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.
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ItemCharacterization of pistil-specific genes encoding proline-rich proteins from Nicotiana alata(University of Melbourne, 1990)This thesis describes investigations into the structure and expression of a group of pistil-specific genes encoding proline-rich proteins in Nicotiana alata. Two cDNA libraries prepared from pistil mRNA of N. alata were screened with a probe encoding a carrot extensin. Low stringency hybridization and washing conditions were used to enhance the chance of detecting DNA sequences encoding high proportions of proline residues but which might be distinct from extensin. Three classes of cDNA clones, corresponding to three proline-rich protein genes (PRP1, PRP2 and PRP3), were isolated from the pistil cDNA libraries. None of these cDNA clones represented a full-length transcript Two genomic clones (PRP3g5 and PRP3g12) corresponding to the PRP3 gene were also characterized. The PRP3g5 clone probably represents a pseudogene of the PRP3 gene family, whereas the clone PRP3g12 is likely to correspond to a functional PRP3 gene. A typical TATA box and several putative polyadenylation signal are present in the sequence of the clone PRP3g12, and the ATG initiation codon of this gene is found to be located in a context which is probably optimum for translation initiation. The clone PRP3g12 does not contain any introns. The three proline-rich proteins predicted from the partial cDNA clones and the genomic clone are all rich in proline residues (29% for PRP1,49% for PRP2 and 30% for PRP3). In addition, PRP1 is also rich in asparagine (14%), and PRP3 is rich in serine (19%). These three genes have a similar codon usage preference for praline: more than 60% of all proline residues are coded by CCA. The 114 amino acid sequence of PRP2 can be divided into two domains; one (nucleotides 1-210) contains 5 repeats of (Pro)4-Ala interspersed with 4 repeats of the pentapeptide Gln-Leu-Pro-Ile-Arg, and the other is composed mainly of tandemly reiterated (Pro)2-5-Gly-Tyr repeats. The first 23 of the 151 amino acid residues in the PRP3 protein predicted from the genomic clone PRP3g12 are hydrophobic and resemble to a signal peptide. The PRP3 resembles extensin in containing six Ser-(Pro)4 repeats which are separated, in most cases, by a Ser-Pro dipeptide. Southern analysis under stringent conditions showed each of the three PRP cDNAs hybridized to a number of restriction fragments for each of the four restriction enzymes used. This indicates that each of these PRP genes belongs to a small multigene family. The carrot extensin genomic clone (pDCSA1) hybridized to a set of restriction fragments different from any of the three PRP genes, suggesting that another gene more homologous to the carrot extensin than any of the three PRP genes is present in N. alata. The hybridization pattern of the clone pDC5A1 is relatively simple, indicating a low copy number of the extensin gene in the genome. Northern analysis indicated that the three PRP genes are different from each other and from the extensin gene. All the three PRP genes are effectively pistil- specific, in contrast to the extensin gene which is expressed in all tissues tested although the level, number and size of the extensin transcripts differs in different tissues of N. alata. The PRP3 gene is developmentally regulated and its maximum expression correlates with the maturity of the pistil. After wounding, the expression of the extensin gene is increased in all tissues tested, whereas, the mRNA levels of both PRP2 and PRP3 decreased in wounded pistils. Wounding had no detectable effect on the expression of the PRP3 gene in stem and leaf tissues, while a small PRP2 transcript (smaller than the major transcript expressed in wounded pistils) is induced in the same tissues after wounding.
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ItemPollen-wall proteins and breeding systems of plants(University of Melbourne, 1978)Quatitative cytochemical methods have been developed to estimate changes during development in the pollen-wall proteins. Acid phosphatase was used as a marker for intine proteins, and non specific esterase for the exine proteins. In marrow-stem kale, Brassica oleracea and ryegrass, Lolium perenne the intine enzyme showed two peaks of accumulation during development, the first corresponding to its synthesis and incorporation in the intine, and the second to accumulation in the pollen cytoplasm at maturity. In contrast, the exine enzyme showed a single peak at pollen maturation. This was correlated with dissolution of the tapetum and consequent loss of its very high esterase activity, coincident with the accumulation of esterase in the exine cavities. In sunflower, Helianthus annuus acid phosphatase and esterase were detected in both exine and intine sites during pollen development. Acid phosphatase was associated with the nexine at pre-vacuolate period with high levels in the tapetum. At mid-vacuolate period the tapetum became plasmodial and enzyme activity was detected around the exine surface and was transferred to the exine cavities by the end of the vacuolate period. Esterase activity was associated with the exine at pre-vacuolate period; subsequently during the vacuolate period, activity was present in the intine, and in the cytoplasm at maturity. Two peaks of accumulation were detected, closely resembling those for the intine marker enzyme in Brassica and Lolium. The developmental cytochemistry of the stigma of Helianthus, Lolium and Secale has been investigated with a view to understanding the nature of selfincompatibility, the site of tube arrest and the route of pollen tube penetration. The callose rejection response in pollen and stigma that has been established for Cosmos and various Crucifers have been found in Helianthus. The rejection reaction is found to occur in the pollen grain and pollen tubes of grasses. The callose produced in germinating self pollen of the grass, Secale cereale has been isolated by a degredative physicochemical procedure. Partial acid hydrolysis, enzyme hydrolysis, sugar analysis and methylation analysis have shown that the material is a 1,3-?-linked glucan; however some evidence points to the presence of 1,4-?-linkages in addition.