One of the distinguishable plant developmental events is the transition from the vegetative to reproductive phase (RP) of development. This stage is preceded by the juvenile to adult transition within the vegetative phase. During the juvenile vegetative phase (JVP) plants are incompetent to initiate reproductive development and are effectively insensitive to photoperiod. With the change to the adult vegetative phase (AVP), plants attain competence to respond to floral inducers, which is required for the transition to the RP. This study exploits Antirrhinum and Arabidopsis as model systems to understand the genetic and environmental factors that regulate floral incompetence during the JVP. Determinants such as irradiance and [CO2] were found to be key modifiers of the JVP. A relationship between photosynthetic assimilate levels and vegetative phase transition was revealed by analysis of carbohydrates in plants at defined developmental stages. Experimental data suggest that carbohydrate levels may be required to reach a specific threshold before plants undergo the transition from a juvenile to an adult phase of vegetative growth. This may be necessary in order to sustain a steady supply of sugars for sufficient bulk flow from the leaves to the shoot apical meristem (SAM), via the phloem, to enable delivery of florigen, which thus renders the SAM competent to flower. Determination of the JVP in Arabidopsis mutants impaired in different genetic pathways has shown that multiple inputs influence the timing of the vegetative phase transition. Carbohydrates have been demonstrated to be involved possibly through their function as nutrients or signals or by their interaction with hormones. Physiological analysis of flowering time mutants has shown that a variety of signals act to promote and enable the juvenile to adult phase transition that involves both floral activators and repressors.

Floral initiation is regulated by an elaborate network of signalling pathways, including the photoperiodic pathway. In Arabidopsis, flowering is promoted through this pathway by activation of FLOWERING LOCUS T (FT) by CONSTANS (CO) in long days. During juvenility plants are incapable of flowering in response to environmental conditions that would normally be favourable. This project studies the molecular basis of floral incompetence during juvenility in the model annual species, Antirrhinum majus and the important commercial tree species, Olea europaea, which has an extended juvenile phase. Photoperiod transfer experiments were used to measure the length of juvenility in plants grown in controlled environment cabinets at different Daily Light Integrals. Analysis of Antirrhinum FT (AmFT) expression during development showed that AmFT expression is minimal during juvenility and increases in all leaves following the end of the juvenile phase. The photoperiodic pathway was shown to be active during juvenility, suggesting that an additional mechanism involving the repression of FT could be involved in the regulation of juvenility. Full length Antirrhinum and Olive cDNAs representing homologues of the Arabidopsis FT repressors TEMPRANILLO 1 (AtTEM1) and AtTEM2, which act antagonistically with CO, were isolated. Molecular and phylogenetic analyses revealed high amino acid identities between Antirrhinum (AmTEM) and Olive (OeTEM) TEM-like proteins and AtTEM1 & 2. AmTEM and OeTEM proteins contain AP2 and B3 domains, consistent with AtTEM1 and AtTEM2, and can be classified as Class I members of the RAV sub-family of B3 transcription factors. AmTEM and OeTEM expression levels were shown to be higher during juvenility suggesting a potential role for TEM in controlling juvenility. A reciprocal relationship between expression levels of AmTEM/AtTEM1 and AmFT/AtFT was revealed in both Antirrhinum and Arabidopsis. Analysis of expression across development showed that AmTEM/AtTEM1 levels decline at around the time juvenility ends corresponding to when AmFT/AtFT levels start to increase. Arabidopsis tem1 mutants over-expressing AmTEM, OeTEM or AtTEM1 exhibited delayed flowering compared to the tem1 mutant, which demonstrated their role in regulating flowering time. Over-expression of AmTEM was shown to increase the length of the juvenile phase, delay the induction of AtCO and AtFT expression and reduce the overall levels of AtFT expression. Conversely, the juvenile phases of tem1 single and tem1/2 double mutants were shown to be shorter than in wild-type plants, with the induction of AtCO and AtFT expression occurring earlier. These findings are consistent with a role for TEM in regulating juvenility, which occurs through the down-regulation of FT and CO, and results in the inability to proceed to reproductive growth.

RNA silencing is a plant antiviral defence mechanism. A hallmark of RNA silencing is the production of small interfering (si) RNAs of 21-25 nucleotides that are generated from the viral genome. Moreover, profiles of viral siRNAs suggest that certain regions, namely hotspots, of the viral genome are more prone to RNA silencing-mediated degradation. African cassava mosaic virus (ACMV) causes cassava mosaic disease which is considered one of the greatest threats to the production of cassava in Africa and throughout the world. Geminiviruses such as ACMV are believed to be both inducers and targets of RNA silencing. The hypothesis of this research is that by targeting siRNA hotspots in the viral genome a novel strategy can be developed to control viral infection. This project has three main objectives. First, it aims to profile siRNAs associated with cassava-infecting DNA viruses via hybridisation and deep sequencing technology. Second, it aims to test the interaction between hotspot vsiRNAs and ACMV. Third, it aims to induce resistance to ACMV, and assess the level of resistance developed, by direct delivery of hotspot vsiRNA in plants. As a preliminarily study, it was demonstrated that synthetic exogenous siRNA targeting one of the siRNA hotspots in the genome of Tomato mosaic virus (ToMV) had a negative impact on virus infection in terms of RNA accumulation and symptom severity in Nicotiana benthmiana plants. In further experiments, different sized siRNAs associated with ACMV infection was detected by both northern blotting and high throughput 454 sequencing of samples from infected plants. Based on the 454 sequencing data obtained sense and anti-sense strand siRNAs have been produced corresponding to siRNA hotspot and coldspot regions. The effects of these exogenous siRNAs on ACMV infection in N. benthamiana plants were investigated. The results revealed that the co-inoculation of the hotspot siRNAs with ACMV reduced viral DNA replication and disease development. ACMV/siRNA co-inoculated plants remained symptomless throughout the 3-month course of the experiments. On the other hand, when ACMV was coinoculated with coldspot targeted siRNA, the viral DNA accumulation was comparable to infection with ACMV alone, and plants displayed typical ACMV infection symptoms. The effect of hotspot siRNA in inducing viral resistance was dose-dependent. The results also revealed that the effectiveness of hotspot siRNA to elicit durable resistance to ACMV infection requires a functional host RNAdependent RNA polymerase 6 (RDR6). In conclusion, these data demonstrate that hotspot vsiRNA could offer a novel strategy for controlling major destructive viral diseases in plants.

Polymyxa graminis and Polymyxa betae are obligate, intracellular, root-infecting organisms of cereals (P. graminis) and members of the Chenopodiacae (P. betae). Between the two species, they transmit approximately 15 economically important plant viruses. These include Soil-borne cereal mosaic virus, Barley yellow mosaic virus and Beet necrotic yellow vein virus and together, cover a world-wide distribution. Recent ribosomal DNA (rDNA) sequence data has shown that temperate isolates of P. graminis belong to two groups or ‘ribotypes’ based on sequence differences in the internal transcribed spacer region. P. betae sequences belong to a third group. These ribotypes appear to differ in host range and ability to transmit viruses. It has been suggested that particular ribotypes have different host specificities or preferences and are involved in the transmission of specific viruses. There were three major areas of work. Firstly the determination of host-virusribotype associations. One approach was to inoculate plants containing virus with specific ribotypes and monitor onward transmission of the virus to a susceptible host. Another approach used plants grown in infested soils to bait any Polymyxa present, then plants were tested for the Polymyxa ribotypes and viruses present. This work confirmed the association between ribotype II and SBCMV and SBWMV, using viruses from a wider range of geographic locations. It also identified a previously unknown role for transmission of Furoviruses by P. graminis ribotype I. Examination of the phylogenetic relationships of Polymyxa ribotypes using rDNA sequences revealed two new ribotype groups. Whilst ribotype groups II, IV and V always form a clade together, the relationship of the other P. graminis ribotypes and P. betae is still not well resolved. Finally, for the first time, infection of Arabidopsis by Polymyxa (P. betae and P. graminis) was demonstrated, showing that this model system could be used to study Polymyxa-host interactions.

Glucosinolates are a group of secondary plant metabolites, which have been shown to play important roles in human health and nutrition. Identification of novel genes or regulators of expression are important for optimising the glucosinolate composition of Brassica crops. This project aimed to develop a HPLC based methodology for quantifying these compounds within Brassica leaf material and to use this to map Quantitative Trait Loci for individual glucosinolates within Brassica oleracea mapping populations. Glucosinolates were analysed using an optimized HPLC-UV method developed in this study for complete separation of desulfated glucosinolates with high resolution for quantification measurements. The reproducibility of the desulfation reaction was improved for robust enzymatic reaction of sulfatase. A data dependent MS and MS/MS methodology was developed to confidently identify seven glucosinolates in the 89 AGDH plant lines distributed between aliphatic and indolic glucosinolate, with different combinations from the parental plants A12DHd and GDDH33. For the quantitative measurements of glucosinolates, an optimized level of glucotropaeolin was used as an internal standard (IS1). In addition, we have demonstrated the first use of a second internal standard (IS2) to significantly improve the reproducibility of the quantitative measurements. Aliphatic glucosinolates were predominant over indolic glucosinolates, where progoitrin has the highest abundance. This methodology was then used to identify Quantitative Trait Loci for individual glucosinolates and for key points in their biosynthesis. A major gene effect was found near the top of B. oleracea LG9 associated with aliphatic glucosinolate synthesis. In addition other Quantitative Trait Loci were identified which corresponded with previous work by other groups and to which individual gene function could be attributed. A number of novel Quantitative Trait Loci were also found which control the synthesis of glucosinolates distributed on the nine chromosomes of C genome. A combination of the quantitative data and genetic analysis of glucosinolate profiles was used to infer the existence of factors at distinct loci and associated these with specific steps in the biosynthesis pathway of glucosinolates in B. oleracea. The assignment of genes or gene regulator functions to Quantitative Trait Loci identified in this study was consistent with known positions of Brassica candidate genes and collinear regions of the Arabidopsis genome. Consequently, this information can be applied to other Brassica species for breeding vegetable crops with modified glucosinolate profiles.

The transition to flowering is one of the major and important phase changes during the plant’s life cycle. It has been shown that multiple signalling pathways are involved in this process. Many of these pathways converge on the floral integrator gene FT. FT is activated in the leaves by an inducing signal (e.g. photoperiod) and then it moves to the shoot apex. At the apex, the FT protein interacts with FD to initiate floral development through transcriptional activation of APETALA1 and SOC1. Several papers have reported that the FT protein moves from leaf to the shoot apex and acts as the flowering stimulus. However, it is not clear whether FT mRNA may also move through the phloem and contribute to the long-distance movement of FT. This study investigated whether FT mRNA has the capacity for long-distance movement, which cis-element(s) is responsible for its mobility and what is the biological significance of FT RNA movement. It also examined which amino acid residue(s) or domain(s) are critical for FT protein function. Utilizing an RNA mobility assay and virus induced RNA silencing, it has been found that FT mRNA, independent of the FT protein and viral sequences, is able to move systemically in Nicotiana benthamiana and Arabidopsis plants. FT RNA also can enter into the shoot apical meristem of tobacco plants. Moreover, the cis–acting element essential for FT RNA movement has been mapped to nucleotides 1-102 of the FT mRNA coding sequence. A protein which specifically binds to FT mRNA only in the vegetative stage of plant development has been identified, suggesting that the FT RNA-protein interaction may be developmentally modulated. Ectopic expression of FT mRNA triggers flowering earlier than wild type plants in inductive short day conditions, suggesting that FT mRNA might also plays a promotory role in floral induction. Using alanine-scaning mutagenesis, 43 FT mutants were constructed using PVX vector and used to inoculate tobacco plants. Viral expression of these FT mutant proteins in N. tabacum Maryland Mammoth tobacco plants resulted in various flowering phenotypes. Analysis of these phenotypes showed that mutant FTV70A stimulated slightly early flowering and significantly improved the number of flowers and seedpods. This raises the possibility that novel FT alleles could be used to speed up breeding process and improve the productivity of crops.

Photosynthetic picoeukaryotes (PPEs) are single celled algae of <3μm diameter, present in both marine and freshwater environments. Marine PPEs have begun to gain increasing recognition as important, ubiquitous primary producers, after largely being overlooked in favour of the more numerous picocyanobacteria for many years. Molecular studies have shown the group to be extremely diverse. However, most molecular studies have used PCR with general primers targeting the nuclear 18S rRNA gene to construct clone libraries and have been dominated by heterotrophic picoeukaryotes. To overcome this problem, more recent molecular studies have targeted the 16S rRNA gene of marine algal plastids, an approach which essentially excludes heterotrophic organisms. In this PhD thesis, the molecular diversity of the PPE community was analysed over broad spatial scales using both 16S and 18S rRNA gene markers to begin to draw global conclusions on the phylogenetic composition of this group and identify the major players in marine CO2 fixation. Moreover, distributions of various PPE classes were also analysed along a range of cruise transects with dot blot hybridisation of PCR amplified DNA using class-specific plastid 16S rRNA gene targeted oligonucleotide probes. All major ocean basins were analysed, encompassing a range of nutrient regimes and latitudes. The dot blot hybridisation approach revealed that the classes Prymnesiophyceae and Chrysophyceae appeared to be ubiquitous and dominated the PPE community throughout large expanses of the global ocean. Furthermore, these classes showed strongly complementary distributions along some of the transects analysed. Clone library construction demonstrated that both classes are comprised of an array of genetic lineages, many with no close cultured counterpart. For one cruise transect, the extended Ellett Line in the North Atlantic Ocean, a fluorescent in situ hybridisation approach was used as a PCR-independent assessment of the PPE community. This approach largely supported the dot blot hybridisation data. Other classes, Cryptophyceae, Pinguiophyceae, Pelagophyceae, Eustigmatophyceae, Pavlovophyceae, Trebouxiouphyceae, Chlorarachniophyceae and Prasinophyceae clade VI, were detected at lower abundances by dot blot hybridisation, with some classes restricted to specific sites. Multivariate statistics indicated that the distribution patterns of PPE classes were influenced by both temperature and nutrient concentrations. However, at the global scale, a large proportion of the variation in dot blot hybridisation data could not be explained by the environmental parameters measured. It is likely that the classes harbour different ecotypes which are individually influenced by environmental factors. Furthermore, biotic parameters not measured in this work e.g. viral lysis, predation or parasitic infection may have been important in controlling the PPE community.

While our knowledge of the functional properties of eukaryotic cellular organelles is quite comprehensive, the mechanisms by which organelles achieve their varied shapes remain poorly understood. By categorising the components which contribute to the architecture of the cell, relationships between structure and function can be established. Many mechanisms have been proposed which try to account for the shape and structure of complex organelles such as mitochondria and, more recently, the endoplasmic reticulum (ER); an organelle which comprises multiple domains, both structurally and functionally. Recently, a class of membrane proteins – the reticulons – have received renewed attention as they are thought to shape the tubular ER through their wedge-like insertion into the lipid bilayer and the formation of scaffolds through homo- and hetero-oligomerisation. Reticulons and reticulon-like proteins have been implicated in many cellular processes and have key roles in many disease states – for which they have received much attention. As much of the research on reticulons has focused on mammals and yeast, the functions of reticulons in plants are poorly characterised. The model organism, Arabidopsis thaliana, contains 21 reticulon genes, expressed throughout all tissues and developmental stages of the plant. This thesis focuses on the ER-shaping properties of a seed-specific reticulon - RTNLB13. Its expression and unique topology were found to contribute to the formation of ER tubules in plants by inducing curvature in the ER membrane. RTNLB13 is restricted to tubular ER and its over-expression induces constrictions within the ER membrane which affects both soluble and membrane protein diffusion. RTNLB13 sits in a ‘w’ conformation in the membrane with its N- and C-termini facing the cytosol. The length and orientation of its transmembrane domains play an important role in protein localisation and inducing membrane curvature. In its native organism, Arabidopsis thaliana, RTNLB13 is expressed during the late stages of embryonic maturation, although a rtn13 knockout yielded no observable phenotype.

This thesis presents the first use of the Partition of Unity Method in quantifying the spatio-temporal dynamics of a fluorescent protein targeted to the chloroplast twin-arginine translocation pathway. The fluorescence loss in photobleaching technique is applied in a modified fashion to the measurement of substrate mobilities in the chloroplast stroma. Our in vivo results address the two suggested protein targeting mechanisms of membrane-binding before lateral movement to the translocon and direct binding to the translocon. A high performance computing C/C++ implementation of the Partition of Unity Method is used to perform simulations of fluoresence loss in photobleaching and allow a compelling comparison to photobleaching data series. The implementation is both mesh-free and particle-less.

The lateral diffusion of Hcf106, a core subunit of the ΔpH-dependent translocon in higher plants, and chlorophyll-containing complexes is thoroughly investigated by Fluorescent Recovery After Photobleaching (FRAP) and Fluorescent Loss in Photobleaching (FLIP). Simple models of a thylakoid network are derived directly from confocal fluorescence microscopy images by Monte Carlo optimisation. These networks are used as the domain for diffusion simulations using the Particle Strength Exchange method, allowing determination of diffusion coefficients for the thylakoid protein Hcf106 and for chlorophyll-containing complexes. Extending the mobility studies to the chloroplast outer membrane, diffusion coefficients are estimated for Toc159, a component of the chloroplast outer membrane translocon TOC.