Activity of Chitin Synthetase during the Development of Fruit Bodies of the Toadstool Coprinus cinereus

Abstract

The final stages of development of mature fruit bodies of the toadstool Coprims cinereus involve the following steps : the rapid elongation of the stipe, apparently by intercalary cell elongation without cell division; the expansion of the cap and maturation of the meiospores; and the release of the spores as the gill tissue progressively autolyses through the action of lysosomal enzymes. This developmental sequence is an autonomous endotrophic process, as an excised young fruit body will develop fully without contact with vegetative cells or exogenous nutrients (Gooday, 1972a,b), but it must involve considerable redistribution of cell materials. The involvement of chitin synthetase (EC 2.4.1.16) in this sequence has been suggested, as development is inhibited by a specific inhibitor of this enzyme, the antibiotic polyoxinD (Gooday, 19726). In the present paper I report direct measurements of chitin synthetase activity during development. Four fruit bodies of each size were harvested from cultures grown on a nutrient agar medium (G. W. Gooday, unpublished work). The caps were removed and the stipes were cut into three equal segments. Each group was weighed and homogenized to a powder under liquid N2 with a pestle and mortar. Each sample was stirred into 5ml of homogenizing medium (0.05 M-Tris-HCI (pH 7.5)I O ~ M M ~ C ~ , mM-EDTA (disodium salt)-0.25~-sucrose). A11 subsequent operations were performed rapidly at 0°C. The homogenate was centrifuged for 20min at IOOOOg (rav. 6cm), the pellet was discarded, and the supernatant was centrifuged for 60min at 105000g, in an 8 x lOml angle rotor in an MSE Superspeed 40 centrifuge. The pellet was resuspended in 5ml of the homogenizing medium (but without the sucrose) and re-centrifuged for 60min at 105000g. The washed microsomal pellet was resuspended in loop1 of the sucrose-free medium and used immediately for the enzyme assay. The assay method was based on that of Glaser & Brown (1957), and the medium consisted of a total of 15p1 containing 5pI of enzyme preparation and final concentrations of: 0.05 M-Tris-HCI, pH7.5, lOm~-MgCl,, 1 mB-EDTA (disodium salt), 2mg of chitodextrinslml, 20m~-N-acetylglucosamine and 0.4m~-UDP-N-acety~ghcosamine containing 13.7nCi of UDP-[U-14C]N-acetylglucosamine (The Radiochemical Centre, Amersham, Bucks., U.K. ; 270mCi/mmol). The components were incubated in a 1 ml plastic centrifuge tube at 25°C for 5min, and the reaction was stopped by immersion in a boiling-water bath for 2min. Theentirecontents of the tube were transferred to theorigin of a t.1.c. plate (Merck pre-spread silica gel F254), followed by two lop1 washes of 50% (v/v) ethanol. The plate was run in propan-1-ol-water (7:3, v/v), dried, and radioautographed overnight with Kodak Microtex X-ray film. Inevery assay all of the radioactivity shown by the radioautographs was located in spots corresponding to the substrate, UDPN-acetylglucosamine (RF 0.40), and the product, chitin (RF 0). These areas were scraped into scintillant [0.7 % 5-(4-biphenylyl)-2-(4-t-butylphenyl)-l -oxa-3,4-diazole and 2.5 % (w/v) Cab-0-Sil in toluene] and their radioactivities counted with a Beckman liquidscintillation counter. The enzyme activity was calculated from the percentage radioactivity (c.p.m.) incorporated into chitin, and expressed as nmol of N-acetylglucosamine incorporated/min. The protein content of the microsomal pellet was determined by the method of Lowry et aI. (1951). Chitin synthetase activity was assayed from fruit bodies that were 6, 20, 28, 43 and 92mm tall (Fig. 1). The 6mm fruit bodies had gills, but not spores, delimited. The 20mm, 28mm and 43mm fruit bodies were progressive stages during rapid elongation, with the spores maturing and the caps expanding. The 92mm fruit bodies were fully elongated, the caps were fully expanded and the first spores were about to be released, but autolysis of the gills, involving protease and chitinase activity (Iten & Matile, 1970), was not yet visible.