School of Botany - Theses
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ItemCell expansion in the elongation mutants of barley (Hordeum vulgare), a model grass(University of Melbourne, 2006)Cell expansion is fundamental to plant growth and morphogenesis, and how individual cells expand influences the morphology of the plant tissues and organs they comprise. This thesis describes a class of barley mutants, the elongation (elo) mutants, in which point mutations in unknown genes affect normal cell expansion. The elo mutants were isolated in a screen for dwarf mutants of barley and a description of five elo lines are presented here. Three elo mutants were recessive (elo1-elo3) and two were semi-dominant (elo4 and elo5). In the semi-dominant lines, the homozygous individuals generally displayed a more severe phenotype than heterozygotes. In order to investigate the functions of the ELO genes, a series of approaches was adopted. Chapter 2 contains a morphological description of each line, including examination of internal morphology of leaves, roots and seeds, and external morphology of leaves and roots. The five elo lines are dwarfed in stature compared to wild-type (WT), with above-ground organs shorter than WT in all lines and roots shorter in all but one of the lines (elo1). A phenotype common to all elo mutants is the presence of radially swollen cells on the leaf epidermis. Leaf epidermal cells were also found to be reduced in length compared to their WT counterparts, indicating that the elo phenotypes result from defects in processes essential to cell expansion in barley. Although all lines had radially swollen leaf epidermal cells, the extent of the aberrant cell expansion phenotype varied between the elo lines, with radial swelling restricted to leaf epidermal cells in some lines and more widespread in others. For example, radial swelling in elo1 was limited to intercostal leaf epidermal cells, while homozygous elo5 seedlings had radially swollen leaf epidermal cells, as well as swelling in all cell types of roots. Developing leaves and roots of two elo mutants (elo3 and elo5) were examined to gain a better understanding of when aberrant cell expansion first occurred. In both lines, radial swelling coincided with the zone of cell elongation. In addition to cell expansion defects, two of the mutations, elo2 and elo5, affected cell division. Additional periclinal cell division planes in leaf epidermal cells resulted in increased cell layers in the leaf epidermis of elo2 and elo5 homozygotes and the aleurone layer of elo2 seeds. Incorrect anticlinal divisions that were oblique rather than perpendicular to the growth axis also occurred in epidermal cell files of homozygous and heterozygous elo 5 leaves. Radial swelling has previously been seen in plants where cellulose biosynthesis is inhibited. To determine whether the radial swelling phenotype of the elo mutants was the result of decreased cell wall cellulose content, differences in the wall polysaccharide composition between WT and the elo mutants were investigated (Chapter 3). A number of techniques were used to determine cell wall composition in WT and elo tissue, including chemical derivitisation of cell wall carbohydrates, FTIR microspectroscopy and immuno-localisation of cell wall epitopes using monoclonal antibodies. Analysis of the cell wall composition of WT and elo leaf blades revealed a reduction in cellulose content in elo leaf cell walls, with a concomitant increase in levels of GAX and pectin. Although little or no difference to cell wall composition was detected in root cell walls, the phenotype and the wall analysis data from leaves indicate that the elo mutations affect cellulose biosynthesis in primary cell walls. To date, no mutants have been described that affect primary wall synthesis in a plant with type II cell walls. Thus, studying the elo mutants provides valuable insights into primary wall synthesis in this group of plants, and allows comparisons to be made with similar processes in plants with a type I cell wall. Transcript analysis of members of the cellulose synthase (CESA) superfamily were also performed in two lines, elo3 and elo5, to determine whether reduced cellulose content could be attributed to a reduction in transcript levels of these genes. Although reduced cellulose content can cause radial swelling, the architecture of the cell wall is also thought to play a part: cellulose microfibrils in elongating cells are inserted into the wall matrix transverse to the growth axis to prevent lateral cell expansion and favour expansion along the growth axis. Decreased cellulose content is also often accompanied by the disordered deposition of cellulose microfibrils into the cell wall. Microfibrils in elongating root cells of two elo lines, elo3 and elo5, were examined to determine whether the radial swelling phenotype was accompanied by altered microfibril deposition in these cells (Chapter 4). Cell walls of elo3 and heterozygous elo5 showed no difference in microfibril organisation compared to WT; however, homozygous elo5 cell walls had a greater proportion of cells with randomly-oriented cellulose microfibrils, indicating that the elo5 mutation affects the correct organisation of cellulose microfibrils in elongating cells. Radial swelling can also result from disruptions to cortical microtubule arrays in elongating cells. For this reason, cortical microtubule arrays were examined in elongating cells of eloS and elo5 roots (Chapter 4). This revealed that cortical microtubule arrays form left-handed helices in elongating cells of these lines, and hence a mutation affecting the orientation of cortical microtubules could be the cause of radial swelling phenotypes in both elo3 and elo5. The precise functions of the ELO proteins in plant growth and development will require identification of the genes that encode them. Progress towards this goal was made by mapping four of the elo genes to regions within the barley genome (Chapter 5). Three of the elo genes, elo1, elo2 and elo5 mapped to the long arm of chromosome 1H and the fourth gene, elo3, mapped to the long arm of chromosome 3H. This represents the first step towards map-based cloning of the elo genes, which is essential to understanding their roles in cell expansion in barley. The final Chapter of this thesis (Chapter 6) summarises the research findings from this study and proposes potential roles for the ELO genes based on their phenotypes. Future research directions are also considered.
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ItemNo Preview AvailableFunctional analysis of the barley cellulose synthase like c (cslc) gene family(University of Melbourne, 2003)Polysaccharides comprise the bulk of the polymeric material in the plant cell wall and occur as structurally independent but interwoven networks of polymers. Microfibrils of cellulose, the most abundant polysaccharide in plants, act as a load-bearing framework. In cereals, which have a type II wall, this cellulosic network is embedded in a matrix composed of polysaccharides such as heteroxylans and (1?3)(1?4)-?-D glucans. Although the precise mechanisms by which plant cells make and assemble these different networks are largely unknown, members of the CESA superfamily (currently classified into cellulose synthase-like (CSLA-CSLH) gene families plus the CESA gene family) are thought to encode catalytic subunits of the enzyme complexes that make many of the constituent polysaccharides. This thesis describes an investigation of the CSLC gene family in barley (Hordeum vulgare L.). Chapter 1 begins by describing the cloning of four CSLC genes from barley (HvCSLC1- HvCSLC4). This work was necessary because limited sequence information for barley was available when this thesis commenced in 2000 and no CSLCs were known from this species. The four genes were identified by a bioinformatics approach and by screening barley cDNA and BAG libraries. The HvCSLC1 sequence is complete and covers the entire 2094 bp of the CSLC coding region. The other three sequences are incomplete because between ~300 and ~600 bp of DNA is missing from the 5' ends of the genes. All of the available barley CSLC ESTs are derived from one or other of these four genes. The location of the HvCSLC genes in the barley genome was determined using blots of DNA from plants in a mapping population. Two HvCSLC genes are tightly linked on chromosome 1H and the other two are on chromosomes 3H and 5H. Quantitative (Q-) PCR was used to monitor expression of each of the four genes in a variety of tissues, including leaf, root, coleoptile and developing endosperm, and in situ PCR to show that CSLC gene transcripts accumulated in all cell types of the coleoptile and root, including the vascular bundle and parenchyma. The data in this chapter point to at least three of the barley CSLC genes being developmentally regulated, suggesting that the products of these genes play specific roles in synthesising polysaccharides in certain cell types rather than a general role in synthesising polysaccharides throughout the plant. The fourth gene, HvCSLC3, is expressed at a low level in most of the tissues tested. It was not possible to correlate the expression of any of these genes to the accumulation of particular polysaccharides. Chapter 3 describes the production of a CSLC-specific antibody and the demonstration that the CSLCs in barley are integral membrane proteins of about 60 kDa that are most likely located in the plasma membrane. This was done using the detergent Triton-X114 to produce a fraction from barley coleoptiles enriched in integral membrane proteins and PEG/dextran two-phase partitioning to produce a fraction from barley suspension cultures enriched in the plasma membrane. The results in this chapter implicate the CSLCs in the synthesis of either cellulose, a (1,4)-?-D glucan, or callose, a (1,3)-?-D glucan. To resolve which of these polysaccharides is produced by the CSLCs, a functional analysis was performed using double-stranded RNA interference (RNAi) to transiently silence the expression of CSLC genes in barley coleoptiles. Chapter 4 presents results from these experiments. Bombarding coleoptiles with a CSLC RNAi construct reduced CSLC transcript levels and brought about a number of phenotypic changes, the most obvious being the development of splits at the coleoptile's apical end. The anticlinal walls separating adjacent epidermal cells were also thinner and wavy. Immunogold labelling with carbohydrate-specific antibodies to the mixed-linkage glucan, a (1,3)(1,4)-?-glucan, arabinoxylan and callose showed increases in the levels of arabinoxylan and mixed linkage glucan on the bombarded surface of the coleoptile compared to the unbombarded surface. Because little or no callose was detected in the walls on either surface, it seems unlikely that the CSLCs play a role in its synthesis. The development of splits in the bombarded coleoptiles suggests a role for the polysaccharide(s) synthesised by CSLCs in cell adhesion. The wavy anticlinal walls are more difficult to interpret and suggest either a role for CSLCs in radial cell expansion or in the limited amount of cytokinesis that occurs during the post-embryonic growth of the coleoptile. The final Chapter considers results from the three experimental chapters as a whole and proposes a model that suggests the CSLCs are involved in making cellulose in localised areas of the cell, such as the anticlinal walls. Clearly farther work on these genes is needed and the thesis concludes by suggesting a number of potentially fruitful areas for future research.
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ItemCharacterisation of wall-associated kinases (WAKs) in grasses( 2016)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.