Elevated Glutathione Biosynthetic Capacity in the Chloroplasts of Transgenic Tobacco Plants Paradoxically Causes Increased Oxidative Stress

Abstract

Glutathione (GSH), a major antioxidant in most aerobic organisms, is perceived to be particularly important in plant chloroplasts because it helps to protect the photosynthetic apparatus from oxidative damage. In transgenic tobacco plants overexpressing a chloroplast-targeted ␥ -glutamylcysteine synthetase ( ␥ -ECS), foliar levels of GSH were raised threefold. Paradoxically, increased GSH biosynthetic capacity in the chloroplast resulted in greatly enhanced oxidative stress, which was manifested as light intensity-dependent chlorosis or necrosis. This phenotype was associated with foliar pools of both GSH and ␥ -glutamylcysteine (the immediate precursor to GSH) being in a more oxidized state. Further manipulations of both the content and redox state of the foliar thiol pools were achieved using hybrid transgenic plants with enhanced glutathione synthetase or glutathione reductase activity in addition to elevated levels of ␥ -ECS. Given the results of these experiments, we suggest that ␥ -ECS-transformed plants suffered continuous oxidative damage caused by a failure of the redox-sensing process in the chloroplast. INTRODUCTIONPlants, like all aerobic organisms, possess an array of hydrophilic and lipophilic antioxidants, such as glutathione, ascorbic acid (vitamin C), phenolic isoflavanoid compounds, ␣ -tocopherol (vitamin E), and the carotenoids, including the xanthophylls (Fryer, 1993; Mullineaux and Creissen, 1996). The reduced forms of these compounds, together with antioxidant enzymes, scavenge reactive oxygen species (ROS) and other products of oxidative reactions. These enzymes include subcellular compartment-specific isoforms of superoxide dismutase (SOD), catalase, ascorbate peroxidase (APX), glutathione S -transferase/glutathione peroxidase (GST/GPX), dehydroascorbate reductase, monodehydroascorbate free radical reductase, and glutathione reductase (GR). Several reduction-oxidation (redox) cycles that scavenge ROS in different subcellular compartments and that involve these enzymes and antioxidants have been proposed (e.g., the ascorbate-GSH cycle). The reducing equivalents for these reactions are derived ultimately from photosynthetic electron transport (Foyer and Halliwell, 1976; Mullineaux and Creissen, 1997). Thus, the degree of reduction of major antioxidant pools is generally considered to reflect the redox status of the tissue in question and is consequently an indicator of oxidative stress.Glutathione, either as GSH or as GSSG (glutathione disulfide; oxidized glutathione), is regarded as a key component of antioxidant defenses in most aerobic organisms, including plants (Foyer et al., 1997). However, the high (i.e., millimolar) concentration of GSH in the chloroplast (Foyer and Halliwell, 1976; Law et al., 1983; Bielawski and Joy, 1986) is in apparent conflict with its proposed roles: the regeneration of ascorbate (Foyer and Halliwell, 1976), reduction of lipid hydroperoxides (Mullineaux et al., 1998), and regulation of chloroplast gene expression by thiol-mediated modulation of RNA polymeras...

Modification of reactive oxygen species scavenging capacity of chloroplasts through plastid transformation

“…Elevated GR activity should lead to more reduced glutathione which would generally be regarded as beneficial for redox homeostasis in chloroplasts (Mittler 2002). However, our finding matches that of Creissen et al (1999) who increased GSH levels by transformation with a gene for a chloroplast-targeted c-glutamylcysteine synthetase (c-ECS) and found increased oxidative stress, which they explained on the basis of impaired redox sensing in the chloroplast. We contend that the increased MV sensitivity of the transplastomic lines over-expressing GR has a similar basis, although it is not reflected in a study in poplar (Foyer et al 1995) in which chloroplast targeted GR did result in improved oxidative stress tolerance.…”

The High Light Response inArabidopsisInvolves ABA Signaling between Vascular and Bundle Sheath Cells

“…Hydrochloric acid (0.1 M) extracts from 100 mg of fresh Arabidopsis tissue ground in liquid nitrogen were prepared and purified over activated charcoal as described by Creissen et al (1999). H 2 O 2 concentrations in purified extracts were determined using an Amplex Red assay kit (Invitrogen) according to the manufacturer's instructions.…”

Greater protection against oxidative damages imposed by various environmental stresses in transgenic potato with higher level of reduced glutathione

“…have extensively reviewed several studies showing the importance of GR in the defense against oxidative stress. In contrast, an increased sensitivity to oxidative stress in transgenic plants with elevated GSH levels by either overexpression of γ-glutamylecysteine synthetase (γ-ECS), glutathione synthetase (GS) or GR has been also reported (Creissen et al 1999, Logan et al 2003.…”