RESEARCH LETTER Hydroquinone and H 2 O 2 di¡erentiallya¡ect the ultrastructure and expression of ligninolytic genes in the basidiomycete Ceriporiopsis subvermispora Alejandro Amoroso 1,2 , Rodrigo A. Mancilla 1,2 , Bernardo Gonz ´ alez 1,3,4 & Rafael Vicu ˜ na 1,2 1 Departamento de Gen ´ etica Molecular y Microbiolog´ ıa, Facultad de Ciencias Biol ´ ogicas, Pontificia Universidad Cat ´ olica de Chile, Santiago, Chile; 2 Instituto Milenio de Biolog´ ıa Fundamental y Aplicada, Santiago, Chile; 3 Center for Advanced Studies in Ecology and Biodiversity, Pontificia Universidad Cat ´ olica de Chile, Santiago, Chile; and 4 Millennium Nucleus on Microbial Ecology and Environmental Microbiology and Biotechnology (EMBA–ICM), Santiago, Chile Correspondence: Rafael Vicu ˜ na, Departamento de Gen ´ etica Molecular y Microbiolog´ ıa, Facultad de Ciencias Biol ´ ogicas, Center for Advanced Studies in Ecology and Biodiversity, Pontificia Universidad Cat ´ olica de Chile, Casilla 114-D, Santiago, Chile. Tel.: 156 2 686 2663; fax: 156 2 222 5515; e-mail: rvicuna@bio.puc.cl Received 30 January 2009; accepted 4 March 2009. First published online 1 April 2009. DOI:10.1111/j.1574-6968.2009.01573.x Editor: Richard Staples Keywords glutathione; hydrogen peroxide; hydroquinone; laccase; ligninolytic fungi; oxidative stress. Abstract The biodegradation of lignin is a highly oxidative process in which various oxidases and peroxidases play a major role. During lignin decay, the generation of aromatic compounds and reactive oxygen species leads to oxidative stress. In this work, the effect of the oxidative compounds H 2 O 2 and hydroquinone in the ligninolytic fungus Ceriporiopsis subvermispora was studied, both at the ultrastructural and at the transcriptional level. Transmission electron microscopy revealed the presence of microvesicles and extensive cytoplasm degeneration after incubation with hydroquinone, but not with H 2 O 2 . Studies of the intracellular redox state of the fungus showed that hydroquinone causes a transient decrease in the reduced glutathione/oxidized glutathione (GSH/GSSG) ratio and an increase in the glutathione-S-transferase mRNA levels. These results suggest that hydroquinone produces oxidative stress in this microorganism. On the other hand, it was observed that hydroquinone, but not H 2 O 2 , affects Mn-dependent peroxide and laccase transcripts levels. We propose that the mechanism by which the fungus reacts against oxidative stress contributes to its selectivity toward lignin during wood decay. Introduction The basidiomycete Ceriporiopsis subvermispora is a white rot fungus that possesses high selectivity toward lignin when growing on wood (Mart´ ınez et al., 2005). Its ligninolytic system is composed of the extracellular enzymes Mn-depen- dent peroxidase (MnP) and laccase (R¨ uttimann et al., 1992), plus an extracellular hydrogen peroxide-producing system, that is required for MnP activity (Urzu ´a et al., 1998). The former catalyzes the H 2 O 2 -dependent oxidation of Mn(II) to Mn(III), which, chelated by organic acids, can oxidize a variety of aromatic compounds (Wariishi et al., 1988, 1989). In turn, laccase catalyzes the one-electron oxidation of phenolic compounds, with a concomitant four-electron reduction of O 2 to H 2 O (Baldrian, 2006). Thus, lignin biodegradation by this fungus (as well as others) is a highly oxidative process. Moreover, during ligninolysis, the genera- tion of aromatic compounds and reactive oxygen species (ROS) gives rise to oxidative stress (Guill´ en et al., 1997, 2000). Studies conducted with several fungi have shown that this condition increases the mRNA levels of MnP, laccase and lignin peroxidase (LiP) (Faison & Kirk, 1985; Li et al., 1995; Fern´ andez-Larrea & Stahl, 1996; Ruiz-Due˜ nas et al., 1999; Min et al., 2001; Belinky et al., 2003; Kim et al., 2006). The latter enzyme possesses the unique ability to withdraw one electron from nonphenolic aromatic substrates, giving rise to cation radicals that undergo fragmentation into smaller compounds (Kirk et al., 1986). Transcripts of these enzymes also increase in the presence of certain aromatic compounds (Yaver et al., 1996; Collins & Dobson, 1997; Mansur et al., 1998; Temp et al., 1999; Scheel et al., 2000; Soden & Dobson, 2001). In this study, the effect of the H 2 O 2 and hydroquinone on the ligninolytic fungal model C. subvermispora was investi- gated. We chose these compounds because H 2 O 2 plays a leading role during the decay of lignin, whereas quinones are FEMS Microbiol Lett 294 (2009) 232–238 c  2009 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved