PHYSIOLOGIA PLANTARUM 117: 85–92. 2003 Copyright C Physiologia Plantarum 2003 Printed in Denmark – all rights reserved ISSN 0031-9317 Molecular cloning of cDNAs for three tau-type glutathione S-transferases in pumpkin (Cucurbita maxima) and their expression properties Masayuki Fujita* and Mohammad Z. Hossain Department of Plant Sciences, Faculty of Agriculture, Kagawa University, Miki-cho, Kita-gun, Kagawa 761-0795, Japan *Corresponding author, e-mail: fujita/ag.kagawa-u.ac.jp Received 4 February 2002; revised 3 June 2002 cDNAs of three major glutathione S-transferases (GSTs), Puga, Pugb and Pugc, in 2,4-D-treated pumpkin (Cucurbita maxima Duch.) callus were individually obtained through im- muno-screenings with corresponding antisera from a pumpkin cDNA library. Comparison of the predicted protein sequences with ones of other plant GSTs reported previously and recog- nition of the presence of a single intron in each genomic gene indicated that they could be classified into tau-type GST group. Hence, Puga, Pugb and Pugc were named CmGSTU1, CmGSTU2 and CmGSTU3, respectively. Expression of the GSTs in various organs of pumpkin plants, and in hypocotyls of seedlings under various stress conditions were examined by Western blotting and Northern blotting analyses. CmGSTU1 Introduction Glutathione S-transferases (GSTs, EC 2.5.1.18) are a family of enzymes that can conjugate glutathione, via its sulphydryl group, to a wide range of hydrophobic, electrophilic compounds. They are ubiquitously distrib- uted in the biosphere and are involved in the cellular detoxification and excretion of many physiological and xenobiotic substances. In plants, many GSTs have been reported to be induced by various stresses such as heavy metals (Hagen et al. 1988), pesticides (Fuerst et al. 1993, Holt et al. 1995), ozone (Clayton et al. 1999), dehy- dration (Kiyosue et al. 1993), low and high temperatures (Boot et al. 1993), pathogens (Taylor et al. 1990) and plant hormones (Hagen et al. 1988, Meyer et al. 1991). Therefore plant GSTs are thought to play vital roles in diversities of stress physiology through some other func- tions as well as GST mediated detoxification. For ex- ample, some GSTs have been demonstrated to possess Abbreviations – CDNB, 1-chloro-2,4-dinitrobenzene; GST, glutathione S-transferase; IPTG, isopropyl-b--thiogalactopyranoside; NAA, naphthalene- 1-acetic acid; pfu, plaque-forming unit. Physiol. Plant. 117, 2003 85 tended to be expressed more in fully expanded mature organs, and CmGSTU2 seemed to be expressed preferentially in leaves and petioles. CmGSTU1 and CmGSTU2 were induced by dehydration. CmGSTU1 was also induced by high tem- perature stress. In this study, the expression pattern of CmGSTU3 could not be determined because the contents were too low to detect with the antiserum. After 2,4- treat- ment, GST activity in the pumpkin callus increased rapidly until day one and then slowly increased at a constant rate up to day 6. The increase seemed to be dependent on post- transcriptional enhancement as well as transcriptional induc- tion. glutathione peroxidase activity (Bartling et al. 1993, Ed- wards 1996, Roxas et al. 1997, Kampranis et al. 2000). In higher plants, many cDNAs and genomic DNAs encoding GSTs and putative GSTs have been isolated. Recently, Edwards et al. (2000) have proposed classifi- cation of plant GSTs into phi-, zeta-, tau- and theta- classes, following the original classification of type I, type II and type III. In this new classification, phi- and tau-classes appear to be specific to plant GSTs, while zeta- and theta-classes consist of mammalian GSTs as well as plant GSTs. The classifications adopted to plant GSTs are based on amino acid sequence identity and conservation of intron:exon placement, not on substrate. Until now, no class-specific GST function has been found. Glutathione peroxidase activity is reported to present in three classes, phi, tau and theta. Pumpkin fruit sarcocarp tissues are known to have