School of Botany - Theses
Now showing items 1-12 of 86
The bryophyte flora of Lord Howe Island: taxonomy, diversity and biogeography
Before this study the known Lord Howe Island bryophyte flora (mosses, liverworts and hornworts) totalled 173 species, consisting of 131 mosses, 40 liverworts and 2 hornworts. For this study I conducted one month of field studies on the island, during which I collected more than 650 specimens, and also studied collections from the island held in Australian herbaria and other collections available in overseas herbaria. Fourteen moss, 32 liverwort and 1 hornwort species are newly reported from Lord Howe Island, including one liverwort new to science. A further 2 moss and 2 liverwort varieties are also new to the island. Twenty-eight moss and 11 liverwort species are discounted from the island’s flora, as well as 3 moss varieties and 1 liverwort variety. As a result, the known bryophyte flora now totals 178 species, consisting of 117 moss species (122 taxa), 58 liverwort species (60 taxa) and 3 hornwort species. These totals exclude 5 moss and 2 liverwort species whose taxonomic status or presence on the island is considered uncertain. One liverwort variety, Heteroscyphus echinellus var. echinellus, is new to Australia. Fourteen bryophyte species and one variety are endemic to the island. Spiridens muelleri, previously thought to be the same as S. vieillardii from New Caledonia, is shown to be a separate species endemic to Lord Howe Island. Chiloscyphus howeanus is also shown to be a legitimate species endemic to the island. Cololejeunea elizabethae is described as a new species, also endemic to the island. Trachyloma wattsii, considered to be endemic to Lord Howe Island, is supported by a molecular analysis as a legitimate species most closely allied to T. planifolium. Confusion about the correct identities of the two Ptychomitrium species on the island is resolved through a revision of the genus for Australia. A previously unrecorded morphological character of Atrichum androgynum is described from a study of Lord Howe Island plants, and a molecular analysis shows that South American plants previously ascribed to A. androgynum do not belong to that species. Hypnodendron vitiense is shown to be paraphyletic, but not as circumscribed by Touw (1971). The Lord Howe Island plants appear to belong to a morphologically cryptic species distinct from H. vitiense s.str, and substantial genetic variation within H. vitiense subsp. australe as currently circumscribed suggests that it might include more than one taxon. Other molecular investigations clarify the relationship between Lord Howe Island populations and mainland Australian populations of a number of moss species. An original and novel investigation of the potential modes of transport of bryophyte propagules to and from the island is made, and a hypothesis is formed about the origins of its bryophyte flora and the biogeographic relationships to the Australian land mass and other western Pacific islands, including New Zealand and New Caledonia. The nearest region of the Australian mainland is shown to be the most likely origin of most of the island’s bryoflora, with the injection of propagules into the high-level jet stream by storms the most likely dispersal mechanism. The presence of numerous otherwise tropical species on the island is probably a result of dispersal by tropical cyclones moving into the Pacific from north-eastern Australia. Migratory birds are shown to be another potential vector for bryophyte dispersal to the island.
Regulation of mucilage production in the Arabidopsis seed coat through MADS-box TF family members
During Arabidopsis seed development, the epidermal cells of the seed coat produce large quantities of pectinaceous mucilage polysaccharides. Following imbition, mucilage is released from these cells and forms a thick protective layer around the germinating seed that can be visualised with Ruthenium Red staining. The seed coat epidermis is therefore an attractive system to investigate the production of cell wall polysaccharides, particularly pectin. Previously, mutations in the transcriptional regulator LEUNIG_HOMOLOG (LUH) have been shown to be associated with a mucilage extrusion defect that is caused by reduced expression of the β-galactosidase MUCILAGE MODIFIED2 (MUM2). To better understand the role of LUH in regulating MUM2, RNA-Seq analysis was performed on whole seeds and identified genes that were differentially expressed in luh mutants. This transcriptomic analysis not only revealed elevated expression of transcription factors with a known role in epidermal cell differentiation, but also several MADS-box transcription factors that perform roles in floral development and silique shattering such SHATTERPROOF1 (SHP1) and SHP2. This thesis presents evidence that SHP1, SHP2 and SEPALLATA3 (SEP3) are a new class of regulators involved in mucilage polysaccharides modification, as loss of their activity result in mucilage extrusion defects. These mucilage defects are enhanced in shp1 shp2 double mutants and correlate with reduced MUM2 expression. While carbohydrate analysis data failed to show an expected increase in galactose residues attached to pectin in shp1 shp2 and sep3 mutants, the decrease of MUM2 in these mutants is shown to contribute to their mucilage defects as partial rescue of mucilage defects in shp1 shp2 and sep3 was achieved with rescue of MUM2 expression. This is further supported by the use of SRDX repressor motif fusions to SHP2 that not only produced mucilage extrusion defects when introduced into Col-0, but also were correlated with the level of reduced MUM2 levels. Finally, the positive regulation of MUM2 by MADS-box TFs was established through a combination of CArG-box motif mutations in the regulatory sequences of MUM2, demonstrating the requirement of MADS-box TFs for proper MUM2 seed coat expression. This was further supported by luciferase-based transactivation assays, which produced increased MUM2 promoter activity on the addition of SHP1, SHP2 and SEP3. In addition, this positive regulation of MUM2 by SHP1, SHP2 and SEP3 was shown to occur in a complex with LUH, mediated by the co-regulator SEUSS (SEU), in protein-protein interaction assays such as yeast 2-hybrid (Y2H), Y3H, and bimolecular fluorescence complementation (BiFC). Overall, this study provides novel, clear evidence that MADS-box TFs can directly bind to MUM2 and mediate its activation with LUH. Future work includes expanding on and integrating MADS-box TFs into a working model that describes the regulatory pathways controlling mucilage polysaccharides production in the Arabidopsis seed coat.
Understanding the mechanism of the synthesis of β-D-(1,3;1,4)-glucans, or mixed linkage glucans (MLGs), by the cellulose synthase like (CSL) F6 protein
The molecular mechanism by which mixed linkage, (1,3;1,4)-β-D-glucan, a non-cellulosic polysaccharide, is synthesised by cellulose synthase-like (CSL) F6 was explored. CSLF6 was shown to be crucial for development in the proposed model grass Brachypodium distachyon through analysis of point mutants. In silico homology modelling of related β-glucan synthases was used to predict domains influencing specificity, informing targeted mutagenesis. CSLF6 from Lolium multiflorum expressed in Escherichia coli was found to produce (1,3;1,4)-β-D-glucan similar to native sources in vitro, allowing biochemical characterisation.
Molecular systematics of siphonous green Algae (Bryopsidales, Chlorophyta)
The evolutionary history of the siphonous green algae (Bryopsidales, Chlorophyta) was investigated using a combination of molecular techniques and phylogenetic inference methods. Analyses of chloroplast genomes of the order revealed the high variability of genome architecture and intron content. Proliferation of nonstandard genes associated with mobile functions (i.e. reverse transcriptase/intron maturase, integrases, etc.) was also observed. Evolutionary relationships of families in the order were investigated by increasing taxon sampling and using chloroplast genome data. The chloroplast phylogenies provided good support for the suborders and most families. Several early branching lineages were also inferred in the Bryopsidineae and Halimedineae. A new classification scheme was proposed for the order, which included the following: establishment of the family Pseudobryopsidaceae fam. nov.; merger of the families Pseudocodiaceae, Rhipiliaceae, and Udoteaceae into a broadly circumscribed Halimedaceae and establishment of tribes for the different lineages found therein; finally, the deep-water genus Johnson-sea-linkia, currently placed in Rhipiliopsis, was reinstated based on the chloroplast phylogenies. Plastid (tufA) and nuclear markers (HSP90) and morphological observations were employed to delimit the Halimeda species found in Western Australia. This facilitated the recognition of Halimeda cuneata and the reinstatement of Halimeda versatilis. Investigation on morphological complexity revealed that simple uniaxial thalli was the ancestral state of the siphonous green algae and was maintained throughout their early evolution. Complex multiaxial thalli evolved afterwards on independent occasions.
Phylogeny of Eremophila and tribe Myoporeae (Scrophulariaceae)
Myoporeae is one of eight tribes recognised in the large, cosmopolitan plant family Scrophulariaceae sensu stricto. Tribe Myoporeae contains seven genera, four of which are endemic to Australia (Calamphoreus, Diocirea, Eremophila, Glycocystis), and the remaining three are distributed in the Caribbean (Bontia); Southern China and Japan (Pentacoelium); and throughout Australia, islands of the Pacific, Hawaii and Mauritius (Myoporum). The largest of these genera, Eremophila, contains over 220 species and is a major component of Australia’s largest biome, the Eremean (arid) zone. A monograph of the tribe was completed just over a decade ago (Chinnock, 2007), which provided an extensive and comprehensive taxonomic framework from which to explore the relationships and evolutionary history of the group. The first phylogenetic study of the Myoporeae (Kelchner, 2003) used two chloroplast markers to better understand generic and species level relationships in the tribe, however, due to a lack of phylogenetic resolution, the results were inconclusive. The aim of this thesis was to generate a comprehensive molecular phylogeny of tribe Myoporeae, utilising the capabilities of high throughput sequencing (HTS) technology. A genome skimming approach was implemented using a custom in-house method of library preparation, to allow for inclusion of the large number of samples required for the study. All three plant genomes (chloroplast, nuclear, mitochondrial) were represented using the genome skimming method, allowing for comparisons to be made between phylogenetic analyses of each genomic dataset. Entire chloroplast genomes (cpDNA) were assembled for 317 taxa, resulting in a well resolved and highly supported phylogeny (see Chapter Three). All allied genera were found to be nested in a paraphyletic Eremophila, with high levels of support. Chinnock’s (2007) sectional classification of Eremophila was only partially supported, with many of the 25 sections scattered throughout numerous clades. For the majority of species included with more than a single sample, a lack of monophyly was observed, which is largely attributed to the effects of introgressive hybridization, incomplete lineage sorting, and/or inappropriate species boundaries. In Chapter Four the entire nuclear ribosomal cistron (nrDNA) was assembled for 355 taxa, then analysed to a produce a moderately supported phylogeny. This phylogeny was largely congruent with the morphology-based taxonomy of the group, though differed markedly from the cpDNA phylogeny of Chapter Three. From a generic perspective, all allied genera were still nested in a polyphyletic Eremophila, while Chinnock’s (2007) sectional classification was better supported by monophyletic lineages (though still in need of revision). An increase in species rank monophyly was also observed relative to the cpDNA analysis, indicating that at least for some species, introgressive hybridization is likely to impact the chloroplast phylogenetic signal. In Chapter Five the mitochondrial genome (mtDNA) was explored, and five regions selected for analysis across a subset of 31 taxa in Myoporeae. The size and prevalence of structural rearrangement within the tribe meant assembly of entire mitogenome(s) was not feasible. Regions selected for analysis displayed low levels of variation, allowing for a moderately well-resolved phylogeny, mostly congruent with the nuclear ribosomal phylogeny of Chapter Four. Overall, construction and comparison of the three genomes in this study allowed for robust interpretation and increased understanding of the complexity in the evolutionary history and phylogenetic relationships of taxa in tribe Myoporeae. Taxonomic revision is needed at generic and sectional levels; however these changes will not be undertaken until further nuclear sequence data allows the relationships of taxa at the basal nodes of the nuclear phylogeny to be resolved. Aside from future taxonomic work, it is anticipated that this study will inform new research on the tribe Myoporeae, including the chemistry of Myoporeae (for pharmacological application); the study of plant:insect interactions between Myoporeae and members of the insect family Miridae; and biogeographic study of Australia’s Eremean zone.
Molecular mechanism of the assembly of (1,3;1,4)-β-D-glucan in Italian ryegrass (Lolium multiflorum) suspension-cultured cells
(1,3;1,4)-β-D-Glucan or mixed linkage β-glucan (MLG) is a major type of non-cellulosic polysaccharide of grass cell walls and is synthesised by the CELLULOSE SYNTHASE-LIKE (CSL) F, H and J proteins with CSLF6 being the predominant isoform. MLG consists of a backbone of unsubstituted and unbranched (1,4)-linked and (1,3)-linked β-glucosyl residues. In cereals, the (1,3)-β-Glc residue always occurs singly, mostly between either two or three (~90%) adjacent (1,4)-β-Glc residues generating characteristic DP3 and DP4 oligosaccharides after digestion with lichenase, an endo-β-glucanase that specifically cleaves MLG. The (1,3)-β-linkages cause molecular “kinks” and their irregular distribution leads to a reduction in molecular alignment between glucan chains, resulting in increased solubility compared to cellulose. The heterogeneity in fine structure of MLG is a key determinant of its solubility in the gastrointestinal tract that imparts its beneficial effects in lowering the risk of several diet-related diseases. The main objective of this project was to understand how grasses make MLG and regulate its fine structure with a view that this knowledge will enable targeted manipulation of MLG to enhance the properties of cereal grains for improved human nutrition and other agro-industrial applications. At the start of this project, it was known that CSLF/H/J proteins were the catalytic subunits of the MLG synthase (MLGS), however, the exact biochemical activity of these proteins had not yet been shown experimentally. It was still unclear how many proteins were required in the MLG biosynthesis and assembly pathway and how they were being regulated. To answer these questions, biochemical, cell biological and molecular approaches were adopted, and primarily applied to our model system, suspension-cultured cells (SCCs) of the grass species Lolium multiflorum (Italian ryegrass). Chapter 1 is a literature review that provides a brief overview of the polysaccharide composition of plant cell walls, the GT2 family of plant polysaccharide synthases to which the CSLF/H/J proteins belong, and ends with a mechanistic model of MLG biosynthesis proposed by Wilson et al (2015), arising, in part, from this study. Chapter 2 is a detailed description of materials and methods used in this project. In Chapter 3, a number of methods were optimised to enable the biochemical characterisation and sub-cellular location of the CSLF6 and CSLH1 proteins to be studied. After evaluating a number of detergents n-dodecyl β-D-maltoside (DDM) was found to effectively solubilise and importantly retain MLGS activity and was also the detergent of choice due to its compatibility with downstream mass spectrometry (MS)-based applications. It was observed that MLGS activity is favoured under alkaline conditions (pH ~9.0) in the presence of both Mg2+ and Ca2+ with the majority of counts incorporated into MLG rather than callose, a (1,3)-β-glucan. Fractionation of microsomal membranes (MM) using sucrose density gradient centrifugation and PEG/Dextran two-phase partitioning showed that most CSLF6 protein is found at the plasma membrane (PM) and not intracellularly, in contrast to CSLH1. The finding of different sub-cellular distributions for these proteins was substantiated by immuno-electron microscopy (Wilson et al., 2015). Western blots of non-denaturing gels on which detergent-solubilised membrane samples were separated revealed that CSLF6 forms higher-order oligomers and an in-gel MLGS activity assay demonstrated that the dimer form of CSLF6 is active. Pre-treatment of membrane proteins with either dithiothreitol (DTT) or hydrogen peroxide (H2O2) favoured CSLF6 monomer or oligomer formation, respectively, indicating that the interactions between CSLF6 polypeptides is likely regulated by redox via the formation of intermolecular disulphide bonds between cysteine (Cys) residues. Also in this chapter, it was discovered that carbonate washing of the PM-enriched (PM-E) fraction increased MLGS specific activity. This was used to good effect in co-immunoprecipitation (co-IP) experiments using the CSLF6-specific antibody to remove contaminant proteins. The combined proteomic analyses of co-IPed proteins and gene network co-expression analyses identified several proteins that likely interact with CSLF6 and impact MLG biosynthesis and/or regulation in some manner: a KORRIGAN-type cellulase (GH9); nucleotide sugar interconverting enzymes relating to UDP-Xyl formation; trafficking-related proteins including RABH1b and ARF-type GTPases, Sec61 and an EPSIN2 protein; cytoskeletal-associated proteins such as a WEB family protein (DUF827); CC1, a CK1 kinase; multiple transferases including an S-adenosyl-L-methionine dependent methyltransferase (DUF248); an O-Fuc transferase-domain containing protein; a CSLD; and multiple unknown proteins. Chapter 4 outlines findings relating to the post-translational modifications (PTMs) of CSLF6 that may be involved in its regulation. A proteomics workflow utilising DTT, IAA and NEM was used to pinpoint which Cys residues were involved in the formation of disulphide bonds and which Cys residues possessed free thiol groups. Only four Cys residues in the LmCSLF6 sequence were detected by MS. C-496 and C-517 were shown to be labelled with IAA, i.e. involved in disulphide bonding while C-246 and C-440 each possessed a free thiol group. A homology model of HvCSLF6 reported by (Schwerdt et al., 2015) and based on the coordinates for a bacterial cellulose synthase (BcsA) (Morgan et al., 2013) offered the possibility of speculating which Cys residues are in close proximity to C-496 and C-517 to potentially form intramolecular disulphide bonds with C-217 or C-643 appearing the most probable. CSLF6 was also identified to be a phospho-protein with three N-terminal phosphorylation sites, Ser-55, Thr-75 and Ser-78, at a similar position within the hypervariable region (HVR1) in which CesA proteins are phosphorylated a short distance downstream of the zinc-finger domain. In addition to observing CSLF6 phospho-sites, many other phospho-proteins were detected including some polysaccharide synthases previously identified to be phosphorylated in other studies, as well as some of the anti-CSLF6 co-IPed proteins identified in Chapter 3 (RABH1b, ARF, EPSIN2, CC1, and an unknown protein). Chapter 5 describes the functional testing of some of the Cys residues and a conserved Thr residue in the gating-loop of CSLF6 using the Nicotiana benthamiana leaf transient expression system. The four Cys residues at the N-terminus of HvCSLF6 were removed by truncation and were shown not be involved in either MLG synthesis or CSLF6 protein dimerisation. Binary vector constructs utilising either the 35S or UBQ10 promoter were tested and it was found that the UBQ10 promoter was preferable as it provided more consistent protein expression of CSLF6 variants. No effect on MLG synthesis or CSLF6 dimer formation was observed when using the UBQ10 promoter to drive the expression of C246S, C496S and C517S mutants. However, T-801 which is highly conserved within all CSLF and CesA proteins was found to be essential for MLG synthesis as no MLG accumulated in infiltrated leaves when it was substituted with Gly. The results of this Chapter indicated that other Cys residues are required to be mutated to elucidate which residues are involved in CSLF6 dimer formation. Chapter 6 briefly summarises the experimental chapters and proposes directions for future experiments in order to fulfil the aims of the research project. In addition, an updated working model of MLG biosynthesis that draws upon the evidence from the experimental chapters and published literature is presented to provoke/guide future experimental approaches.
Defining the roles of essential genes in the malaria parasite life cycle
The combination of drug resistance, lack of an effective vaccine and ongoing conflict and poverty mean that malaria remains a major global health crisis. Understanding metabolic pathways at all parasite life stages is important in prioritising and targeting novel anti-parasitic compounds. To overcome limitations of existing genetic tools to investigate all the parasite life stages, new approaches are vital. This project aimed to develop a novel genetic approach using post meiotic segregation to separate genes and bridge parasites through crucial life stages. The unusual heme synthesis pathway of the rodent malaria parasite, Plasmodium berghei, requires eight enzymes distributed across the mitochondrion, apicoplast and cytoplasm. Deletion of the ferrochelatase (FC) gene, the final enzyme in the pathway, confirms that heme synthesis is not essential in the red blood cell stages of the life cycle but is required to complete oocyst development in mosquitoes. The lethality of FC deletions in the mosquito stage makes it difficult to study the impact of these mutations in the subsequent liver stage. To overcome this, I combined locus-specific fluorophore expression with a genetic complementation approach to generate viable, heterozygous oocysts able to produce a mix of FC expressing and FC deficient sporozoites. In the liver stage, FC deficient parasites can be distinguished by fluorescence and phenotyped. Parasites lacking FC exhibited a severe growth defect from early to mid-stages of liver development in-vitro and could not infect naïve mice, confirming liver stage arrest. These results validate the heme pathway as a potential target for prophylactic drugs targeting liver stage parasites. Energy metabolism in malaria parasites varies remarkably over the parasite life cycle. Parasites depend solely on anaerobic glycolysis at blood stage but need Krebs cycle, the electron transport chain, and mitochondrial ATP synthase during mosquito stage development. Again, reverse genetic approaches to study the hepatic stage of Plasmodium have been thwarted because parasites with defects in energy pathways are unable to complete the mosquito stage. I used the genetic complementation approach established to study heme biosynthesis to bridge parasites lacking the β subunit of mitochondrial ATP synthase through mosquito stage and studied their development in the liver stage. ATPase knockouts were indistinguishable from wildtype in in-vitro liver stage assays of size, nuclear content, and merosome production. Robust progression to blood stage confirmed the dispensability of mitochondrial ATP synthesis in liver stages. I extended this approach to explore the essentiality of upstream mitochondrial electron transport and Krebs cycle during the liver stage. I speculate that energy metabolism in the liver stage resembles that in the blood stage, relying predominantly on glycolysis for ATP production. There are numerous genetic tools to manipulate the blood stage malaria parasite genome in general, but existing genetic tools to generate viable parasites with defects in blood stage essential genes are limited. To overcome this limitation, I have developed a novel strategy in which I first insert a complementary copy of the essential gene-of-interest, and then delete the endogenous gene, and then take advantage of meiosis and segregation during the mosquito stage to create haploid knockout sporozoites. I genotype the parasites along the way by fluorescence microscopy. As proof of principle, I created complemented knockouts of the blood stage essential 1-deoxy-D-xylulose-5- phosphate reductoisomerase (DXR) gene, crossed these with wildtype parasites, and then tracked the progeny through in-vitro and in-vivo liver development. Precomplementation proved difficult, perhaps due to inappropriate expression of important metabolic genes. Additionally, problems with apparent silencing of the fluorophore tags compromised my ability to genotype cross progeny preventing any firm conclusion on the function of isoprenoid precursor pathway of liver stage parasites. Nevertheless, my success in generating a blood stage essential gene knockout via precomplementation provides encouragement that this novel reverse genetic strategy can be implemented to investigate the role of blood stage-essential genes in sporozoite and liver stages of malaria parasites.
Pathogenicity genes of Leptosphaeria maculans, the fungus that causes blackleg disease of canola (Brassica napus)
Blackleg disease caused by the fungus Leptosphaeria maculans is the most serious disease of canola (Brassica napus) worldwide. Current approaches to control blackleg are through the use of agronomic techniques such as crop rotations and sowing disease-resistant canola varieties; however, L. maculans has overcome major gene resistance in commercially released cultivars. Effective control strategies require knowledge of plant defence and fungal pathogenicity mechanisms. Fungal pathogenicity genes are used to establish infection or to avoid plant defences and have the potential to be fungicide targets. The focus of this Ph.D. was to identify and characterise pathogenicity genes in L. maculans. In this thesis five L. maculans mutants were identified by screening L. maculans T-DNA mutants for reduced pathogenicity on B. napus. One of these mutants was identified using TAIL-PCR. Unfortunately, traditional PCR-based techniques such as TAIL-PCR and plasmid rescue are not always successful in identifying T-DNA insertion sites. To overcome this, next generation Illumina sequencing was used to identify the T-DNA insertion sites in the remaining four mutants. The next generation sequencing revealed not only the T-DNA insertion sites but also chromosomal rearrangements, deletions and single nucleotide polymorphisms (SNPs). The decreasing cost of next generation sequencing (NGS) makes this is a cost and time-effective method of identifying T-DNA insertion sites. Two putative pathogenicity genes were identified; one a predicted glutathione synthase gene caused by a T-DNA insertion, the other a transcription factor identified in several of the T-DNA mutants caused by a SNP identified using NGS. The transcription factor identified has homologs that are also involved in pathogenicity in two other ascomycete plant pathogens. This represents a new direction in which to pursue our understanding of the genes required for fungi to cause disease. Transcription factors potentially provide multiple targets for drug or fungicide use. Fungicide targets could also include any of the genes regulated downstream of the transcription factor. The identification and characterisation of pathogenicity genes will ultimately lead to an increased understanding of plant-pathogen interactions and provide new potential targets for the development of antifungals or other molecular-based strategies to control blackleg disease of canola and other plant diseases.
Functional analysis of the apical polar ring its role in secretion and motility of Toxoplasma parasites
Human parasites Toxoplasma and Plasmodium species belong to the phylum Apicomplexa and are some of the most successful groups of human parasites on the planet. Part of this success can be attributed to the cytoskeletal components that afford them structural stability and flexibility required to efficiently attach to and invade host cells. As members of the superfamily Alveolata, they possess a pellicle comprised of a set of flattened vacuoles pressed up against the plasma membrane, with proteinaceous support network and actin actin-based motility system. In addition to this, Toxoplasma also possesses an apical complex which is a tubulin based structure comprised of a set of apical polar rings and a conoid, which is a tight-knit tubulin based structure that is evolutionarily derived from ancestral flagella components. The apical complex is biologically significant because it is the entry point the parasite uses to enter a host cell in order to parasitize it, and this process is conserved in Plasmodium species. However, unlike other organelles, the proteins of the apical complex have no known conserved targeting signals so identification of proteins that target here has been slow to progress. A Toxoplasma protein homologous to a predicted cytoskeletal Tetrahymena thermophila protein was identified and localized to the apical complex, which we call RNG2. RNG2 was functionally characterized by inducible knock down and found that RNG2 played a role in the cGMP signalling pathway upstream of calcium dependent activation of CDPKs, which severely impacted microneme secretion, conoid extrusion and even other downstream processes, particularly internal calcium release. In addition to this, I used various calcium and cyclic di-nucleotide signalling agonists and inhibitors to investigate novel regulation patterns of micronemes and dense granules. RNG2 and other cGMP and calcium signalling proteins, PKG, CDPK1 and CDPK3 all show altered secretion of dense granules showing for the first time a regulatory mechanism of dense granules based on calcium.
Classification and phylogeny of the plant genus Dianella Lam. ex Juss.
The global distribution of Dianella Lam. ex Juss. (flax lilies, Asphodelaceae, Asparagales) extends from south-eastern Africa, Madagascar and India, to south-east Asia (north to Japan), Australia, Pacific Islands from Micronesia to Tahiti, Pitcairn Islands, New Zealand, New Caledonia, Norfolk Island and Hawaii. It occurs throughout Australia (excluding the central arid region), where there is the greatest diversity of 42 taxa (19 species and 23 varieties) (Australian Plant Census 2016). Of those, three taxa also extend to south-east Asia, and a further 17 species occur outside Australia (Australian Plant Census 2016; Kew World Checklist of Selected Families, compiled by Govaerts et al. 2016). Plants are characterised by two leaf forms: basal strap-like leaves and cauline leaves on aerial stems (+/- extravaginal branching units). The showy flowers have a characteristic struma between the anther and filament, while the fruit is a fleshy berry, typically in shades of purple. The name Dianella is attributed to the Greek goddess Diana, the mythical goddess of the hunt. A cpDNA phylogeny by Wurdack & Dorr (2009) found Dianella to be monophyletic and sister to the monotypic genus Eccremis from South America. However, there has been no comprehensive phylogenetic analysis of taxa within Dianella, which has the potential to reveal not only taxonomic relationships but biogeographic patterns and the evolutionary history of the group, including the role of polyploidy. Furthermore, species delimitations, including complexes of varieties, have been based only on morphology from field observation and herbarium samples and require further study. Using three chloroplast markers (trnQUUG–5'rps16, 3'rps16–5'trnK(UUU) and rpl14– rps8–infA–rpl36) and two nuclear markers, (ITS 4, ITS 5, 18SE-ETS and DIAN-ETS) molecular phylogenetic analyses using Bayesian and Maximum Parsimony are presented (Chapters 2, 3, 4 and 5). Accessions include the majority of Australian and extra-Australian Dianella. The related outgroup genera Eccremis, Stypandra, Thelionema and Herpolirion were also included. The cpDNA and nrDNA phylograms were relatively congruent and a combined data set produced the most resolution. The combined results (Chapter 5) differed from those of Wurdack & Dorr (2009) in showing Stypandra with a sister relationship to Herpolirion + Thelionema. Within Dianella, resolved clades largely related to biogeographic regions, such as the Hawaiian Islands and New Caledonia, Norfolk Island related to New Zealand and Australian bioregions, revealing for example an early divergence between eastern and Western Australian lineages, congruent with the pattern for other Australian biota. Of the four Australian species complexes described by Rodney Henderson in The Flora of Australia, volume 45, the D. caerulea complex was found to be monophyletic except for two varieties that clustered with other far-north Queensland taxa, and two D. caerulea var. caerulea samples that are morphologically distinct when compared to taxa in the complex. Of the other species complexes, D. revoluta, D. pavopennacea and D. longifolia are each polyphyletic. Their relationships indicate biogeographic patterns, such as for D. longifolia accessions, which were resolved in two separate clades, one clade from the Kimberley and Northern Territory, and one clade from eastern and southern Australia. For extra-Australian Dianella, the widespread D. ensifolia was also polyphyletic occurring in multiple clades with distinct taxonomic units able to be recognised. Chromosome counts available from the literature were plotted on the phylogeny for Dianella and indicated that polyploidy has arisen multiple times, particularly in taxa of some of the Australian species complexes and in D. ensifolia sensu lato. These results indicate the need to recognise new species and to resurrect other taxa for Australian and extra-Australian Dianella. Chapter six is a morphometric, multivariate analysis (using phenetic clustering and ordination methods) of Hawaiian Dianella to determine the number of species on the islands. Field collections were made on Oahu, Maui, Hawaii and Kauai to examine populations in situ, develop species concepts and collect plant material for the dataset. The results indicate that five operational taxonomic units should be recognised including the current taxon D. sandwicensis. Fruit morphology is unique, with distinctive fruit dye colour and fruit surface colour in some taxa. D. lavarum, a narrow endemic that inhabits recent dry lava flows, observed in the Hawaii Volcanoes National Park, is to be resurrected. A review of herbarium specimens confirmed its distributional range extends to Maui, which is in agreement with Otto Degener who originally described the species. The D. caerulea complex was also analysed further in Chapter 7, based on extensive fieldwork in Queensland, New South Wales, Victoria and Western Australia, using multivariate analysis of a morphological data set. Morphometric clusters were largely in agreement with Henderson's varieties, but it is recommended that some be raised to species level. D. caerulea var. assera and D. caerulea var. producta, which appear to be sister taxa based on the shared character extravaginal branching, were each found to include morphological variation. It is recommended that these taxa be recognised as species with three subgroups recognised in D. caerulea var. assera, and five subgroups in D. caerulea var. producta; however, further field sampling is required for taxonomic revision.
Characterisation of wall-associated kinases (WAKs) in grasses
Wall-associated kinases (WAKs) are members of the receptor-like kinase family, an important class of plant-specific plasma membrane proteins considered as potential signalling molecules. WAKs in the model eudicot Arabidopsis thaliana have been proposed to be involved in cell expansion and in response to pathogens and mineral toxicities. WAK proteins also regulate turgor pressure by forming interactions with pectins and other proteins (i.e. glycine-rich protein), suggesting a possible mechanism for WAK involvement in cell wall signalling pathways. In contrast, few reports exist on the role of WAKs in grasses. In this thesis, WAKs from barley (Hordeum vulgare) and Brachypodium distachyon (two model commelinid monocot grass species), were investigated using molecular and biochemical approaches. Selected candidate WAKs were characterised to gain an understanding of their expression patterns, function, and interactions at the cell wall. Chapter III describes the identification of WAK proteins in barley (43) and Brachypodium (115). Through analysis of protein structure and motifs, galacturonan-binding domain, EGF-like domain, and a protein kinase domain were identified as typical motifs of WAKs from barley and Brachypodium. Phylogenetic studies of the identified WAKs revealed that 5 AtWAKs, 32 HvWAKs and 107 BdWAKs clustered into one large clade. Within this large clade, AtWAKs formed an exclusive sub-clade, whereas the HvWAKs and BdWAKs were interspersed amongst several sub-clades. Our result suggest that the grass WAKs likely diverged from the common ancestor after the divergence of monocots and dicots. 10 HvWAKs and 12 BdWAKs were selected for further study based on sequence analysis and technical considerations. Expression profiling studies were performed and described in Chapter III. In several tissues of barley and Brachypodium, various expression levels of the selected WAK genes were observed with a differential expression pattern. In coleoptiles, a rapidly expanding tissue in early development, three WAKs (HvWAK2, BdWAK2, BdWAK7) displayed the highest expression level, with an expression peak at 48 h post-germination. The expression pattern of these genes correlated with the growth of the coleoptile, implying a potential role of these genes in regulating cell expansion. In addition to the expression profiling, experiments were conducted to study the expression of selected WAK genes under stress conditions. As described in Chapter IV, six WAKs (HvWAK14, HvWAK11, BdWAK2, BdWAK7, BdWAK8, BdWAK10) showed significantly increased expression levels upon salicylate (SA) treatment, while four WAKs (HvWAK7, HvWAK14, BdWAK2, BdWAK10) were induced upon salt treatment. In combination with expression profiling results, HvWAK2 and BdWAK2 were chosen as candidate genes for further investigation. Following over-expression of BdWAK2 in Nicotiana leaves, a phenotype reminiscent of hypersensitive cell death was observed (Chapter IV). This phenotype was diminished by either truncation or mutation to the kinase domain of BdWAK2, implicating the kinase activity of BdWAK2 as the cause of the cell death. This result, combined with the fact that expression of BdWAK2 was significantly induced under stressed (both SA and salt treatment) conditions, suggests BdWAK2 may have a significant role in defence responses in Brachypodium. Based on the expression data described in Chapter III, HvWAK2 was thought to be a development-related WAK gene involved in cell expansion. In Chapter V, the sub-cellular location and potential homo-dimerisation of HvWAK2 was investigated. Upon expression with a fluorescent tag in both Nicotiana and onion (Allium sepa), HvWAK2 was observed on the plasma membrane. In addition, using a BiFC approach, homo-dimerisation of HvWAK2 was shown. In order to investigate the nature of the attachment of BdWAK2 and HvWAK2 to the cell wall, an in vitro polysaccharide binding assay was performed (Chapter V). The binding assay indicated that, similar to AtWAK2, both BdWAK2 and HvWAK2 form attachments to pectins, but not other classes of cell wall polysaccharides. Through these findings, WAKs such as BdWAK2 are proposed to have dual intracellular signaling roles modulated via interactions with pectins in the cell wall. Along with a summary of the characteristics of grass WAKs, the final chapter (Chapter VI) discusses how the data obtained for grass WAKs in this study correlates with existing models of WAK signalling mechanisms, and provides a description for more targeted approaches for future work on this large gene family.