Gary Creissen

Photosynthetic electron transport regulates the expression of cytosolic ascorbate peroxidase genes in Arabidopsis during excess light stress.

Exposure of Arabidopsis plants that were maintained under low light (200 mumol of photons m-2 sec-1) to excess light (2000 mumol of photons m-2 sec-1) for 1 hr caused reversible photoinhibition of photosynthesis. Measurements of photosynthetic parameters and the use of electron transport inhibitors indicated that a novel 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 (H2O2) associated with photoinhibition of photosynthesis, resulted in a rapid increase (within 15 min) in mRNA levels of two cytosolic ascorbate peroxidase genes (APX1 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 recovery from photooxidative stress, transcripts for cytosolic glutathione reductase (GOR2) increased, emphasizing the role of glutathione in this stress.

Evidence for a Direct Link between Glutathione Biosynthesis and Stress Defense Gene Expression in Arabidopsis

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 γ-GLUTAMYLCYSTEINE SYNTHETASE 1 (GSH1), which encodes chloroplastic γ-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.

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

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 γ-glutamylcysteine synthetase (γ-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 γ-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 γ-ECS. Given the results of these experiments, we suggest that γ-ECS–transformed plants suffered continuous oxidative damage caused by a failure of the redox-sensing process in the chloroplast.

Changes in oxidative processes and components of the antioxidant system during tomato fruit ripening

Abstract. 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.

Synthesis and properties of glutathione reductase in stressed peas

We have subjected peas (Pisum sativum L.) to four different oxidative stresses: cold conditions (4 °C) in conjunction with light, treatment with paraquat, fumigation with ozone, and illumination of etiolated seedlings (greening). In crude extracts of leaves from stressed plants, an increase (up to twofold) in activity of glutathione reductase (GR) was observed which was consistent with previous reports from several laboratories. In all cases, except for ozone fumigation, the increase in activity was not due to an elevation in the steady-state levels of GR protein. None of the applied stresses had any effect on steady-state levels of GR mRNA. In contrast to the small increase in GR activity, the Km of GR for glutathione disulphide showed a marked decrease when determined for extracts of stressed leaves, compared with that from unstressed plants. This indicates that GR from stressed plants has an increased affinity for glutathione disulphide. The profile of GR activity bands fractionated on non-denaturing acrylamide gels varied for extracts from differently stressed leaves and when compared with GR from unstressed plants. The changes in GR-band profiles and the alteration in the kinetic properties are best explained as changes in the isoform population of pea GR in response to stress.

Pseudomonas sax Genes Overcome Aliphatic Isothiocyanate–Mediated Non-Host Resistance in Arabidopsis

Natural-product effectors of disease resistance in Arabidopsis reveal complementary disabling mechanisms in the pathogen. 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.

Cloning and characterisation of a cytosolic glutathione reductase cDNA from pea (Pisum sativum L.) and its expression in response to stress

A second glutathione reductase (GR) cDNA has been cloned and sequenced from pea (Pisum sativum L. cv. Birte). This new GR cDNA (GOR2) does not encode a preprotein with a transit peptide and therefore is most likely to represent a cytosolic GR. It is significantly different at the DNA level from the previously cloned chloroplastidial/mitochondrial pea GR (GOR1), but retains the features characteristic of GRs from all sources and has GR activity when expressed in Escherichia coli. GOR2 maps to linkage group 6 on the pea genome map and it seems likely that this is the only locus for this gene. In contrast to GOR1, transcript levels of GOR2 increase in the recovery (post-stress) phases of both drought and chilling by about ten- and three-fold respectively. GOR2 therefore may play a role in the restoration of the post-stress redox state of the cytosolic glutathione pool.

Cytosolic ascorbate peroxidase from Arabidopsis thaliana L. is encoded by a small multigene family

A second cytosolic ascorbate peroxidase (cAPX; EC 1.11.1.11) gene from Arabidopsis thaliana has been characterised. This second gene (designated APX1b) maps to linkage group 3 and potentially encodes a cAPX as closely related to that from other dicotyledonous species as to the other member of this gene family (Kubo et al, 1993, FEBS Lett 315: 313–317; here designated APX1a), which maps to linkage group 1. In contrast, the lack of sequence similarity in non-coding regions of the genes implies that they are differentially regulated. Under non-stressed conditions only APX1a is expressed. APX1b was identified during low-stringency probing using a cDNA coding for pea cAPX which, in turn, was recovered from a cDNA library by immunoscreening with an antiserum raised against tea plastidial APX (pAPX). No pAPX cDNAs were recovered, despite the antiserum displaying specificity for pAPX in Western blots.

Isolated plant mitochondria import chloroplast precursor proteins in vitro with the same efficiency as chloroplasts.

Most chloroplast and mitochondrial proteins are synthesized with N-terminal presequences that direct their import into the appropriate organelle. In this report we have analyzed the specificity of standard in vitro assays for import into isolated pea chloroplasts and mitochondria. We find that chloroplast protein import is highly specific because mitochondrial proteins are not imported to any detectable levels. Surprisingly, however, pea mitochondria import a range of chloroplast protein precursors with the same efficiency as chloroplasts, including those of plastocyanin, the 33-kDa photosystem II protein, Hcf136, and coproporphyrinogen III oxidase. These import reactions are dependent on the Δϕ across the inner mitochondrial membrane, and furthermore, marker enzyme assays and Western blotting studies exclude any import by contaminating chloroplasts in the preparation. The pea mitochondria specifically recognize information in the chloroplast-targeting presequences, because they also import a fusion comprising the presequence of coproporphyrinogen III oxidase linked to green fluorescent protein. However, the same construct is targeted exclusively into chloroplastsin vivo indicating that the in vitromitochondrial import reactions are unphysiological, possibly because essential specificity factors are absent in these assays. Finally, we show that disruption of potential amphipathic helices in one presequence does not block import into pea mitochondria, indicating that other features are recognized.

Role of hydrogen peroxide and the redox state of ascorbate in the induction of antioxidant enzymes in pea leaves under excess light stress

In this work we used two different pea cultivars, JI281 is a semidomesticated land race of pea from Ethiopia whereas JI399 is a typical domesticated pea variety. Exposure of pea leaves to excess light (EL) for 1 h caused a reversible photoinhibition of photosynthesis as showed by changes in Fv / Fm. Although little difference existed between the two pea genotypes with respect to photoinhibition, after 60 min of EL the decline in Fv / Fm was higher in JI281 than in JI399 leaves. As a consequence of EL, H2O2 increased in both pea cultivars, whereas lipid peroxidation and protein oxidation slightly increased, although differences between cultivars were minimal. The redox state of ascorbate shifted towards its oxidized form under EL stress in both cultivars. Transcript levels of genes coding antioxidant enzymes varied with EL in both cultivars, but the response was more pronounced in JI399. The induction observed during EL was maintained or increased after the stress period, as occurred for cytGR and chlMDHAR. GR protein accumulation and activity correlated with the transcript accumulation in JI399, but not in JI288. In this work, a possible role for H2O2 and redox status of ascorbate in the photoxidative stress signalling is discussed.