1643 F.AMICARELLI, A.BONFIGLI, S.COLAFARINA, A.M.CIMINI, B.PRUITI, P.CESARE, M.P.CERU  , C. DI ILIO 2 , G.PACIONI 1 AND M.MIRANDA*  Departments of Basic and Applied Biology and Environmental Sciences, University of L’Aquila, I-67010 Coppito, L’Aquila, Italy  Department of Biomedical Sciences, Faculty of Medicine, University ‘ G. D’Annunzio’, I-66100 Chieti, Italy Truffles live in an environment poor in oxygen, and are a good model for investigation of enzymatic adaptation to microaerobic conditions. We studied the antioxidant and glutathione dependent enzymatic endowment of Tuber truffles. Superoxide dismutase, catalase, glutathione peroxidase Se dependent, glutathione reductase, glyoxalase 1 and glyoxalase 2 are expressed and correlate with the microaerobic metabolism, growth rate and mycorrhizal symbiosis of truffles. A very low or undetectable glutathione S-transferase activity was found. For comparison the same enzyme activities were investigated in Agaricus bisporus which is epigeous and non- mycorrhizal. Truffles are hypogeous and ectomycorrhizal. They live under microaerobic conditions, and are expected to show both morphological, physiological and biochemical adaptations to an environment poor in oxygen. Very limited information is available concerning truffle metabolism and in particular oxidative metabolism (Pacioni, Ragnelli & Miranda, 1995). The interest in truffles (Tuber spp.) is due to their commercial value and the environmental conditions in which they live, symbiotic, microaerobic and hypogeous. We have previously shown that Tuber melanosporum, T. aestivum, T. brumale, T. magnatum and T. excavatum have tyrosinase (EC 114181) and laccase (EC 11032) ac- tivities (Miranda et al., 1997). These activities are oxygen dependent so that, even if the oxygen level within the soil is low, polyphenol oxidation should be accompanied by superoxide and hydrogen peroxide production (Riley, 1988). It was previously shown that Tuber spp. show respiratory adaptations similar to those of yeast under anaerobic conditions, such as an abnormal respiratory chain and deranged mitochondria (Miranda et al., 1997). The question arises whether this also occurs under microaerobic environmental conditions and if respiratory failure, the occurrence of melanin synthesis, might justify the expression of antioxidant defences ? Moreover, due to the inefficient respiratory chain and para- anaerobic conditions, glycolysis should be very active in truffles, and the presence of the enzymes involved in glycolysis has been demonstrated (Cardoni et al., 1995). As a consequence, the byproduct of glycolysis methylglyoxal (MG) should be * Corresponding author. present at relatively high levels in the truffle tissues, so that, being cytotoxic and genotoxic (Thornalley, 1990) the cells should scavenge it. Glyoxalase 1 (GLO 1) and glyoxalase 2 (GLO 2), EC 4415 and EC 3126 respectively, are the enzymes involved in MG scavenging by using reduced glutathione (GSH) and releasing -lactate and GSH and correlate with cell proliferation (Uotila, 1973 ; Mannervik, 1980 ; Thornalley, 1990). Since GSH levels required to detoxify cells from MG depend on the glutathione recycling enzymes and from those GSH dependent enzymes other than glyoxalases, we also investigated the levels of glutathione-S- transferase (GST, EC 25118), glutathione peroxidase selenium dependent (GSH-PX Se, EC 11119) and glutathione reductase (GSSG-RX, EC 16427). The occur- rence of the peroxisomal marker enzyme palmitoylCoA oxidase (EC 13993) was also investigated. Catalase (CAT, EC 11116) and (Cu-Zn) superoxide dismutase (SOD, EC 11511) were studied by both assaying the enzyme activity of truffle extracts andor immunoblotting and electrophoresis. Furthermore, since the symbiosis between fungi and plants may affect also the expression of stress proteins, such as catalase and peroxidase (Martin et al., 1995), we compared the endowment of antioxidant and detoxicant enzymes of truffles, that are hypogeous ectomycorrhizal fungi, with that of Agaricus bisporus which is epigeous and non-mycorrhizal. SOD occurrence has been reported in Agaricus brunnescens (Royse & May, 1982). As truffles grow more slowly than A. bisporus the activities of GSH-dependent and recycling enzymes may also correlate with growth rate while the antioxidant defence to the H  O  release by the symbiotic plant (Martin et al., 1995) and to fatty acid oxidation (Griffin, 1994) in addition to melanin synthesis. Mycol. Res. 103 (12) : 1643–1648 (1999) Printed in the United Kingdom Glutathione dependent enzymes and antioxidant defences in truffles : organisms living in microaerobic environments