GR is predominantly found in chloroplast. However, a small amount of the enzyme isoforms is also found in mitochondria, cytosol, and per-oxisomes (Edwards et al. 1990; Jimmenez et al. 1997). In leaves, bulk of GR activity is found in chloroplast, whereas root plastids exhibit a lower

Fig. 8.1 Schematic diagram showing molecular functioning of glutathione reductase (GR)

proportion of enzyme cellular activity (Foyer and Halliwell 1976). In higher plants, three types of GR occur in the cytosol, chloroplast, and mitochondria, respectively (Creissen et al. 1992; Kubo et al. 1993; Tang and Webb 1994; Creissen and Mullineaux 1995; Mullineaux et al. 1996; Kaminaka et al. 1998). Cytosolic isoforms of GRs from rice (RGRC2; Kaminaka et al. 1998) and pea (GOR2; Stevens et al. 2000) have been identified through sub-cellular fractionation; in addition, chloroplastic GRs have been identified from Arabidopsis (AT-2; Kubo et al. 1993) and pea (GOR1; Creissen et al. 1992, 1995).

Changes in the GR isoform population between and within sub-cellular compartments in response to stress were observed in pea (Edwards et al. 1994) and maize (Anderson et al. 1995). Different GR isoforms can be stimulated by different environmental signals and have different functions in the response of plant to stress (Stevens et al. 1997). About 80% of GR activities in leaf tissues are accounted for by chloroplastic isoforms (Edwards et al. 1990). Scavenging of AOS and maintaining a high ratio of reduced to oxidized glutathione by chloroplastic GR (GR1) are necessary in oxygenic photosynthesis (Foyer et al. 1995; Kornyeyev et al. 2003).

4.2 Primary Structure

A cDNA encoding GR was cloned by immuno-screening from Arabidopsis thaliana. The amino acid sequence deduced from the nucleotide sequence coincides with the N-terminal amino acid sequence of the major isozyme (GR II) purified from leaves of A. thaliana. The polypeptide comprises of an N-terminal leader sequence of 74 amino acids, which has features of chloro-plast-targeting peptides, and a mature polypep-tide of 491 residues with a molecular mass of 52.7 kDa, which shows homology with GRs from other species. The Km for GSSG is 44 mM and that for NADPH is 5.0 mM for GR II at 25°C. The pH optimum for GR II was 7.5-8.0. The native molecular mass of GR II was is kDa, indicating that GR II is a homodimer. GR II had an isoelec-tric point of 4.8. The cDNA hybridizes with a 2.1-kb poly(A)+ RNA from leaves of A. thaliana. Genomic Southern analysis indicates that the gene corresponding to the cDNA is likely a single-copy gene (Kubo et al. 1993).

GR is a homodimeric FAD-containing enzyme which belongs to the family of NADPH-dependent oxidoreductases is universal in occurrence and is found in both prokaryotic as well as eukaryotic organisms. This homodimeric protein constitutes of subunits with molecular weight of about 55 kDa. In the absence of thiols GR exhibits a propensity to form tetramers and larger forms. Although these larger forms show catalytic activity, GSH that is its product, maintains the enzyme in its dimeric form under cellular conditions. GR maintains a high GSH/GSSG ratio in cells (Alscher 1989) . It forms a salient part of ROS-scavenging system in concert with SOD and the enzymes of the well-known ascorbate-glutathi-one cycle (Foyer and Halliwell 1976). GR catalyses the reduction of GSSG which consists of two GSH linked by a disulphide bridge to GSH. GSH formed plays a vital role in the ASH-GSH cycle, maintenance of the sulphydryl group and also acts as a substrate for GSTs. Both GR and GSH play key roles in determining the tolerance of a plant under various abiotic stresses. The GSH pool maintained by GR is important for active protein function. In addition, millimolar concentrations of GSH act as an all-important redox buffer, forming a barrier between protein cystine groups and ROS. GSH also functions in limiting the metal-induced oxidative stress as from it are derived the principal heavy-metal complexing peptides of plants - the phytochelatins. One of the main features of GR is that it is thermostable.

Following is the diagram depicting the basic reaction catalysed by GR (Fig. 8.1).

As far as the mechanism of reduction of GSSG to GSH is concerned, GR acts in a ping-pong fashion in which the NADPH binds and transfers a hydride to FAD, then leaves before diglutathi-one binds. In other words, the two substrates are mutually exclusive. The catalytic cycle of GR comprises of two phases; a reductive half-reaction phase during which FAD, the prosthetic group of GR, is reduced by NADPH, and the oxidative half-reaction phase in which the resulting dithiol reacts with the glutathione disulphide and the final electron acceptor, GSSG, is reduced to two GSH at the GR active site. The H2 O2 scavenging, in particular, is carried out by catalase, various per-oxidases and the ascorbate-glutathione pathway.

The ascorbate-glutathione pathway, also known as Halliwell-Asada pathway, operates in different cellular locations like chloroplast, mitochondria, cytosol, peroxisomes, and apoplast. This cycle involves four main enzymes namely GR, APX, MDHAR, and MDAR. This pathway also includes a network of different metabolites with redox properties for the ROS detoxification which further help in averting the ROS-accrued oxidative damage in plants. The diagrammatic summary of this pathway is indicated in Fig. 8.2.

The studies regarding the GR have shown an increased GR activity in various plant species under different types of abiotic stresses. From the studies using transgenic plants, it has been proved that GR plays a prominent role in conferring resistance to oxidative stress caused by drought, ozone, heavy metals, high light, salinity, cold stress, etc. An increased GR activity has been reported in the roots of C. arientinum under salt stress, whereas Eyidogan and Oz (2005) have established elevated GR activity in the leaf tissue of the same plant under the salt stress conditions. There has also been found an enhanced GR activity in A. thaliana, Vigna mungo, Triticum aestivum, Capsicum annuum. and Brassica juncea following the cadmium treatments. Sharma and Dubey (2005) have found an increased GR

Fig. 8.2 Diagrammatic summary indicating the network of different metabolites with redox properties for the ROS detoxification which further help in averting the ROS-accrued oxidative damage in plants

Fig. 8.2 Diagrammatic summary indicating the network of different metabolites with redox properties for the ROS detoxification which further help in averting the ROS-accrued oxidative damage in plants activity in Oryza sativa seedlings during drought conditions.

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