Planta (1996)200:186 194 P l a n t a ~) Springer-Verlag 1996 Characterisation of a glutathione reductase gene and its genetic locus from pea (Pisum satirum L.) Philip Mullineaux, Corine Enard*, Roger Hellens, Gary Creissen John lnnes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, UK Received: 2 January 1996/Accepted: 1 March 1996 Abstract. A cDNA encoding the chloroplast/mitochon- drial form of glutathione reductase (GR; EC 1.6.4.2) from pea (Pisum sativum L.) was used to map a single GR locus, named GORI. In two domesticated genotypes of pea (cv. Birte and JI 399) it is likely that the GOR1 locus contains a single gene. However, in a semi-domesticated land race of pea (JI 281) two distinct but closely related sets of GR gene sequences were detected at the GOR1 locus. The extra GR sequences in JI 281 represent either a second intact gene or a partial or pseudogene copy. A GR gene was cloned from cv. Birte, sequenced and its structure analysed. No feature of the transcription or structure of the gene suggested a mechanism for generating any more than one form of GR. From these data plus previously published biochemical evidence we suggest that a second, distinct gene encoding for the cytosolic form of GR should be present in peas. The GORl-encoded GR mRNA can be detected in all main organs of the plant and no alternative spliced species was presen t which could perhaps account for the generation of multiple isoforms of GR. The mis- match between the number of charge-separable isoforms in pea and the proposed number of genes suggests that different GR isoforms arise by some form of post-trans- lational modification. Key words: Gene structure - Genetic locus - Gene trans- cripts Glutathione reductase (isoforms) - Pisum The pea glutathione reductase gene sequences will appear in the EMBL/Genbank databases with the accession number X90996 * Present address: Laboratoire de Pathologie Vegetale, Institut Na- tional Recherche Agronomique, 16 rue Claude Bernard, F-75231 Paris Cedex 05, France Abbreviations: GR = glutathione reductase; PCR = polymerase chain reaction; RACE = rapid amplification of cDNA ends; RFLP = restriction fragment length polymorphism Correspondence to: P. Mullineaux; FAX: 44 (1603)456844; E-mail: mullineaux@bbsrc.ac.uk Introduction Glutathione (GSH) is often found as the major free thiol compound in a wide range of organisms from the bacter- ial, animal and plant kingdoms (Meister and Anderson 1983; Smith et al. 1990). Glutathione plays a central role in many processes; it is involved in detoxification of xenobiotics and heavy metals (Timmerman 1989; Steffens 1990), in regulation of gene expression in response to biotic and abiotic stress (Wingate et al. 1988; Herouart et al. 1993; Wingsle and Karpinski 1995), in sulphur trans- port (Meister and Anderson 1983; Polle and Rennenberg 1994) and as an antioxidant in both enzymic and non- enzymic reactions (Sies 1985; Alscher 1989). In plants, GSH is thought to play a key role as part of the ascorbate/ glutathione cycle in ameliorating the effects of reactive oxygen intermediates generated during photooxidative stress, although a similar cycle involving only ascorbate has been recently proposed (Foyer and Halliwell 1976; Asada 1994). Glutathione reductase (GR; EC 1,6.4.2) is a flavo- protein oxidoreductase which catalyses the reduction of oxidised glutathione (glutathione disulphide; GSSG) to GSH using NADPH as the electron donor (Scruton et al. 1990; Creissen et al. 1994). Glutathione reductase is there- fore crucial in maintaining the antioxidant capacity of the cell (Meister and Anderson 1983; Creissen et al. 1994). This enzyme may also be involved in the regulation of steady-state levels of glutathione in bacteria and plants, although how this is achieved remains obscure (Kunert et al. 1990; Broadbent et al. 1995). Glutathione reductase has been purified from seven plant species and its properties studied: maize (Mahan and Burke 1987), pea (Kalt-Torres et al. 1984; Connell and Mullet 1986; Bielawski and Joy 1986; Edwards et al. 1990), spinach (Halliwell and Foyer 1978; Tanaka et al, 1988), Arabidopsis (Kubo et al. 1993), Scots pine (Wingsle 1989), eastern white pine (Anderson et al. 1990) and red spruce (Hausladen and Alscher 1994). The chloroplastic isoform of GR has been the most studied, especially from peas where it constitutes 77% of total leaf GR (Kalt-Torres et al. 1984; Connell and Mullet