Ágnes Gallé

Plant glutathione peroxidases: Emerging role of the antioxidant enzymes in plant development and stress responses

The plant glutathione peroxidase (GPX) family consists of multiple isoenzymes with distinct subcellular locations which exhibit different tissue-specific expression patterns and environmental stress responses. Contrary to most of their counterparts in animal cells, plant GPXs contain cysteine instead of selenocysteine in their active site and while some of them have both glutathione peroxidase and thioredoxin peroxidase functions, the thioredoxin regenerating system is much more efficient in vitro than the glutathione system. At present, the function of these enzymes in plants is not completely understood. The occurrence of thiol-dependent activities of plant GPX isoenzymes suggests that - besides detoxification of H2O2 and organic hydroperoxides - they may be involved in regulation of the cellular redox homeostasis by maintaining the thiol/disulfide or NADPH/NADP(+) balance. GPXs may represent a link existing between the glutathione- and the thioredoxin-based system. The various thiol buffers, including Trx, can affect a number of redox reactions in the cells most probably via modulation of thiol status. It is still required to identify the in vivo reductant for particular GPX isoenzymes and partners that GPXs interact with specifically. Recent evidence suggests that plant GPXs does not only protect cells from stress induced oxidative damage but they can be implicated in plant growth and development. Following a more general introduction, this study summarizes present knowledge on plant GPXs, highlighting the results on gene expression analysis, regulation and signaling of Arabidopsis thaliana GPXs and also suggests some perspectives for future research.

Glutathione transferase activity and expression patterns during grain filling in flag leaves of wheat genotypes differing in drought tolerance: Response to water deficit.

Total glutathione S-transferase (GST, EC 2.5.1.18) and glutathione peroxidase (GPOX) activity were measured spectrophotometrically in Triticum aestivum cv. MV Emese and cv. Plainsman (drought tolerant) and cv. GK Elet and Cappelle Desprez (drought-sensitive) flag leaves under control and drought stress conditions during the grain-filling period, in order to reveal possible roles of different GST classes in the senescence of flag leaves. Six wheat GSTs, members of 3 GST classes, were selected and their regulation by drought and senescence was investigated. High GPOX activity (EC 1.11.1.9) was observed in well-watered controls of the drought-tolerant Plainsman cultivar. At the same time, TaGSTU1B and TaGSTF6 sequences, investigated by real-time PCR, showed high-expression levels that increased with time, indicating that the gene products of these genes may play important roles in monocarpic senescence of wheat. Expression of these genes was also induced by drought stress in all of the four investigated cultivars, but extremely high transcript amounts were detected in cv. Plainsman. Our data indicate genotypic variations of wheat GSTs. Expression levels and early induction of two senescence-associated GSTs under drought during grain filling in flag leaves correlated with high yield stability.

Salicylic acid increased aldose reductase activity and sorbitol accumulation in tomato plants under salt stress

Increased aldose reductase (ALR) activities were detected in the leaf tissues of tomato plants grown for 3 weeks in culture medium containing 10−7 or 10−4 M salicylic acid (SA), and in the roots after the 10−4 M SA pretreatment. The ALR activity changed in parallel with the sorbitol content in the leaves of the SA-treated plants. Salt stress elicited by 100 mM NaCl enhanced the accumulation of sorbitol in the leaves of control plants and as compared with the untreated control the sorbitol content in the SA-pretreated leaves remained elevated under salt stress. DEAE cellulose anionexchange column purification of the protein precipitated with 80 % (NH4)2SO4 revealed two enzyme fractions with ALR activity in both the leaf and the root tissues. The fraction of the leaf extract that was not bound to the column reacted with glucose and glucose-6-P as substrates, whereas glucose was not a substrate for the bound fraction or for root isoenzymes. The root enzyme was less sensitive to salt treatment: 50 mM NaCl caused 30 % inhibition in the leaf extract, whereas the enzyme activity of the root extract was not affected. It is suggested that increased ALR activity and sorbitol synthesis in the leaves of SA-treated tomato plants may result in an improved salt stress tolerance.

Isohydric and anisohydric strategies of wheat genotypes under osmotic stress: Biosynthesis and function of ABA in stress responses

Changes in water potential (ψw), stomatal conductance, abscisic acid (ABA) accumulation, expression of the major genes involved in ABA biosynthesis, activities of abscisic aldehyde oxidase (AO, EC 1.2.3.1) and antioxidant enzymes were studied in two wheat cultivars with contrasting acclimation strategies subjected to medium strength osmotic stress (-0.976MPa) induced by polyethylene glycol (PEG 6000). Because the biosynthetic pathway of ABA involves multiple gene products, the aim of this study was to unravel how these genes are regulated in isohydric and anisohydric wheat genotypes. In the root tissues of the isohydric cultivar, Triticum aestivum cv. Kobomugi, osmotic stress increased the transcript levels of 9-cis-epoxycarotenoid dioxygenase (NCED) gene, controlling the rate limiting step of ABA biosynthesis. Moreover, this cultivar exhibited a higher basal activity and a higher induction of aldehyde oxidase isoenzymes (AAO2-AAO3), responsible for converting ABAldehyde to ABA. It was found that the fast activation of the ABA biosynthesis in the roots generated an enhanced ABA pool in the shoot, which brought about a faster closure of the stomata upon increasing osmotic stress and, as a result, the plants could maintain ψw in the tissues close to the control level. In contrast, the anisohydric genotype, cv. GK Öthalom, exhibited a moderate induction of ABA biosynthesis in the roots, leading to the maintenance but no increase in the concentration of ABA on the basis of tissue water content in the leaves. Due to the slower response of their stomata to water deficit, the tissues of cv. GK Öthalom have to acclimate to much more negative water potentials during increasing osmotic stress. A decreased activity of superoxide dismutase (SOD) was found in the leaves and roots of both cultivars exposed to osmotic stress, but in the roots elevated activities of catalase (CAT), peroxidase (POX), glutathione reductase (GR) and glutathione transferase (GST) were detected in the isohydric cultivar, suggesting that this genotype was more successful in the elimination of reactive oxygen species caused by the stress conditions.

Physiological and molecular responses to heavy metal stresses suggest different detoxification mechanism of Populus deltoides and P. x canadensis

Plants have divergent defense mechanisms against the harmful effects of heavy metals present in excess in soils and groundwaters. Poplars (Populus spp.) are widely cultivated because of their rapid growth and high biomass production, and members of the genus are increasingly used as experimental model organisms of trees and for phytoremediation purposes. Our aim was to investigate the copper and zinc stress responses of three outstanding biomass producer bred poplar lines to identify such transcripts of genes involved in the detoxification mechanisms, which can play an important role in the protection against heavy metals. Poplar cuttings were grown hydroponically and subjected to short-term (one week) mild and sublethal copper and zinc stresses. We evaluated the effects of the applied heavy metals and the responses of plants by detecting the changes of multiple physiological and biochemical parameters. The most severe cellular oxidative damage was caused by 30μM copper treatment, while zinc was less harmful. Analysis of stress-related transcripts revealed genotype-specific differences that are likely related to alterations in heavy metal tolerance. P. deltoides clones B-229 and PE 19/66 clones were clearly more effective at inducing the expression of various genes implicated in the detoxification process, such as the glutathione transferases, metallothioneins, ABC transporters, (namely PtGSTU51, PxMT1, PdABCC2,3), while the P. canadensis line M-1 accumulated more metal, resulting in greater cellular oxidative damage. Our results show that all three poplar clones are efficient in stress acclimatization, but with different molecular bases.

Glutathione-Related Enzyme System: Glutathione Reductase (GR), Glutathione Transferases (GSTs) and Glutathione Peroxidases (GPXs)

The glutathione-related enzymes are usually considered to accompany the main non-enzymatic antioxidative compounds of the ascorbate–glutathione cycle. Taking into account that the redox processes are not spontaneous in cells, but the adequate reaction velocity and appropriate specificity are achieved by the catalyzing activity of enzymes, special attention has raised toward the glutathione-utilizing enzymes. Glutathione reductase (GR) is a NADPH-dependent oxidoreductase which catalyzes the conversion of oxidized glutathione (GSSG) to reduced glutathione (GSH). Some members of the diverse glutathione transferase (GST) enzyme family have GSH-dependent thiol transferase activity and participate in the recycling of antioxidants (ascorbate, flavonoids, quinones), while other isoenzymes, due to their S-transferase activity, are involved in the detoxification mechanisms using GSH as co-substrate. A significant portion of GST isoenzymes also has glutathione peroxidase activity and can convert lipid peroxides and other peroxides to less harmful compounds. The plant glutathione peroxidase enzymes (GPXs) may be involved in the detoxification of H2O2 and organic hydroperoxides and in the regulation of the cellular redox homeostasis by maintaining the thiol/disulfide balance. Most of plant GPXs prefer to use thioredoxin (TRX) instead of glutathione as a reducing agent, and it is thought that the GPXs may represent a link between the glutathione- and the thioredoxin-based system. The GR, GPX and some GST isoenzymes have Cys in their active center and thus are directly regulated by redox status. This chapter summarizes their roles in stress responses as antioxidant enzymes, in determining the redox status of cells, and emphasizes their connection to redox signaling mechanisms.