The formation of viable pollen relies upon a complex interaction of genes in time and space within the anther. One of the most important maternal tissues involved in the production of functional pollen is the tapetum, which is a highly active tissue that plays a major secretory role during pollen development. This project involved the molecular analysis of genes that are expressed in the anther tapetum and are critical for functional pollen development. A number of these are thought to be regulated by, or interact with MALESTERILITY1 (MS1), a transcriptional regulator of male gametogenesis (Yang et al., 2007) or ABORTED MICROSPORE (Xu et al., 2010). Work involved analysis of an ABC transporter (At3g13220), which has been shown to be critical for viable pollen formation and confirmed as directly regulated by AMS (Xu et al., 2010). Another protein, POB2, which appears to be involved in ubiquitin-based proteolytic breakdown, is thought to interact with the MS1 protein. POB2 was identified from a previous screen of a stamen specific yeast-2-hybrid library using the MS1 protein. This interaction has been subsequently confirmed in this work by further yeast two hybrid analyses and bifunctional fluorescent complementation. Further work involved verification of this interaction in vitro and in planta by pull-downs and transient expression of proteins in E. coli and Nicotiana benthamiana respectively. Other work focused on identifying factors that regulate MS1 expression; this identified novel male sterile mutants derived from screening fast neutron mutagenised seed carrying the MS1Prom:MS1-GFP functional fusion protein. Microscopic observation of the fluorescent reporter showed changes in the stage specific expression of MS1 in some of these mutants. Backcrossing of the male sterile mutants with the parental plants (carrying the MS1Prom:MS1-GFP fusion construct) and the ms1 mutant confirmed one as a new mutant and the other three as being allelic to the ms1 mutation. Gene mapping of this mutant was subsequently conducted and suggest that it may be located on chromosome 3. These results are providing insight into the regulatory network of MS1 and AMS during anther development.

The ornamental industry has become an important economic force in recent years, in the UK alone this industry is estimated to be around £2.1 billion, while the international trade is around £60-75 billion (Chandler and Tanaka, 2007). The continued success of the floriculture industry depends on the introduction of new species/cultivars with major alterations in key agronomic characteristics, such as resistance to pathogens, novel flower colour and patterns or control of male fertility. Lilium, one of the most important bulbous ornamental crops, is an attractive and popular cut flower. However, the production of vast quantities of pollen that stains easily and is toxic to animals is not always desirable. The control of pollen release without affecting the appearance of the flower is therefore an important breeding goal. Lilium is also susceptible to several fungal pathogens, including Botrytis cinerea, which infects leaves, stem and flowers leading to a reduction of yield. New cultivars have tended to rely upon selective breeding as a mechanism for trait development. However approaches that utilise transgenes to manipulate traits of interest provide alternative opportunities for the ornamental industry provided that transformation and regeneration can be achieved efficiently. A rapid, highly efficient and reproducible Agrobacterium-mediated transformation for Lilium has been developed. Successful transient GUS expression in callus, shoots and basal plate discs was achieved using A. tumefaciens strain AGL1 containing plasmid pBI121 harbouring intron-containing GUS and NPTII genes in cultivars "Beverly's Dream", "Star Gazer", "Night Flyer", "Acapulco", "Sweet Surrender" and Lilium leichtlinii. Based on the same transformation protocol, transgenic plants of cv. "Star Gazer" overexpressing the RCH10 chitinase gene from rice were generated. In vitro sporulation assays of these plants showed different levels of resistance to Botrytis cinerea correlated to the level of relative expression of the transgene. This is the first report of induced pathogen resistance in any Lilium cultivar by transgenic approach. Experiments were also conducted to modify fertility and pollen release in Lilium by translating regulatory gene information from Arabidopsis to Lilium. Transgenic plants of cv. "Star Gazer" either overexpressing or silencing the AtMYB26 gene, were generated. RNAi lines showed a delay in anther dehiscence suggesting that pollen development pathways could be conserved between Arabidopsis and Lilium. In addition, partial sequences of the putative orthologues of AtMS1 and AtMYB26 in Lilium were identified and cloned for future research.

Abscission is an important process in the life cycle of a plant. It takes place in predetermined sites called Abscission Zones (AZs). In the previous study of our group, a potential abscission-related gene At1g64405 (G2) was identified of particular interest using a transcriptional analysis. The aim of this study was to characterize this gene in detail. Expression analysis of G2 was carried out by fusing its promoter with GUS or GFP. Reporter gene expression was detected specifically in floral organ AZ and cortical cells surrounding the sites of lateral roots emergence. Crosses were then carried out between G2:GUS plants and three important abscission mutants: ida, 35S:IDA and bop1/bop2 in order to further investigate the expression pattern of G2. The results, together with the bioinformatics analysis, indicate that G2 is specifically expressed in AZ and is an abscission-related gene, and reveal an inverse correlation between the expression of G2 and IDA. A gene manipulation strategy was then undertaken to generate the ectopically expressed and silenced lines of G2. Overexpression of G2 was achieved by fusing G2 to a 35S promoter whereas the null lines were obtained by an RNAi strategy. 35S:G2 plants displayed unusual root hair morphology while down-regulating G2 generated plants where pollen partially failed to develop. 35S:IDA mutants displayed phenotypes with earlier abscission, extended AZ, and the ectopic secretion of AGPs at the site of organ shedding. Ectopic expression of G2 in 35S:IDA plants partially suppressed these features. A bioinformatics analysis was performed to study G2 protein sequence in order to find out potential functional domains and four motifs were selected that may be important for protein function. The potential role of G2 will be discussed in detail in this thesis.

The Arabidopsis thaliana MALE STERILITY1 (MS1) gene, encodes a plant homeodomain (PHD) transcription factor critical for viable pollen formation (Wilson et al., 2001). In the ms1 mutant, there are alterations in the production of pollen wall materials, as well as a failure of tapetal programmed cell death (PCD) (Vizcay-Barrena and Wilson, 2006). This ultimately results in the failure to produce viable pollen. Large numbers of genes are down-regulated in the ms1 mutant indicating that MS1 plays a key role in regulating late tapetal expression and pollen wall deposition (Ito et al., 2007; Yang et al., 2007). Two putative MS1 interacting proteins At1g58210/NET2A (termed as Y2H54) and AT2G46260/ LRB1 (termed as POB2) were identified from a previous Arabidopsis stamen specific yeast-2-hybrid screen, using a truncated version of the MS1 protein without the PHD motif. POB2 and MS1 were found co-localised in the nucleus, while Y2H54 was specifically located at the plasma membrane. Further confirmation of the interaction using Förster resonance energy transfer (FRET) assay methods showed that POB2 failed to interact with MS1 in planta, however, the association between the two proteins occurred in vitro, as confirmed by protein pull-down assays. Additionally, enhanced general plant growth and floral development were seen in the overexpression lines of Y2H54. However, no significant phenotypes were observed in the RNAi silencing lines. Chromatin Immunoprecipitation (ChIP) analysis uncovered that MS1 directly regulated the expression of MYB DOMAIN PROTEIN 99 (MYB99) by binding to its promoter. Other putative MS1 direct targets identified by ChIP include 3-KETOACYL-COA SYNTHASE 7 (KCS7), 3-KETOACYL-COA SYNTHASE 15 (KCS15), SPERMIDINE HYDROXYCINNAMOYL TRANSFERASE (SHT) and TAPETUM-SPECIFIC METHYLTRANSFERASE 1 (TSM1). Histone extraction and western blotting assays suggest a role for MS1 in facilitating detrimethylation of H3 marks. H3K36me3 deposition was enhanced at MYB99 in ms1 compared with the wild type, suggesting that MS1 may regulate MYB99 via H3K36me3. A new model for the MS1 regulatory network in pollen wall formation has therefore been proposed.

The plant hormone gibberellin (GA) regulates many developmental processes during a plant’s life cycle, including root and hypocotyl growth. Bioactive GAs promote GA-responsive growth and development by targetting DELLA proteins for degradation. Whilst the early steps of GA signalling are well understood it is not yet clear how the DELLA proteins alter the expression of GA-responsive genes. As other steps of the signalling pathway are encoded by multi-gene families it is possible that genetic redundancy is masking the transcription factors that act downstream of DELLAs. Using a chemical screen based on DELLA protein’s control of GA biosynthesis, 28 chemicals which blocked the GA-mediated downregulation of GA20ox1::GUS activity were identified. Using GA-mediated RGA degradation as a marker, 11 chemicals were identified as acting downstream of DELLAs in the GA signalling pathway. One of the chemicals (N23) identified in the screen was found to induce agravitropic root growth, a response more often associated with perturbation of auxin signalling. However, N23 had no effect on auxin signalling based on the characterisation of its effect on auxin-inducible genes and AUX/IAA degradation. The mode of action of N23 requires further investigation. However, N23 represents a potential for studying the role of GA in modulating gravitropism. The compound N16 potentially perturbs GA signalling by altering GA transport. It was found to block the uptake of both radiolabelled and fluorescent labelled GA into the root. Five days of exposure to N16 was required before any inhibition was observed on Col-0 roots but root elongation in ga1-3 seedlings was inhibited after only 24 hours suggesting that roots of wild type plants are saturated for GA. The site of action of N16 was not identified, but a putative oligopeptide transporter OPT6 was which is rapidly downregulated in the roots in response to GA application was investigated as a potential novel GA transporter. However, GA uptake assays in yeast strains overexpressing OPT6 proved inconclusive.

The gall forming aphid Pemphigus spyrothecae is a plant parasite that colonises the leaf petiole of the black poplar Populus nigra and its hybrids and varieties. Models of habitat selection are described and discussed in context with galling aphids. Habitat quality and aphid fitness can be quantified easily. Measures of relative reproductive fitness may be used to determine selection pressures driving habitat selection. The poplar trees differed significantly in budburst phenology, but Pemphigus spyrothecae stem mothers hatched on all trees within two days. Structural differences among and within trees were examined. Within and between host plant variation accounted for a dramatic difference in aphid fecundity. Larger leaves or shoots supported galls containing a higher number of offspring and a lower rate of gall failure. On singly galled shoots, gall position on a shoot did not affect reproductive outcome. Most stem mothers located their galls at the top of the petiole, closest to the base of the leaf, but gall position on the petiole did not affect the number of offspring produced. No relationship between stem mother size and fecundity could be detected. When competitor density increased on the same shoot or petiole, average fitness declined. Gall order on the petiole affected the growth rate of the gall tissue, but not the enclosed colony. However, distal galls (furthest from the leaf) were, on average, heavier and had more offspring than proximal galls. Predators are known to be important in regulating insect populations. Although, predators, on average, approached larger galls more often, this was not statistically significant. Due to the restrictive assumptions of models based on the Ideal Free Distribution, no qualitative fit of the distribution pattern of Pemphigus spyrothecae aphids was attempted. However, the characteristics of this aphid system were found to approximate an unequal competitors model. The availability of closed leaves on shoots at budburst, when stem mothers emerge, appeared to be the most important factor (of those examined) explaining the population distribution seen in spiral galling aphids.

Morphological and growth observations made on landraces of African yam bean (AYB) used in this study confirm that this species is the most morphologically variable in the genus (Potter, 1992). Morphological characters such as seed colour, stem colour, internode length, leaf size and number of leaves per plant were found to vary between landraces. Growth and development was controlled by both genotype and environment. Flowering was observed only when plants were grown at 25°C, rather than at 30°C, with a 12 h photoperiod. Tuber formation occurred only in AYBS and not in other landraces. Growth rates differed between landraces and between environments with plants grown in the soil displaying faster growth than those grown in pots. The response to the environment (pot and soil experiment) differed between landraces, i.e. AYB1 performed better than AYB2 in the pot experiment, whereas it was surpassed by AYB2 in the soil experiment. Clonal propagation protocols were developed using nodal explants/propagules to reproduce material with a high level of genetic uniformity from existing shoot meristems. Clonal propagation was investigated using macro (leafy stem cuttings) and micro (in vitro propagation from nodal stem segments) approaches. Axenic shoot cultures have been achieved from stem nodal segments sterilised with 10% "Domestos" bleach and grown in MS-based medium fortified with cytokinins. Amongst the cytokinins used, BAP (6-benzylaminopurine) was found to be more suitable than TDZ (N-phenyl-N'-1,2,3thidiazol-5-ylurea) and 2iP (6-(y, y-dimethylallylamino)purine) at both culture establishment and shoot multiplication stages, although optimisation of the protocol for shoot multiplication requires further study. There was persistent callus proliferation at both the establishment/initiation of cultures and the multiplication stage and the use of other plant growth regulators, such as GA3 (Gibberellic acid) and TIBA (2,3,5-triiodobenzoic acid), known to counter callus growth in cultures, did not give positive results. Although in vitro adventitious root formation was erratic, some shoots were able to root when exposed to auxins (IBA [indole-3-butyric acid] and NAA [αnaphthaleneacetic acid]) and were established in compost. IBA was preferable to NAA, as it induced more root formation. Overall, AYB cuttings produced adventitious roots relatively easily with or without auxins. Auxins at low concentrations induced rapid formation of roots in high numbers. Unlike in vitro rooting, adventitious rooting of cuttings was as high as 100% without any auxin treatment, suggesting a possibility of other factors involved in the rooting process in vitro. A cheap source of nitrogen for AYB is in the form of biological nitrogen fixation. AYB nodulated profusely with strains of both a slow growing Bradyrhizobium sp. and a fast growing Rhizobium sp., plants forming nitrogen fixing nodules with strains ORS302, CP279 and NGR234. Nitrogen fixed from the atmosphere accounted for 79-98% of the plant nitrogen and supported plant growth by an increase of up-to 1547% of dry matter in shoots.

Natural products play a pivotal role in medicine especially in the cancer arena. Many drugs that are currently used in cancer chemotherapy originated from or were inspired by nature. The toxicity and growth inhibitory activity of novel extracts of five different species of Malaysian rainforest plants, which are Melodinus species, Daphniphyllum scortechinii, Ficus fistulosa, Kopsia arborea and Tabernaemontana corymbosa, were investigated. The preliminary screening and chemical structure of jerantinine B highlighted this compound to be selected as a focus of this PhD study. Jerantinine B (JB) is one of seven novel Aspidosperma indole alkaloids isolated from the leaf extract of Tabernaemontana corymbosa. JB was previously evaluated to be one of the most potent cytotoxic jerantinine among all jerantinines against vincristine-sensitive and vincristine-resistant human oral epidermoid carcinoma cell lines. Furthermore, structural similarity of JB with the lower half of the vincristine chemical structure is therefore highlighted this compound to be a valuable candidate for biological evaluation. Herein, detailed biological evaluation of JB and its acetate derivative (jerantinine B acetate) on various human-derived carcinoma cell lines are reported. Our preliminary investigation showed significant inhibition of cell growth in sensitive and vincristine-resistant cancer cell lines, accompanied by significant inhibition of cell counts and colony formation associated with induction of apoptosis in human cancer cell lines after exposure to jerantinine B. Significant dose-dependent upregulation of apoptosis biomarkers (cleaved PARP and caspase 3) was shown and further confirmed apoptosis. Inhibition of cancer cell migration and invasion was observed after exposure of cells to JB. Profound G2/M cell cycle arrest was observed after treatment of cancer cell lines with JB. Tubulin polymerisation was significantly inhibited by JB and JB acetate. Morphological characterisations of mitotic arrest and apoptosis including microtubule disruption, multi-nucleation, DNA fragmentation, and membrane blebbing were obviously demonstrated by confocal microscopy in JB-treated cells. Indeed, significant interference in the dynamicity of microtubules caused by JB was observed and was relatively similar to that caused by vincristine and colchicine. Binding affinity of JB to heterodimeric tubulin protein was confirmed, measured and compared to vincristine and colchicine. Both JB and colchicine were shown to exhibit a cooperative binding response compared to vincristine which was characterised by no cooperative interaction with tubulin protein. The high-resolution crystal structure was obtained finally showing that JB acetate binds to the colchicine site on microtubules. Polo-like kinase 1 (PLK-1; an early trigger for the G2/M transition) was also dose-dependently inhibited by JB. Investigating a secondary mechanism by which jerantinine induces apoptosis, inhibits microtubule assembly and overcomes vincristine resistance via production of reactive oxygen species was considered. JB induced significant levels of ROS in treated cancer cells including vincristine-resistant cells, possibly contributing to their growth inhibitory and apoptotic destiny. Vincristine was unable to induce reactive oxygen production in vincristine-resistant cells. JB acetate demonstrated enhanced chemical stability compared to JB which could be the reason behind the greater potency of jerantinine B acetate compared to JB. Improvement in the targeting JB acetate to HER2-overexpressing breast cancer cells was attempted following conjugation of JB acetate to the HER2 affibody; potency was enhanced 2.25-fold when cells were exposed to the conjugate compared with JB acetate alone. The jerantinine alkaloid family represent a promising class of novel alkaloids which may produce putative clinical candidate molecules with broad-spectrum antitumour activity.

Our planet is growing rapidly in population and with that comes a demand for resources. To address issues in food security, scientists are looking to the underground parts of plants for novel mechanisms that will eventually lead to enhanced crop traits. Scientists are examining the underlying genetic frameworks to identify which genes play key roles in specific developmental processes. In this study we examined the model plant Arabidopsis thaliana as the roots are easily visualised, the genome has been sequenced, and there are many tools broadly available to work with. This thesis has used a multidisciplinary approach to uncover the signalling cascades revolving around the small signalling peptide RALF34, which is significantly involved in primary and lateral root growth. We have demonstrated, in the following chapters that RALF peptides are inherent to normative lateral root initiation, potentially regulated through shoot derived auxin. Furthermore, RALF4 and 34 peptides play a strong role in restricting primary root growth, and that together these peptides have an additive effect on cell elongation. Lastly, we identify several leucine-rich repeat receptor-like proteins, kinase proteins, and cell wall remodelling enzymes, which putatively play unique and diverse roles during primary and lateral root development. Taken together, this thesis provides novel and unique insights into new signalling pathways during root growth, which may in future aid in agronomic enterprises.

The problem consisted of thoroughly exploring Tarrant County, Texas, in an attempt to collect and study critically as many species of Spermatophytes as possible. In addition a thorough examination was made of herbarium specimens assembled from the region by other botanists.