Ascorbate-glutathione Pathway Research Articles

ROS regulation in Dunaliella salina by fulvic acid: induction of enzymes related to the ascorbate–glutathione pathway and antioxidant metabolites

ROS regulation in Dunaliella salina by fulvic acid: induction of enzymes related to the ascorbate–glutathione pathway and antioxidant metabolites

Mechanistic insights of salicylic acid-mediated salt stress tolerance in Zea mays L. seedlings

Elevated sodium level (Na+) poses significant threat to crop plant physio-biochemical processes, leading to impaired growth followed by decline in productivity. Addressing this challenge, requires an eco-friendly and cost-effective strategy that enhances plant salt stress tolerance capacity. In this context, the exogenous source of plant growth regulators (PGRs) proved to be an efficient approach. Of various PGRs, salicylic acid (SA) is an emerging signaling molecule that boosts plant stress endurance mechanism. This study investigates SA-mediated salt stress tolerance in maize (Zea mays L.) seedlings, by examining morpho-physiological and biochemical traits. Maize seedlings were subjected to varying levels of salt stress (0, 25, 50, 75, 100, and 150 mM NaCl) for a period of 10-days. The results revealed that, a substantial decline in germination percentage, shoot and root length, plant biomass, vigour index, and various other physiological parameters under salt stress causing concentrations. Conversely, salt stress increased oxidative stress indicators, including hydrogen peroxide (H2O2) and malondialdehyde (MDA), osmolytes and elemental concentrations as well as antioxident enzymes (SOD, CAT, POX, APX, GR, AsA). However, the exogenous supplementation of SA at 0.1 mM significantly restored most morpho-physiological attributes in maize under salt stress conditions. This suggests that SA actively triggers the ascorbate-glutathione (AsA-GSH) pathway and other key enzymes, leading to sodium extrusion and improving antioxidant defense in maize seedlings. This finding provides valuable insights for maize farmers that employing SA could lead to improved maize production in saline soils.

Expression of the alfalfa gene MsMDHAR in Arabidopsis thaliana increases water stress tolerance.

The ascorbate-glutathione pathway plays an essential role in the physiology of vascular plants, particularly in their response to environmental stresses. This pathway is responsible for regulating the cellular redox state, which is critical for maintaining cell function and survival under adverse conditions. To study the involvement of the alfalfa monodehydroascorbate reductase (MsMDHAR) in water stress processes, Arabidopsis thaliana plants constitutively expressing the sequence encoding MsMDHAR were developed. Transgenic events with low and high MsMDHAR expression and ascorbate levels were selected for further analysis of drought and waterlogging tolerance. Under water stress, Arabidopsis transgenic plants generated higher biomass, produced more seeds, and had larger roots than wild type ones. This higher tolerance was associated with increased production of waxes and chlorophyll a at the basal level, greater stomatal opening and stability in regulating the relative water content and reduced H2O2 accumulation under stress conditions in transgenic plants. Overall, these results show that MsMDHAR is involved in plant tolerance to abiotic stresses. The data presented here also emphasises the potential of the MsMDHAR enzyme as a plant breeding tool to improve water stress tolerance.

Physio-biochemical and transcriptomics analyses reveal molecular mechanisms of enhanced UV-B stress tolerance in rice induced by titanium dioxide nanoparticles

ABSTRACT Rice (Oryza sativa L.) stands out as the world's most vital staple food crop, yet is susceptible to UV-B radiation stress. This study investigates the physiological and transcriptome responses in rice exposed to titanium dioxide nanoparticles (TiO2 NPs) under UV-B radiation. Results demonstrate that TiO2 NPs, applied alone (TN) or in combination with UV-B stress (UV+TN), significantly enhance rice plant growth and physiological parameters. Reactive oxygen species (ROS) levels, elevated under UV-B stress, are significantly reduced by TN and UV+TN treatments, thereby regulating antioxidants particularly involved in ascorbate-glutathione pathway. Transcriptomics analysis of identified DEGs in UV+TN, utilizing KEGG pathway analysis, reveals significant enrichment in various pathways. These pathways include glutathione metabolism, pyruvate metabolism, starch and sucrose metabolism, regulation of basal transcription factors, plant hormonal signal transduction pathways, cellular processes associated with energy, and the MAPK signaling pathway. Overall, TiO2 NPs application modulates diverse biological and metabolic pathways, enhancing UV-B stress tolerance in rice.

Fusarium Head Blight Infection Induced Responses of Six Winter Wheat Varieties in Ascorbate-Glutathione Pathway, Photosynthetic Efficiency and Stress Hormones.

Fusarium head blight (FHB) is one of the most studied fungal diseases of wheat, causing massive grain yield and quality losses. This study aimed to extend previous studies on the physiological and biochemical responses of winter wheat to FHB stress in a controlled environment by focusing on the ascorbate-glutathione pathway (AsA-GSH), photosynthetic efficiency, and stress hormone levels, thus providing insight into the possible interactions of different defense mechanisms during infection. The activity of AsA-GSH metabolism was increased in FHB resistant varieties, maintaining the redox state of spikes, and consequently preserving functional photosystem II. Furthermore, carotenoids (Car) were shown to be the major pigments in the photosystem assembly, as they decreased in FHB-stressed spikes of resistant and moderately resistant varieties, compared to controls. Car are also the substrate for the synthesis of abscisic acid (ABA), which acts as a fungal effector and its elevated content leads to increased FHB susceptibility in inoculated spikes. The results of this study contributed to the knowledge of FHB resistance mechanisms and can be used to improve the breeding of FHB resistant varieties, which is considered to be the most effective control measure.

Modulation in gene expression and enzyme activity suggested the roles of monodehydroascorbate reductase in development and stress response in bread wheat

Monodehydroascorbate reductase (MDHAR) is a crucial enzymatic antioxidant of the ascorbate-glutathione pathway involved in reactive oxygen species scavenging. Herein, we identified 15 TaMDHAR genes in bread wheat. Phylogenetic analysis revealed their clustering into three groups, which are also related to the subcellular localization in the peroxisome matrix, peroxisome membrane, and chloroplast. Each TaMDHAR protein consisted of two conserved domains; Pyr_redox and Pyr_redox_2 of the pyridine nucleotide disulfide oxidoreductase family. The occurrence of diverse groups of cis-regulatory elements in the promoter region and their interaction with numerous transcription factors suggest assorted functions of TaMDHARs in growth and development and in light, phytohormones, and stress responses. Expression analysis in various tissues further revealed their importance in vegetative and reproductive development. In addition, the differential gene expression and enhanced enzyme activity during drought, heat, and salt treatments exposed their role in abiotic stress response. Interaction of MDHARs with various antioxidant enzymes and biochemicals related to the ascorbate-glutathione cycle exposed their synchronized functioning. Interaction with auxin indicated the probability of cross-talk between antioxidants and auxin signaling. The miR168a, miR169, miR172 and others interaction with various TaMDHARs further directed their association with developmental processes and stress responses. The current study provides extensive information about the importance of TaMDHARs, moreover, the precise role of each gene needs to be established in future studies.

Galaxolide-contaminated soil and tolerance strategies in soybean plants using biofertilization and selenium nanoparticle supplementation.

The current study aimed to address the response of soybean (Glycine max) plants to biofertilization and selenium supplementation treatments under galaxolide contamination of soil. In this regard, a pot experiment was carried out where the soybean plants were treated with the plant growth-promoting Actinobacteria (Actinobacterium sp.) as a biofertilizer (PGPB treatment) and/or selenium nanoparticles (Se treatment; 25 mg L-1) under two non-polluted and galaxolide-polluted soils (250 mg galaxolide per kg of soil) to assess the modifications in some plant physiological and biochemical traits. Although higher accumulation of oxidative biomarkers, including hydrogen peroxide (+180%), malondialdehyde (+163%), and protein oxidation (+125%), indicating oxidative stress in galaxolide-contaminated plants, an apparent decline in their contents was observed in response to biofertilization/supplementation treatments in contaminated soil, especially. It was mainly related to the higher detoxification of ROS in PGPB- and Se-treated plants under galaxolide-induced oxidative stress, in which the direct ROS-scavenging enzymes (+44 -179%), enzymatic (+34 - 293%) and non-enzymatic (+35 - 98%) components of the ascorbate-glutathione pathway, and antioxidant molecules (+38 - 370%) were more activated than in control plants. In addition, a higher accumulation of detoxification activity markers, including phytochelatins (+32%) and metallothioneins (+79%), were found in the combined treatments (PGPB+Se) under galaxolide contamination. Moreover, combined treatment with PGPB and Se ameliorated the levels of chlorophyll a content (+58%), stomatal conductance (+57%), the maximum efficiency of photosystem II (PSII) (+36%), and photorespiratory metabolism (including +99% in glycolate oxidase and +54% in hydroxypyruvate reductase activity) in leaves under galaxolide contamination, which resulted in higher photosynthesis capacity (+36%) and biomass production (+74%) in galaxolide-stressed plants as compared to control group. In conclusion, the application of beneficial Actinobacteria and selenium nanoparticles as biofertilization/supplementation is expected to be useful for improving plant toleration and adaptation against galaxolide contamination.

Melatonin and strigolactone mitigate chromium toxicity through modulation of ascorbate-glutathione pathway and gene expression in tomato

Chromium (Cr) is considered one of the most hazardous metal contaminant reducing crop production and putting human health at risk. Phytohormones are known to regulate chromium stress, however, the function of melatonin and strigolactones in Chromium stress tolerance in tomato is rarely investigated. Here we investigated the potential role of melatonin (ML) and strigolactone (SL) on mitigating Chromium toxicity in tomato. With exposure to 300 μM Cr stress a remarkable decline in growth (63.01%), biomass yield (50.25)%, Pigment content (24.32%), photosynthesis, gas exchange and Physico-biochemical attributes of tomato was observed. Cr treatment also resulted in oxidative stress closely associated with higher H2O2 generation (215.66%), Lipid peroxidation (50.29%), electrolyte leakage (440.01%) and accumulation of osmolytes like proline and glycine betine. Moreover, Cr toxicity up-regulated the transcriptional expression profiles of antioxidant, stress related and metal transporter genes and down-regulated the genes related to photosynthesis. The application of ML and SL alleviated the Cr induced phytotoxic effects on photosynthetic pigments, gas exchange parameters and restored growth of tomato plants. ML and SL supplementation induced plant defense system via enhanced regulation of antioxidant enzymes, ascorbate and glutathione pool and transcriptional regulation of several genes. The coordinated regulation of antioxidant and glyoxalase systems expressively suppressed the oxidative stress. Hence, ML and SL application might be considered as an effective approach for minimizing Cr uptake and its detrimental effects in tomato plants grown in contaminated soils. The study may also provide new insights into the role of transcriptional regulation in the protection against heavy metal toxicity.

Analysis of physiological and proteomic changes in marigold (Calendula officinalis) in response to short term cold stress

C. officinalis, pot marigold, is an important plant growing in China, Europe and USA which can withstand harsh winters in Kashmir Himalayan region in India. We investigated the cold stress tolerance mechanism in Calendula officinalis by assessing various physiological dimensions and two dimensional (2D) proteomic analysis on exposure to 4 °C at different time intervals. Seeds of C. officinalis were grown at 25 °C for two weeks, transferred to growth chamber set at 4 °C and tissues were analyzed every after 24 h for one week for physiological and proteomic alterations. Differentially expressed proteins were subjected to MALDI-TOF-MS analysis. The plant shows an increased dry weight with an increased relative water content maintaining the cell turgidity and photosynthetic activity during cold stress. The enhancement of ascorbate-glutathione pathway and accumulation of various non-enzymatic antioxidants (AsA, GSH and GSSG) protects the plant against the oxidative damage during cold stress. Total of 24 proteins spots exhibit significant changes at least after 24 h cold stress. The up-regulated proteins in response to cold stress belonged to photosynthesis, respiration, stress resistance, fatty acid metabolism, antioxidant defense, plant development and signal transduction. Three proteins corresponding to spot 90, 136 and 356 were found to be unique which may play a crucial role in cold tolerance mechanism in C. officinalis. Besides, some cold responsive proteins identified in C. officinalis are also involved in salt, heavy metal and abiotic stress response, which strongly suggest their cross-tolerance to biotic and abiotic stress. The transcript expression of some selected cold responsive proteins demonstrates a correlation with the proteomic data, which suggests that the proteomic data is reliable and can be used for further studies. Furthermore, this is the first report of proteomic study on C. officinalis in response to cold stress that opens novel insights in understanding the mechanism of cold tolerance in ornamental plants, which are important source of gene pool for plant breeding initiatives leading to the development of cold resilient commercial crops.

Biochemical and structural characterization of a robust and thermostable ascorbate recycling monodehydroascorbate reductase (MDHAR) from stress adapted pearl millet

Ascorbate (AsA) is a crucial antioxidant in plants, and its recycling is necessary for protecting cells from oxidative damage and imparting stress tolerance. The monodehydroascorbate reductase (MDHAR) enzyme of the ascorbate-glutathione pathway plays a vital role in recycling AsA from monodehydroascorbate (MDHA) radical. Pennisetum glaucum (Pg), also known as pearl millet, is known to be more tolerant to abiotic stress than other food crops, such as rice. However, the contribution of MDHAR from this sessile plant to its unique stress tolerance mechanism is not well understood. In this study, we isolated a gene encoding the MDHAR enzyme from heat stress-adapted pearl millet and characterized it using enzyme kinetics, thermal stability assays, and crystal structure determination. Our results indicate that PgMDHAR is a more robust enzyme than its rice counterpart (Oryza sativa; Os). We solved the crystal structure of PgMDHAR at 1.8 Å and found that the enzyme has a more compact structure and greater stability than OsMDHAR. Using hybrid quantum mechanics and molecular mechanics calculations, we demonstrate that the structure of PgMDHAR contributes to increased stability towards bound FAD. Overall, the higher structural stability and affinity for NADH demonstrated by PgMDHAR are expected to impart improved stress tolerance. Our findings suggest that transgenic food crops expressing MDHAR from stress-adapted pearl millet may exhibit better tolerance to oxidative stress in the unpredictable climatic conditions prevalent today.

Plant dehydroascorbate reductase moonlights as membrane integrated ion channel

Plant dehydroascorbate reductases (DHARs) are only known as soluble antioxidant enzymes of the ascorbate-glutathione pathway. They recycle ascorbate from dehydroascorbate, thereby protecting plants from oxidative stress and the resulting cellular damage. DHARs share structural GST fold with human chloride intracellular channels (HsCLICs) which are dimorphic proteins that exists in soluble enzymatic and membrane integrated ion channel forms. While the soluble form of DHAR has been extensively studied, the existence of a membrane integrated form remains unknown. We demonstrate for the first time using biochemistry, immunofluorescence confocal microscopy, and bilayer electrophysiology that Pennisetum glaucum DHAR (PgDHAR) is dimorphic and is localized to the plant plasma membrane. In addition, membrane translocation increases under induced oxidative stress. Similarly, HsCLIC1 translocates more into peripheral blood mononuclear cells (PBMCs) plasma membrane under induced oxidative stress conditions. Moreover, purified soluble PgDHAR spontaneously inserts and conducts ions in reconstituted lipid bilayers, and the addition of detergent facilitates insertion. In addition to the well-known soluble enzymatic form, our data provides conclusive evidence that plant DHAR also exists in a novel membrane-integrated form. Thus, the structure of DHAR ion channel form will help gain deeper insights into its function across various life forms.

The impact of cadmium stress on the ascorbate-glutathione pathway and ascorbate regeneration in tea plants

Ascorbic acid (AsA) and glutathione (GSH) contribute to defense responses under abiotic stresses. The present study explored the ascorbate-glutathione cycle and ascorbate regeneration under high concentration (30 mM) of cadmium in the tea plant (Camellia sinensis L.). The tea leaves showed speckles and necrosis from the third day of Cd treatment. The content of superoxide anion (O2.-) and hydrogen peroxide (H2O2) in the leaves were significantly higher until the seventh day after Cd treatment. The content of O2.- and H2O2 were the highest on the fifth day (212.7 and 153.6 % of the control, respectively). The AsA content increased (86.9 %) on the first day after Cd treatment and decreased significantly in the subsequent days, while GSH showed a reverse trend. The enzymatic activity assays showed that dehydroascorbate reductase (DHAR) and glutathione reductase (GR) involved in AsA regeneration were downregulated considerably after Cd foliar application. In contrast, the activities of ascorbate peroxidase (APX) and monodehydroascorbate reductase (MDHAR) increased on the first day and then declined. Reverse-transcription quantitative PCR showed upregulation of glutathione synthetase (CsGSHS), γ-glutamylcysteine synthetase (Csγ-ECS), and CsMDHAR of the AsA regeneration pathway and downregulation of CsDHAR and CsGR. The expressions of GDP-L-galactose phosphorylase (CsGGP), L-galactose-1-phosphate phosphatase (CsGPP), and L-galactono- 1,4-lactone dehydrogenase (CsGaILDH) of the L-galactose pathway were also downregulated. The results indicated that AsA, which can respond to Cd stress of plants by increasing antioxidant ability, was consumed to scavenge ROS; moreover, Cd stress inhibited AsA synthesis and regeneration, which made that tea plants suffering severe damage.

Biochar-Mediated Control of Metabolites and Other Physiological Responses in Water-Stressed Leptocohloa fusca.

We investigated biochar-induced drought tolerance in Leptocohloa fusca (Kallar grass) by exploring the plant defense system at physiological level. L. fusca plants were exposed to drought stress (100%, 70%, and 30% field capacity), and biochar (BC), as an organic soil amendment was applied in two concentrations (15 and 30 mg kg-1 soil) to induce drought tolerance. Our results demonstrated that drought restricted the growth of L. fusca by inhibiting shoot and root (fresh and dry) weight, total chlorophyll content and photosynthetic rate. Under drought stress, the uptake of essential nutrients was also limited due to lower water supply, which ultimately affected metabolites including amino and organic acids, and soluble sugars. In addition, drought stress induced oxidative stress, which is evidenced by the higher production of reactive oxygen species (ROS) including hydrogen peroxide (H2O2), superoxide ion (O2-), hydroxyl ion (OH-), and malondialdehyde (MDA). The current study revealed that stress-induced oxidative injury is not a linear path, since the excessive production of lipid peroxidation led to the accumulation of methylglyoxal (MG), a member of reactive carbonyl species (RCS), which ultimately caused cell injury. As a consequence of oxidative-stress induction, the ascorbate-glutathione (AsA-GSH) pathway, followed by a series of reactions, was activated by the plants to reduce ROS-induced oxidative damage. Furthermore, biochar considerably improved plant growth and development by mediating metabolites and soil physio-chemical status.

Effect of L-cysteine treatment to induce postharvest disease resistance of Monilinia fructicola in plum fruits and the possible mechanisms involved

Plum is an important stone fruit in China, but the fruit is easily perishable and susceptible to infection by pathogens. Traditionally, synthetic fungicides are used to control diseases. However, the side effects of fungicides should not be ignored. Cysteine, generally recognized as safe (GRAS) amino acid, has been reported to play roles in the plant abiotic stress response, but little is known about the role of cysteine to control postharvest diseases in fruits. Therefore, this study was designed to investigate the effect of L-cysteine treatment on control of postharvest brown rot in artificially inoculated plum fruits and the possible biocontrol mechanisms involved. Postharvest plum fruits were inoculated with 1, 10, 100 and 1000 mg L−1 L-cysteine. 100 mg L−1 L-cysteine treatment effectively controlled brown rot in artificially inoculated plum fruits by inducing resistance. Furthermore, 100 mg L−1 L-cysteine treatment increased the activities of glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH), enhanced the content of NADPH of the pentose phosphate pathway, as well as improved the contents of H2O2 and some amino acids in the artificially inoculated plum fruits. 100 mg L−1 L-cysteine treatment also elevated the antioxidant content (AsA, GSH) and the antioxidant enzymes activities (APX, GR, MDAR, DHAR) of the ascorbate-glutathione (AsA-GSH) pathway. The protective effects of L-cysteine treatment on postharvest plum fruits likely be due to activating some defense-related responses of the fruit against infection. L-cysteine treatment is a safe promising method for controlling postharvest brown rot in plum fruits.

Alleviation of salinity induced growth and photosynthetic decline in wheat due to biochar and jasmonic acid application involves up-regulation of ascorbate-glutathione pathway, glyoxylase system and secondary metabolite accumulation

Combined application of biochar and jasmonic acid for inducing salt stress tolerance in wheat. A 100 mM salt stress was applied fifteen days after germination. biochar (BC, 5%) and jasmonic acid (JA, 5 μM) was applied in individual and in joint applications. The results revealed that growth traits, contents of δ-Amino levulinic acid, glutamate 1-semialdehyde, total chlorophylls and carotenoids, nutrients, nitrate reductase and leaf gas exchange traits were reduced under salt stress. Salt stress increased oxidative stress biomarkers and enzymes of glyoxylase system considerably. It was found that the single and joint application of biochar or jasmonic acid alleviated the decline with maximal alleviation observed in biochar + jasmonic acid treated plants by preventing the oxidative damage and boosting the activities of antioxidant enzymes and enzymes of glyoxylase system. The accumulation of osmolytes and secondary metabolites was more evident under joint biochar and jasmonic acid treatments than individual under NaCl stress. The contents of reducing agents (ascorbate and glutathione) were orchestrated under joint biochar and jasmonic acid application. Nevertheless, the decline in the activity of nitrate reductase and nutrient contents was maximally alleviated under joint application of BC and JA. Therefore, it can be concluded that combined application of biochar and jasmonic acid can be exploited for improved growth and photosynthesis and salinity stress tolerance in wheat and perhaps in other cereal crops.

Co-over expression of Ascorbate Glutathione pathway enzymes improve mercury tolerance in tomato

The genetic modification of plants for the removal of inorganic pollutants from contaminated soil and water bodies is an emerging area for addressing environmental concerns. This approach is based on the ability of plants to take up and accumulate heavy metals, with efficiency being dependent on the underlying mechanisms of heavy metal accumulation and tolerance. A robust antioxidant pathway is determinantal for heavy metal uptake and accumulation and, therefore, in this study, we evaluated the transgenic tomato plants installed with Ascorbate Glutathione (ASA-GSH) pathway genes for uptake, accumulation, and response to mercury (Hg). We observed that ASA-GSH overexpressing lines were resilient to Hg stress as they displayed higher photosynthetic activity and increased photosynthetic gas exchange parameters with a concomitant decrease in ion leakage under Hg stress. Additionally, transgenic lines accumulated high osmolytes and showed enhanced activity of antioxidant enzymes. Moreover, the results of SEM and confocal microscopy confirmed least damage to plant tissue in ASA-GSH overexpressing lines compared to wild-type under Hg-stress which was further supported by Atomic absorption study that revealed a significant decline in Hg accumulation in the leaves of transgenic lines compared to wild-type under stress conditions. In conclusion, pyramiding of ASA-GSH pathway genes in tomato plants is an efficient approach for the development of Hg-resistant tomato plants and the reclamation of Hg-contaminated sites.

Nitric oxide mediated regulation of ascorbate-glutathione pathway alleviates mitotic aberrations and DNA damage in Allium cepa L. under salinity stress

Allium cepa L. is an important medicinal and food plant enormously affected by salinity in terms of its growth and quality. This experiment investigates ameliorative potential of NO donor sodium nitroprusside (SNP) on chromosomal aberrations and physiological parameters in A. cepa L. roots exposed to salinity stress. Roots with different concentrations of NaCl (25, 50, and 100 mM) alone, and in combination with 100 µM SNP were analyzed for mitotic aberrations, DNA damage, proline, malondialdehyde (MDA) content, and ascorbate-glutathione (AsA-GSH) cycle after 120 h of salinity treatments. Results revealed that salinity stress increased chromosomal aberrations, MDA, proline accumulation, and severely hampered the AsA-GSH cycle function. The comet assay revealed a significant (p ≤ 0.05) enhancement in tail length (4.35 ± 0.05 µm) and olive tail moment (3.19 ± 0.04 µm) at 100 mM NaCl exposure. However, SNP supplementation decreased total percent abnormalities, while increased the prophase, metaphase, anaphase, and telophase indexes. Moreover, ascorbate peroxidase and glutathione reductase activities increased with AsA/DHA and GSH/GSSG ratios, respectively. Results suggest that SNP supplementation alleviates salinity stress responses by improving AsA-GSH cycle and proline accumulation. Based on present findings, NO supplementation could be recommended as a promising approach for sustainable crop production under salinity stress.

Physiological, Biochemical and Molecular Response of Different Winter Wheat Varieties under Drought Stress at Germination and Seedling Growth Stage

Due to climate change in recent years, there has been an increasing water deficit during the winter wheat sowing period. This study evaluated six Croatian winter wheat varieties’ physiological, biochemical, and molecular responses under two drought stress levels at the germination/seedling growth stage. Lipid peroxidation was mainly induced under both drought stress treatments, while the antioxidative response was variety-specific. The most significant role in the antioxidative response had glutathione along with the ascorbate-glutathione pathway. Under drought stress, wheat seedlings responded in proline accumulation that was correlated with the P5CS gene expression. Expression of genes encoding dehydrins (DHN5, WZY2) was highly induced under the drought stress in all varieties, while genes encoding transcription factors were differentially regulated. Expression of DREB1 was upregulated under severe drought stress in most varieties, while the expression of WRKY2 was downregulated or revealed control levels. Different mechanisms were shown to contribute to the drought tolerance in different varieties, which was mainly associated with osmotic adjustment and dehydrins expression. Identifying different mechanisms in drought stress response would advance our understanding of the complex strategies contributing to wheat tolerance to drought in the early growth stage and could contribute to variety selection useful for developing new drought-tolerant varieties.

Hydrogen sulfide attenuates intracellular oxidative stress via repressing glycolate oxidase activities in Arabidopsis thaliana

BackgroundHydrogen sulfide (H2S) has been proposed to exert anti-oxidative effect under many environmental stresses; however, how it influences oxidative stress remains largely unclear. ResultsHere, we assessed the effects of H2S on oxidative stress responses such as salicylic acid (SA)-dependent cell death, which triggered by increased H2O2 availability in Arabidopsis thaliana catalase-deficient mutants cat2 displaying around 20% wild-type catalase activity. H2S generation and its producing enzyme L-cysteine desulfhydrase (LCD/DES) were found to transient increase in response to intracellular oxidative stress. Although introducing the mutation of des1, an important LCD, into the cat2 background produced little effect, H2S fumigation not only rescued the cell death phenotype of cat2 plant, but also attenuated SA accumulation and oxidation of the glutathione pool. Unexpectedly, the activities of major components of ascorbate–glutathione pathway were less affected by the presence of H2S treatment, but decreased glycolate oxidase (GOX) in combination with accumulation of glycolate implied H2S treatment impacts the cellular redox homeostasis by repressing the GOX-catalyzed reaction likely via altering the major GOX transcript levels.ConclusionsOur findings reveal a link between H2S and peroxisomal H2O2 production that has implications for the understanding of the multifaceted roles of H2S in the regulation of oxidative stress responses.

Quantitative Measurement of Ascorbate and Glutathione by Spectrophotometry.

Ascorbate and glutathione are key chemical antioxidants present at relatively high concentrations in plant cells. They are also reducing cofactors for enzymes that process hydrogen peroxide in the ascorbate-glutathione pathway. Due to these two related biochemical functions, the compounds form an interface between reactive oxygen species and sensitive cellular components. Therefore, their status can provide reliable and direct information on cell redox state, signaling, and plant health. While several methods exist for quantification of ascorbate and glutathione, simple enzyme-dependent assays allow them to be measured easily and inexpensively in common extracts. This chapter describes a protocol to measure total contents, as well as the major oxidized and reduced forms, of both compounds in plant tissues.