Science Collected Works - Theses
Permanent URI for this collection
Search Results
1 - 1 of 1
-
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.