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.

New insecticidal natural products are required to find compounds with higher intrinsic activities to lower field application rates, and with novel modes of action to combat insect pest species which have developed resistance to current commercial insecticides. Using a taxonomic approach, studies on plants of the Myrtaceae family led to the isolation and characterisation of a range of insecticidal natural products 1 - 9 (figure 1). These compounds are all structurally related as they contain a tetramethylcyclohexenedione group, attached to either a terpene or a phloroglucinol moiety. Seven of the nine compounds (1 - 6, 9) are novel. For compounds 7 and 8, no synthesis had been previously reported. The compounds are active against a range of insect species, although in general they are less active than commercial natural products. Further tests show some of the compounds are potent antifeedants. Synthesis of seven of the nine natural products (1 - 5, 7, 8) by short, convergent, stereospecific and high yielding routes was achieved. The synthetic routes were devised to mimic the postulated biosynthesis of the compounds. Reaction of syncarpic acid, an aldehyde and pyrrollidine formed a Mannich base, which on elimination gave a key alkylidene intennediate. This key intermediate was reacted, with either terpenes in Diels-Alder reactions or with phloroglucinols in aromatic alkylation reactions, to synthesise seven of the natural products.

Flower initiation and development to anthesis in Dutch iris were investigated using three cultivars, 'Wedgwood', 'Ideal' and 'Professor Blaauw'. In particular, environmental effects on flower initiation and on flower abortion were studied. With the aid of a thermal-time model, the rate of initiation (the reciprocal of time to initiation) was shown to be linearly related to the temperature used to store dry bulbs, provided that this was constant. When bulbs were transferred from one temperature to another, however, the rates of initiation differed from those expected. It was concluded that the sequence of temperatures was important and a possible mechanism for flower induction was discussed. Following the start of flower initiation per se, a different optimum temperature for flower-organ differentiation was observed. The effect of both pre-planting and post-planting temperatures on the growth and development of the flower were investigated. The proportion of bulbs with successful flower development to anthesis increased with mean flower and stem dry weight. The heaviest flower and stems were produced by those bulbs with the greatest leaf weight. This was attributed to the increased availability of current photosynthates in plants with the largest foliage frames. Flower development was affected by an interaction between time of planting, bulb size and glasshouse temperature for a given light integral. With later bulb plantings, between January and March, higher light integrals were required for the same flower development at moderately high temperatures (16-18°C). This higher light requirement was detected with the largest bulbs first and not until the last planting date with smaller bulbs. At higher temperatures (20°C) flower development was poor regardless of the light integral. At lower temperatures (14°C) the same flower development achieved regardless of the light integral within the range tested. Further investigation in controlled environments enabled the detection of a photoperiodic effect at the moderately high temperatures. Daughter-bulb growth was promoted by long photoperiods and high temperatures increasing sink strength there for assimilates with a corresponding decrease in flower development. The partitioning of assimilates under high and low light was examined by determining the distribution of 14C-labelled assimilates during growth in the glasshouse. Daughter bulbs under low light had a higher relative specific activity than those under high light, at the time when the flower was most prone to abortion. Application of cytokinin to the flower bud resulted in a reduced daughter bulb weight, but heavier flower buds. It was concluded that flower development was affected by the total current assimilate available and the partitioning of these assimilates between daughter bulbs and flower bud.

In this thesis, the distribution and state of Posidonia oceanica meadows along the Turkish coast of the north eastern Mediterranean was investigated. The study area was a unique site due to the presence of eastern geographical limit of Posidonia oceanica meadows in Mediterranean Sea. It was aimed to assess the possible reasons of the absence and/or regression in relation to environmental changes. The study was conducted from September 2004 to January 2007 in 5 main regions between Anamur/Mersin and Samandag/Hatay. The regions were determined according to the presence/absence of Posidonia oceanica meadows / 1 region including the boundary meadow at the geographical distribution limit, 1 region western and 3 regions eastern to that boundary meadow. To describe the hydrological characteristics of the study area temperature, salinity and light were measured as abiotic descriptors. Additionally sedimentological analysis was carried out to check the suitability of the substrate type for Posidonia oceanica colonization. The main differences between the regions were found in temperature and light conditions. Both, the recordings of temperature loggers at 10 and 15 meter depths and the sea surface temperature retrieved from satellite (NOAA-AVHRR) showed that the eastern stations with no Posidonia oceanica meadows were warmer than the western stations. Consequently in this study the maximum tolerable temperature limit for Posidonia oceanica growth was determined as 28.4&deg / C from the data collected by the temperature loggers placed to natural meadows near to the boundary meadow. Generally, the whole study area had a limiting light condition for seagrass growth due to low penetration depths of the 10% of surface irradiation. This was a generalized minimum light requirement for growth of seagrasses. Especially in Mersin and Iskenderun bays the minimum required light level did not reached deeper than 10 meter depths in coastal stations. The biological parameters of the Posidonia oceanica meadows were investigated under 3 complementary methods in the first two regions. The structural descriptors were measured in-situ. The lowest shoot density and leaf lengths were found to be in boundary meadow. This meadow had also the lowest depth limit among other stations. The functional descriptors were examined by the lepidochronological and phenological analysis in laboratory. Most of the measurements varied either with respect to depth or among stations according to the abiotic factors of the stations. The lepidochronological cycles obtained from sheath thicknesses were further correlated with abiotic descriptors via a mathematical model. Finally, the first transplantation experiment of Posidonia oceanica along Turkish coasts has been conducted in 2 regions where presently no meadows exist. The survival of cuttings in one station indicated the success of the methodology, while the failure in other stations provided information on the possible reasons of absence/degradation of natural meadows in the area. These included the destructive impact of bottom trawling, the high pressure of grazing (the potential causes of Lessepsian migration) and the long term changes in climate resulting in alterations of environmental conditions such as increasing temperature and reduced light penetration.