Downloaded from www.microbiologyresearch.org by IP: 54.70.40.11 On: Thu, 01 Aug 2019 18:46:14 Journal of General Microbiology (1 982), 128, 2667-2674. Printed in Great Britain 2667 Biosynthesis of Alkali-insoluble Cell-wall Glucan in Schizophyllum commune Protoplasts By A. S. M. SONNENBERG, J. H. SIETSMA* AND J. G. H. WESSELS Department of Developmental Plant Biology, Biological Centre, University of Groningen, Haren, The Netherlands (Received 23 March 1982) Biosynthesis of cell-wall glucans was studied during regeneration of protoplasts from Schizophyl- lum commune. Double-labelling experiments using [ SC]glucose and [3H]glucose indicated a larger pool size of precursors for an alkali-insoluble (1 -+ 3)-P-glucan than for the other cell-wall components, (1 -+ 3)-wglucan and chitin. Pulse-chase experiments established the existence of a water-soluble, partly alkali-soluble (1 t 3)-P-glucan as a precursor for the alkali-insoluble wall glucan, containing only (1 -+ 3)-P-linkages. Polyoxin D, an inhibitor of chitin synthase, com- pletely arrested accumulation of alkali-insoluble (1 -+ 3)-P-glucan. This antibiotic did not inhibit the synthesis of the water-soluble glucan, but prevented the incorporation of this material into the alkali-insoluble glucan/chitin complex. Cycloheximide added at the start of regeneration prevented the synthesis of the alkali-insoluble (1 -+ 3)-P-glucan and of the water-soluble glucan precursor, whereas no effect on the formation of the alkali-insoluble glucan was observed when cycloheximide was added 3 h after the onset of the regeneration. INTRODUCTION The hyphal wall of the basidiomycete Schizophyllum commune grown in culture for 5 d con- tains a water-soluble (1 + 3)-P/( 1 -+ 6)-P-glucan (mucilage), an alkali-soluble (1 -+ 3)-a-D-glUCan (S-glucan)and an alkali-insoluble complex of (1 -+ 3)-P/( 1 -+ 6)-fi-~-glucan (R-glucan) with chitin (Wessels et al., 1972; Sietsma & Wessels, 1977). The insolubility of the glucan in the alkali- insoluble complex has been explained by the presence of covalent linkages between this glucan and chitin (Sietsma & Wessels, 1979). In the present study regeneration of protoplasts was used as a system to study biosynthesis of these wall components. It has been shown previously that protoplasts of S. commune are fully capable of regenerating a new wall and reverting to normal hyphal growth (de Vries & Wessels, 1975; van der Valk & Wessels, 1976). These earlier studies showed (i) the synthesis of S-glucan and chitin started after a very short lag period, but synthesis of alkali-insoluble glucan was delayed for several hours ; (ii) polyoxin D, an antibiotic inhibiting chitin synthase, prevented synthesis of both chitin and R-glucan and also the reversion to hyphal growth; and (iii) cyclohex- imide prevented both the formation of R-glucan and the reversion to hyphal growth, while the formation of chitin and S-glucan was seemingly unimpaired for several hours. The present results give evidence for a water-soluble (1 -+ 3)-fi-D-glucan functioning as precursor for the alkali-insoluble (1 + 3)-P-D-glUCan in a glucan/chitin complex and emphasize the importance of secondary polysaccharide modifications in wall construction. METHODS Growth of' mycelium and preparation qf'protoplasts. Dikaryotic fruiting mycelium of Schizophyllum commune, obtained by mating strains 1-40 (CBS 344.81) and 1-50 (CBS 342.81), was grown at 24 "C on minimal medium (Dons et al., 1979) supplemented with 15 g agar 1-' in Petri dishes placed upside down. Basidiospores accumulat- ing on the lids were collected regularly and stored at -40 "C in sterile water, Mycelium was grown from 0022-1287/82/0001-0487 $02.00 0 1982 SGM