Induction of morphological changes in Ustilago maydis cells by octyl gallate E. Sierra-Campos, 1 M. A. Valdez-Solana, 2 D. Matuz-Mares, 1 I. Vela ´ zquez 1 and J. P. Pardo 1 Correspondence J. P. Pardo pardov@bq.unam.mx 1 Departamento de Bioquı ´mica, Facultad de Medicina, Universidad Nacional Auto ´ noma de Me ´ xico, Apartado Postal 70-159, Coyoaca ´n 04510, Me ´ xico D. F., Mexico 2 Departamento de Microbiologı ´a, Escuela Nacional de Ciencias Biolo ´ gicas, IPN, Casco de Santo Tomas, Mexico Received 23 May 2008 Revised 27 October 2008 Accepted 5 November 2008 The effects of octyl gallate on Ustilago maydis yeast cells were analysed in relation to its capacity to oxidize compounds (pro-oxidant actions). All phenolic compounds tested inhibited the alternative oxidase (AOX). However, only octyl gallate induced a morphological change in yeast cells and collapsed the mitochondrial membrane potential. In contrast to octyl gallate, propyl gallate and nordihydroguaiaretic acid caused only a negligible cell change and the membrane potential was not affected. Our findings show that structurally related phenolic compounds do not necessarily exert similar actions on target cells. Preincubation of U. maydis cells with trolox inhibited the change to pseudohyphal growth produced by octyl gallate. These results suggest that in addition to the inhibitory action of octyl gallate on the AOX, this compound induces a switch from yeast to a mycelium, probably through the formation of lipid peroxides. INTRODUCTION Gallic acid, a natural plant triphenol that constitutes tannin, and some esters of gallate, especially propyl, octyl and lauryl gallate, are widely used as scavenging reactive oxygen species (ROS). The antioxidant effect of poly- phenolic compounds is closely related to their hydrogen donor activity (Serrano et al., 1998), and this is the case for gallate esters, which inhibit lipid peroxidation by donating hydrogen to peroxyl and alkoxyl radical intermediates. However, gallic acid compounds show various cytotoxic and antiproliferative effects on tissues and cells. Alkyl gallates, including octyl gallate, induce apoptosis with DNA fragmentation in hepatocytes (Inoue et al., 1994; Nakagawa et al., 1997) and some tumour cells (Serrano et al., 1998); they also show trypanocidal effects (Koide et al., 1998), and free radicals of propyl gallate inhibit the activity of some redox enzymes (Brzhevskaia et al., 1966). These cytotoxic effects of gallate compounds are assumed to be due to the pro-oxidant action, not to their antioxidant capacity. Alkyl gallates also have antifungal activities against Saccharomyces cerevisiae, Zygosaccharomyces bailii, Aspergillus niger and Candida albicans (Kubo, 1999; Fujita & Kubo, 2002b; Hirasawa & Takada, 2004). In addition, propyl gallate inhibits the growth of micro- organisms by inhibiting respiration and nucleic acid synthesis (Boyd & Beveridge, 1979). Similarly, nordihy- droguaiaretic acid (NDGA) strongly inhibits succinate cytochrome c reductase by depleting the thiol groups (Shi & Pardini, 1995). Therefore, these natural compounds could serve as useful alternatives or additives to conven- tional antifungals (Kim et al., 2006). Corn smut disease is caused by the phytopathogenic basidiomycete Ustilago maydis. The disease results in stunted maize plant growth and reduced yield, leading to severe economic losses (Martinez-Espinoza et al., 2002). U. maydis is a dimorphic micro-organism, capable of producing different morphological forms (yeast or mycelium) in response to environmental stimulus. The change in morphology in U. maydis results from the activation of two conserved signalling pathways: a mito- gen-activated protein kinase (MAPK) signalling cascade and a cyclic AMP-dependent pathway (D’Souza & Heitman, 2001). Both pathways are thought to transduce environmental signals, such as nutrient availability, pres- ence of lipids, air exposure, acidic pH, and pheromones from cells of opposite mating type, during the transition from budding to filamentous growth (Bo ¨lker et al., 1995; Klose et al., 2004; Martı´nez-Espinoza et al., 2004). It has been shown that U. maydis is able to express a rotenone-insensitive NADH dehydrogenase and an altern- ative oxidase (AOX), which may play an important role in the yield of ATP synthesis (Jua ´rez et al., 2004) due to their non-proton motive character. It is also likely that the composition of the fungal electron transport chain depends on the metabolic state of the cell, and on developmental Abbreviations: AOX, alternative oxidase; DCFDA, dichlorofluorescein diacetate; DHR123, dihydrorhodamine 123; NDGA, nordihydroguaiare- tic acid; ROS, reactive oxygen species. Microbiology (2009), 155, 604–611 DOI 10.1099/mic.0.020800-0 604 020800 G 2009 SGM Printed in Great Britain