The time of bolting is an important factor in lettuce production because it affects the yield and quality of the harvested crop. Bolting is promoted by higher temperatures and is an increasing problem for growers with the current trend for warmer summers. Lettuce plants that are in the early stages of bolting are visibly indistinguishable from non-bolting plants, however there are changes in the biosynthesis of secondary metabolites which are produced to protect the young floral bud from insect attack. These compounds give the lettuce plant a bitter taste and render the crop unsaleable. The development of late bolting varieties, which would have a greater ‘holding ability’ in the field, would result in reduced crop losses and an extension to the growing season. In many plants, the timing of the transition from vegetative growth to flowering is controlled by environmental cues which serve to communicate growth conditions favourable for sexual reproduction and seed maturation. Studies in Arabidopsis have led to the identification of several different pathways that come together to regulate flowering time. Little research has been done on these response pathways in lettuce, however, research has shown that components of these pathways are conserved between Arabidopsis and other crop species. The aim of this project is to isolate genes regulating flowering time in lettuce in order that novel alleles of these genes can be used to manipulate bolting time. A lettuce BAC library has been screened and homologues of eight Arabidopsis flowering time genes, principally from the autonomous pathway, have been isolated. Functional orthologues of FLOWERING LOCUS T (FT) and the autonomous pathway gene, FLK have been characterised in lettuce, suggesting that there is conservation of the genes involved in flowering time in Arabidopsis and lettuce. Lettuce lines with a range of bolting times, including lines which bolt significantly later than wild-type have been identified from EMS mutagenised populations of cultivated lettuce and a diversity set of wild lettuce. Homozygous lines from a Lactuca sativa cv. Larissa EMS population with a reproducible late bolting phenotype when tested under commercial growing conditions have been identified. These lines have been made available to Rijk Zwaan® for inclusion in future breeding programs aimed at delaying bolting and improving the ‘holding’ ability’ of commercial lettuce crops. Genomic sequence of selected lettuce flowering time genes have been compared between the late bolting lines and wild-type looking for polymorphisms that may account for the late bolting phenotype. Polymorphisms within these genes were identified in some of the late bolting lines, however through analysing the polymorphism in segregating backcross populations they have been shown not to be causing the late bolting phenotype. Transcriptome sequencing has also been performed to identify polymorphisms in other, possibly novel, genes which may be causing the late bolting phenotype, as yet, no mutation segregating within the late bolting lines has been identified.

Meiotic recombination in eukaryotes requires two orthologues of the E. coli RecA proteins, Rad51 and Dmc1. Both genes play an important role in the binding of single strand DNA, homology search, strand invasion and strand exchange resulting in Holliday junctions which are resolved into crossovers or non-crossovers events. Even though both genes are well characterized in a variety of organisms including plants, very little information is available from hexaploid wheat. In most diploid plant species, deletion of either the RAD51 or DMC1 orthologues leads to sterility but wheat being a polyploid, offers a unique opportunity to examine the effects of the deletion of specific homoeologue, while maintaining a degree of fertility. The transcript expression profiling of RAD51 and DMC1 genes in Arabidopsis, rice and wheat using available microarray databases indicated higher levels of expression in mitotically and meiotically active tissues compared to other tissues. However, the possible function of the DMC1 gene in mitotic-active tissues needs to be investigated further. Previously cDNA sequences of TaRAD51 and TaDMCl of hexaploid wheat were cloned and reported. In this study, it has been demonstrated that the reported TaRAD51A1 and TaRAD51A2 cDNA sequences are (D) and (A) homoeologues of TaRAD51 respectively and TaDMCl cDNA sequence is (D) homoeologue of the TaDMC1. This study also found that the amino acid sequences and evolutionary relationships of RAD51 and DMC1 cDNA homoeologues are highly conserved across eukaryotes. Functional characterization of TaRAD51 and TaDMCl gene homoeologues was undertaken in planta using Forward Genetics, Reverse Genetics and Complementation methods. Forward and Reverse Genetic screening of a subset of a Highbury mutant population could not identify any mutants that have deletions in TaRAD51 and TaDMC1 genes. However, Reverse Genetics screening of Paragon mutant population identified mutant lines that tested as having deletions for all the three homoeologues of TaRAD51 and TaDMCl. However, most likely due to high mutational load and a deleterious phenotype, only a few mutant lines survived. Phenotypic and cytogenetic analysis indicated the probable functional redundancy of TaRAD51 (B) homoeologue in meiosis, although the unknown size of the deletion and limited phenotype makes it impossible to completely certain of this. The single mutants for TaDMC1 (B) and (D) indicated a reduction in pollen viability and ear fertility compared to wild-type. The cytological examination of these mutants indicated low levels of abnormal diakinesis, resulting in the formation of dyads. However, the single mutants were still able to produce normal tetrads. This suggests that there is a possible dosage effect of these homoeologues in hexaploid wheat. Unless deletion lines for the (A) and (D) homoeologues of TaRAD51 and (B) homoeologue of TaDMC1 can be recovered and characterized the above assumptions will remain inconclusive. The results of complementation assays using over-expressing CaMV35S::TaRAD51(D)±GFP constructs demonstrated a very low (-14% and -2%, respectively, with +GFP and -OFP constructs) functional complementation in terms of seed set compared to 0% in homozygous Atrad51 mutants. One explanation of these results is that the wheat genes are not complete functional orthologues for the inactivated Arabidopsis genes. The functional complementation experiments could not be performed for TaDMC1 gene because of time limitation, although the transformants were produced in AtDMC1/atdmc1 background. Finally, overexpression of the TaRAD51 gene suggests 2-fold increase in genetic distances in Arabidopsis using CaMV35S::TaRAD51(D) construct. This was done by crossing the appropriate transformant with fluorescent tetrad lines. However the results need to be confirmed by a large scale analysis.

Folate malnutrition is a major problem in many countries around the world especially in Asia and Africa. Stable foods such as rice contain very low amounts of folate. White rice which is the most popular form for human consumption contains less than 80 µg folate per 100 g. Given that it forms a major part of the South East Asian diet, rice represents an important target for enhancing folate levels. The objectives of this thesis is study the mechanisms that regulate total folate levels in rice grains and attempt to enhance and stabilise folate in rice endosperm. Three main strategies were adopted. First, the natural variation of folate biosynthesis gene expression was probed using RT- qPCR. Second, functional genomic approaches were used to manipulate the activity of rice folylpolyglutamate synthetase (FPGS), the enzyme which adds glutamate residues to folate. Third, genetic engineering was used to express FPGS enzymes and mammalian folate binding proteins in rice endosperm. RT-qPCR revealed that the variation in folate biosynthesis transcript abundance was closely correlated with total folate levels among rice varieties. High transcript abundance of all folate biosynthesis genes was associated with high total folate levels in Moroberekan rice mature seed. Comparative genomic studies revealed that rice FPGS is encoded by two distinct genes, FPGS Os03g02030 and FPGS Os10g35940. Transcript abundance of FPGS Os03g02030 appeared higher than that of FPGS Os10g35940 in seed, whilst, transcript abundance of FPGS Os10g35940 was higher in leaf. To determine the function of the FPGS Os03g02030 gene in rice seed, a TDNA knock out line was characterised. Disrupting Os03g02030 gene expression resulted in delayed seed maturation and decreased mono- and polyglutamylated folate pools in mutant seed. RT-qPCR detected an increase in the transcript abundance of folate biosynthesis genes in seed of the knock out plant, whereas the folate deglutamylating enzyme y-glutamyl hydrolase (GGH) mRNA level was reduced. A potential feedback mechanism to maintain folate abundance during rice development was uncovered through the alternative functional FPGS Os10g35940 activity and reduction of folate breakdown. Protein-bound folate forms are better protected from oxidative degradation resulting in greater folate stability (Suh et al. 2001). Two rice FPGS and mammalian folate binding proteins was successfully introduced into rice endosperm using Agrobacterium based transformation in an attempt to retain and stabilise folate pool within rice endosperm. Analysis in terms of folate abundance and bioavailability will form part of future studies.

Quantitative profiling of mono- and polyglutamyl folates in rice was achieved using the microbiological assay (MA) and a newly developed liquid chromatography tandem mass spectrometry (LC-MS/MS) method. MA was used to screen 51 rice cultivars for their total folate content and LC-MS/MS was employed to measure naturally occurring mono- and polyglutamated forms of the vitamin in wild type, FPGS Os03g02030 knockout and transgenic lines with overexpressed FPGS genes and with folate binding protein from cow’s milk (cFBP) and rat’s liver (GNMT). Natural variation among rice cultivars in terms of total folate content was measured using MA screening and the validated LC-MS/MS technique of simultaneous profiling of mono- and polyglutamated folates through MeOHAA/PO4 extraction revealed that the naturally-occurring species in wild type rice are 5-CH3-H4PteGlu, 5/10-CHO-H4PteGlu, 5-CH3-H4PteGlu4, 5-CH3-H4PteGlu5 and 5/10-CHO-PteGlu5. There was a general decrease in these folate forms in the FPGS Os03g02030 knockout rice line while a dramatic increase was observed in overexpressed FPGS, cFBP and GNMT compared to Nipponbare in terms of 5-CH3-H4PteGlu4, 5/10-CHO-H4Pteglu5, 5-CH3-H4PteGlu6, and 5/10-CHO-H4Pteglu6 levels, resulting in a 2.5 to 8.8-fold increase in the total folate pool in the unpolished grains of rice. This study looked at the role of the two FPGS genes (Os03g02030 and Os10g35940) found in rice and the possible effect of introducing folate binding proteins (cFBP and GNMT) in terms of the overall folate profile in rice which can be exploited in breeding programmes designed to enhance folate content in staple crops like rice.

The rhizosphere has a rich fungal microbiome, including parasites, commensals and mutualists. An important group in the rhizosphere are assumed to be the arbuscular mycorrhizal fungi (AMF), which live in symbiosis with around 80% of plant species. AMF have been shown to increase plant yield, biomass, disease resistance, and shoot P. Plants exchange carbon in the form of sugars for nutrients assimilated by AMF. There is little known about AMF in association with Miscanthus giganteus, a productive bioenergy crop grown in the UK and abroad. Work was carried out to characterise the abundance, organisation, importance, function and stability over space and time of rhizosphere fungi and AMF in M. giganteus roots. Field samples from Lincolnshire were analysed using staining and molecular techniques, including small subunit rRNA gene terminal restriction fragment length polymorphism, clone libraries and amplicon pyrosequencing, and meta-transcriptomics. M. giganteus was also grown in a number of pot experiments, with various treatments including fungal inoculations and fungicide application. A number of fungal phyla were found in the roots, particularly Ascomycota, the composition of which shifted over time and exhibited diurnal patterns of activity. Fungi enhanced plant growth by a third, and were functionally active in the roots in the meta-transcriptome. AMF communities were found at much lower relative abundances in roots, and inoculation with AMF did not enhance M. giganteus growth. The work highlights the importance of the whole root mycobiome to plant growth and health, and the relatively small role Glomeromycota play in M. giganteus comparison with other fungi. The work also demonstrated the dynamic nature of fungal activity over hours, months, and years, and the complex interactions the fungal community has with environmental variables.

Economic, political and environmental factors have prioritized the need for research on phosphate (Pi) acquisition efficiency (PAE), Pi use efficiency (PUE) and Pi fertilizer uptake efficiency in crops. However, the coordination of molecular responses to Pi starvation and the mechanisms of Pi starvation tolerance have been investigated predominantly in model plants but remain elusive in grain crops, especially in wheat. This project investigates transcriptional profiles in wheat, particularly in the roots, as a response to nutrient availability focusing on phosphate (Pi). Furthermore, appropriate screening approaches and the difficulties in crop improvement, particularly for wheat, are discussed. Pi acquisition by plants is mediated by members of Pi transporter families. The roles of these Pi transporters in Pi partitioning and re-translocation is complex and the knowledge about their functioning in wheat still limited. Here, members of the Pht1 family in wheat were identified, their expression profiles determined when exposed to different nutrient regimes in roots and ear tissues at various developmental stages and their potential role as targets for genetic improvement discussed. In addition to Pi transporters, regulatory genes including transcription factors, signalling pathways and apparently other Pi-responsive genes with unknown function are also of critical importance. Therefore, the genome-wide responses to limited nutrient availability were investigated for the first time in roots of field-grown wheat exposed to limited nutrient availability resulting in the identification of several candidate genes for PAE/PUE improvement on the molecular level. These data were validated against other studies and across a wider wheat germplasm. Furthermore, the correlation of candidate gene expression to the nutritional status, Pi availability and PAE/PUE properties revealed four potential target genes which may be major contributors to genotypic diversity of this trait. However, there are still some agronomic bottlenecks which impede implementing Pi efficient crops and the application of molecular tools and marker genes.

The oomycete Hyaloperonospora arabidopsidis (Hpa) is the causal agent of downy mildew of Arabidopsis thaliana; a system that can be used as a model for the study of plant-pathogen interactions. In order for successful colonisation, biotrophic pathogens such as Hpa suppress or evade plant defences through secretion of effector proteins into the plant to manipulate and disrupt the host immune system. Alignment of oomycete effector proteins has revealed a conserved amino acid sequence at the N-terminus with the consensus sequence RxLR (arginine, any, leucine, arginine), thus allowing the use of Bioinformatic approaches to identify putative effector proteins in the Hpa genome. Studying effector action and their targets in the host may help elucidate important components of the plant defence response, eventually leading to more durable crops. Expression of the Hpa effector HaRxL21 in planta has been shown to alter host susceptibility to Hpa, Botrytis cinerea and Pseudomonas syringae. Here the interaction targets of HaRxL21 are presented and interaction with the transcriptional co-repressor TOPLESS (TPL) has been validated in planta using BIFC and Co-IP. Using deletion and mutation analysis, the specificity of the interacting protein domains has been identified as between the CTLH domain of TPL and Leucine residues within the EAR motif of HaRxL21. Microarrays have revealed effects of HaRxL21 on host transcription, particularly up regulation of genes involved in ABA signalling and a decreased induction of SA responsive genes upon SA induction. Finally, work has been carried out to determine the biochemical function of HaRxL21, showing an increased stability of TPL in the presence of this effector.

This thesis describes work focused on the chemical synthesis and herbicidal activity of the natural product phyllostictine A, a molecule of unique structure and unknown mode of action. Chapter 1 serves to introduce the natural product and describe the known activity of the natural product. Furthermore, it discusses literature methods for the construction of a-methylene-b-lactams, a key component of phyllostictine A. Chapter 2 describes work towards the construction of the macrocyclic rings found in phyllostictine A. As a result a-methylene-b-lactams have been shown, for the first time, to participate in RCM reactions. Formation of 11- and 12-membered trisubstituted membered rings was possible, however, the nature of the nitrogen substituent has a large impact. For example, 12-membered rings 124, 130 were formed in 37% yield when a para-methoxyphenyl group was utilised, while simple ethyl substitution could only achieve yields of 20%. Boc protected lactam 169 produced only linear dimer 170. The synthesis of tetrasubstituted alkenes via RCM was attempted with lactam 145, however, resulted in an unexpected rearrangement product. Not only was the RCM sensitive to the nitrogen substituent but also the size of ring being formed. For example, 11-membered rings produced significant amounts of the 22-membered dimers 89 and 87. Throughout the RCM reactions performed in this thesis held a preference for the Z-alkene. The trend was confirmed both by NMR shift analysis and X-ray crystallography. Chapter 3 describes work towards the synthesis of 4,4-disubstituted a-methylene-b-lactam subunit of phyllostictine A. Three methods: epoxide rearrangement, carbonylation of methyleneaziridines and carbonylation of 2-bromo-allyl-propenes were explored. Chapter 4 describes the herbicidal activity of phyllostictine A against the single celled algae C. reinhardtii. ED50 data was obtained for phyllostictine A against C. reinhardtii for the first time. Furthermore, it was shown to be comparable to the commercial herbicide glyphosate. Six novel a-methylene-b-lactams synthesised in Chapter 2 were tested for herbicidal activity against C. reinhardtii which has enabled us to develop preliminary structure-activity relationships.

Brassica napus is a commercially important crop worldwide and its use is quickly increasing due to its beneficial oil products and biofuel demands. Yield can be lost through infection by a fungal pathogen, Leptosphaeria maculans, the causal agent of stem canker (blackleg). An early indication of the presence of stem canker is a lesion (leaf spot) on the cotyledons or early leaves. The leaf spot stage of the disease was used in this work to ascertain if biological control agents applied both individually and in combination decreased the lesion area and also to quantify the amount of L. maculans DNA present using quantitative polymerase chain reaction (qPCR). The natural production of antibiotics by some bacteria is a commonly found form of antagonistic biological control. Bacillus amyloliquefaciens and Pseudomonas chlororaphis spp. aureofaciens 30.84 evaluated in this work both produce antibiotics and were assayed for their ability to provide control of Leptosphaeria maculans. Known active strains and field isolates of Bacillus and Pseudomonas were tested as potential biocontrol agents in vitro and then used in in planta assays. The in planta assays using bacterial isolates applied individually indicated that all the bacteria gave statistically significant control of L. maculans at the leaf spot stage. Those isolates with highest activity were further evaluated in combination, to determine if improved control of leaf spot occurred. Firstly, however, it was important to confirm the two bacteria would be compatible and antibiotics would be produced. To this aim, an in vitro assay using mutant Chromobacterium violaceum confirmed Pseudomonas chlororaphis spp. aureofaciens upregulated antibiotic production using acyl-homoserine lactones, signalling molecules. Consequently, it was vital that the Bacillus applied with it did not produce lactonase which would denature these molecules. PCR was used to confirm the enzyme was not present. It was, however, shown using in planta assays that combinations of Bacillus and Pseudomonas did not halt the infection or growth of L. maculans, but appeared to lead to increased lesion size. Colonisation of the cotyledons by the bacterial biological control agents applied onto the cotyledons was monitored by washing recovery, serial dilution, plating and colony counting along with qPCR of the DNA. All bacteria colonised successfully when applied individually. However, the populations decreased from the quantity at time zero when they were applied in combination, indicating they were unable to colonise the cotyledons successfully under those circumstances. To quantify Leptosphaeria infection, the concentration of ergosterol, a fungal sterol, was quantified to measure the colonisation of cotyledons. Concentrations were assessed using high performance liquid chromatography (HPLC). This assay was not successful no free ergosterol could be detected. This was probably due to L. maculans either having small amounts of ergosterol in its cell membranes, or having most of the ergosterol esterfied and unsuitable for quantification using this method. Polymerase chain reaction (PCR) was used to ascertain the presence of fungal hyphae within asymptomatic regions of cotyledons. It was found that the fungal DNA was detected within all areas of the cotyledon irrespective of whether the leaf spot could be seen. This result highlights the unreliability of the common method of visually assessing the presence and/or severity of L. maculans infection using leaf spot area. To monitor the populations of bacteria and the fungus in real time, DNA was extracted from the cotyledons and quantified using quantitative PCR (qPCR). The amount of L. maculans DNA isolated decreased when the BCAs were applied individually, and increased when the BCAs were applied in combination (when compared with the amount isolated from the control cotyledons). These results confirmed earlier, non-molecular assessments. To provide a benchmark for biocontrol activity, fungicides used in the control of leaf spot on oilseed rape were tested under the standard experimental conditions. Whilst control was obtained, it was not as effective as when used in the field, probably due to the formulations being optimised for field conditions. Fungicides targeted at wheat pathogens were also tested for control against L. maculans. Field application rates of these fungicides were not successful, as all damaged the epidermis of the cotyledon, resulting in death of the plant. Application of ¼ field rate still induced epidermal damage in all cotyledons except those sprayed with Q8Y78 (now called Refinzar®), where a necrotic lesion could be seen without pycnidia, at day 15 after inoculation.

The legume podborer, Maruca vitrata (Lepidoptera: Crambidae) is a serious pest of legumes throughout Asia and Africa. Previous workers identified (E,E)-10,12-hexadecadienal (EE10,12-6:Ald), (E,E)-10,12-hexadecadienol (EE10,12-16:OH) and (E)-10-hexadecenal (E10-16:Ald) as sex pheromone components of female M. vitrata. They developed a lure that attracts male moths in the field in Benin but they failed to attract male moths in the laboratory, or to develop a lure that attracts male moths in Asia or the rest of Africa. Synthetic lures also attracted significant number of female moths. The objectives of this study were to re-examine the sex pheromone of female M. vitrata with the aim of developing synthetic lures that attracted male moths in a wind-tunnel and in the field in both Africa and Asia and determining the reason for attraction of female moths to synthetic pheromone lure. Procedures were developed for mass rearing M. vitrata of African and Indian strains using synthetic diet without loss of vigour and reproductive potential. The female sex pheromone was re-examined using gas chromatography linked to mass spectrometry (GC-MS) and gas chromatography coupled with electroantennography (GC-EAG). In this study two new components, (E)-10-hexadecenol E10-16:OH and (Z,Z,Z,Z,Z)-3,6,9,12,15-tricosapentaene (ZZZZZ3,6,9,12,15-23:H), were identified as a part of the M. vitrata pheromone blend along with (E,E)-10,12-hexadecadienol (EE10,12-16:Ald). Laboratory windtunnel tests showed attraction of male moths to EE10,12-16:Ald and blends with E10-16:OH or ZZZZZ3,6,9,12,15-23:H equal to that of natural female extract for the first time. Field trials were conducted in West Africa in Burkina Faso, Ghana, Nigeria, Benin and at two locations in India. Although variable trapping results were obtained, in Burkina Faso the major component, EE10,12-16:Ald alone, caught significantly more moths than other synthetic blends consisting major and minor components. A similar result was found in India and the trap catch was significantly increased when E10-16:OH was added to the major component in a ratio of 10:90. Synthetic blends also captured female moths in West Africa but not in India. The role of the newly identified pheromone components and possible reasons for the female moth capture are discussed.