reveal that, while it can mediate some phot-regulated responses when expressed in the phot1-5 phot2-1 double mutant the protein is not functional in phototropism providing a unique phototropin that could be used to investigate the downstream control of this response. These findings highlight key differences between the mode of action of plant and algal phototropins that have so far gone unrecognised.

The aim of this thesis is to investigate the evolution of cultivated flax (Linum usitatissimum L.). Specifically, it focuses on the adaptation of flax to European latitudes during the spread of agriculture. The two main objectives of this study are to determine 1) the role of hybridization with the flax wild relative (pale flax; Linum bienne Mill.) in the adaptation to northerly latitudes and 2) the link between the adaptation and the emergence of fibre flax varieties. Flowering time genes play an important role in adaptation to northerly latitudes. Homologs of three flowering genes from Arabidopsis thaliana (LFY, TFL1 and TOC1) were identified and sequenced in flax. This included seven flax paralogs of TFL1, which shared gene structure, exonic sequences, conservation of amino acids in the active site, and expression patterns with the phosphatidylethanolamine binding protein family that includes TFL1. Sixteen different haplotypes of LuTFL1 were discovered amongst sequence data derived from a diversity collection of flax accessions. Haplotypes in cluster I are associated with samples from Southern Europe and the Near East, while those in cluster III are associated with Central and Northern Europe. The LuTFL1 gene was under selection or selective sweep that occurred in cultivated flax. Haplotype I was inherited from pale flax during domestication, while haplotype III was inherited post-domestication following hybridization with pale flax from around the Bosphorus. Within sequence data of LuTFL2, ten haplotypes were discovered and these too were correlated with latitude. Contrary to the previous case however, there is no evidence of selection in LuTFL2. LuTFL1 and LuTFL2 might be in linkage disequilibrium. A collection of RADseq markers was developed to investigate the genetic structure of flax and measure the genetic migration between wild and cultivated species. Of all the RADseq markers, 219 were characterized with single SNP and used for further analyses. The genetic diversity of cultivated flax was found to be similar to that of pale flax. The population structure inferred from SNP data showed genetic separation of dehiscent and non-dehiscent varieties, but no distinction between intermediate, fibre and oil varieties of cultivated flax. The population is structured from south to north within landraces, intermediate and fibre varieties. There is strong evidence for admixture from pale flax to cultivated flax, especially from the populations in the area of domestication. High levels of genetic migration are shown from all populations of pale flax towards both the southern and northern cultivated flax. RADseq SNP alleles that are associated with northerly latitudes in cultivated flax were introduced through post-domestication hybridization with pale flax of various geographic origin. These combined molecular results indicate that pale flax contributed to the adaptation of cultivated flax in the European climate through post-domestication gene flow. Considering archaeological and phenotypic data from the literature, the adaptation to northerly latitude might have led to the emergence of fibre varieties. This inference is supported by a custom-made programme called PGROWTH, which models the impact of FT and TFL1 gene expression on flax architecture. These two genes play an important role in plant development and therefore might have an impact on flax architecture. This study suggests the need of carrying out transgenic, functional experiments with LuTFL1 constructs in the future.

In this thesis I present a study on floral determination in the short day plant Pharbitis nil. Shoot apical meristems are determined if, following induction, they form floral organs even if isolated in non-inductive conditions. Five day old P. nil is fully induced by a 48 h dark period. P. nil apices are determined with respect to carpels 24 h after the 48 h inductive treatment if cultured in glucose medium but not until 5 d after induction if cultured in sucrose. I found that similar differential effects existed in the determination times of the other floral organs by excising shoot apices periodically through a 48 h dark treatment and the following 24 h of continuous light, culturing them in glucose or sucrose medium and measuring the degree of floral development. I cultured apices with glucose analogues instead of glucose. The analogue 3 oxymethylglucose, which is transported into the cell but not phosphorylated, mimicked the effect of glucose so that glucose entry into the cell probably acts as a signal for floral development. Glucose was as prevalent as sucrose in the sap extracted from seedlings, regardless of induction. Structural homologues of the Arabidopsis thaliana genes LEAFY, AGAMOUS and CRABS CLAW were cloned in P. nil. The homologues PnAG1, PnAG2, PnCRC2 and PnLFY1 were found to be expressed more strongly in induced than in non-induced apices in vivo and more strongly in induced apices cultured in glucose medium than in sucrose medium using semi- quantitative RT-PCR. I conclude that PnAG1, PnAG2, PnCRC2 and PnLFY1 are floral homeotic genes and that glucose is involved in signalling for floral development and signals for the increased expression of these genes. Finally a model of floral determination, based on these conclusions, is proposed.

The LFY gene, which has now been isolated in at least 17 species, is important in the transition from vegetative to reproductive growth. The floral meristems of lfy mutants exhibited increased inflorescence characteristics, and constitutive expression of the gene was sufficient to convert lateral inflorescence meristems to solitary flowers in Arabidopsis, tobacco and Populus. Previous work on the determinate plant, Silene coeli-rosa, which required 7 LD for 100% flowering, concentrated on changes to the cell cycle and peptide composition of the shoot apex during floral evocation. A partial cDNA clone of a Silene LFY homologue (SFL) has been isolated. SFL shows strong homology to other LFY homologue proteins within the two conserved domains, with up to 88% identical amino acids. It contains highly acidic and basic domains, a glutamine rich region and leucine repeats: all putat5ive transcriptional activation domains. Expression studies using quantitative PCR show that SFL was not induced by non-inductive SD conditions, or by a continuous light treatment that inhibited flowering. This is in contrast to the expression patterns observed in vegetative Arabidopsis, pea, petunia, Impatiens, tobacco and tomato, but consistent with the expression of Antirrhinum homologue which is restricted to the floral meristems. During the 7 LD induction period, SFL transcripts were first detected after 5 LD, a treatment which resulted in 81% of plants flowering, under the conditions used. Fewer than 5 LD failed to induce flowering or SFL expression. SFL was also expressed in apices subjected to an inductive 7 LD treatment followed by 48h darkness, which delayed flowering and suppressed the synchronisation of the cell cycle which occurs immediately prior to floral initiation. In situ hybridisation revealed the spatial expression of SFL in Silene. No SFL was detected prior to D7 during floral induction or in non-inductive SD controls. On experimental D7, SFL mRNA was restricted to the flanks of the primary apical dome and in D8 apices expression had spread throughout the dome. Importantly, this pattern of expression differs to that observed in the other two determinate species in which LFY has been studied, namely tobacco and Impatiens.

The cell cycle comprises the four phases of, G1, S-phase, G2 and mitosis. Two critical transitions are G1/S and G2/M; the latter is regulated by WEE1 kinase and CDC25 phosphatases. The scope of this thesis was to investigate the regulation of the G2/M transition of the cell cycle by WEE1 and CDC25, and how these genes interface with plant growth regulators in Arabidopsis thaliana. In Arabidopsis roots, the frequency of lateral roots was found to be increased by ectopic expression of Schizosaccharomyces pombe (Sp)cdc25e and reduced by Arath;WEE1 expression. I examined the effect of Arath;WEE1 and Spcdc25 on induction of shoots and roots in Arabidopsis hypocotyls in vitro. Hypocotyl explants from two over-expressing WEE1 lines , three T-DNA insertion lines and two expressing cdc25 (Spcdc25e) lines together with wild type (WT) were cultured on two-way gradients of kinetin (Kin) and naphthyl acetic acid (NAA). Below a threshold concentration of NAA (100 ng ml-1), WEE1 repressed morphogenesis in vitro, whereas at all NAA/Kin combinations Spcdc25 promoted morphogenesis (particularly root formation) over and above that in WT. Loss of function wee1-1 cultures were very similar to WT. Quantitative data indicated a significant increase in the frequency of root formation in Spcdc25e cultures compared with WT particularly at low Kin concentrations, and WEE1oe’s repressive effect was overcome by NAA but not Kin. In conclusion, WEE1 has a repressive effect on morphogenesis in vitro that can be overcome by auxin whereas Spcd25 by-passes a cytokinin requirement for the induction of morphogenesis in vitro. The role of CDC25 and WEE1 in DNA damage responses was also analysed. Two over-expressing Arath;CDC25 lines and T-DNA mutants showed no difference to WT either in standard conditions or zeocin-supplemented treatments. However, root length was longer in Arath;CDC25oe lines treated with hydroxyurea (HU) and lateral root number was increased compared to WT. This suggests a differential response of Arath;CDC25oe in the DNA replication (HU-induced) and DNA damage (zeocin-induced) checkpoints (Chapter 5). Finally the roles of WEE1 and CDC25 in cell cycle regulation were examined using tobacco TBY-2 cell cultures expressing Arath;WEE1, Nicotiana tabacum (Nicta)WEE1 or Arath;CDC25. Whilst Nicta;WEE1 lengthened G2 of the cell cycle, Arath;WEE1 had an unusual effect of shortening G2 phase and Arath;CDC25 had no observable effect (Chapter 6).

Enzymatic, covalent attachment of the Small Ubiquitin-like Modifier (SUMO) protein to a substrate protein, SUMOylation, is a stress inducible post-translational modification conserved throughout eukaryotes. SUMO conjugation to proteins alters protein interactions, regulating signalling pathways in the cell, and modulating response. SUMO proteases process SUMO into its mature form as a prerequisite to conjugation, in addition to providing reversibility to the SUMOylation pathway by cleaving SUMO from substrate proteins. Salicylic acid (SA) is a key hormone in propagating defense activation and signalling against biotrophic pathogens in plants. An investigation into the role of SUMO proteases OVERLY TOLERANT to SALT1 and -2 (OTS1 and -2) in SA regulation was performed using Arabidopsis thaliana mutants and transgenic over expressing lines. OTS1 and -2 were required for the restriction of SA biosynthesis and signalling in unchallenged plants. Further, SA treatment promoted OTS1 degradation and accumulation of SUMO conjugates, suggesting a positive relationship between SUMO conjugation and SA synthesis. Mutants of the SUMO E3 ligase SAP and MIZ1 (SIZ1) possess reduced levels of SUMO conjugates whilst displaying elevated SA content and activated defenses. This apparent contradiction was investigated using single siz1 and triple ots1 ots2 siz1 mutants, which were found to possess comparable SA related phenotypes to the ots1 ots2 double mutant. Finally it was concluded that there is more to the regulation between SA biosynthesis and SUMOylation than the presence or absence of SUMOylated proteins, and further, that promotion of SUMO conjugates by SA may facilitate modulation of other signalling pathways.

A chloroplast protease capable of processing the precursor of ribulose bisphosphate carboxylase small subunit to the mature size has been purified 350-fold from stromal extracts of pea leaves. The enzyme has a molecular weight of about 180,000 daltons, a pH optimum near 9.0, and is inhibited by metal-chelators but not by serine- or thiol-protease inhibitors. The partially purified enzyme is also capable of processing the precursors of wheat and barley pre-plastocyanin to the mature size, and is therefore neither precursor- nor species-specific. The enzyme displays a high degree of reaction specificity in that it has failed to cleave all protein substrates tested other than precursors destined for the chloroplast. The small subunit precursor (molecular weight 20,000) is processed to the mature size (molecular weight 14,000) via an intermediate of molecular weight 18,000. The second cleavage can be inhibited by pre- incubation of the precursor with iodoacetate. A preliminary investigation into the basis for the specificity of the small subunit precursor processing reaction has been carried out. Proline, lysine and arginine residues in the small subunit precursor polypeptide chain have been replaced by amino acid analogues of these residues. The abnormal precursors are very poor substrates for the purified processing enzyme, and are imported into intact isolated chloroplasts at much-reduced rates. The significance of these observations is discussed with reference to the primary structure of the small subunit precursor.