Malate production, sugar metabolism, and redox homeostasis in the leaf growth zone of Rye (Secale cereale) increase stress tolerance to aluminum stress: A biochemical and genome-wide transcriptional study.

Global soil acidification is increasing, enlarging aluminum (Al) availability in soils, leading to reductions in plant growth. This study investigates the effect of Al stress on the leaf growth zones of Rye (Secale cereale, cv Beira). Kinematic analysis showed that the effect of Al on leaf growth rates was mainly due to a reduced cell production rate in the meristem. Transcriptomic analysis identified 2272 significantly (log2fold > |0.5| FDR < 0.05) differentially expressed genes (DEGs) for Al stress. There was a downregulation in several DEGs associated with photosynthetic processes and an upregulation in genes for heat/light response, and H2O2 production in all leaf zones. DEGs associated with heavy metals and malate transport were increased, particularly, in the meristem. To determine the putative function of these processes in Al tolerance, we performed biochemical analyses comparing the tolerant Beira with an Al sensitive variant RioDeva. Beira showed improved sugar metabolism and redox homeostasis, specifically in the meristem compared to RioDeva. Similarly, a significant increase in malate and citrate production, which are known to aid in Al detoxification in plants, was found in Beira. This suggests that Al tolerance in Rye is linked to its ability for Al exclusion from the leaf meristem.

Subcellular compartmentalization of aluminum reduced its hazardous impact on rye photosynthesis.

Aluminum (Al) toxicity limits crops growth and production in acidic soils. Compared to roots, less is known about the toxic effects of Al in leaves. Al subcellular compartmentalization is also largely unknown. Using rye (Secale cereale L.) Beira (more tolerant) and RioDeva (more sensitive to Al) genotypes, we evaluated the patterns of Al accumulation in leaf cell organelles and the photosynthetic and metabolic changes to cope with Al toxicity. The tolerant genotype accumulated less Al in all organelles, except the vacuoles. This suggests that Al compartmentalization plays a role in Al tolerance of Beira genotype. PSII efficiency, stomatal conductance, pigment biosynthesis, and photosynthesis metabolism were less affected in the tolerant genotype. In the Calvin cycle, carboxylation was compromised by Al exposure in the tolerant genotype. Other Calvin cycle-related enzymes, phoshoglycerate kinase (PGK), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), triose-phosphate isomerase (TPI), and fructose 1,6-bisphosphatase (FBPase) activities decreased in the sensitive line after 48 h of Al exposure. Consequentially, carbohydrate and organic acid metabolism were affected in a genotype-specific manner, where sugar levels increased only in the tolerant genotype. In conclusion, Al transport to the leaf and compartmentalization in the vacuoles tolerant genotype's leaf cells provide complementary mechanisms of Al tolerance, protecting the photosynthetic apparatus and thereby sustaining growth.

Starch biosynthesis contributes to the maintenance of photosynthesis and leaf growth under drought stress in maize.

To understand the growth response to drought, we performed a proteomics study in the leaf growth zone of maize (Zea mays L.) seedlings and functionally characterized the role of starch biosynthesis in the regulation of growth, photosynthesis and antioxidant capacity, using the shrunken-2 mutant (sh2), defective in ADP-glucose pyrophosphorylase. Drought altered the abundance of 284 proteins overrepresented for photosynthesis, amino acid, sugar and starch metabolism, and redox-regulation. Changes in protein levels correlated with enzyme activities (increased ATP synthase, cysteine synthase, starch synthase, RuBisCo, peroxiredoxin, glutaredoxin, thioredoxin and decreased triosephosphate isomerase, ferredoxin, cellulose synthase activities, respectively) and metabolite concentrations (increased ATP, cysteine, glycine, serine, starch, proline and decreased cellulose levels). The sh2 mutant showed a reduced increase of starch levels under drought conditions, leading to soluble sugar starvation at the end of the night and correlating with an inhibition of leaf growth rates. Increased RuBisCo activity and pigment concentrations observed in WT, in response to drought, were lacking in the mutant, which suffered more oxidative damage and recovered more slowly after re-watering. These results demonstrate that starch biosynthesis contributes to maintaining leaf growth under drought stress and facilitates enhanced carbon acquisition upon recovery.

Maize roots and shoots show distinct profiles of oxidative stress and antioxidant defense under heavy metal toxicity.

Heavy metal accumulation in agricultural land causes crop production losses worldwide. Metal homeostasis within cells is tightly regulated. However, homeostasis breakdown leads to accumulation of reactive oxygen species (ROS). Overall plant fitness under stressful environment is determined by coordination between roots and shoots. But little is known about organ specific responses to heavy metals, whether it depends on the metal category (redox or non-redox reactive) and if these responses are associated with heavy metal accumulation in each organ or there are driven by other signals. Maize seedlings were subjected to sub-lethal concentrations of four metals (Zn, Ni, Cd and Cu) individually, and were quantified for growth, ABA level, and redox alterations in roots, mature leaves (L1,2) and young leaves (L3,4) at 14 and 21 days after sowing (DAS). The treatments caused significant increase in endogenous metal levels in all organs but to different degrees, where roots showed the highest levels. Biomass was significantly reduced under heavy metal stress. Although old leaves accumulated less heavy metal content than root, the reduction in their biomass (FW) was more pronounced. Metal exposure triggered ABA accumulation and stomatal closure mainly in older leaves, which consequently reduced photosynthesis. Heavy metals induced oxidative stress in the maize organs, but to different degrees. Tocopherols, polyphenols and flavonoids increased specifically in the shoot under Zn, Ni and Cu, while under Cd treatment they played a minor role. Under Cu and Cd stress, superoxide dismutase (SOD) and dehydroascorbate reductase (DHAR) activities were induced in the roots, however ascorbate peroxidase (APX) activity was only increased in the older leaves. Overall, it can be concluded that root and shoot organs specific responses to heavy metal toxicity are not only associated with heavy metal accumulation and they are specialized at the level of antioxidants to cope with.

Vermicompost Supply Modifies Chemical Composition and Improves Nutritive and Medicinal Properties of Date Palm Fruits From Saudi Arabia.

To meet the increased demand for phytochemicals, plant cultivation in soil amended with biofertilizers has been developed. Here, we aimed to use vermicompost as an environmentally safe biofertilizer to enhance the nutritive and medicinal value of five common cultivars of Saudi date palm; namely Phoenix dactylifera L. var. Ajwa, Hulwa, Ruthana, Sefri, and Luban. To determine changes in the fruit nutritive composition, primary metabolites, antioxidants, phenolic compounds and mineral profiles were analyzed in the fruits from non-fertilized and vermicompost-fertilized date palms. We also tested how changes in the fruit chemical compositions due to vermicompost fertilization affected their medicinal potentials. Applying vermicomposts generally increased primary metabolites, vitamins, and mineral content as well as the medicinal potential of the date palm fruits. This positive effect is possibly explained by the role of vermicomposts in improving soil health and fertility. Furthermore, clustering analyses and principal component analysis (PCA) indicated cultivar-specific responses. PCA analysis also revealed that the bioactivities of the date palm fruit extracts and their antioxidants tended to display correlated output values. One of the highly accumulated phenolic compounds, ß-D-glucogallin, was extracted and purified from P. dactylifera L. var. Ajwa fruits and showed significant antioxidant, anticancer, antibacterial, antimutagenic, and antiprotozoal activities. Overall, applying vermicompost is an innovative approach to increase the nutritive quality and medicinal potential of date palm fruits.

Metalaxyl Effects on Antioxidant Defenses in Leaves and Roots of Solanum nigrum L.

Overuse of pesticides has resulted in environmental problems, threating public health through accumulation in food chains. Phytoremediation is a powerful technique to clean up contaminated environments. However, it is necessary to unravel the metabolic mechanisms underlying phytoremediation in order to increase the efficiency of this process. Therefore, growth, physiological and biochemical responses in leaves and roots of Solanum nigrum L. exposed to the commonly used fungicide metalaxyl were investigated. This species shows characteristics that make it valuable as a potential tool for the remediation of organic pollutants. We found that once inside the plant, metalaxyl altered carbon metabolism, which resulted in a reduction of growth and lower biomass accumulation due to impairment of carbohydrate production (total soluble sugar, starch, rubisco) and increased photorespiration (glycolate oxidase, Gly/Ser ratio). A significant increase of antioxidant defenses (polyphenols, flavonoids, tocopherols, ascorbate, glutathione, superoxide dismutase, catalase, peroxidases, monodehydroascorbate- and dehydroascorbate reductase, gluthatione reductase) kept reactive oxygen species (ROS) levels under control (superoxide anion) leaving cell membranes undamaged. The results suggest that enhancing carbon assimilation and antioxidant capacity may be target parameters to improve this species' phytoremediation capacities. Highlights • Metalaxyl inhibits growth by reducing photosynthesis and inducing photorespiration • Elevated antioxidant defenses protect metalaxyl-treated plants from oxidative damage • Ascorbate and glutathione are key antioxidants in metalaxyl tolerance.

Neither artificial light at night, anthropogenic noise nor distance from roads are associated with oxidative status of nestlings in an urban population of songbirds.

Increasing urbanization is responsible for road-related pollutants and causes an unprecedented increase in light and noise pollution, with potential detrimental effects for individual animals, communities and ecosystems. These stressors rarely act in isolation but studies dissecting the effects of these multiple stressors are lacking. Moreover, studies on urban stressors have mainly focused on adults, while exposure in early-life may be detrimental but is largely ignored. To fill this important knowledge gap, we studied if artificial light at night, anthropogenic noise and road-related pollution (using distance from roads as a proxy) explain variation in oxidative status in great tit nestlings (Parus major) in an urban population. Artificial light at night, anthropogenic noise and distance from roads were not associated with variation of the nine studied metrics of oxidative status (superoxide dismutase-SOD-, glutathione peroxidase-GPX, catalase-CAT-, non-enzymatic total antioxidant capacity-TAC-, reduced glutathione-GSH-, oxidized glutathione-GSSG-, ratio GSH/GSSG, protein carbonyls and thiobarbituric acid reactive substances-TBARS). Interestingly, for all oxidative status metrics, we found that there was more variation in oxidative status among individuals of the same nest compared to between different nests. We also showed an increase in protein carbonyls and a decrease of the ratio GSH/GSSG as the day advanced, and an increase of GPX when weather conditions deteriorated. Our study suggests that anthropogenic noise, artificial light at night and road-related pollution are not the most important sources of variation in oxidative status in great tit nestlings. It also highlights the importance of considering bleeding time and weather conditions in studies with free-living animals.

Zinc-induced differential oxidative stress and antioxidant responses in Chlorella sorokiniana and Scenedesmus acuminatus.

Algae are frequently exposed to toxic metals, and zinc (Zn) is one of the major toxicants present. We exposed two green microalgae, Chlorella sorokiniana and Scenedesmus acuminatus, to sub-lethal concentrations (1.0 and 0.6mM) of Zn for seven days. Algal responses were analysed at the level of growth, oxidative stress, and antioxidants. Growth parameters such as cell culture yield and pigment content were less affected by Zn in C. sorokiniana, despite the fact that this alga accumulated more zinc than S. acuminatus. Also, C. sorokiniana, but not S. acuminatus, was able to acclimatize during long-term exposure to toxic concentrations of the test metals (specific growth rate (µ) was 0.041/day and total chlorophyll was 14.6mg/mL). Although, Zn induced oxidative stress in both species, C. sorokiniana experienced less stress than S. acuminatus. This could be explained by a higher accumulation of antioxidants in C. sorokiniana, where flavonoids, polyphenols, tocopherols, glutathione (GSH) and ascorbate (ASC) content increased. Moreover, antioxidant enzymes glutathione S transferase (GST), glutathione reductase (GR), superoxide dismutase (SOD), peroxidase (POX) and ascorbate peroxidase (APX), showed increased activities in C. sorokiniana. In addition to, and probably also underlying, the higher Zn tolerance in C. sorokiniana, this alga also showed higher Zn biosorption capacity. Use of C. sorokiniana as a bio-remediator, could be considered.

Mixture toxicity of copper, cadmium, and zinc to barley seedlings is not explained by antioxidant and oxidative stress biomarkers.

The analysis of metal mixture toxicity to plants is complicated by mutual interactions. In the present study, mixture effects of zinc (Zn), cadmium (Cd), and copper (Cu) on barley (Hordeum vulgare L.) root elongation were analyzed using oxidative stress parameters. The hypothesis was that toxic mixture effects on plant growth are better explained by biochemical parameters than by exposure information, because the former excludes interactions among metals for root uptake. Barley seedlings were exposed for 5 d or 14 d to these metals in nutrient solutions, added in isolation and as mixtures. Root elongation in Cu+Cd mixtures was well predicted from free metal ion concentrations in solution, using concentration addition (CA) or independent action (IA) reference models. In contrast, Zn acted antagonistically when combined with Cu and/or Cd, relative to both CA and IA. This protective effect of Zn correlated with the biomarkers measured in the long-term experiment; oxidative stress (indicated by malondialdehyde level, for example) decreased after addition of Zn. In addition, it was found that some biomarkers were sensitive to both Cu and Cd dosed in isolation, but not to Cu+Cd mixtures. Overall, the exposure explained mixture effects better than most of the 16 measured biomarkers (i.e., the biochemical effects). It is concluded that these biomarkers are not robust indicators for metal mixture toxicity, potentially because different metals have different parallel modes of action on growth that are insufficiently indexed by the biomarkers. Environ Toxicol Chem 2017;36:220-230. © 2016 SETAC.

Future Climate CO2 Levels Mitigate Stress Impact on Plants: Increased Defense or Decreased Challenge?

Elevated atmospheric CO2 can stimulate plant growth by providing additional C (fertilization effect), and is observed to mitigate abiotic stress impact. Although, the mechanisms underlying the stress mitigating effect are not yet clear, increased antioxidant defenses, have been held primarily responsible (antioxidant hypothesis). A systematic literature analysis, including "all" papers [Web of Science (WoS)-cited], addressing elevated CO2 effects on abiotic stress responses and antioxidants (105 papers), confirms the frequent occurrence of the stress mitigation effect. However, it also demonstrates that, in stress conditions, elevated CO2 is reported to increase antioxidants, only in about 22% of the observations (e.g., for polyphenols, peroxidases, superoxide dismutase, monodehydroascorbate reductase). In most observations, under stress and elevated CO2 the levels of key antioxidants and antioxidant enzymes are reported to remain unchanged (50%, e.g., ascorbate peroxidase, catalase, ascorbate), or even decreased (28%, e.g., glutathione peroxidase). Moreover, increases in antioxidants are not specific for a species group, growth facility, or stress type. It seems therefore unlikely that increased antioxidant defense is the major mechanism underlying CO2-mediated stress impact mitigation. Alternative processes, probably decreasing the oxidative challenge by reducing ROS production (e.g., photorespiration), are therefore likely to play important roles in elevated CO2 (relaxation hypothesis). Such parameters are however rarely investigated in connection with abiotic stress relief. Understanding the effect of elevated CO2 on plant growth and stress responses is imperative to understand the impact of climate changes on plant productivity.

High Salinity Induces Different Oxidative Stress and Antioxidant Responses in Maize Seedlings Organs.

Salinity negatively affects plant growth and causes significant crop yield losses world-wide. Maize is an economically important cereal crop affected by high salinity. In this study, maize seedlings were subjected to 75 mM and 150 mM NaCl, to emulate high soil salinity. Roots, mature leaves (basal leaf-pair 1,2) and young leaves (distal leaf-pair 3,4) were harvested after 3 weeks of sowing. Roots showed the highest reduction in biomass, followed by mature and young leaves in the salt-stressed plants. Concomitant with the pattern of growth reduction, roots accumulated the highest levels of Na(+) followed by mature and young leaves. High salinity induced oxidative stress in the roots and mature leaves, but to a lesser extent in younger leaves. The younger leaves showed increased electrolyte leakage (EL), malondialdehyde (MDA), and hydrogen peroxide (H2O2) concentrations only at 150 mM NaCl. Total antioxidant capacity (TAC) and polyphenol content increased with the increase in salinity levels in roots and mature leaves, but showed no changes in the young leaves. Under salinity stress, reduced ascorbate (ASC) and glutathione (GSH) content increased in roots, while total tocopherol levels increased specifically in the shoot tissues. Similarly, redox changes estimated by the ratio of redox couples (ASC/total ascorbate and GSH/total glutathione) showed significant decreases in the roots. Activities of enzymatic antioxidants, catalase (CAT, EC 1.11.1.6) and dehydroascorbate reductase (DHAR, EC 1.8.5.1), increased in all organs of salt-treated plants, while superoxide dismutase (SOD, EC 1.15.1.1), ascorbate peroxidase (APX, EC 1.11.1.11), glutathione-s-transferase (GST, EC 2.5.1.18) and glutathione reductase (GR, EC 1.6.4.2) increased specifically in the roots. Overall, these results suggest that Na(+) is retained and detoxified mainly in roots, and less stress impact is observed in mature and younger leaves. This study also indicates a possible role of ROS in the systemic signaling from roots to leaves, allowing leaves to activate their defense mechanisms for better protection against salt stress.

Nutritional Status as the Key Modulator of Antioxidant Responses Induced by High Environmental Ammonia and Salinity Stress in European Sea Bass (Dicentrarchus labrax).

Salinity fluctuation is one of the main factors affecting the overall fitness of marine fish. In addition, water borne ammonia may occur simultaneously with salinity stress. Additionally, under such stressful circumstances, fish may encounter food deprivation. The physiological and ion-osmo regulatory adaptive capacities to cope with all these stressors alone or in combination are extensively addressed in fish. To date, studies revealing the modulation of antioxidant potential as compensatory response to multiple stressors are rather lacking. Therefore, the present work evaluated the individual and combined effects of salinity challenge, ammonia toxicity and nutritional status on oxidative stress and antioxidant status in a marine teleost, European sea bass (Dicentrarchus labrax). Fish were acclimated to normal seawater (32 ppt), to brackish water (20 ppt and 10 ppt) and to hypo-saline water (2.5 ppt). Following acclimation to different salinities for two weeks, fish were exposed to high environmental ammonia (HEA, 20 mg/L representing 50% of 96h LC50 value for ammonia) for 12 h, 48 h, 84 h and 180 h, and were either fed (2% body weight) or fasted (unfed for 7 days prior to HEA exposure). Results show that in response to decreasing salinities, oxidative stress indices such as xanthine oxidase activity, levels of hydrogen peroxide (H2O2) and lipid peroxidation (malondialdehyde, MDA) increased in the hepatic tissue of fasted fish but remained unaffected in fed fish. HEA exposure at normal salinity (32 ppt) and at reduced salinities (20 ppt and 10 ppt) increased ammonia accumulation significantly (84 h-180 h) in both feeding regimes which was associated with an increment of H2O2 and MDA contents. Unlike in fasted fish, H2O2 and MDA levels in fed fish were restored to control levels (84 h-180 h); with a concomitant increase in superoxide dismutase (SOD), catalase (CAT), components of the glutathione redox cycle (reduced glutathione, glutathione peroxidase and glutathione reductase), ascorbate peroxidase (APX) activity and reduced ascorbate (ASC) content. On the contrary, fasted fish could not activate many of these protective systems and rely mainly on CAT and ASC dependent pathways as antioxidative sentinels. The present findings exemplify that in fed fish single factors and a combination of HEA exposure and reduced seawater salinities (upto 10 ppt) were insufficient to cause oxidative damage due to the highly competent antioxidant system compared to fasted fish. However, the impact of HEA exposure at a hypo-saline environment (2.5 ppt) also defied antioxidant defence system in fed fish, suggesting this combined factor is beyond the tolerance range for both feeding groups. Overall, our results indicate that the oxidative stress mediated by the experimental conditions were exacerbated during starvation, and also suggest that feed deprivation particularly at reduced seawater salinities can instigate fish more susceptible to ammonia toxicity.

High environmental ammonia elicits differential oxidative stress and antioxidant responses in five different organs of a model estuarine teleost (Dicentrarchus labrax).

We investigated oxidative status and antioxidant profile in five tissues (brain, liver, gills, muscle and kidney) of European sea bass (Dicentrarchus labrax) when exposed to high environmental ammonia (HEA, 20 mg/L~1.18 mM as NH4HCO3) for 12 h, 2 days, 3.5 days, 7.5 days and 10 days. Results show that HEA triggered ammonia accumulation and induced oxidative stress in all tissues. Unlike other organs, hydrogen peroxide (H2O2) and malondialdehyde (MDA) accumulation in liver were restored to control levels. This recovery was associated with a concomitant augmentation in superoxide dismutase (SOD), catalase (CAT), components of glutathione redox cycle (glutathione peroxidase GPX, glutathione reductase, reduced glutathione), ascorbate peroxidase activity and reduced ascorbate content. On the contrary, in brain during prolonged exposure many of these anti-oxidant enzymes were either unaffected or inhibited, which resulted in persistent over-accumulation of H2O2 and MDA. Branchial and renal tissue both involved in osmo-regulation, revealed an entirely dissimilar compensatory response; the former rely mainly on the ascorbate dependent defensive system while the glutathione catalytic pathway was activated in the latter. In muscle, GPX activity first rose (3.5 days) followed by a subsequent drop, counterbalanced by simultaneous increment of CAT. HEA resulted in a relatively mild oxidative stress in the muscle and kidney, probably explaining the modest anti-oxidative responses. Our findings exemplify that oxidative stress as well as antioxidant potential are qualitatively diverse amongst different tissues, thereby demonstrating that for biomonitoring studies the screening of adaptive responses at organ level should be preferred over whole body response.

Anti-oxidative defences are modulated differentially in three freshwater teleosts in response to ammonia-induced oxidative stress.

Oxidative stress and the antioxidant response induced by high environmental ammonia (HEA) were investigated in the liver and gills of three freshwater teleosts differing in their sensitivities to ammonia. The highly ammonia-sensitive salmonid Oncorhynchus mykiss (rainbow trout), the less ammonia sensitive cyprinid Cyprinus carpio (common carp) and the highly ammonia-resistant cyprinid Carassius auratus (goldfish) were exposed to 1 mM ammonia (as NH4HCO3) for 0 h (control), 3 h, 12 h, 24 h, 48 h, 84 h and 180 h. Results show that HEA exposure increased ammonia accumulation significantly in the liver of all the three fish species from 24 h-48 h onwards which was associated with an increment in oxidative stress, evidenced by elevation of xanthine oxidase activity and levels of hydrogen peroxide (H2O2) and malondialdehyde (MDA). Unlike in trout, H2O2 and MDA accumulation in carp and goldfish liver was restored to control levels (84 h-180 h); which was accompanied by a concomitant increase in superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase activity and reduced ascorbate content. Many of these defence parameters remained unaffected in trout liver, while components of the glutathione redox cycle (reduced glutathione, glutathione peroxidase and glutathione reductase) enhanced to a greater extent. The present findings suggest that trout rely mainly on glutathione dependent defensive mechanism while carp utilize SOD, CAT and ascorbate as anti-oxidative sentinels. Hepatic cells of goldfish appear to utilize each of these protective systems, and showed more effective anti-oxidative compensatory responses towards HEA than carp, while trout were least effective. The present work also indicates that HEA exposure resulted in a relatively mild oxidative stress in the gills of all three species. This probably explains the almost complete lack of anti-oxidative responses in branchial tissue. This research suggests that oxidative stress, as well as the antioxidant potential clearly differ between salmonid and cyprinid species.

The thiol compounds glutathione and homoglutathione differentially affect cell development in alfalfa (Medicago sativa L.).

Glutathione (GSH) is an important scavenger of Reactive Oxygen Species (ROS), precursor of metal chelating phytochelatins, xenobiotic defence compound and regulator of cell proliferation. Homoglutathione (hGSH) is a GSH homologue that is present in several taxa in the family of Fabaceae. It is thought that hGSH performs many of the stress-defence roles typically ascribed to GSH, yet little is known about the potential involvement of hGSH in controlling cell proliferation. Here we show that hGSH/GSH ratios vary across organs and cells and that these changes in hGSH/GSH ratio occur during dedifferentiation and/or cell cycle activation events. The use of a GSH/hGSH biosynthesis inhibitor resulted in impaired cytokinesis in isolated protoplasts, showing the critical importance of these thiol-compounds for cell division. However, exposure of isolated protoplasts to exogenous GSH accelerated cytokinesis, while exogenous hGSH was found to inhibit the same process. We conclude that GSH and hGSH have distinct functional roles in cell cycle regulation in Medicago sativa L. GSH is associated with meristemic cells, and promotes cell cycle activation and induction of somatic embryogenesis, while hGSH is associated with differentiated cells and embryo proliferation.

Ability of ellagic acid to alleviate osmotic stress on chickpea seedlings.

Seed germination and growth of seedlings are critical phases of plant life that are adversely affected by various environmental cues. Water availability is one of the main factors that limit the productivity of many crops. This study was conducted to assess the changes in the sensitivity of chickpea seedlings to osmotic stress by prior treatment of chickpea seeds with a low concentration (50 ppm) of ellagic acid. Ellagic acid was isolated and purified from Padina boryana Thivy by chromatographic techniques. After ellagic acid treatment, seeds were germinated for 10 days under different osmotic potentials (0, -0.2, -0.4, -0.6 and -0.8 MPa) of polyethylene glycol (PEG) solutions. Ellagic acid treatment accelerated the germination and seedling growth of chickpea under osmotic stress conditions. Consistent with the accelerated growth, ellagic acid-treated seedlings also showed a significant increase in the total antioxidant capacity (FRAP) as well as an increase in the compatible solutes (proline and glycine betaine) content. Additionally, treated seedlings revealed lower lipid peroxidation levels (MDA), electrolyte leakage (EL) and H2O2. Flavonoid and reduced glutathione (GSH) content, and the activity of antioxidant enzymes [catalase (CAT), peroxidase (POX), superoxide dismutase (SOD), glutathione reductase (GR)] and enzymes of the shikimic acid pathway [phenylalanine ammonia lyase (PAL) and chalcone synthase (CHS)] all showed a remarkable increase with ellagic acid pretreatment compared to untreated seedlings especially under mild osmotic stress values (-0.2 and -0.4 MPa). These results suggested that treatment with ellagic acid could confer an increased tolerance of chickpea seedlings to osmotic stress, through reducing levels of H2O2 and increasing antioxidant capacity.

The response of the foliar antioxidant system and stable isotopes (δ(13)C and δ(15)N) of white willow to low-level air pollution.

In this study we aimed to determine and elucidate the effect of ambient air pollution on the foliar antioxidant system and stable carbon and nitrogen isotopes of white willow (Salix alba L.). We grew white willow in uniform potting soil in the near vicinity of sixteen air quality monitoring stations in Belgium where nitrogen dioxide (NO2), ozone, sulfur dioxide and particulate matter concentrations were continuously measured. The trees were exposed to ambient air during six months (April-September 2011), and, thereafter, the degree of lipid peroxidation and foliar content of antioxidant molecules (ascorbate, glutathione, polyphenols, flavonoids), antioxidant enzymes (superoxide dismutase, ascorbate peroxidase, peroxidase) and foliar stable carbon (δ(13)C) and nitrogen (δ(15)N) isotopes were measured. We found that lipid peroxidation was caused by air pollution stress, arising from high ambient NO2 concentrations, as shown by an increased amount of malondialdehyde. The antioxidant system was activated by increasing the amount of polyphenols at monitoring stations with a high atmospheric NO2 and low O3 concentration, while no increase of key enzymes (e.g., ascorbate, glutathione) was observed. The δ(13)C also decreased with increasing NO2 concentrations and decreasing O3 concentrations, probably reflecting a decreased net photosynthesis and/or a concomitant decrease of (13)CO2 in the atmosphere. Shade also influenced foliar δ(13)C and the content of leaf ascorbate and glutathione.

Dihydrolipoic acid reduces cytochrome b561 proteins.

Cytochrome b561 (Cyt-b561) proteins constitute a family of trans-membrane proteins that are present in a wide variety of organisms. Two of their characteristic properties are the reducibility by ascorbate (ASC) and the presence of two distinct b-type hemes localized on two opposite sides of the membrane. Here we show that the tonoplast-localized and the putative tumor suppressor Cyt-b561 proteins can be reduced by other reductants than ASC and dithionite. A detailed spectral analysis of the ASC-dependent and dihydrolipoic acid (DHLA)-dependent reduction of these two Cyt-b561 proteins is also presented. Our results are discussed in relation to the known antioxidant capability of DHLA as well as its role in the regeneration of other antioxidant compounds of cells. These results allow us to speculate on new biological functions for the trans-membrane Cyt-b561 proteins.

Cytochromes b561: ascorbate-mediated trans-membrane electron transport.

SIGNIFICANCE: Cytochromes b561 (CYB561s) constitute a family of trans-membrane (TM), di-heme proteins, occurring in a variety of organs and cell types, in plants and animals, and using ascorbate (ASC) as an electron donor. CYB561s function as monodehydroascorbate reductase, regenerating ASC, and as Fe³âº-reductases, providing reduced iron for TM transport. A CYB561-core domain is also associated with dopamine ß-monooxygenase redox domains (DOMON) in ubiquitous CYBDOM proteins. In plants, CYBDOMs form large protein families. Physiological functions supported by CYB561s and CYBDOMs include stress defense, cell wall modifications, iron metabolism, tumor suppression, and various neurological processes, including memory retention. CYB561s, therefore, significantly broaden our view on the physiological roles of ASC. RECENT ADVANCES: The ubiquitous nature of CYB561s is only recently being recognized. Significant advances have been made through the study of recombinant CYB561s, revealing structural and functional properties of a unique "two-heme four-helix" protein configuration. In addition, the DOMON domains of CYBDOMs are suggested to contain another heme b. CRITICAL ISSUES: New CYB561 proteins are still being identified, and there is a need to provide an insight and overview on the various roles of these proteins and their structural properties. FUTURE DIRECTIONS: Mutant studies will reveal in greater detail the mechanisms by which CYB561s and CYBDOMs participate in cell metabolism in plants and animals. Moreover, the availability of efficient heterologous expression systems should allow protein crystallization, more detailed (atomic-level) structural information, and insights into the intra-molecular mechanism of electron transport.

Effect of elevated CO2 and temperature on the oxidative stress response to drought in Lolium perenne L. and Medicago sativa L.

Studies addressing the combined impact of multiple climate factors on plant abiotic stress responses are still scarce. We investigated physiological and molecular (antioxidant), responses to water deficit, in grassland-model species, Lolium perenne L. and Medicago lupulina L., under future climate conditions, i.e. elevated CO2 (+CO2, +375 ppm) and elevated temperature (+T, +3 °C). Elevated CO2, but not warming, significantly increased biomass (gDW) in L. perenne, but not in M. lupulina. Photosynthesis (A(sat)) and stomatal conductance (g(s)), were differently affected by climate in each species, L. perenne generally being more sensitive. Elevated CO2 increased lipid peroxidation levels in M. lupulina, but not in L. perenne, and had no effect on protein oxidation and little effect on antioxidant levels. Drought stress caused severe inhibition in biomass and photosynthesis, most severely in L. perenne, and strongly increased oxidative damage. Elevated CO2 protected against the drought-induced damage. Decreased activities of APX and POX may indicate lower levels of oxidative challenge (relaxation) at the level of H2O2 production. Polyphenols, tocopherols and antioxidant capacity, increased under drought stress, in all climate conditions. Elevated CO2, increased reduced ascorbate (ASC) and reduced glutathione (GSH), and their redox status, in both species, although to different levels. Changes in activities of key ASC/GSH cycle enzymes, under stress and climate treatments, showed weak correlations with ASC and GSH levels, indicating the complexity of this network. Together this work supports the idea that redox changes are involved in responses to climate changes, in the absence and presence of water-deficit stress.