Science Collected Works - Theses
Permanent URI for this collection
Search Results
1 - 3 of 3
-
ItemThe role of NaGSL1 in callose synthesis in the pollen tubes of nicotiana alata(University of Melbourne, 2005)Plant cell walls are dynamic structures that play a key role in plant growth and development and in the responses of plants to stress. Callose, a 1,3-?-glucan, is found in the cell wall at specialized locations throughout plant development, and is deposited in response to wounding and pathogen challenge. The enzyme callose synthase (UDP-Glc: 1,3-?-D-glucan 3-?-D-glucosyl transferase; EC 2.4.1.34 ), or CalS, is responsible for the production of callose. Until quite recently, CalS was unique among plant polysaccharide synthases for being both active and stable in vitro. Despite these biochemical advantages, the proteins that comprise this enzyme in plants have not been definitively identified. A family of genes, the glucan-synthase-like (GSL) genes, has been proposed to encode plant CalS enzymes based on the similarity of these genes to genes believed to encode the fungal 1,3-?-glucan synthase (Saxena and Brown, 2000; Cui et al., 2001; Doblin et al., 2001; Hong et al., 2001a). A number of studies have provided evidence to support a role for GSL genes in callose synthesis at both the biochemical (Cui et al., 2001; Hong et al., 2001a; Li et al., 2003) and molecular (Cui et al., 2001; Doblin et al., 2001; Hong et al., 2001a; Jacobs et al., 2003; Nishimura et al., 2003) levels; however, definitive proof of function for the GSL genes has not yet been shown. In this thesis, pollen tubes of Nicotiana alata Link et Otto are used as an experimental system to study callose synthesis. Pollen tubes form upon germination of pollen grains, and usually grow through the style to deliver the sperm to the ovaries. N. alata pollen tubes can also be grown in culture. The wall of N. alata pollen tubes is made predominantly (86%) of callose (Li et al., 1999), and a highly active, developmentally regulated CalS enzyme has been biochemically characterized from pollen tubes (Schl�pmann et al., 1993; Li et al., 1997; Turner et al., 1998; Li et al., 1999). An N. alata GSL gene, NaGSLl, is abundantly expressed specifically in pollen grains and tubes and is therefore a candidate to encode the pollen-tube CalS (Doblin et al., 2001). This thesis aims to determine if the polypeptide encoded by the NaGSL1 gene, NaGSL1, is the pollen-tube CalS and, following a positive identification, to investigate the regulation of NaGSL1 and thus of callose synthesis in N. alata pollen tubes. This required the production of a full-length NaGSL1 cDNA and antibodies against a bacterially expressed region of NaGSL1, and the successful production of these molecular tools is described in Chapter 2. A biochemical link between NaGSL and the pollen-tube CalS is established in Chapter 3. The pollen-tube CalS was enriched several hundred fold using continuous-density-gradient centrifugation and product entrapment, essentially as described by Turner et al. (1998). Polypeptides from each stage of the enrichment were analysed by SDS-PAGE. The most abundant polypeptide in the product-entrapped material had a molecular weight of 220 kDa and was identified as NaGSL1 from tryptic peptides using both peptide mass fingerprinting (PMF) by MALDI-TOF MS, and MS/MS. Labelling with the anti-GSL antibodies showed that NaGSL1 is enriched through the CalS-enrichment process and abundant in the product-entrapped material, with the relative abundance of NaGSL1 correlating with CalS activity. This data thus provided a strong biochemical link between NaGSL1 and CalS activity, leading to the conclusion that NaGSL1 is the pollen-tube CalS. PMF was also used to investigate the identity of other polypeptides in the product-entrapped material. The only other plant polypeptides identified, a 103-kDa plasma membrane H+-ATPase and a 60-kDa ? subunit of the mitochondrial ATPase, were deduced to be contaminants. All other polypeptides were present in low abundance, and so could not be identified. Therefore, it appears that NaGSL1 does not require the presence of another protein for CalS activity. Once it was established that NaGSL1 is the pollen-tube CalS (Chapter 3), the regulation of NaGSL1 was investigated in Chapter 4 by tracking the abundance and location of the NaGSLl polypeptide through pollen-tube growth using the anti-GSL antibodies, and then relating these data to levels of CalS activity. Because the pollen- tube CalS can be activated in vitro by trypsin (Schl�pmann et al., 1993; Li et al., 1997, 1999), CalS activity assays were conducted without trypsin (measuring the amount of active enzyme) and with trypsin (measuring the total amount of enzyme). NaGSL1 was present in low abundance 30 min after pollen-grain hydration, the same time at which CalS activity is first detected. As the NaGSL1 mRNA is present in mature pollen grains well before germination, the production of NaGSLl appears to be regulated at translation. The amount of the NaGSL1 protein increased over the first 16 h of pollen-tube growth, and paralleled the increases in the amount of CalS over this time. After continuous-gradient-density centrifugation of membranes from 4-h and 16-h pollen tubes, NaGSL1 was present in fractions enriched for intracellular membranes and in fractions enriched for plasma membrane (PM). Without trypsin activation, CalS was predominantly in the PM-enriched fractions. Trypsin treatment increased the CalS activity in the PM-enriched fractions and also revealed a previously inactive CalS in the fractions enriched for intracellular membranes. The relative abundance of NaGSL1 in different fractions detected with the anti-GSL antibodies correlated with the level of CalS activity after trypsin treatment, that is, with the total CalS activity. The subcellular location of NaGSL1 were determined by immuno-electron-microscopy (immunoEM). Both the results from membrane fractionation and from immunoEM show that NaGSL1 is located predominantly in the ER and Golgi during the early stages of pollen-tube growth, and is located predominantly in a population of vesicles and the PM during the later stages of pollen-tube growth. Western blot analysis and CalS activity assays were used to analyse the mechanism of trypsin activation and determine if this occurred via the removal of a sizeable autoinhibitory domain from NaGSL1. The results are described in Chapter 5 and show that there was no consistent correlation between the production by trypsin of lower-molecular-weight forms of NaGSL1 and a high level of CalS activity. Activation of NaGSL1 was concluded not to be due to cleavage of a sizeable autoinhibitory domain. Whilst it is possible that trypsin activation is due to the removal of a di- or tri-peptide from the C-terminus of NaGSL1, it seems more likely that trypsin is acting upon a separate, inhibitory protein and causing its dissociation from NaGSL1. This inhibitory protein would not be present in a fully activated, product-entrapped CalS preparation, in agreement with the results of Chapter 3. Heterologous expression was used to investigate the function and properties of NaGSL1, and this is described in Chapter 6. Yeast was selected as the heterologous host as it contains homologous genes, the FKS genes, believed to encode a 1,3-?-glucan synthase (Douglas et al., 1994a). When the full-length NaGSL1 cDNA was transformed into a range of yeast fks mutants, neither full nor partial complementation were observed, even though the full-length NaGSL1 polypeptide was expressed in yeast and targeted (at least partially) to the PM. The heterologously expressed NaGSL1 did not display in vitro CalS activity, and the addition of trypsin or pollen- tube homogenate did not stimulate CalS activity. Instead a low-molecular-weight, non-proteinaceous component of the yeast lysate appeared to inhibit the pollen-tube CalS. The failure of NaGSL1 to complement fks mutants and to display in vitro CalS activity when expressed in yeast may be due to the yeast biosynthetic machinery being unable to correctly fold, post-translationally modify and/or regulate the plant protein. The lack of fks complementation may therefore relate to differences in the production and/or regulation of the plant GSL and fungal FKS proteins, even though they appear to have the same catalytic function. Heterologous expression of NaGSL1 in plant cells other than pollen tubes may be required to investigate the molecular mechanism of NaGSL1 and its activation.
-
ItemCharacterization of arabinogalactan-proteins (AGPs) from the pistil of Nicotiana alata(University of Melbourne, 1995)Arabinogalactan-proteins (AGPs) are common components of most plant tissues, plant secretions, and suspension cultured plant cells. AGPs are a family of proteoglycans that consist of mainly carbohydrate (usually >90% w/w) rich in galactose (-60%) and arabinose (-30%) residues and a protein backbone rich in hydroxyproline (Hyp), Ala, and Ser. Various physiological functions have been proposed for the AGPs, such as maintaining water balance in the plant, providing nutrients for growing pollen tubes, responding to pathogen infection, and cell-cell recognition. More recently, membrane bound AGPs have also been implicated as determinants of cellular identity. Although the structure of the carbohydrate moiety of the AGPs is relatively well studied, little is known about the protein backbone, partly because of the difficulties in the separation of individual AGPs and subsequent cloning of the genes encoding these protein backbones. This in turn has hindered our understanding of the role that AGPs play in plant growth and development. Using a combination of ion-exchange and gel filtration chromatography, a major group of buffer-soluble AGPs have been isolated from the pistils of Nicotiana alata. Carbohydrate was removed by anhydrous hydrogen fluoride and the deglycosylated AGP protein backbones were fractionated by reversed-phase HPLC into three major fractions; an unbound fraction, and two fractions which eluted from the column with retention times of 25 min (RT25) and 30-40 min (RT35). Amino acid analysis of the two bound fractions showed that the RT25 backbone was "typical" of AGPs in that it contained mainly Hyp, Ala, and Ser, while the RT35 fraction was "atypical" of AGPs and was rich in Asx, Glx, and Ala. Internal amino acid sequences were obtained from the RT25 protein backbone after protease digestion. Gene-specific oligonucleotides were designed according to the peptide sequence and polymerase chain reaction (PCR) was carried out. The PCR product was then used to screen a style cDNA library, and a cDNA clone, AGPNa1, was obtained (Du et al., 1994). The AGPNa1 cDNA (712 bp) encodes a protein of 132 amino acid residues and is consistent with our concept of a "classical" AGP dominated by Hyp/Pro, Ala, and Ser residues. The predicted AGP protein backbone contains an N-terminal secretion sequence, which agrees with the known extracellular location of the pistil AGPs. The middle part of the deduced backbone is rich in Pro, Ala, and Ser. The abundance of hydroxyl amino acid residues is consistent with the extensive O- glycosylation of the AGPs. The C-terminus of the protein is very hydrophobic. Whether this hydrophobic tail serves as a membrane anchor or is proteolytically removed in vivo is unclear. The AGPNa1 is a single-copy gene and is expressed in all plant tissues examined, which suggests that it may have a general role in plant growth and development. Using a similar strategy, internal amino acid sequences were obtained from the RT35 protein backbone and a cDNA, AGPNa3, was isolated (Du et al., 1995). The AGPNa3 cDNA (762 bp) encodes a protein of 169 amino acid residues with three domains, an N-terminal signal sequence, a central Pro-rich domain, and a C- terminal Cys-rich domain. The AGPNa3 thus encodes an AGP that is "non- classical" in that it is rich in Asx and Glx but relatively poor in Hyp. AGPNa3 is also a single-copy gene. Northern blot analysis showed that the AGPNa3 gene is only expressed in the mature pistils of N. alata but not in other tissues. Within the pistil, the AGPNa3 gene is primarily expressed in the stigma, indicating a possible role in the pollination process. To further investigate the function of AGPs, transgenic plants with sense or antisense constructs containing the AGPNa1 cDNA were generated. As expected, the sense plants produced higher amounts of the AGPNa1 transcript in the pistil than the control plants; one of the antisense plants had significantly reduced expression of AGPNa1. However, all transgenic plants grew normally and were not noticeably different from untransformed plants. Future transformation experiments to generate transgenic plants containing the antisense AGPNa3 gene and double knockout plants with reduced expression of both AGPNa1 and AGPNa3 gene will be valuable in revealing further the functions of AGPs.
-
ItemCallose synthesis in pollen tubes from nicotiana alata(University of Melbourne, 1994)Pollen tubes are the gametophytic stage of the flowering plant life cycle, and transport the germ cells through the specialised sporophytic tissues of the pistil to the egg cells in the ovary. Pollen tubes are formed by a single cell that grows by extension at its tip and that synthesises a unique wall and regular transverse plugs. The wall and plugs are comprised mostly of callose, a (1,3)-?-D-glucan with a few 6-linked branches. This thesis describes a callose synthase (uridine diphosphate glucose: (1,3)-?-D-glucan 3-?-D-glucosyltransferase, EC 2.4.1.34) activity of membranes prepared from cultured Nicotiana alata pollen tubes. The insoluble product synthesised by this enzyme is a linear (1,3)-?-D-glucan with a degree of polymerisation of at least 1200, and it forms microcrystalline fibrils of approximately 8 nm diameter and 150 nm length. Pollen-tube callose synthase has some distinct properties that differ from those of the wound-induced callose synthase activity in preparations from suspension-cultured cells of N. alata or other sporophytic tissues: the novel pollen-tube activity is not dependent on Ca2+ or other divalent cations, has a high Km (1.5-2.5 mM) for the substrate uridine-diphosphate glucose (UDP-Glc) and is increased ten-fold by treatment of the membranes with trypsin in the presence of detergent. The Ca2+-independence of this synthase is in accordance with the low cytoplasmic Ca2+ concentrations previously reported in the subapical region of the pollen-tube tip where callose is synthesised. Extraction and analysis of metabolites from growing pollen tubes showed that UDP-Glc is the major sugar nucleotide present, with a calculated cytoplasmic concentration of 3.5 mM; the high Km of pollen-tube callose synthase thus coincides with the high cytoplasmic UDP-Glc concentration in these cells. Callose is deposited at a constant rate of 1.4 to 1.7 nmol Glc.min-1 in tubes from 1 mg pollen from 3 h after germination, and the intracellular pool of UDP-Glc (1.6 nmol in tubes from 1 mg pollen) is therefore turned over in 1 min or less if UDP-Glc is a substrate for callose synthesis in vivo. Metabolic labelling with [14C] sucrose shows that incorporation of radioactivity into wall material is linear over time, while incorporation into the pools of glucose monophosphates and UDP-Glc reaches saturation within 1 min. The specific activity of extracted UDP-Glc corresponds to that of glucan deposited. Results from metabolic labelling are thus consistent with UDP-Glc being the substrate for callose synthesis in vivo. The rate of synthesis of (1,3)-?-glucan by non-trypsin-treated pollen-tube membranes incubated with a ?-glucoside activator and cytoplasmic concentrations of UDP-Glc was slightly greater than the rate of (l,3)-B-glucan deposition in growing pollen tubes. The pollen-tube callose synthase characterised in vitro can therefore account for synthesis of the callose backbone in vivo, and the significance of the latent activity uncovered by proteolytic activation therefore remains to be determined.