Land plants are sessile and have developed sophisticated mechanisms that allow for both immediate and acclimatory responses to changing environments. Partial exposure of low light-adapted Arabidopsis plants to excess light results in a systemic acclimation to excess excitation energy and consequent photooxidative stress in unexposed leaves. Thus, plants possess a mechanism to communicate excess excitation energy systemically, allowing them to mount a defense against further episodes of such stress. Systemic redox changes in the proximity of photosystem II, hydrogen peroxide, and the induction of antioxidant defenses are key determinants of this mechanism of systemic acquired acclimation.

Exposure of Arabidopsis plants that were maintained under low light (200 pmol of photons m-2 sec-I) to excess light (2000 pmol of photons m-2 sec-I) for 1 hr caused reversible photoinhibition of photosynthesis. Measurements of photosynthetic parameters and the use of electron transport inhibitors indicated that a nove1 signal transduction pathway was initiated at plastoquinone and regulated, at least in part, by the redox status of the plastoquinone pool. This signal, which preceded the photooxidative burst of hydrogen peroxide (H202) associated with photoinhibition of photosynthesis, resulted in a rapid increase (within 15 min) in mRNA levels of two cytosolic ascorbate peroxidase genes ( A f X 1 and APX2). Treatment of leaves with exogenous reduced glutathione abolished this signal, suggesting that glutathione or the redox status of the glutathione pool has a regulatory impact on this signaling pathway. During recovety from photooxidative stress, transcripts for cytosolic glutathione reductase (GOR2) increased, emphasizing the role of glutathione in this stress.

The mutant regulator of APX2 1-1 (rax1-1) was identified in Arabidopsis thaliana that constitutively expressed normally photooxidative stress-inducible ASCORBATE PEROXIDASE2 (APX2) and had $50% lowered foliar glutathione levels. Mapping revealed that rax1-1 is an allele of g-GLUTAMYLCYSTEINE SYNTHETASE 1 (GSH1), which encodes chloroplastic g-glutamylcysteine synthetase, the controlling step of glutathione biosynthesis. By comparison of rax1-1 with the GSH1 mutant cadmium hypersensitive 2, the expression of 32 stress-responsive genes was shown to be responsive to changed glutathione metabolism. Under photo-oxidative stress conditions, the expression of a wider set of defense-related genes was altered in the mutants. In wild-type plants, glutathione metabolism may play a key role in determining the degree of expression of defense genes controlled by several signaling pathways both before and during stress. This control may reflect the physiological state of the plant at the time of the onset of an environmental challenge and suggests that changes in glutathione metabolism may be one means of integrating the function of several signaling pathways.

Analysis of the oxidative processes taking place during fruit ripening in a salad tomato variety (Lycopersicon esculentum Mill. cv. Ailsa Craig) revealed changes in oxidative and antioxidative parameters. Hydrogen peroxide content, lipid peroxidation and protein oxidation were measured as indices of oxidative processes and all were found to increase at the breaker stage. The levels of the aqueous-phase antioxidants, glutathione and ascorbate, increased during the ripening process and these increases were associated with significant changes in their redox status, becoming more reduced as ripening progressed. Changes in the activities of superoxide dismutase, catalase and the enzymes involved in the ascorbate-glutathione cycle during ripening indicated that the antioxidative system plays a fundamental role in the ripening of tomato fruits.

Most plant-microbe interactions do not result in disease; natural products restrict non-host pathogens. We found that sulforaphane (4-methylsulfinylbutyl isothiocyanate), a natural product derived from aliphatic glucosinolates, inhibits growth in Arabidopsis of non-host Pseudomonas bacteria in planta. Multiple sax genes (saxCAB/F/D/G) were identified in Pseudomonas species virulent on Arabidopsis. These sax genes are required to overwhelm isothiocyanate-based defenses and facilitate a disease outcome, especially in the young leaves critical for plant survival. Introduction of saxCAB genes into non-host strains enabled them to overcome these Arabidopsis defenses. Our study shows that aliphatic isothiocyanates, previously shown to limit damage by herbivores, are also crucial, robust, and developmentally regulated defenses that underpin non-host resistance in the Arabidopsis-Pseudomonas pathosystem.

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...

Summary N-terminal presequencas from cDNAs encoding mitochondrion-or chloroplast-specific proteins are able, with variable efficiencies, to target preprotsins to their respective organelles, in the few cases studied in which a nuclearencoded protein is found in both these orgeneiles, each compartment-specific isoform is encoded by a separate gene. Glutathione reductase (GR) from peas is encoded by a single nuclear gene and yet GR is distributed between chloroplasts, mitochondda and the cytosoL Previous sequence analysis of a full-length GR cDNA revealed the presence of a putative plastid transit peptide. However, expression of this cDNA in transgenic tobacco resulted in substantially elevated GR activities in beth chloroplasts and mitochondria in four independent lines examined. There was no effect on expression of the endogenous tobacco GR genes. Replacement of the GR presequenca with presequencas from pea rbcS (chloroplast) and Nicotiana plumbaginifolia Mn-SOD (mitochonddon) resulted in targeting of GR only into the appropriate organeile. Expression of a fusion protein between the amino terminal region of GR and phosphinothricin acatyl transferase resulted in targeting of the foreign proteinto chloroplasts and mitochondria. Thus, the pea GR presequence is capable of co-targeting this enzyme or a foreign protein to chloroplasts and miti:¢hondria in vivo. This is the first example of co-targeting by a higher plant preprotein.

When low-light-grown Arabidopsis rosettes are partially exposed to excess light (EL), the unexposed leaves become acclimated to excess excitation energy (EEE) and consequent photo-oxidative stress. This phenomenon, termed systemic acquired acclimation (SAA), is associated with redox changes in the proximity of photosystem II, changes in foliar H2O2 content and induction of antioxidant defences. The induction of extra-plastidial antioxidant systems is important in the protection of the chloroplast under EL conditions. A larger range of transcripts encoding different antioxidant defence enzymes may be induced in the systemically acclimated leaves and these include those encoded by the glutathione peroxidase (GPX2) and glutathione-S-transferase (GST) genes, which are also highly induced in the hypersensitive response and associated systemic acquired resistance (SAR) in incompatible plant-pathogen interactions. Furthermore, the expression of the SAR-inducible pathogenesis-related protein gene, PR2, is enhanced in SAA leaves. Wounded leaf tissue also shows enhanced systemic induction of a cytosolic ascorbate peroxidase gene (APX2) under EL conditions. These and other considerations, suggest H2O2 and other reactive oxygen species (ROS) could be the common factor in signalling pathways for diverse environmental stresses. These effects may be mediated by changes in the level and redox state of the cellular glutathione pool. Mutants with constitutive expression of a normally EL-inducible APX2 gene have much reduced levels of foliar glutathione. The expression of APX1 and APX3, encoding cytosolic and peroxisome-associated isoforms, respectively, are also under phytochrome-A-mediated control. The expression of these genes is tightly linked to the greening of plastids in etiolated seedlings. These data suggest that part of the developmental processes that bring about the acclimation of leaves to high light includes the configuration of antioxidant defences. Therefore, the linkage between immediate responses of leaves to EL, acclimation of chloroplasts to EEE and the subsequent changes to leaf form and function in high light could be mediated by the activity of foliar antioxidant defences and changes in the concentration of ROS.