Hedgerows are increasingly being recognised as important in terms of landscape biodiversity. In Britain, recent hedgerows are not valued as highly as ancient hedgerows due to distinct differences in species-richness and historical significance. However, other potential ecological functions of species-poor hedgerows may have been overlooked, especially their potential role as corridors and habitats that help overcome the effects of habitat fragmentation. This thesis examines the spatial and temporal distributions of plant species in recent hedgerows in close proximity to remnant ancient woodland seed sources, and evaluates immigration, colonization and establishment processes that occur at this interface. Transects were used to sample plant species distributions across the transition between remnant ancient woodland communities and adjoining Enclosure Act hedgerows in Northamptonshire. An analysis of the distribution of herbaceous plant species within the seed bank across the woodland- hedgerow transition is also presented. A census of colonizing woody plant species within the Experimental Hawthorn Hedgerows at Monks Wood, in Cambridgeshire was used to investigate the: I) influence of seed source availability, ii) immigration potential, iii) colonization and iv) establishment success under three hedgerow management regimes. Woodland herbaceous plant species were found to have limited spatial and temporal dispersal strategies and species successfully dispersing into adjoining species-poor recent hedgerows were those able to reproduce vegetatively. The seed bank analysis showed that species of disturbed landscapes were dispersing into the woodlands, but were not successfully colonizing. Woody plant species dispersal was found to be operating at a larger spatial scale than herbaceous plant species. The abundance of seed sources and the availability of potential bird dispersers were found to correlate with the colonization success of woody plants within the Experimental Hedgerows, but the survival and establishment was related to hedgerow structure. The implications of these findings in relation to seed source-sink dynamics, corridor theory, habitat function, conservation and landscape planning were considered

Using targeted expression of a constitutively active repressor of GA signalling Susana Ubeda-Tomas et al., (2007) demonstrated that GA action in endodermal cells is necessary for correct root growth. However, GUS studies have shown the final and penultimate GA-biosynthetic genes are not expressed in the endodermis, indicating movement of GAs may be required. This study used the targeted mis-expression of GA degrading enzymes in Col-0 and the attempted targeted rescue of GA biosynthetic and signalling mutants, using the corresponding GA metabolic or signalling component, to gain an insight into the localisation of important GA biosynthesis and signalling sites. This study has demonstrated that GA12 can be made by epidermal, cortical and endodermal cells. However, the ground tissue of the elongation zone does not contain GA12 due to the early GA biosynthetic enzymes only being expressed within cells with a close proximity to the QC. Subsequently the 20-oxidation converts GA12 to GA15, to GA24 to GA9. These reactions mobilise GA allowing it to move from the meristematic region to the elongation zone. GA20ox and GA3ox activity is required in both the meristematic region and the elongation zone for correct root growth to occur. In addition, GA metabolic components are subject to tissue specific GA feedback regulation as a result of post-transcriptional processing and/or post-translational modifications to their protein stability. GA perception in any tissue of the elongation zone can promote complete cell elongation, suggesting that any one tissue can elongate it neighbours, or that each cell is capable of releasing a signal to ensure they all elongate proportionally. The transcriptional network within the endodermis has a disproportionately important role in GAs regulation of cell division within the root proximal meristem but GA action in other cell types is also required. The cambian and bundle sheath cells in aerial tissue like the endodermis in the root contain an important transcriptional network that promotes GA dependant growth.

Early studies of the tomato ethylene signalling network using yeast two-hybrid screen previously identified three novel proteins (IntCR22, 242 and 266) that could interact with a putative ethylene kinase LeCTR2 (Lin et al., 2003). In this study, it has been demonstrated that IntCR22 is a cytoplasmic UDP-glycosyltransferase and IntCR266 is a chloroplast metallo-proteinase homologue to the Arabidopsis FtSH5/VAR1, whereas IntCR242 encodes a novel chloroplast protein with a C-terminal histidine-rich domain. In order to gain more insight into the tomato ethylene signalling mechanism, the sub-cellular localization and protein-protein interactions of the tomato ethylene signalling components have been investigated by fluorescent protein labelling and yeast two-hybrid experiments. Three tomato ethylene receptors (ETR1, NR and ETR4) and a downstream regulator EIN2 have been found in the endoplasmic reticulum (ER). Three putative downstream MAPKK kinases (CTRs) could interact with the C-terminus of the ethylene receptor possibly on the cytoplasmic side of the ER, whereas a novel ethylene signalling component GREEN-RIPE was located in the Golgi. It was therefore concluded from the localization study that IntCR242 and IntCR266 were false positives from the yeast two-hybrid screen and could not interact in vivo with the ethylene signalling components. The results presented in this study, in line with previous ethylene research suggest a possible involvement of the plant endomembrane system in the ethylene signalling network. However, the question as to how the ethylene signal moves from the ER localized receptors to promote activation of genes for the transcription factors within the nucleus remains unsolved.

Despite widespread conservation efforts, global heathland area has substantially decreased in recent decades. Heathland habitats require low nitrogen availability in order to persist. Over the past 150 years, however, nitrogen deposition has increased markedly. Early observational studies and research using artificial N applications have identified N deposition as the primary driver of heathland succession into grassland or woodland, and N enrichment is considered a threat to heathland sustainability. This study investigated soil fertility and vegetation composition at 25 lowland heathland sites in low rainfall regions of mainland Britain within a modelled wet N deposition range of 1.85 to 10.90 kg N ha-1 y-1. A bioassay approach was used to quantify relationships between soil fertility and N deposition, heathland patch size and the management regimes. This study discovered significant positive relationships between N enrichment and C. vulgaris shoot mass, N and P concentrations. No relationship between N enrichment and N : P mass ratio was found suggesting no N induced shift to P limitation. It was determined that soil phosphomonoesterase activity was not up-regulated in response to N enrichment. This suggests that the soil P reserves are sufficient to satisfy demand under current N deposition loads. Heathland patch size was negatively related to C .vulgaris shoot dry-mass which was used as a proxy for soil fertility. Measured atmospheric ammonia concentrations were not related to C. vulgaris growth and shoot chemistry. No relationships were found between any variable tested and heathland vegetation composition suggesting that local factors, such as management intervention, may be substantial determinants of vegetation composition. This study presents relationships between temperature at origin and C. vulgaris growth from populations located along a latitudinal gradient in Western Europe. The findings of this thesis have implications for current heathland management, and for future management under a climate change scenario.

Radiocaesium (134Cs, 137Cs) is of environmental concern because of its incorporation into the food chain and prolonged emission of harmful radiation. Plants take up caesium via cation transporters which cannot discriminate between radioactive and stable caesium (133Cs). Around 80% of angiosperms live in symbiosis with arbuscular mycorrhizal (AM) fungi that deliver mineral nutrients to their hosts. Contrasting effects of AM fungi on caesium accumulation by plants have been reported. The ultimate aim of this thesis was to determine whether AM fungi reduced caesium accumulation in Medicago truncatula by down regulating the expression of plant genes encoding specific potassium transporters through improving potassium nutrition of their hosts. Accumulation of potassium and stable caesium by non mycorrhizal and mycorrhizal Medicago truncatula was studied, and the effects of caesium and AM fungi on plant gene expression were investigated. In these experiments, shoot potassium concentrations of non mycorrhizal and mycorrhizal plants were identical. However, in some experiments AM associations decreased shoot caesium concentrations. These observations were also true for five other plant species studied. Colonisation of Medicago truncatula with Glomus sp. influenced expression of some genes encoding cation transport proteins, but the expression profile did not suggest improved potassium nutrition. The presence of caesium also affected the expression of several putative cation transporters, but the consequences of these changes are unknown. A reduced colonisation rate of Medicago truncatula by Glomus intraradices was observed at caesium concentrations that exist in the rhizosphere. In conclusion, in these experiments, AM fungi did not improve plant potassium nutrition, and there was no evidence that AM fungi reduced caesium accumulation by down regulating expression of plant genes encoding potassium transporters. Although colonisation by AM fungi can reduce shoot caesium concentrations, this was not always observed. Thus, fungal inoculation cannot be relied upon to deliver crops with reduced radiocaesium concentrations.

Hawaiian Skirt (HWS) is an F-box gene in Arabidopsis that plays a key role in plant floral organ development. HWS has been identified due to sepal fusion along their basal margins resulting in failure to shed its floral organs (Gonzalez-Carranza et al., 2007). Similar phenotypic characteristics can be seen in the ectopically expressed microRNA miR164 (Mallory et al., 2004; Lauf et al., 2004) and in the double mutants cup-shaped cotyledon 1 cucJ/cuc2 (Aida et at., 1997). Previous studies carried out by Gonzalez-Carranza et al. (unpublished) using genetic crosses between hws-1 and other floral mutants has revealed that HWS may play a crucial role in the microRNA biogenesis. In an effort to identify potential substrates of HWS and to identify the role of HWS in the miRNA pathway, a population of EMS mutagenized hws-1 was used for isolation and characterization of suppressors of hws-l. Screening a number of EMS mutagenized hws-l populations has identified several suppressor lines that are currently under study. From the identified mutants, two lines 43.1 and 80.5 were selected for further characterization analysis. These suppressor lines rescue the distinctive sepal fusion phenotype of hws-l as well as displaying other phenotypic characteristics. Characterization of the suppressor lines has identified that 43.1 is an allele of HST gene, which is involved in miRNA biogenesis, and 80.5 is an allele of AS2 gene, which is an adaxial cell fate determinant. Expression analyses have revealed that loss of HWS gene function leads to the repression of both 43.1 and 80.5. Genetic analyses have also confirmed that loss of HWS gene function results in an upregulation of CUC1 and CUC2 gene expression. The results obtained in this project have shown that HWS is involved in miRNA, adaxial-abaxial and Organ boundary signalling, concluding that HWS may have a wider function in different signalling pathways than previously proposed.

Flooding events are becoming more common throughout the world as a result of climate change, resulting in reduced crop yields. It was recently discovered that plants sense low oxygen (O2) (associated with flooding) through regulated proteolysis of the group VII Ethylene Response Factor transcription factors (ERFVIIs), via the Cys-Arg/N-end rule pathway of ubiquitin mediated proteolysis, which also senses another gas, nitric oxide (NO). The N-terminal (Nt) Cys of physiological (e.g. ERFVIIs) and artificial substrates was shown to be key for N-end rule function, and work in mammalian systems suggested that oxidation of Nt-Cys by O2 and NO was a required prerequisite for subsequent Nt arginylation by arginyl tRNA transferases (ATEs). However the exact mechanism of Nt-Cys oxidation has not been discovered. The primary aim of this thesis was to define the mechanism of in vivo Nt-Cys oxidation essential in determining the stability of Arg/N-end rule protein substrates, including ERFVIIs. In this study a novel approach was developed to investigate the oxidation of Nt-Cys in vivo using transgenic Arabidopsis expressing Cys-2 reporter proteins (derived from the transgenes 35S::MC-polyG-HA-GUS and Ubi1::MCGGAIL-GUS). Cys-2 of the reporter proteins is made Nt constitutively by co-translational Methionine Aminopeptidase (MAP) activity. Biochemical techniques were combined with analytical chemistry to investigate the in vivo oxidation of Nt-Cys using liquid chromatography mass spectrometry (LC-MS). In addition, synthetic peptides representing the Nt-sequence of the in vivoreporter protein were used to define the oxidative and nitrosylative modifications occurring at Nt-Cys in vitro. Findings of this study include the successful development of two new in vivo O2 sensor artificial N-end rule substrates, in the plant genetic model Arabidopsis thaliana. A further outcome revealed that the ERFVII RELATED TO APETALA 2.12 (RAP2.12) is stabilised in the shoot and root apical meristem to a greater extent than in other regions of seedlings in response to hypoxia, indicating that meristematic cells could be important oxygen sensory zones. Cys-2 reporter proteins were used in in vivo studies to attempt to identify the nature of the Nt residues following N-end rule action. Although Nt-peptides derived from affinity purified Cys-2 reporter protein were not identified by LC-MS, it was possible to demonstrate that ubiquitination is required on Cys-2 reporter proteins before 26S proteasome degradation. Combined results from Nt-Cys in vitro synthetic peptides and Cys-2 reporter proteins substrates of the Arg/N-end rule pathway, provide indirect evidence that post translational modifications (PTMs)not defined before occur in vivo. Using synthetic peptides it was possible to show evidence for Nt-Cys-sulfenamide formation after oxidation of Nt-Cys. This finding suggests that Cys-sulfenamide formation, a previously recognised reversible modification preventing irreversible oxidation to Cys-sulfonic acid, could occur at Nt-Cys of in vivoprotein substrates. An important finding of this study was the observednonreactionbetween the nitrosylated Nt-Cys and H2O or conversely oxidised Nt-Cys and NO. As Plant Cysteine Oxidases (PCOs) do not require NO to oxidise Nt-Cys, this result raises further uncertainty as to how NO is involved in the oxidation of Nt-Cys, suggesting that NO may be involved in the enzymatic activation of PCOs or the arginylation of Nt-Cys by ATEs. The findings of this study did not identify the Nt-peptide of the Cys-2 reporter protein, and hence the Nt-Cys oxidation state of an in vivo substrate of the Arg/N-end rule pathway remains unidentified. Despite this the result that Nt-Cys can be oxidised to Cys-sulfenamide is novel and an important discovery to the field of reactive Cys biology and chemistry.

In the present study the use of haploid plants and tissues was considered in relation to plant genetic manipulation. Haploid plants can be exploited directly, in the synthesis of true breeding lines. Alternatively, haploid plants and tissues may provide material for further experimentation involving protoplast fusion. Both approaches were investigated. Cyclamen persicum an attractive flowering plant is grown commercially from seed produced following open cross-pollination. As a result, Cyclamen is highly heterozygous, but the resulting variation is commercially undesirable. Inbreeding depression prevents the recovery of commercial inbred lines. Anther culture as an alternative approach for the recovery of true breeding lines was attempted. In order to test the efficiency of the culture procedure and conditions, anther culture of N. tabacum was also attempted, since this species is known to be highly responsive to anther culture. Despite the recovery of very many allodihaploid N. tabacum, plants from anther culture, no success was achieved with Cyclamen, and the possible reasons for this are discussed. It has recently been proposed that limited gene transfer might be achieved by somatic hybridisation if diploid protoplasts of a crop species were fused with haploid protoplasts of a wild type species, and novel allotriploid somatic hybrid plants recovered. Haploid protoplasts can be isolated from anther culture derived plants, however the range of species responsive to anther culture is limited. Tetrads, formed as a result of meiosis in the pollen mother cells, were investigated as an alternative source of haploid protoplasts for fusion studies. Somatic hybrids were recovered following fusion between N. tabacum leaf mesophyll (2n) and N. glutinosa tetrad (n) protoplasts. The somatic hybrids were fertile, and the progeny of the first backcross to N. tabacum were obtained. These results, and potential limitations to somatic hybridisation are considered in the context of plant breeding.

Screening of an En element population of Arabidopsis thaliana resulted in the identification of a mutant known as snakeskin (sks) in which the epidermal cell-cell linkages appear to be significantly perturbed (Marchant and Bennett, unpublished results). As a result of the breakdown of epidermal cell-to-cell linkages there are changes in epidermal cell architecture and distribution. These include alterations in pavement epidermal cell shape, stomatal distribution and trichome morphology. The defect in epidermal cell-to-cell adhesion may be a consequence of reduced cell expansion resulting in increased cell division as a compensation mechanism. This results in altered stomatal index and density and reduced root and hypocotyl elongation. A change in the ability of a cell to expand may be attributed to defects in cell wall composition or to the cytoskeleton that determines where the cell wall components arc localised during expansion. The sks mutant was found to have changes in its cellulose, xyloglucan and pectin fractions of the cell wall resulting in defects in cell wall adhesion, strength and expansion. To gain a greater understanding of the sks mutation the molecular basis of the SKS gene map based cloning was undertaken employing a combination of SSLPs, InDels and SNP markers. The SKS gene was found to be located on the bottom arm of chromosome 1 on BAC clone TIl 111. SALK insert lines have been utilised in a candidate gene approach in addition to fine mapping the mutation with SNP markers.

The N-end rule pathway of targeted proteolysis is regulated by Group VII ethylene response factors (ERFVII’s). The aim of this research work was to analyse the relationship between substrates (ERFVII’s) of the N-end rule pathway and genes, which have promoters containing a double ‘GCCGCC’ Ethylene-Responsive Element Binding Protein (EBP) cis-element. Several genes were identified containing double EBP elements. Cloning and transformation of the promoters from five of these genes (PYL, ERD4, AT1G14810, AT3G13440 and AT5G44420) carrying two copies of the GCC-boxes present in the 5' UTR (5’ untranslated region) or promoter region was conducted into Arabidopsis wild-type (Col-0) and prt6-1 mutant plants. Expression driven by these promoters in the leaves and flowers of transgenic plants was analysed through GUS staining to reveal promoter activities. Enhanced promoter activity was seen in prt6-1 lines (mutated in the E3-ligase of the N-end rule pathway) in comparison to Col-0. Further, cDNA of leaves and flowers of Col-0 and prt6-1 were analysed by q-RT-PCR (quantitative real-time PCR) for expression of PYL7, ERD4, AT1G14810, AT3G13440; t-test analysis showed a significant difference (p-value<0.05) only in leaves of Col-0 and prt6-1 for PYL7. Analysis of the genetic relationship between N-end rule pathway and genes containing GCC-boxes was also performed by analysing double mutant combinations of prt6-1 and mutants of genes containing the EBP elements (pyl7prt6-1, erd4prt6-1 and abi5prt6-1) and Col-0 under different concentration of salt to determine the effect of stress due to salinity on the regulation of genes. At 125mM salt concentration significant difference was identified in highest number of mutant lines in comparison to Col-0. An analysis of the in-vivo binding of the ERFVII RAP2.3 to the promoter of GCC-boxes containing genes was performed through Chromatin Immuno-precipitation assay (ChIP). The t-test analysis on qChIP-PCR data indicated significant difference between IgG and HA-IPs for both ABI5 and PYL7 performed on normoxic 35S:MA-RAP2.3-HA in Col-0 line. Further, in-vivo localization of ERFVII’s HRE1 and RAP2.2 conditional stability was analysed using promERFVII’s:MC/MA-ERFVII’s-YFP constructs in Col-0 and prt6-1. This thesis suggests that genes PYL7, ERD4, AT1G14810, AT3G13440 that have double ‘GCCGCC’ EBP elements are downstream targets of the N-end rule pathway. Further analysis via ChIP suggests that RAP2.3 interacts with the ‘GCCGCC’ binding site of promoters in the ABI5 and PYL7 genes, however further work is needed to confirm this. Additionally, sub-cellular localization of promERFVII’s:MC/MA-ERFVII’s-YFP studies suggest the location of HRE1 and RAP2.2 in nuclei of early stage root tips studied on 4-days old etiolated seedlings.