We screened a cDNA library generated from harvested and stored sporophores of Agaricus bisporus and identified 19 genes with higher transcript levels than at the time of harvest. Five of these genes had no detectable mRNA levels prior to detachment from the mycelium. Sequence analysis of ten clones revealed significant similarities to known genes, these code for proteins involved in polymer breakdown and metabolism, cell wall synthesis, stress tolerance, cytochrome P450 activity and DNA binding. The diversity of functions of these genes suggests the changes in the sporophore after harvest involve several different physiological processes.

Serpula lacrymans is a basidiomycete cord-forming wood decay fungus which reallocates nitrogen within an extensive perennial mycelial system in response to spatial discontinuities in external nutrient supply. Intracellular stored protein is mobilised by conversion to amino acids at nutrient-poor sites within a mycelium or when a whole mycelium is starved. Intracellular peptidase and proteinase activities of the mycelium were investigated with the aim of identifying proteases specifically activated in response to a nitrogen demand. Mycelium for enzyme extraction grown as surface mats in static liquid culture was homogenised, and the extract used in assays for proteinase and peptidase with various synthetic peptide substrates conjugated to 4-nitroaniline. Activities against different substrates were characterised with respect to pH, inhibitor sensitivity, requirements for divalent metal ions, isoelectric point, and by changes in activities in starved mycelium. Four different activities were found, comprising two peptidases one of which had metalloprotease characteristics, a serine-type proteinase, and a proteinase active at pH 2.5 which was not affected by any of the inhibitors tried. Both the latter were most active in starved mycelium. Isoelectric focusing showed peaks with activities corresponding to the serine-type proteinase and one of the manganese-activated peptidases. Possible roles for these enzymes in nitrogen reallocation during mycelial foraging are discussed.

The edible mushroom, Agaricus bisporus, when cultivated commercially derives its nitrogen from the proteins present in compost, much of which is firmly bound to the lignin-humic complex. The proteinases involved in mobilization of nitrogen were studied in both the filtrates of liquid culture and mushroom compost. Proteinase activities were detected in liquid culture filtrates with a range of substrate specificities. One activity with chymotrypsin-like specificity was found by Western blotting to be the 27 kDa serine proteinase previously purified from senescent sporophores. Serine proteinase activity and level in the filtrates rapidly increased during mycelial growth on liquid medium containing humic fraction (extracted from mushroom compost) as sole nitrogen source. Activity and level remained low in filtrates of cultures grown on glutamate or casein. Non-denaturing gel analysis revealed the serine proteinase to be the most prominent proteolytic activity in compost filtrates. Regulation of the activity in compost was indicated by a doubling during the early stages of sporophore growth.