Distribution of (l->3)- - and (l->4)- -Glucan Synthases Along the Hyphae of Saprolegnia monoica

Abstract

Protoplasts from hyphae of Saprolegnia monoica, collected from lytic medium after different times of incubation, represented a sequential fractionation of mycelium from apical to distal zones. This method gives the opportunity to study the intrahyphal localization of cell wall glucan synthases in relation to apical growth. (1 +4)-P-Glucan synthases were mainly found in early protoplasts (i.e. from apical zones) while the highest (1 +3)+glucan synthase activities were recovered in late protoplasts (i.e. from the subapical zones). Early protoplasts also exhibited the highest rate of cell wall synthesis during regeneration. The distribution of the enzymes along the fungal hyphae might provide a clue to the mechanisms of cell wall polysaccharide deposition during apical growth. METHODS Culture methods. Saprolegnia monoicaPringsheim (no. 539 67 Dick) obtained from CBS, Baarn, The Netherlands, was maintained on a wheat flour medium. Mycelia were grown in liquid medium (Machlis, 1953) at 23 "C for 48 h and harvested by filtration. Protopfast production. Mycelia (48 h old) were converted to protoplasts during incubation in a lytic medium pH 5.8, containing 5 mg Driselase ml-1 (Fluka, Buchs, Switzerland), 1 mg cellulase ml-1 and 0.5 M-sorbitol as 0022-1287/84/0001-1635 $02.00 0 1984 SGM Journal of Botany 40, 449-460. RAYMOND, Y., FICHER, G. B. & MACLACHLAN, G. A. (1978). Tissue slice and particulate p glucan synthetase activities from Pisum epicotyls. Plant Physiology 61, 938-942. SIETSMA, J. H. (1969). Protoplast formation and cell wall composition of some Oomycete species. PhD thesis, University of Amsterdam. UPDEGRAFF, D. M. (1969). Semi-micro determination of cellulose in biological materials. Analytical Biochemistry 32, 420-424.

Structural and Morphological Diversity of (1→3)-β-d-Glucans Synthesized in Vitro by Enzymes from Saprolegnia monoïca. Comparison with a Corresponding in Vitro Product from Blackberry (Rubus fruticosus)

“…Also, in the cytosol of fungal hyphae, the Ca 2+ concentration is known to be higher at the elongating apex than in the nongrowing parts of the hyphae (63,64). From these observations, and in agreement with the data of Girard and Fe `vre (65), it has been proposed that the enzyme working at pH 9 would be more specifically located in the growing apical part of the hyphae, whereas the enzyme with a pH optimum of 6 would have a subapical localization (46). This hypothesis is in keeping with a recent report that shows that (1f3)-β-D-glucans are present in all regions of both elongating and nonelongating hyphae of the Oomycete Achlya bisexualis, a close relative of S. monoı ¨ca (66).…”

A novel 1,3-β-glucan synthase from the oomycete Saprolegnia monoica

“…Its activity was higher in protoplasts released after 45 min than in protoplasts collected after 90 min lysis, suggesting that the enzyme may be located in the apical part of the hypha. This contrasts strongly with the Ca 2+ , Mn 2+ and Mg 2+ inhibition of the former characterized enzyme and with its subapical localization (Girard & Fèvre, 1984). A concentration gradient of free Ca 2+ occurs in the cytosol of growing hyphae with the concentration of the divalent cation highest at the apex (Jackson & Heath, 1993; Levina et al , 1995).…”

“…It is therefore possible that l,3- β -GSl and l,3- β -GS2 are found at different sites in the hypha since the previously described glucan synthase (i.e. l,3- β -GSl) was found associated with subapical cytoplasm (Girard & Fevre, 1984).…”

“…The digestion of a fungal colony by lytic enzymes enables a sequential fractionation of mycelium as protoplast production has been considered as a sequential release of cytoplasm from apical to subapical parts (Isaac et al , 1978; Girard & Fèvre, 1984). Protoplasts were collected after 45 and 90 min lysis, washed in the osmotic buffer, homogenized, and membranes were collected then CHAPS-solubilized.…”

Use of Molecular Cytology to Study the Structure and Biology of Phytopathogenic and Mycorrhizal Fungi

“…Studies of the Oomycete Saprolegnia monoica have shown ␤-1,3-glucan synthase activity is highest near the hyphal apex (Girard and Fè vre, 1984). Interestingly the activity of ␤-1,4-glucan synthase is highest in a subapical zone, suggesting that cellulose microfibril deposition is not concentrated within the apical dome.…”