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Species recognition is an integral component of mate selection and must occur in all sexually reproducing organisms to avoid costly hybridisation. Species recognition abilities may be comprised of both innate components and experience during ontogeny through the learning of visual, acoustic, and other sensory species-specific cues. But how greatly is the ability to recognise one‟s own species (conspecifics) over others (heterospecifics) dependent on the phylogeographic relationship of the array of potential species as social partners and to what extent is the discriminatory behaviour modulated by subject ontogeny versus species identity? Using a model system, which is widely studied in all disciplines of avian research, the zebra finch (Taeniopygia guttata castanotis), I aimed to investigate the visual and acoustic cues involved in conspecific recognition by both female and male individuals of this species. I used an array of previously untested phylogeographically relevant estrildid heterospecifics as my stimuli and tested subjects of diverse experimental ontogenetic treatments. By scoring a wide-selection of measured behavioural responses my research indicates that female and male zebra finches prefer live conspecifics over live phylogeographically relevant heterospecific stimuli and this preference is more consistent by females than males. Female zebra finches rely on both visual and acoustic features of potential social partners for accurate species discrimination; in this regard video playbacks or the diverse colour morphs of domesticated zebra finches may be useful tools for further experimentation. Additionally, females display significant individuality in their behavioural responses which may be relevant for pair bonding decisions made by both sexes. I further documented that normally-reared zebra finches will prefer song playbacks of their own species but that both rearing in an indoor restricted acoustic environment of conspecifics or cross-fostering to another species will reduce discrimination preferences, although the results may depend on the behavioural metrics analysed. This dissertation is presented as a general overview with details of my specific contributions towards the work included in this thesis, followed by discrete review and data chapters, and a final general discussion.

Anthocyanin concentration is an important determinant of the colour of many fruits. In apple (Malus x domestica), centuries of breeding have produced numerous varieties in which levels of anthocyanin pigment vary widely and change differently in response to environmental and developmental stimuli. The apple fruit cortex is usually colourless, although germplasm does exist where the cortex is highly pigmented due to the accumulation of anthocyanins, carotenoids or chlorophylls. From studies in a diverse array of plants species, it is apparent that anthocyanin biosynthesis is controlled at the level of transcription. In this thesis the transcript levels of the anthocyanin biosynthetic genes in a red-fleshed apple are compared with a white-fleshed cultivar. A MYB transcription factor (TF), MdMYB10, was isolated and is shown to be similar in sequence to known anthocyanin regulators in other species. Further, this TF is shown to induce anthocyanin accumulation in both heterologous and homologous systems, generating pigmented patches in transient assays in tobacco leaves and highly pigmented apple plants following stable transformation with constitutively expressed MdMYB10. Efficient induction of anthocyanin biosynthesis in transient assays by MdMYB10 was dependent on the co-expression of two bHLH proteins from apple, MdbHLH3 and MdbHLH33. A strong correlation between expression of MdMYB10 and apple anthocyanin levels during fruit development suggests that this TF is responsible for controlling anthocyanin biosynthesis in apple fruit; in the red-fleshed cultivar and in the skin of other varieties, there is an induction of MdMYB10 expression concurrent with colour formation during development. Mutations in the genes encoding for either the biosynthetic or transcriptional regulation of the anthocyanin pathway have been linked to colour phenotypes. Generally this is a loss of function resulting in a reduction or a change in the distribution of anthocyanin. The upstream regulatory region of MdMYB10 was investigated and found to contain a rearrangement. This modification is responsible for increasing the level of anthocyanin throughout the plant to produce a striking phenotype that includes red foliage and red fruit flesh. It consists of a series of multiple repeats, forming a minisatellite-like structure that comprises five direct tandem repeats of a 23 base pair sequence. This MdMYB10 rearrangement is present in all the red foliage apple varieties and species tested, but in none of the white fleshed varieties. Transient assays demonstrated that the 23 bp sequence motif is a target of the MdMYB10 protein itself, and the number of repeat units correlates with an increase in transactivation by MdMYB10 protein. The repeat motif is capable of binding MdMYB10 protein in electrophoretic mobility shift assays. Taken together, these results indicate that a rearrangement in the promoter of MdMYB10 has generated an autoregulatory locus and this autoregulation is sufficient to account for the increase in MdMYB10 transcript levels and subsequent ectopic accumulation of anthocyanins throughout the plant. Characterisation of MdMYB10 and the rearrangement in the promoter region has implications for the development of new varieties through classical breeding or a biotechnological approach. Understanding whether this mutation is simply an allele of other recently published apple MYB TFs, or if this is the only R2R3 MYB TF involved in apple anthocyanin response, is a challenge for future research.

The cell cycle is one of the fundamental processes in nature, and is primarily concerned with the faithful replication of cellular contents, followed by even division to produce two identical daughter cells. It is made up of five discrete biochemical steps, comprising the interphase (G1, S and G2) and the mitotic phase (mitosis and cytokinesis), with two major regulatory checkpoints at G1 and G2. The focus of this research is the G2 checkpoint, which ensures the successful achievement of DNA replication, prior to the initiation of mitosis. Arrest or progression is principally mediated by the CDK1/cyclin B1 complex; phosphorylation of CDK1 by wee1 kinase prevents progression to mitosis, and subsequent dephosphorylation by the CDC25 phosphatases, initially the B isoform, leads to mitotic onset. The aim of this research was the biophysical and/or biochemical characterisation of the molecular complexes that form at the G2 checkpoint to regulate entry into mitosis. CDK1 and cyclin B1 were separately expressed and purified from baculovirus-infected Sf9 cells. The wee1/14-3-3β complex was also expressed and purified, incorporating either full length wee1 or a truncated version from which the N-terminal domain of wee1 was deleted. Both exhibited wee1 kinase activity, at equivalent levels, p<0.001, with a 2.4 fold increase in kinase activity when wee1 is bound by 14-3-3β, p>0.001. Tryptic digestion of the complex indicated that its architecture was likely to be flexible and open, particularly within the N-terminal domain of wee1. CD analyses indicated that the wee1/14-3-3β complex was folded, with 30-40% α-helical content and 10-20% β-sheet content. Dissociation experiments were unsuccessful, however, indicating a high strength of interaction between wee1 and 14-3-3β. The empirical stoichiometry of the complex was determined as 1:1; subsequent native molecular weight determination suggested that the minimal functional unit is likely to be a 2:2 wee1/14-3-3β arrangement. It was proposed that the structural architecture of this complex may be similar to the serotonin N-acetyltransferase/14-3-3ζ complex. Experiments to determine the structure experimentally, using either TEM or x-ray crystallography, were unsuccessful, as the complex appeared to exhibit a high degree of flexibility in solution. CDC25B was also expressed and purified, and was found to co-purify with a putative Sf9 14-3-3 protein. Consequently, it was re-cloned to co-express with 14-3-3β, and subsequent analysis of the resulting CDC25B/14-3-3β complex indicated that the empirical stoichiometry was 1:1, with the functional organization likely to be a 2:2 arrangement. It was proposed that the structural arrangement of this complex is most likely to be similar to that of the wee1/14-3-3β complex.

Whole document restricted, see Access Instructions file below for details of how to access the print copy. / Four topics concerning the cell walls of commelinid monocotyledons were investigated. First, the distribution of (1→3),(1→4)-β-glucans and (1→3)-β-glucans in the walls of different cell types in vegetative organs of barley (Hordeum vulgare) was determined using immunogold labelling with monoclonal antibodies. All walls were labelled with the antibody to (1→3),(1→4)-β-glucans, except those of the outer root cap. Two types of labelling distribution were found over primary walls: only adjacent to the plasma membrane, and throughout the wall and middle lamella. Small amounts of labelling occurred with the (1→3)-β-glucan antibody, mostly over plasmodesmata. Both antibodies labelled cell plates. Second, changes in location and density of (1→3),(1→4)-β-glucans in specific walls of barley coleoptiles during development were investigated by immunogold labelling and analysed quantitatively. In non-lignified walls from 2.5- to 5-days after germination, three different patterns of change were recognized. In most walls, there was an increasing amount of label located evenly throughout the walls (Type A pattern). Some walls showed a decrease in labelling density in the area beneath the cuticle but an increase in the area adjacent to the plasma membrane (Type B), whereas others showed decreasing amounts of label located evenly throughout the walls (Type C). In lignified walls (Type D) the label was confined mainly to the compound middle lamella. Third, the location of (1→3),(1→4)-ß-glucans in walls of vegetative organs from 9 families of the Poales (s.l.) was determined by immunogold labelling. Three types of wall-labelling pattern were identified depending on the labelling of specific walls. Type 1 (Poaceae and Flagellariaceae) had the heaviest labelling of non-lignified walls and Type 2 (Restionaceae and Xyridaceae) the heaviest labelling of lignified walls. Type 3 (Cyperaceae and Juncaceae) had only very light labelling of all walls. Walls of the Typhaceae, Sparganiaceae, and Bromeliaceae were unlabelled. Fourth, the ferulic and p-coumaric acids ester-linked to non-lignified primary walls from 11 families of commelinid monocotyledons were quantified. These acids were present in the walls of all species, with trans-ferulic acid the most abundant. ‘Driselase’ digestion and mild acid hydrolysis of isolated walls released ferulolyl oligosaccharides with structures that indicated the ferulic acid was ester-linked to glucuronoarabinoxylans.