The expression, accumulation and kinetic properties of 1 -aminocyclopropane- 1 -carboxylic acid (ACC) oxidase (ACO), the enzyme which catalyses the final step in the ACC-dependent pathway of ethylene biosynthesis in plants, is examined. The investigation is divided into three sections: (i) identification of two ACO genes in apple leaf tissue, designated MD-ACO2 and MD-ACO3, (ii) kinetic analyses of each of the three isoforms of ACO in apple (MD-ACO1, MD-ACO2 and MD-ACO3) expressed in E. coli, and (iii) temporal and developmental expression in vivo of each of the ACO genes and accumulation of the corresponding gene products in leaf and fruit tissue. The coding regions of putative ACO gene transcripts were generated from leaf tissue using RT-PCR. Sequence alignments and interrogation of the expressed sequence tags (ESTs) database indicated that the entire open reading frame (ORF) sequences were encoded by two distinct genes, and these are designated MD-ACO2 and MD-ACO3. A third ACO gene had been identified in apple by other research workers previously, and designated MD-ACO1. Differences are obvious in the number of base-pairs (bp) constituting the entire ORF of MD-ACO1 (942 bp), MD-ACO2 (990 bp) and MD-ACO3 (966 bp). MD-ACO1 and MD-ACO2 share a close nucleotide sequence identity of 93.9 % in the ORF but diverge in the 3' untranslated regions (3' -UTR) (69.5 %). In contrast, MD-ACO3 shares a lower sequence identity with both MD-ACO1 (78.5 %) and MD-ACO2 (77.8 %) in the ORF, and in the 3'-UTR (MD-ACO1, 68.4 %; MD-ACO2, 71 %). A comparison of the gene structures show that the endonuclease restriction sites are unique to each individual MD-ACO sequence. Genomic Southern analysis, using probes spanning the 3'-UTR and the 3'-end of the coding region confirmed that MD-ACO3 is encoded by a distinct gene. However, while the distinction between MD-ACO1 and MD-ACO2 is not as definitive, different gene expression patterns adds credence to their distinctiveness. Each of the three deduced amino acid sequences contain all of the residues hitherto reported to be necessary for maximal ACO activity. Expression of MD-ACO1, MD-ACO2 and MD-ACO3 as fusion proteins in E. coli was induced using isopropy1-β-D-thiogalactopyranoside (IPTG), the recombinant proteins purified by nickel-nitrilotriacetic acid (NiNTA) affinity chromatography and the products had predicted masses determined from the nucleotide sequences, including the His-tag of 38.53 kDa (MD-ACO1), 40.39 kDa (MD-ACO2) and 39.3 kDa (MD-ACO3). Polyclonal antibodies were raised against the MD-ACO3 fusion in rabbit for western blot analysis. Antibodies had been raised previously against recombinant MD-ACO1, and while it was considered likely the MD-ACO2 would be recognized by the MD-ACO1 antibodies, MD-ACO2 does not appear to be recognized in vivo by the antibody. Analyses of the kinetic properties of the three apple ACOs was undertaken. Apparent Michaelis constants (Km) of 89.39 μM (MD-ACO1), 401.03 μM (MD-ACO2) and 244.5 μM (MD-ACO3) have been determined which suggests differences in the affinity of each enzyme for the substrate ACC. Maximum velocity (Vmax) was determined for MD-ACO1 (15.15 nmol), MD-ACO2 (12.94 nmol) and MD-ACO3 (18.94 nmol). The catalytic constant (Kcat) was determined for MD-ACO1 (6.6 x 10-2), MD-ACO2 (3.44 x 10-2) and for MD-ACO3 (9.14 x 10-2), with kcat/Km(μM s-1) values of 7.38 x 10-4 μM s-1 (MD-ACO1), 0.86 x 10-4 M s-1 (MD-ACO2) and 3.8 x 10-4 μM s-1 (MD-ACO3). The optimal pH for MD-ACO1 was 7.0 - 7.5, MD-ACO2 7.5 - 8.0 and MD-ACO3 7.0 - 8.0. All three isoforms exhibited absolute requirements for the co-substrate ascorbate in vitro with optimal activity at 30 mM. Similarly, ferrous iron (FeSO4.7H20; of 15 - 25 μM) and sodium bicarbonate (NaHCO3; of 30 mM) were required for optimal activity, and were the same for all isoforms. No significant difference in thermostability was found in this study between the MD-ACO isoforms at the P = 0.05 level. However, the activities of the enzyme differed significantly between temperatures over time. In vivo expression of each of the ACO genes in leaf tissue determined using RT-PCR and cDNA Southern analysis reveal that both MD-ACO2 and MD-ACO3 are expressed in young leaf tissue and in mature leaf tissue. While MD-ACO3 is expressed predominantly in young leaf tissue, MD-ACO2 (in common with MD-ACO1) is expressed predominantly in mature fruit tissue. None of the MD-ACOs were observed to be senescence associated genes (SAG). MD-ACO3 protein accumulated predominantly in young leaf tissue and less intensely in both mature leaf tissue and young fruit tissue, while MD-ACO1 accumulated only in mature fruit tissue. For the developmental studies, samples were collected at approximately 11 am in this study. MD-ACO2 and MD-ACO3 were also expressed in leaf tissue collected over a 24 h period in the spring and also in the autumn. For both genes transcripts accumulated in the presence of fruit but tended to disappear in the absence of fruit. These results show that MD-ACO1, MD-ACO2 and MD-ACO3 are differentially expressed, and that MD-ACO3 is encoded by a gene distinct from MD-ACO1 and MD-ACO2. MD-ACO1 and MD-ACO2 are either allelic forms of the same gene or closely clustered. Although there is some variation in kinetic properties which may reflect different physiological environments, they do not vary greatly.

In the Shoot Apical Meristem (SAM) the position at which leaf primordia arise on the periphery, and their subsequent differentiation, have been shown to be (at least in part) to be directed by genetic programs of development. A candidate gene associated with this regulation is TERMINAL EAR 1 (TE 1) a maize gene identified by the irregular phyllotaxy of its mutant lines. Unlike most other genes associated with meristem function, TE 1 is a novel RNA binding gene of the RRM type. It has been shown to have orthologues in a variety of plants including Arabidopsis thaliana as well as unicellular eukaryotes including MEI2, a gene whose product is associated with the regulation of meiosis in Schizosaccharomyces pombe. In order to more fully understand TE1's role, a functional characterisation of two of the so-called Mei2-like genes was undertaken in the model plant A. thaliana. These genes are called Terminal Ear-Like 1 and 2 (TEL1 and TEL2). Constitutive overexpression of the cDNA of TEL2 using the Cauliflower Mosaic Virus 35S promoter (CaMV35S) revealed a phenotype involving an apparently prolonged vegetative phase. However this was only observed in a limited number of lines of the total screened, and the next generation did not reiterate this phenotype. These difficulties were overcome using the LhGpOP construct system for ectopic misexpression in specific domains as well as inducible ubiquitous expression. Ectopic expression of either TEL cDNA is shown to lead to a pleiotrophic spectrum of phenotypes, which in general, were associated with reduced determinant development outside the apical meristems and as well as a delayed overall developmental progression. This provided some evidence that the normal function of TEL genes within the apical meristems is the repression of differentiation associated with the regulation of plant growth and architecture.

Shoot morphogenesis in Eucalyptus occidentalis and Metrosideros excelsa was analysed at the morphological, physiological and molecular levels to understand the regulation of phase change and the floral transition. Study of the regulation of these developmental plant processes is limited in woody species due to their long juvenile phase. Six ecotypes of E. occidentalis were grown to two predetermined architectures (free branching or single stem). Free branching plants of ecotype 13648 displayed adult shoot phenology (lanceolate leaves) earlier than single stem counterparts. In addition, changes in leaf morphology in free branching plants were accompanied with changes in leaf anatomy and gas exchange signifying that in E. occidentalis complexity of shoot architecture had a significant effect on rate of phase change. Flowering was observed in all but one ecotype irrespective of architecture demonstrating that vegetative phase change and floral transition are temporally uncoupled in this species. To understand the floral transition at the molecular level in E. occidentalis, partial homologues of the inflorescence meristem identity gene TERMINAL FLOWER1 and floral meristem identity genes LEAFY and APETALA1 were isolated. The expression patterns of these meristem identity genes during development of free branching and single stem plants were analysed by quantitative real-time PCR. Increased levels of expression of EOLFY and EOAP 1 (relative to α -TUBULIN) were displayed at more proximal nodes in free branching plants than in single stem plants. Elevated floral meristem identity gene expression levels correlated with flower initiation. Further, effects of architecture and environment on gene expression were monitored in E. occidentalis. The overriding effect of shoot architecture on the floral transition was observed under warm long day and ambient environments. Elevated levels of EOLFY and EOAP 1 were correlated with floral bud score and EOAP1 was found to be a reliable marker of floral transition in E. occidentalis. Low levels of EOTFLI expression were detected in buds irrespective of their position on the plant leading to the suggestion that this might have contributed to the precocious flowering observed in this species. In contrast to E. occidentalis, M excelsa attained adult shoot phenology (pubescent leaves) faster when grown as single stem plants than as free branching plants. It appears that growth as height is required for vegetative phase change in this species. However, floral transition occurred only once single stem plants were allowed to branch. Vegetative phase change and the transition to flowering seem to be coordinated in this species with the former being a pre-requisite for the latter.

Alcohol and tobacco are drugs of abuse which are legal to sell and consume in most western societies. Addiction to these two substances has major social and health implications worldwide. The brain structure known to mediate addictive behaviour is the dopaminergic mesocorticolimbic system. Dopaminegic neurons arise from the ventral tegmental area, project to the nucleus accumbens and interact with the amygdala and the prefrontal cortex. Chronic alcoholism elicits marked damage in the prefrontal cortex with significant loss of neurons and glia. The key components of addiction, tolerance and dependence, are thought to be the result of semipermanent adaptive changes in gene expression. Gene expression profiling of the mesocorticolimbic system from human alcoholics and alcohol-dependent animals has revealed highly region-specific alterations. How these molecular changes result in the development of alcohol dependence in humans is not fully understood. Complicating factors in human alcoholism include a high comorbidity with smoking, socioeconomic factors and the prevalence of underlying psychological pathologies. Gene expression profiling of the prefrontal cortex of six alcoholics and six controls resulted in the identification of functional gene groups sensitive to alcoholism. Mitochondrial function was found down regulated while mRNA levels of genes involved in stress response and cell protection were elevated. These results correlate with the pathology of the prefrontal cortex in chronic alcoholism. Some of the control cases used for gene expression profiling were later identified as chronic smokers, while all of the alcoholics were heavy smokers. To date the heavy co-morbidity of alcoholism with smoking has not been taken into account. Thus the expression of selected genes were investigated by realtime PCR in an extended case set of non-smoking alcoholics, smoking alcoholics, smoking non-alcoholics and non-smoking, non-alcoholics. This study revealed that alcoholism itself had a significant impact on the expression of midkine, the high affinity glial glutamate transporter, member 1 and the tissue inhibitor of the metalloproteinase 3. Heavy smoking itself led to a small but significant elevation of MDK mRNA levels as well as an increase in variation of excitatory amino acid transporter 1 and metalloproteinase inhibitor, member 3 expression. Apolipoprotein D however was induced by chronic smoking but not by alcohol dependence. These results highlight the need of careful case selection in future studies on gene expression in the human alcoholic brain. Peptide antibodies were produced to midkine and a polyclonal antibody against the excitatory amino acid transporter 1 was obtained from a collaborating laboratory. Western blots utilizing these antibodies revealed a marked increase in midkine and excitatory amino acid transporter 1 protein in alcoholics compared to non-smoking and non-drinking controls. In coronal sections of human prefrontal cortex of alcoholics and non-smoking non-drinking controls, immunofluorescence of midkine was obtained from nuclei throughout the layers of the cortex and from the cell bodies of a distinct set of astrocytes in cortical layer II. Double staining with glial fibrillary acidic protein revealed that a portion of midkine-positive nuclei were localised in glial cells. There was no difference in immunostaining of alcohol and control sections with midkine. In summary these results indicate that midkine protein is induced in the prefrontal cortex of the chronic alcoholic. However, this increase in protein may not be strong enough to be visualised by immunohistochemistry. Midkine induction may be reflective of reparative processes in the prefrontal cortex of the chronic alcoholic. Excitatory amino acid transporter 1 staining in non-alcoholic, non-smoking control cases were obtained as a confluent band in cortical layer II and sparsely in deeper cortical layers. Excitatory amino acid transporter 1 immunoreactivity overlapped partially with glial fibrillary acidic protein labelling. In chronic alcoholics, excitatory amino acid transporter 1 staining in the area between the cortical layer II and VI was significantly increased. At withdrawal, glutamate levels may reach toxic levels in the cortex. The increase in cells expressing excitatory amino acid transporter 1 throughout the cortical layers may indicate a protective measure of this brain region in the chronic alcoholic. Additionally, layer specific expression of midkine and excitatory amino acid transporter 1 in the prefrontal cortex of the healthy individual may implicate a specialised role of these astrocytes.

Dothistromin (DOTH) is a secondary metabolite produced by the fungal peanut pathogen Passalora arachidicola and pine needle pathogen Dothistroma septosporum. The chemical structure of DOTH is similar to a precursor of aflatoxin (AF) and sterigmatocystin (ST), which are secondary metabolites produced by Aspergillus species. A size fractionated genomic library was made and 11 putative DOTH genes were identified in P. arachidicola. The DOTH genes in P. arachidicola were compared to DOTH genes in D. septosporum as well as to AF and ST genes in Aspergillus species. The DOTH gene products in P. arachidicola showed 73 - 96% amino acid identity to DOTH genes in D. septosporum and 50 - 69% amino acid identity to AF/ST genes in Aspergillus. The DOTH biosynthesis genes in P. arachidicola had similar gene organization and direction of transcription to DOTH biosynthesis genes in D. septosporum and is similar in that 11 putative DOTH genes are separated into three mini-clusters. This differs from the AF/ST clusters in which 25 AF/ST genes are tightly clustered in a 70 kb region. Identification of transcription factor binding sites upstream of DOTH genes in P. arachidicola and D. septosporum suggested similar co-regulation of DOTH gene expression in P. arachidicola and D. septosporum. Tandem and inverted repeat sequences were identified in intergenic regions in the P. arachidicola DOTH gene cluster, but the distribution of those repeats appears to be random. This suggests that the fragmentation of the DOTH biosynthesis gene cluster is not due to retrotransposon activity or recombination between repeat sequences. The DOTH biosynthesis gene clusters in P. arachidicola and D. septosporum could be ancestral to AF/ST biosynthesis clusters in Aspergillus species.

The fungal endophyte Epichloë festucae forms a largely mutualistic association with the ryegrass species Lolium perenne. E. festucae produces a range of bio-protective alkaloids that protect the host grass from herbivory by both mammals and insects. One such alkaloid, Lolitrem B, is a potent mycotoxin and the causative agent of ryegrass staggers in livestock. Ten genes required for biosynthesis of lolitrem B are encoded in the ltm gene cluster. The ltm genes are expressed in a plant-specific manner, with high levels of expression in planta and very low levels of expression in culture. The mechanism regulating ltm gene expression is unknown but it is predicted to involve signalling from the host plant. The ltmM gene was chosen for use in the investigation of ltm gene regulation because the flanking regions do not contain retrotransposon sequence, which surrounds much of the ltm gene cluster. To identify fungal genes involved in the plant-induced expression of ltmM, a mutagenesis and screening system was developed using a PltmM-gusA ‘knock-in’ construct to detect expression from the ltmM promoter. Agrobacterium tumefaciens-mediated T-DNA mutagenesis was used to create a set of mutants with random insertions in the genome. Mutants were then screened for altered PltmM-gusA expression, both in culture and in planta. Three mutants were identified with increased PltmM-gusA expression in culture, however, no mutants were identified with loss of PltmM-gusA expression in planta. This indicates that a mechanism of repression is involved in the plant-induced expression of ltmM, either directly or indirectly. TM mutants of interest were also observed for altered symbiosis phenotypes. Mutants were identified with reduced colonisation rates and altered hyphal growth in planta. Integration sites were identified for two colonisation mutants and the disrupted genes are predicted to be the CTP:cholinephosphate cytidylyltransferase (CCT) gene PCT1 and the mitogen-activated protein kinsase kinase (MAPKK) gene mkk2.

Memory formation in Drosophila melanogaster is composed of two pathways that are genetically distinct, and functionally independent of each other. These are short-term and long-term memory. Short-term memory is a transient phenomenon, located in the cytoplasm of the neuronal cells, which requires no alteration of gene expression. The formation of long-term memory requires a change in gene expression, therefore chromatin-modifying complexes may play an integral part. The mushroom-bodies of Drosophila are a distinct bilateral brain structure and are essential for the formation and recollection of long-term memory. Therefore, an alteration in gene expression within the mushroom bodies is essential to the formation of long-term memory. Disruption of a gene within the mushroom-bodies that resulted in an alteration in the formation of long-term memory would indicate that the gene is involved in long-term memory. In order to investigate the role of the two chromatin-modifying proteins, HDACX and pr-Set7, whose role in memory function is unknown, RNA interference was used to knockdown expression of their respective mRNA. Published GAL4 lines were used to drive down expression in the mushroom bodies. The efficacy of the knockdown on levels of mRNA was measured by quantitative RT-PCR. The effect of these knockdowns on the formation of long-term memory was assayed using conditioned courtship. Additionally, the actual spatial and temporal expression of the GAL4 drivers was investigated using fluorescent proteins, and analysed using fluorescent microscopy. Both pr-set7 and HDACX appear to play a role in long-term memory function. The RNAi-induced knockdown of the individual mRNAs caused impairment in long-term memory formation, although the exact mode of action is still to be elucidated. The levels of mRNA from these knockdowns were reduced within the head, although not to the extent expected. The fluorescent microscopy analysis indicated that the expression of mushroom-body specific GAL4 drivers was more widespread than previously reported.

Content removed from thesis due to copyright restrictions: Schwelm, A., Barron, N. J., Zhang, S. & Bradshaw, R. E. (in press). Early expression of aflatoxin-like dothistromin genes in the forest pathogen Dothistroma septosporum. / Dothistroma septosporum causes pine needle blight, a foliar disease currently causing epidemics in the Northern hemisphere. D. septosporum synthesizes dothistromin, a mycotoxin similar in structure to the aflatoxin (AF) precursor versicolorin B. Orthologs of AF genes, required for the biosynthesis of dothistromin, have been identified along with others that are speculated to be involved in the same pathway. The dothistromin genes are located on a mini-chromosome in Dothistroma septosporum but, in contrast to AF genes, not in a continuous cluster. The aim of this study was to increase knowledge of the biological role of dothistromin, which was previously a suspected pathogenicity factor. To identify putative roles of dothistromin, the dothistromin gene expression was investigated and green fluorescence protein (GFP) reporter gene strains of D. septosporum were developed. Expression analyses of dothistromin genes revealed co-regulation. More surprisingly, dothistromin is produced at an early stage of growth and gene expression is highest during exponential growth. This is fundamentally different to the late exponential/stationary phase expression usually seen with secondary metabolites such as AF. Strains with a dothistromin gene (dotA) promoter-regulated GFP confirmed early expression of the toxin genes, even in spores and germtubes. Parallel studies with transformants containing a GFP-DotA fusion protein suggest spatial organization of dothistromin biosynthesis in intracellular vesicles. The early expression of dothistromin genes led to the hypotheses that dothistromin is either required in the early stage of the plant/fungi interaction, or for inhibiting the growth of competing fungi. Constitutive GFP strains helped to determine that dothistromin is not a pathogenicity factor. However, a putative role of dothistromin in competition with other fungi, including pine-colonizing species, was detected, supporting the second hypothesis. It was shown that dothistromin-producing strains appear to have a competitive advantage which is lacking in dothistromin-deficient strains. However, some competitors were not affected and have potential as biocontrol agents. In summary, this work has led to the discovery of an unusual pattern of regulation of a secondary metabolite, has made substantial progress in identifying the biological role of dothistromin, and has indicated potential for biocontrol of Dothistroma needle blight.

Content removed from thesis due to copyright restrictions: Schwelm, A., Barron, N. J., Zhang, S. & Bradshaw, R. E. (in press). Early expression of aflatoxin-like dothistromin genes in the forest pathogen Dothistroma septosporum. / Dothistroma septosporum causes pine needle blight, a foliar disease currently causing epidemics in the Northern hemisphere. D. septosporum synthesizes dothistromin, a mycotoxin similar in structure to the aflatoxin (AF) precursor versicolorin B. Orthologs of AF genes, required for the biosynthesis of dothistromin, have been identified along with others that are speculated to be involved in the same pathway. The dothistromin genes are located on a mini-chromosome in Dothistroma septosporum but, in contrast to AF genes, not in a continuous cluster. The aim of this study was to increase knowledge of the biological role of dothistromin, which was previously a suspected pathogenicity factor. To identify putative roles of dothistromin, the dothistromin gene expression was investigated and green fluorescence protein (GFP) reporter gene strains of D. septosporum were developed. Expression analyses of dothistromin genes revealed co-regulation. More surprisingly, dothistromin is produced at an early stage of growth and gene expression is highest during exponential growth. This is fundamentally different to the late exponential/stationary phase expression usually seen with secondary metabolites such as AF. Strains with a dothistromin gene (dotA) promoter-regulated GFP confirmed early expression of the toxin genes, even in spores and germtubes. Parallel studies with transformants containing a GFP-DotA fusion protein suggest spatial organization of dothistromin biosynthesis in intracellular vesicles. The early expression of dothistromin genes led to the hypotheses that dothistromin is either required in the early stage of the plant/fungi interaction, or for inhibiting the growth of competing fungi. Constitutive GFP strains helped to determine that dothistromin is not a pathogenicity factor. However, a putative role of dothistromin in competition with other fungi, including pine-colonizing species, was detected, supporting the second hypothesis. It was shown that dothistromin-producing strains appear to have a competitive advantage which is lacking in dothistromin-deficient strains. However, some competitors were not affected and have potential as biocontrol agents. In summary, this work has led to the discovery of an unusual pattern of regulation of a secondary metabolite, has made substantial progress in identifying the biological role of dothistromin, and has indicated potential for biocontrol of Dothistroma needle blight.