Glutathione Transferases Are Involved in the Genotype-Specific Salt-Stress Response of Tomato Plants.

Glutathione transferases (GSTs) are one of the most versatile multigenic enzyme superfamilies. In our experiments, the involvement of the genotype-specific induction of GST genes and glutathione- or redox-related genes in pathways regulating salt-stress tolerance was examined in tomato cultivars (Solanum lycopersicum Moneymaker, Mobil, and Elán F1). The growth of the Mobil plants was adversely affected during salt stress (100 mM of NaCl), which might be the result of lowered glutathione and ascorbate levels, a more positive glutathione redox potential (EGSH), and reduced glutathione reductase (GR) and GST activities. In contrast, the Moneymaker and Elán F1 cultivars were able to restore their growth and exhibited higher GR and inducible GST activities, as well as elevated, non-enzymatic antioxidant levels, indicating their enhanced salt tolerance. Furthermore, the expression patterns of GR, selected GST, and transcription factor genes differed significantly among the three cultivars, highlighting the distinct regulatory mechanisms of the tomato genotypes during salt stress. The correlations between EGSH and gene expression data revealed several robust, cultivar-specific associations, underscoring the complexity of the stress response mechanism in tomatoes. Our results support the cultivar-specific roles of distinct GST genes during the salt-stress response, which, along with WRKY3, WRKY72, DREB1, and DREB2, are important players in shaping the redox status and the development of a more efficient stress tolerance in tomatoes.

Crosstalk between the redox signalling and the detoxification: GSTs under redox control?

Reactive oxygen species (ROS), antioxidants and their reduction-oxidation (redox) states all contribute to the redox homeostasis, but glutathione is considered to be the master regulator of it. We aimed to understand the relationship between the redox potential and the diverse glutathione transferase (GST) enzyme family by comparing the stress responses of two tomato cultivars (Solanum lycopersicum 'Moneymaker' and 'Ailsa Craig'). Four-week-old plants were treated by two concentrations of mannitol, NaCl and salicylic acid. The lower H2O2 and malondialdehyde contents indicated higher stress tolerance of 'Moneymaker'. The redox status of roots was characterized by measuring the reduced and oxidized form of ascorbate and glutathione spectrophotometrically after 24 h. The redox potential of 'Ailsa Craig' was more oxidized compared to 'Moneymaker' even under control conditions and became more positive due to treatments. High-throughput quantitative real-time PCR revealed that besides overall higher expression levels, SlGSTs were activated more efficiently in 'Moneymaker' due to stresses, resulting in generally higher GST and glutathione peroxidase activities compared to 'Ailsa Craig'. The expression level of SlGSTs correlated differently, however Pearson's correlation analysis showed usually strong positive correlation between SlGST transcription and glutathione redox potential. The possible redox regulation of SlGST expressions was discussed.

Pest and disease management by red light.

Light is essential for plant life. It provides a source of energy through photosynthesis and regulates plant growth and development and other cellular processes, such as by controlling the endogenous circadian clock. Light intensity, quality, duration and timing are all important determinants of plant responses, especially to biotic stress. Red light can positively influence plant defence mechanisms against different pathogens, but the molecular mechanism behind this phenomenon is not fully understood. Therefore, we reviewed the impact of red light on plant biotic stress responses against viruses, bacteria, fungi and nematodes, with a focus on the physiological effects of red light treatment and hormonal crosstalk under biotic stress in plants. We found evidence suggesting that exposing plants to red light increases levels of salicylic acid (SA) and induces SA signalling mediating the production of reactive oxygen species, with substantial differences between species and plant organs. Such changes in SA levels could be vital for plants to survive infections. Therefore, the application of red light provides a multidimensional aspect to developing innovative and environmentally friendly approaches to plant and crop disease management.

Compensation of Mutation in Arabidopsis glutathione transferase (AtGSTU) Genes under Control or Salt Stress Conditions.

Glutathione transferases (GSTs) play a crucial role in detoxification processes due to the fact of their glutathione (GSH) conjugating activity, and through glutathione peroxidase or dehydroascorbate reductase (DHAR) activities, they influence the redox state of GSH and ascorbate (AsA). The plant-specific tau (GSTU) group is the largest class of Arabidopsis GSTs, and their members are involved in responses to different abiotic stresses. We investigated the effect of salt stress on two-week-old Arabidopsis thaliana wild-type (Col-0), Atgstu19 and Atgstu24 mutant plants after applying 150 mM NaCl for two days. The Atgstu19 seedlings had lower GST activity and vitality both under control conditions and after salt stress than the wild-type, but the level of total ROS was similar to the Col-0 plants. The GST activity of the knockout Atgstu24 mutant was even higher under control conditions compared to the Col-0 plants, while the ROS level and its vitality did not differ significantly from the wild-type. Analysis of the AtGSTU expression pattern revealed that the mutation in a single AtGSTU gene was accompanied by the up- and downregulation of several other AtGSTUs. Moreover, elevated AsA and GSH levels, an altered GSH redox potential and increased DHAR and glutathione reductase activities could help to compensate for the mutation of AtGSTU genes. The observed changes in the mutants suggest that the investigated isoenzymes influence the redox homeostasis under control conditions and after NaCl treatment in Arabidopsis seedlings. These data indicate for the first time the more general role of a temporary shift of redox status as part of GST mechanisms and regulation.

Genome-wide identification of the glutathione transferase superfamily in the model organism Brachypodium distachyon.

The detoxification of harmful metabolites can determine the effectiveness of plant stress responses. Scavenging some of these toxic stress by-products through the reduced form of glutathione is catalysed by members of the glutathione transferase (GST) enzyme superfamily. The involvement of these enzymes was studied in the model organism Brachypodium distachyon (L.)P.Beauv. Bd21 and in its derivative Bd21-3, a more drought tolerant line. Osmotic stress treatment resulted in a decrease in the water potential of both Brachypodium genotypes, the difference between the control and treated plant's ψw decreased by the last sampling day in Bd21-3, suggesting some degree of adaptation to the applied osmotic stress. Increased GST activity revealed a severe defence reaction against the harmful imbalance of the redox environment. Screening for the gene sequences led to the identification of 91 full-length or partial GST sequences. Although purple false brome has a relatively small genome, the number of identified GST genes was almost as high as the number predicted in wheat. The estimation of GST expression showed stress-induced differences: higher expression levels or the fast induction of BdGSTF8, BdGSTU35 and BdGSTU42 gene products presumably indicate a strong detoxification under osmotic stress.

The Arabidopsis glutathione transferases, AtGSTF8 and AtGSTU19 are involved in the maintenance of root redox homeostasis affecting meristem size and salt stress sensitivity.

The tau (U) and phi (F) classes of glutathione transferase (GST) enzymes reduce the glutathione (GSH) pool using GSH as a co-substrate, thus influence numerous redox-dependent processes including hormonal and stress responses. We performed detailed analysis of the redox potential and reactive oxygen species levels in longitudinal zones of 7-day-old roots of Arabidopsis thaliana L. Col-0 wild type and Atsgtf8 and Atgstu19 insertional mutants. Using redox-sensitive cytosolic green fluorescent protein (roGFP2) the redox status of the meristematic, transition, and elongation zones was determined under control and salt stress (3-hour of 75 or 150 mM NaCl treatment) conditions. The Atgstu19 mutant had the most oxidized redox status in all root zones throughout the experiments. Using fluorescent dyes significantly higher superoxide radical (O2-) levels was detected in both Atgst mutants than in the Col-0 control. Salt treatment resulted in the highest O2- increase in the Atgstf8 root, while the amount of H2O2 elevated most in the case of Atgstu19. Moreover, vitality decreased in Atgstu19 roots more than in wild type under salt stress. Our results indicate that AtGSTF8 and especially the AtGSTU19 proteins function in the root fine-tuning the redox homeostasis both under control and salt stress conditions.

Diurnal changes in tomato glutathione transferase activity and expression.

Although the participation of glutathione transferases (GSTs) in light-dependent pathways and the circadian changes in the whole detoxification system have been studied, there are fewer results regarding the exact daily fluctuation of GSTs. In the present study, it was demonstrated that light up-regulated, while dark period decreased the plant GST activity and the expression of the selected tau group GST genes in tomato. These findings provide additional information on our current knowledge on the circadian rhythm of GSTs in plants and could help in further defining detoxification processes.

Plant Glutathione Transferases and Light.

The activity and expression of glutathione transferases (GSTs) depend on several less-known endogenous and well-described exogenous factors, such as the developmental stage, presence, and intensity of different stressors, as well as on the absence or presence and quality of light, which to date have received less attention. In this review, we focus on discussing the role of circadian rhythm, light quality, and intensity in the regulation of plant GSTs. Recent studies demonstrate that diurnal regulation can be recognized in GST activity and gene expression in several plant species. In addition, the content of one of their co-substrates, reduced glutathione (GSH), also shows diurnal changes. Darkness, low light or shade mostly reduces GST activity, while high or excess light significantly elevates both the activity and expression of GSTs and GSH levels. Besides the light-regulated induction and dark inactivation of GSTs, these enzymes can also participate in the signal transduction of visible and UV light. For example, red light may alleviate the harmful effects of pathogens and abiotic stressors by increasing GST activity and expression, as well as GSH content in leaves of different plant species. Based on this knowledge, further research on plants (crops and weeds) or organs and temporal regulation of GST activity and gene expression is necessary for understanding the complex regulation of plant GSTs under various light conditions in order to increase the yield and stress tolerance of plants in the changing environment.

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.

The role of Arabidopsis glutathione transferase F9 gene under oxidative stress in seedlings.

Arabidopsis thaliana contains 54 soluble glutathione transferases (GSTs, EC 2.5.1.18), which are thought to play major roles in oxidative stress responses, but little is known about the function of individual isoenzymes. The role of AtGST phi 9 (GSTF9) in the salt- and salicylic acid response was investigated using 2-week-old Atgstf9 and wild type (Wt) plants. Atgstf9 mutants accumulated more ascorbic acid (AsA) and glutathione (GSH) and had decreased glutathione peroxidase (GPOX) activity under control conditions. Treatment of 2-week-old seedlings with 10⁻7 M salicylic acid (SA) for 48 h resulted in elevated H2O2level and enhanced GST activity in Atgstf9 plants, 10⁻5 M SA treatment enhanced the malondialdehyde and dehydroascorbate contents compared to Wt. 50 and 150 mM NaCl increased the GST activity, AsA and GSH accumulation in Atgstf9 seedlings more pronounced than in Wt plants. We found that the Atgstf9 mutants had altered redox homeostasis under control and stress conditions, in which elevated AsA and GSH levels and modified GST and GPOX activities may play significant role. The half-cell potential values calculated from the concentration of GSH and GSSG indicate that this GST isoenzyme has an important role in the salt stress response.

Hardening with salicylic acid induces concentration-dependent changes in abscisic acid biosynthesis of tomato under salt stress.

The role of salicylic acid (SA) in the control of abscisic acid (ABA) biosynthesis is controversial although both plant growth regulators may accumulate in tissues under abiotic and biotic stress conditions. Hardening of tomato plants to salinity stress with 10(-4)M SA ("high SA") resulted in an up-regulation of ABA biosynthesis genes, zeaxanthin epoxidase (SlZEP1), 9-cis-epoxycarotenoid dioxygenase (SlNCED1) and aldehyde oxidases (SlAO1 and SlAO2) in the roots and led to ABA accumulation both in root and leaf tissues. In plants pre-treated with lower concentration of SA (10(-7)M, "low SA"), the up-regulation of SlNCED1 in the roots promoted ABA accumulation in the root tissues but the hormone concentration remained at control level in the leaves. Salt stress induced by 100mM NaCl reduced the transcript abundance of ABA biosynthetic genes and inhibited SlAO activity in plants hardened with "high SA", but the tissues maintained root ABA level over the untreated control. The combined effect of "high SA" and ABA under salt stress led to partially recovered photosynthetic activity, reduced ethylene production in root apices, and restored root growth, which is one of the main features of salt tolerance. Unlike "high SA", hardening with "low SA" had no influence on ethylene production, and led to reduced elongation of roots in plants exposed to 100mM NaCl. The up-regulation of carotenoid cleavage dioxygenases SlCCD1A and SlCCD1B by SA, which produce apocarotenoids, may open new pathways in SA sensing and signalling processes.

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 supergene family in tomato: Salt stress-regulated expression of representative genes from distinct GST classes in plants primed with salicylic acid.

A family tree of the multifunctional proteins, glutathione transferases (GSTs, EC 2.5.1.18) was created in Solanum lycopersicum based on homology to known Arabidopsis GSTs. The involvement of selected SlGSTs was studied in salt stress response of tomato primed with salicylic acid (SA) or in un-primed plants by real-time qPCR. Selected tau GSTs (SlGSTU23, SlGSTU26) were up-regulated in the leaves, while GSTs from lambda, theta, dehydroascorbate reductase and zeta classes (SlGSTL3, SlGSTT2, SlDHAR5, SlGSTZ2) in the root tissues under salt stress. Priming with SA exhibited a concentration dependency; SA mitigated the salt stress injury and caused characteristic changes in the expression pattern of SlGSTs only at 10(-4) M concentration. SlGSTF4 displayed a significant up-regulation in the leaves, while the abundance of SlGSTL3, SlGSTT2 and SlGSTZ2 transcripts were enhanced in the roots of plants primed with high SA concentration. Unexpectedly, under high salinity the SlDHAR2 expression decreased in primed roots as compared to the salt-stressed plants, however, the up-regulation of SlDHAR5 isoenzyme contributed to the maintenance of DHAR activity in roots primed with high SA. The members of lambda, theta and zeta class GSTs have a specific role in salt stress acclimation of tomato, while SlGSTU26 and SlGSTF4, the enzymes with high glutathione conjugating activity, characterize a successful priming in both roots and leaves. In contrast to low concentration, high SA concentration induced those GSTs in primed roots, which were up-regulated under salt stress. Our data indicate that induction of GSTs provide a flexible tool in maintaining redox homeostasis during unfavourable conditions.

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.

Different peroxidase activities and expression of abiotic stress-related peroxidases in apical root segments of wheat genotypes with different drought stress tolerance under osmotic stress.

One-week-old seedlings of Triticum aestivum L. cv. Plainsman V, a drought tolerant; and Cappelle Desprez, a drought sensitive wheat cultivar were subjected gradually to osmotic stress using polyethylene glycol (PEG 6000) reaching 400 mOsm on the 11th day. Compared to controls cv. Plainsman V maintained the root growth and relative water content of root tissues, while these parameters were decreased in the drought sensitive cv. Cappelle Desprez under PEG-mediated osmotic stress. Simultaneously, H(2)O(2) content in 1-cm-long apical segment of roots comprising the proliferation and elongation zone, showed a transient increase in cv. Plainsman V and a permanent raise in cv. Cappelle Desprez. Measurements of the transcript levels of selected class III peroxidase (TaPrx) coding sequences revealed significant differences between the two cultivars on the 9th day, two days after applying 100 mOsm PEG. The abundance of TaPrx04 transcript was enhanced transitionally in the root apex of cv. Plainsman V but decreased in cv. Cappelle Desprez under osmotic stress while the expression of TaPrx01, TaPrx03, TaPrx19, TaPrx68, TaPrx107 and TaPrx109-C decreased to different extents in both cultivars. After a transient decrease, activities of soluble peroxidase fractions of crude protein extracts rose in both cultivars on day 11, but the activities of cell wall-bound fractions increased only in cv. Cappelle Desprez under osmotic stress. Parallel with high H(2)O(2) content of the tissues, certain isoenzymes of covalently bound fraction in cv. Cappelle Desprez showed increased activity suggesting that they may limit the extension of root cell walls in this cultivar.

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.