Characterisation of pea cytosolic glutathione reductase expressed in transgenic tobacco

Abstract

Expression in transgenic tobacco (Nicotiana tabacum L.) of a pea (Pisum sativum L.) GOR2 cDNA, encoding an isoform of glutathione reductase (GOR2), resulted in a 3- to 7-fold elevation of total foliar glutathione reductase (GR) activity. The enzyme encoded by GOR2 was confirmed to be extraplastidial in organelle fractionation studies and was considered most likely to be localised in the cytosol. A partial purification of GOR2 was achieved but a standard affinity chromatography step, using adenosine-2',5'-diphosphate-Sepharose and often employed in the purification of GR from diverse sources, was unsuccessful with this isoform. Preparative isoelectric focussing was employed as part of the purification procedure of GOR2 and a complete separation from plastidial/mitochondrial glutathione reductase (GOR1) was achieved. The isoform GOR2 was shown to have a slower migration on non-denaturing polyacrylamide gels compared with GOR1 and properties typical of GR enzymes from plant sources.

Enhanced sensitivity to oxidative stress in transgenic tobacco plants with decreased glutathione reductase activity leads to a decrease in ascorbate pool and ascorbate redox state

“…To date, two GR genes in pea and tobacco, GOR1 and GOR2, have been characterized (Creissen et al 1992;Stevens et al 1997Stevens et al , 2000. GOR1 encodes a protein co-targeted into chloroplasts and mitochondria (Creissen et al 1992 and GOR2 encodes a protein which is extraplastidial and most likely to be located in the cytosol (Stevens et al 2000).…”

Oxidative Stress and Acclimation Mechanisms in Plants

“…Only two Arabidopsis GR genes have been characterized, however (Stevens et al 2000). A peroxisomal GR gene has not been characterized to date, although a peroxisomal GR protein has been described (Corpas et al 2001).…”

Molecular cloning of glutathione reductase cDNAs and analysis of GR gene expression in cowpea and common bean leaves during recovery from moderate drought stress

“…Most of the ascorbate-glutathione cycle enzymatic activities, including GR, are located in chloroplasts, cytosol, mitochondria and peroxisomes (Edwards et al, 1990;Jimenez et al, 1997). However, only two forms of GR genes have been identified to date, a cytosolic isoform (cGR) and an isoform dualytargeted (dtGR) to both chloroplasts and mitochondria (Lee et al, 1998;Stevens et al, 2000;Rudhe et al, 2002;Chew et al, 2003).…”