School of Botany - Theses
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ItemArabidopsis arabinogalactan proteins containing lysine rich domains(University of Melbourne, 2004)Plant cell walls play a pivotal role in determining the final shape and function of plant cells and tissues. Primary plant cell walls are composed of polysaccharides (pectins, cross-linking glycans and cellulose) and some proteins (approximately 10 % (w/w); Bacic et al., 1988; Carpita and Gibeaut, 1993; Cosgrove, 1997). The proteins are both enzymic and structural and include a complex group known as the hydroxyproline (Hyp)-rich glycoprotein (HRGP) superfamily. This family includes the extensins, arabinogalactan-proteins (AGPs), proline (Pro)/Hyp-rich glycoproteins and the solanaceous lectins (Showalter, 1993; Nothnagel, 1997; Johnson et al., 2003b). This thesis will focus on the most highly glycosylated proteoglycans of the HRGP family, the AGPs. AGPs are located in the extracellular matrix, and are associated with the plasma membrane and the cell wall. They consist mainly of carbohydrate chains covalently attached to a Hyp-rich protein backbone that is typically less than 10 % (w/w) of the molecule. In some cases the Hyp-rich domain of the protein backbone is interrupted by a short basic region. This small subclass of AGPs are know as the lysine (Lys)-rich AGPs and have been identified and characterised from Arabidopsis thaliana (hereafter referred to as Arabidopsis; Caspar, 1998; Gilson et al., 2001; Schultz et al., 2002), Nicotiana alata (Gilson et al., 2001), Lycopersicon esculentum (Pogson and Davies, 1995; Li and Showalter, 1996), Pinus taeda (Zhang et al., 2003) and Cucumis sativums (Park et al., 2003). AtAGP17, AtAGPl8 and AtAGP19, identified from Arabidopsis, contain a protein backbone that consists of an N-terminal secretion signal sequence, a Pro/Hyp, Ala, Ser, and Thr-rich central domain that is interrupted by a short Lys-rich domain, and a C-terminal hydrophobic signal sequence that is likely to be a GPI membrane anchor signal sequence. To determine the function of the Lys-rich AGPs, Arabidopsis was selected as a model system to take advantage of the publicly available genomic resources. Arabidopsis resources used in this project to characterise the Arabidopsis Lys-rich AGPs included microarray data, mutant lines, genome sequence and EST databases. In situ hybridisation, green fluorescent protein technology, promoter: :GUS experiments, RNA gel blot analysis and antibodies were used to assess the location and expression of the Lys-rich AGPs in Arabidopsis tissues. Antibodies generated against the Lys-rich region of AGP 17 and AGP 18 were used to determine the location of these AGPs and in future studies will make it possible to isolate individual AGPs in order to characterise their carbohydrate components. Experiments suggest that Lys-rich AGPs have lower expression profiles than many other AGPs, although they were expressed in most tissue types examined. Although the precise function(s) that AGPs perform are unknown, they are implicated in diverse developmental roles such as differentiation, cell-cell recognition and embryogenesis (Knox, 1995). A reverse genetics approach to isolate knockout mutants was used to investigate the function of the Lys-rich AGPs. An Arabidopsis insertion mutant from the Feldmann lines was discovered to be resistant to Agrobacterium tumefaciens transformation (rati; Nam et al., 1999). It was shown that rat1 is deficient in its ability to bind Agrobacterium tumefaciens (hereafter referred to as Agrobacterium). Scanning electron microscopy showed that lack of binding to the root surface does not result from an inhibition of cellulose synthesis by the bacterium. Cellulose microfibrils are secreted by Agrobacterium and are used for close adhesion to the root surface (Matthysse, 1994; Matthysse and McMahan, 1998). Characterisation of the rat1 mutant showed that the phenotype is due to down-regulation of the Lys-rich AGP AGP 17 in roots as a result of a T-DNA insertion in the promoter of AGP17. Another phenotype of the rat1 mutant is delayed germination of rat1 seeds. Furthermore, the mucilage extruded from seeds during the imbibition process differs from the wildtype, suggesting differences in pectic methyl esterification. The association between Lys-rich AGPs and pectin in seed mucilage is supported by the discoveries that pectin associates (Yamada, 2000) and copurifies with AGPs (Shea et ah, 1989; Serpe and Nothnagel, 1994, 1995). Lys-rich AGPs may be involved in cross-linking cell wall components, either through the basic residues of their Lys-rich domain or by their carbohydrate epitopes. To understand the basis for the rat1 phenotype, key players in the defence response were investigated. Based on changes in PR1 gene expression and decreases in free salicylic acid levels upon Agrobacterium infection, I suggest a mechanism by which AGP 17 allows Agrobacterium to rapidly reduce the systemic acquired resistance (SAR) response during the infection process.