Halliwell-Asada Cycle Research Articles

Deciphering lead tolerance mechanisms in a population of the plant species Biscutella auriculata L. from a mining area: Accumulation strategies and antioxidant defenses

The uptake and distribution of Pb and the mechanisms involved in the metal tolerance have been investigated in a mine population of Biscutella auriculata. Seedlings were exposed to 125 μM Pb(NO3)2 for 15 days under semihydroponic conditions. The results showed an increase in the size of Pb-treated seedlings and symptoms of toxicity were not observed. ICP-OES analyses showed that Pb accumulation was restricted to root tissue. Imaging of Pb accumulation by dithizone histochemistry revealed the presence of the metal in vacuoles and cell wall in root cells. The accumulation of Pb in vacuoles could be stimulated by an increase in phytochelatin PC2 content. Pb did not promote oxidative damage and this is probably due the increase of antioxidative defenses. In the leaves, Pb produced a significant increase in superoxide dismutase activity, while in roots an increase in catalase and components of the Foyer– Halliwell–Asada cycle were observed. The results indicated that Biscutella auriculata has a high capacity to tolerate Pb and this is mainly due to a very efficient mechanism to sequester the metal in roots and a capacity to avoid oxidative stress. This species could therefore be very useful for phytostabilization and repopulation of areas contaminated with Pb.

Regulation of boron toxicity responses via glutathione-dependent detoxification pathways at biochemical and molecular levels in Arabidopsisthaliana

The fine-tuned regulation of the Halliwell-Asada cycle (ascorbate-glutathione pathway) in Arabidopsis thaliana under boron (B) toxicity was shown in our previous report. In this study, we investigated the expression levels of some members of the glutathione S-transferase (GST) superfamily, such as phi (GSTF2, GSTF6, GSTF7, and GSTF8), tau (GSTU19), and zeta (GSTZ1) classes in Arabidopsis thaliana that were exposed to 1 mM boric acid (1B) and 3 mM boric acid (3B). Additionally, the expression levels of genes for glutathione (GSH) and phytochelatin biosynthesis as well as miR169 and miR156 were evaluated in Arabidopsis thaliana exposed to 1B and 3B. Moreover, changes in the levels of total GST activity; GSH; and total, protein-bound, and nonprotein thiols were spectrophotometrically determined. GSH levels and nonprotein thiol content did not change significantly following both B-toxicity conditions. Expression levels of GSH1 and GSH2 stayed stable under 1B toxicity; however, GSH1 expression increased significantly under 3B conditions in Arabidopsis thaliana. The expression levels of four genes from phi class members of GST were not dramatically changed under B-toxicity conditions. However, the transcript levels of miR169, ATGSTU19, and ATGSTZ1 were significantly increased after 1B and 3B exposure. These GST genes may have a role in the dramatic increase of total GST activity under toxic B. To the best of our knowledge, this is the first report displaying an integrative view of high-B-induced regulation of GSH-dependent enzymatic machinery at different biological organization levels in Arabidopsis thaliana.

Time course of antioxidant enzymes in the paraquat-resistant and tolerant Heary fleabane (<i>Conyza bonariensis</i>) biotypes in response to ozone exposure and paraquat application

In order to investigate the molecular mechanism of oxidative stress resistance due to either paraquat or ozone in plants, two paraquat biotypes (resistant and sensitive) of Conyza bonariensis were used. We monitored some biochemical parameters (total peroxydase, guaiacol peroxydase, ascorbate peroxydase and superoxyde dismutase) following a paraquat fumigation (0.001%, 4 hours), and a pulse of elevated ozone (200 ppb, 5 hours) or intermediate ozone (50 ppb, 4 hours). The mechanisms of protection induced by ozone exposure were not similar to the pathway leading to paraquat resistance, due to a different stimulation response system to antioxidants.© 2019 International Formulae Group. All rights reservedKeywords: Antioxidants, Halliwell-Asada cycle, Ozone, Paraquat, Conyza bonariensis

Ozone primes changes in phytochemical parameters in the medicinal herb Hypericum perforatum (St. John’s wort)

Plants produce secondary metabolites promoting adaptation to changes in the environment and challenges by pathogenic microorganisms. Ozone (O3), a crucial gaseous air pollutant, has been reported to alter the chemical composition of plants and this also involves changes in bioactive secondary compounds. The objective of this work was to assess under controlled conditions the possible role of O3 (110 ppb, 5 h) as an elicitor of antioxidant compounds in Hypericum perforatum L. (St. John’s Wort, SJW) plants at two stages, vegetative (VS, three-months old) and reproductive (RS, six-months-old) stage. At the end of the treatment, we found an early and transient burst of hydrogen peroxide (H2O2, +35% in comparison to controls), a less efficient metabolism of the Halliwell-Asada cycle (as confirmed by the unchanged levels of ascorbate) and the up-regulation of the reduced/oxidized glutathione ratio (about 2-fold higher than control), which represent a typical initial stress response in VS plants. After 24 h of exposure, a significant reduction of H2O2 levels was observed that could be dependent on the activation of peroxidase activity (+26%), accumulation of total phenols, flavonoids and anthocyanins (+45, +58 and +2%, respectively) and increased antioxidant potential. A modification of the pool of soluble phenolics (at the quantitative and qualitative levels) was observed in VS and RS plants, so confirming its involvement in detoxification mechanisms. Our results show the potential of using O3 to enhance the concentration of several antioxidant phytochemicals and consequently to increase the beneficial properties of SJW plants at different phenological stages.

What about the detoxification mechanisms underlying ozone sensitivity in Liriodendron tulipifera?

Liriodendron tulipifera (known as the tulip tree) is a woody species that has been previously classified as sensitive to ozone (O3) in terms of visible leaf injuries and photosynthetic primary reactions. The objective of this work is to give a thorough description of the detoxification mechanisms that are at the basis of O3 sensitivity. Biochemical and molecular markers were used to characterize the response of 1-year-old saplings exposed to O3 (120ppb, 5hday-1, for 45 consecutive days) under controlled conditions. O3 effects resulted in a less efficient metabolism of Halliwell-Asada cycle as confirmed by the diminished capacity to convert the oxidized forms of ascorbate and glutathione in the reduced ones (AsA and GSH, respectively). The reduced activity of AsA and GSH regenerating enzymes indicates that de novo AsA biosynthesis occurred. This compound could be a cofactor of several plant-specific enzymes that are involved in the early part of the phenylpropanoid and flavonoid biosynthesis pathway, as confirmed by the significant rise of PAL activity (+75%). The induction of the defence-related secondary metabolites (in particular, rutin and caffeic acid were about threefold higher) and the concomitant increase in transcript levels of PAL and CHS genes (+120 and 30%, respectively) suggest that L. tulipifera utilized this route in order to partially counteract the O3-induced oxidative damage.

The harsh life of an urban tree: the effect of a single pulse of ozone in salt-stressed Quercus ilex saplings.

Ozone (O3) and salinity are usually tested as combined factors on plant performance. However, the response to a single episode of O3 in plants already stressed by an excess of NaCl as occurs in the natural environment has never been investigated, but is important given that it is commonly experienced in Mediterranean areas. Three-year-old Quercus ilex L. (holm oak) saplings were exposed to salinity (150 mM NaCl, 15 days), and the effect on photosynthesis, hydric relations and ion partitioning was evaluated (Experiment I). In Experiment II, salt-treated saplings were exposed to 80 nl l-1 of O3 for 5 h, which is a realistic dose in a Mediterranean environment. Gas exchanges, chlorophyll fluorescence and antioxidant systems were characterized to test whether the salt-induced stomatal closure limited O3 uptake and stress or whether the pollutant represents an additional stressor for plants. Salt-dependent stomatal closure depressed the photosynthetic process (-71.6% of light-saturated rate of photosynthesis (A380)) and strongly enhanced the dissipation of energy via the xanthophyll cycle. However, salt-treated plants had higher values of net assimilation rate/stomatal conductance (A/gs) than the controls, which was attributable to a greater mesophyll conductance gm/gs and carboxylation efficiency (higher gm/maximal rate of Rubisco carboxylation (Vcmax)), thus suggesting no damage to chloroplasts. O3 did not exacerbate the effect of salinity on photosynthesis, however a general enhancement of the Halliwell-Asada cycle was necessary to counteract the O3-triggered oxidative stress. Despite the 79.4% gs reduction in salt-stressed plants, which strongly limited the O3 uptake, a single peak in the air pollutant led to an additional burden for the antioxidant system when plants had been previously subjected to salinity.

New strategy for drug discovery by large-scale association analysis of molecular networks of different species.

The development of modern omics technology has not significantly improved the efficiency of drug development. Rather precise and targeted drug discovery remains unsolved. Here a large-scale cross-species molecular network association (CSMNA) approach for targeted drug screening from natural sources is presented. The algorithm integrates molecular network omics data from humans and 267 plants and microbes, establishing the biological relationships between them and extracting evolutionarily convergent chemicals. This technique allows the researcher to assess targeted drugs for specific human diseases based on specific plant or microbe pathways. In a perspective validation, connections between the plant Halliwell-Asada (HA) cycle and the human Nrf2-ARE pathway were verified and the manner by which the HA cycle molecules act on the human Nrf2-ARE pathway as antioxidants was determined. This shows the potential applicability of this approach in drug discovery. The current method integrates disparate evolutionary species into chemico-biologically coherent circuits, suggesting a new cross-species omics analysis strategy for rational drug development.

Redox homeostasis via gene families of ascorbate-glutathione pathway

The imposition of environmental stresses on plants brings about disturbance in their metabolism thereby negatively affecting their growth and development and leading to reduction in the productivity. One of the manifestations of abiotic and biotic stress conditions is the enhanced production of reactive oxygen species (ROS) which can be hazardous to cells. Therefore, in order to protect themselves against toxic ROS, plant cells employ the anti-oxidant defense system. The ascorbate-glutathione pathway (Halliwell-Asada cycle) is an indispensible component of the ROS homeostasis mechanism of plants. This pathway entails the antioxidant metabolites: ascorbate, glutathione and NADPH along with the enzymes linking them. The ascorbate-glutathione pathway is functional in different subcellular compartments and all the enzymes of this pathway exist as multiple isoforms. The expression of different isoforms of the enzymes of ascorbate-glutathione pathway is developmentally as well as spatially regulated. Moreover, various abiotic and biotic stress conditions modulate the expression of the enzyme- isoforms differently. It is the intricate regulation of expression of different isoforms of the ascorbate-glutathione pathway enzymes that helps in the maintenance of redox balance in plants under various abiotic and biotic stress conditions. The present review provides an insight into the gene families of the ascorbate-glutathione pathway, shedding light on their role in different abiotic and biotic stress conditions as well as in the growth and development of plants.

Mechanisms of resistance to paraquat in plants

The aim of this brief review is to draw information from studies of the mechanism of evolved resistance in weeds, together with information from laboratory studies of paraquat tolerance in model plants. Plants having mutations that limit paraquat uptake into cytoplasm, that confer various stress tolerances or that have transgenes that co-express two or more of the chloroplast Halliwell-Asada cycle enzymes can all exhibit enhanced tolerance to paraquat. However, none of these mechanisms correspond to the high-level resistances that have evolved naturally in weeds. Most, but not all, of the evidence from studies of paraquat-resistant biotypes of weeds can reasonably be reconciled with the proposal of a single major gene mechanism that sequesters paraquat away from chloroplasts and into the vacuole. However, the molecular details of this putative mechanism remain ill-defined.

A Fogging System Improves Antioxidative Defense Responses and Productivity in Tomato

Crops cultivated during summer in greenhouses and screen-covered structures (screenhouses) are negatively affected by stressful temperatures and vapor pressure deficit (VPD), which in turn influence yield and product quality. Fogging systems have been proposed as cooling methods to mitigate the adverse effects of high evaporation and excess temperatures in greenhouses. To evaluate the possible action of the fogging system on antioxidant response in cherry tomato (Solanum lycopersicum) fruit production, we studied the behavior of reactive oxygen species (ROS) scavenger enzymes such as superoxide dismutase, catalase, guaiacol peroxidase, enzymes involved in the ascorbate-glutathione cycle (Halliwell-Asada cycle), and compounds with antioxidant functions such as ascorbate, glutathione, proline, and polyamines. Fogging-screenhouse treatments, SF (a fogging system inside the screenhouse) and SFS (fogging system with a plastic sheeting, to maintain the microclimate created by the fogging system in the screenhouse) showed the best performance in terms of radiation with a percentage reduction of incident radiation on the crop of 18% and 37% and a mean reduction of maximum VPD values of 0.4 and 0.2 kPa, respectively, compared with the unfogging treatment (S). These improved environmental conditions, besides promoting the highest activities of ROS-scavenging enzymes and Halliwell-Asada cycle, the redox state of the ascorbate, and a low proline:free putrescine ratio, would explain the increase in commercial weight of fruit by 21% and 17% in SF and SFS, respectively, with respect to S.

Plant type ferredoxins and ferredoxin‐dependent metabolism

Ferredoxin (Fd) is a small [2Fe-2S] cluster-containing protein found in all organisms performing oxygenic photosynthesis. Fd is the first soluble acceptor of electrons on the stromal side of the chloroplast electron transport chain, and as such is pivotal to determining the distribution of these electrons to different metabolic reactions. In chloroplasts, the principle sink for electrons is in the production of NADPH, which is mostly consumed during the assimilation of CO2 . In addition to this primary function in photosynthesis, Fds are also involved in a number of other essential metabolic reactions, including biosynthesis of chlorophyll, phytochrome and fatty acids, several steps in the assimilation of sulphur and nitrogen, as well as redox signalling and maintenance of redox balance via the thioredoxin system and Halliwell-Asada cycle. This makes Fds crucial determinants of the electron transfer between the thylakoid membrane and a variety of soluble enzymes dependent on these electrons. In this article, we will first describe the current knowledge on the structure and function of the various Fd isoforms present in chloroplasts of higher plants and then discuss the processes involved in oxidation of Fd, introducing the corresponding enzymes and discussing what is known about their relative interaction with Fd.

Transcriptional changes of antioxidant responses, hormone signalling and developmental processes evoked by the Brassica napus SHOOTMERISTEMLESS during in vitro embryogenesis

Previous work showed that alterations in Brassica napus (Bn) SHOOTMERISTEMLESS (BnSTM) expression levels influence microspore-derived embryogenesis in B. napus. While over-expression of BnSTM increased microspore-derived embryo (MDE) yield and quality, down-regulation of BnSTM repressed embryo formation [16]. Transcriptional analyses were conducted to investigate the molecular mechanisms underpinning these responses. The induction of BnSTM resulted in a heavy transcriptional activation of genes involved in antioxidant responses, hormone signalling and developmental processes. Several antioxidant enzymes, including catalases, superoxide dismutases, and components of the Halliwell-Asada cycle were induced in embryos ectopically expressing BnSTM and contributed to the removal of reactive oxygen species (ROS). These changes were accompanied by elevated levels of ascorbate and glutathione, which have been shown to promote embryonic growth and development. Induction or repression of BnSTM altered the early cytokinin response, whereas late responses, modulated by Type-A Arabidopsis response regulators (ARRs), were induced in MDEs over-expressing BnSTM. Major differences between transgenic MDEs were also observed in the expression pattern of several auxin transporters and key developmental factors required for normal embryogenesis. While some of these factors, BABYBOOM1 (BBM1) and SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK), play a key role during early embryogeny, others, CYP78A5, LEAFY COTYLEDON1 and 2 (LEC1 and LEC2), as well as WOX2 and 9, are required for proper embryo development. Collectively these results demonstrate the involvement of BnSTM in novel developmental processes which can be utilized to enhance invitro embryogenesis.

Manganese-excess induces oxidative stress, lowers the pool of antioxidants and elevates activities of key antioxidative enzymes in rice seedlings

Manganese (Mn) is an essential element for plant growth but in excess, specially in acidic soils, it can become phytotoxic. In order to investigate whether oxidative stress is associated with the expression of Mn toxicity during early seedling establishment of rice plants, we examined the changes in the level of reactive oxygen species (ROS), oxidative stress induced an alteration in the level of non-enzymic antioxidants and activities of antioxidative enzymes in rice seedlings grown in sand cultures containing 3 and 6 mM MnCl2. Mn treatment inhibited growth of rice seedlings, the metal increasingly accumulated in roots and shoots and caused damage to membranes. Mn treated plants showed increased generation of superoxide anion (O2 .−), elevated levels of H2O2 and thiobarbituric acid reactive substances (TBARS) and decline in protein thiol. The level of nonprotein thiol, however, increased due to Mn treatment. A decline in contents of reduced ascorbate (AsA) and glutathione (GSH) as well as decline in ratios of their reduced to oxidize forms was observed in Mn-treated seedlings. The activities of antioxidative enzymes superoxide dismutase (SOD) and its isoforms Mn SOD, Cu/Zn SOD, Fe SOD as well as guaiacol peroxidase (GPX) increased in the seedlings due to Mn treatment however, catalase (CAT) activity increased in 10 days old seedlings but it declined by 20 days under Mn treatment. The enzymes of Halliwell-Asada cycle, ascorbate peroxidase (APX) monodehydoascorbate reductase (MDHAR), dehyroascorbate reductase (DHAR) and glutathione reductase (GR) increased significantly in Mn treated seedlings over controls. Results suggest that in rice seedlings excess Mn induces oxidative stress, imbalances the levels of antioxidants and the antioxidative enzymes SOD, GPX, APX and GR appear to play an important role in scavenging ROS and withstanding oxidative stress induced by Mn.

Does iodine biofortification affect oxidative metabolism in lettuce plants?

Plants produce low levels of reactive oxygen species (ROS), which form part of basic cell chemical communication; however, different types of stress can lead to an overexpression of ROS that can damage macromolecules essential for plant growth and development. Iodine is vital to human health, and iodine biofortification programs help improve the human intake through plant consumption. This biofortification process has been shown to influence the antioxidant capacity of lettuce plants, suggesting that the oxidative metabolism of the plant may be affected. The results of this study demonstrate that the response to oxidative stress is variable and depends on the form of iodine applied. Application of iodide (I(-)) to lettuce plants produces a reduction in superoxide dismutase (SOD) activity and an increase in catalase (CAT) and L-galactono dehydrogenase enzyme activities and in the activity of antioxidant compounds such as ascorbate (AA) and glutathione. This did not prove a very effective approach since a dose of 80μM produced a reduction in the biomass of the plants. For its part, application of iodate (IO (3) (-) ) produced an increase in the activities of SOD, ascorbate peroxidase, and CAT, the main enzymes involved in ROS detoxification; it also increased the concentration of AA and the regenerative activities of the Halliwell-Asada cycle. These data confirm the non-phytotoxicity of IO (3) (-) since there is no lipid peroxidation or biomass reduction. According to our results, the ability of IO (3) (-) to induce the antioxidant system indicates that application of this form of iodine may be an effective strategy to improve the response of plants to different types of stress.

Response of antioxidant enzymes in Nicotiana tabacum clones during phytoextraction of heavy metals

Tobacco, Nicotiana tabacum, is a widely used model plant for growth on heavy-metal-contaminated sites. Its high biomass and deep rooting system make it interesting for phytoextraction. In the present study, we investigated the antioxidative activities and glutathione-dependent enzymes of different tobacco clones optimized for better Cd and Zn accumulation in order to characterize their performance in the field. The improved heavy metal resistance also makes the investigated tobacco clones interesting for understanding the plant defense enzyme system in general. Freshly harvested plant material (N. tabacum leaves) was used to investigate the antioxidative cascade in plants grown on heavy metal contaminated sites with and without amendments of different ammonium nitrate and ammonium sulfate fertilizers. Plants were grown on heavily polluted soils in north-east Switzerland. Leaves were harvested at the field site and directly deep frozen in liquid N(2). Studies were concentrated on the antioxidative enzymes of the Halliwell-Asada cycle, and spectrophotometric measurements of catalase (CAT, EC 1.11.1.6), ascorbate peroxidase (APX, EC 1.11.1.11), superoxide dismutase (SOD, EC 1.15.1.1), glutathione peroxidase (GPX, EC 1.11.1.9), glutathione reductase (GR, EC 1.6.4.2), glutathione S-transferase (GST, EC 2.5.1.18) were performed. We tried to explain the relationship between fertilizer amendments and the activity of the enzymatic defense systems. When tobacco (N. tabacum) plants originating from different mutants were grown under field conditions with varying fertilizer application, the uptake of cadmium and zinc from soil increased with increasing biomass. Depending on Cd and Zn uptake, several antioxidant enzymes showed significantly different activities. Whereas SOD and CAT were usually elevated, several other enzymes, and isoforms of GST were strongly inhibited. Heavy metal uptake represents severe stress to plants, and specific antioxidative enzymes are induced at the cost of more general reactions of the Halliwell-Asada cycle. In well-supplied plants, the glutathione level remains more or less unchanged. The lack of certain glutathione S-transferases upon exposure to heavy metals might be problematic in cases when organic pollutants coincide with heavy metal pollution. When planning phytoremediation of sites, mixed pollution scenarios have to be foreseen and plants should be selected according to both, their stress resistance and hyperaccumulative capacity.

Oxidative Stress and Antioxidants in Tomato (Solanum lycopersicum) Plants Subjected to Boron Toxicity

Boron (B) toxicity triggers the formation of reactive oxygen species in plant tissues. However, there is still a lack of knowledge as to how B toxicity affects the plant antioxidant defence system. It has been suggested that ascorbate could be important against B stress, although existing information is limited in this respect. The objective of this study was to analyse how ascorbate and some other components of the antioxidant network respond to B toxicity. Two tomato (Solanum lycopersicum) cultivars ('Kosaco' and 'Josefina') were subjected to 0.05 (control), 0.5 and 2 mm B. The following were studied in leaves: dry weight; relative leaf growth rate; total and free B; H(2)O(2); malondialdehyde; ascorbate; glutathione; sugars; total non-enzymatic antioxidant activity, and the activity of superoxide dismutase, catalase, ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase, glutathione reductase, ascorbate oxidase and l-galactose dehydrogenase. The B-toxicity treatments diminished growth and boosted the amount of B, malondialdehyde and H(2)O(2) in the leaves of the two cultivars, these trends being more pronounced in 'Josefina' than in 'Kosaco'. B toxicity increased ascorbate concentration in both cultivars and increased glutathione only in 'Kosaco'. Activities of antioxidant- and ascorbate-metabolizing enzymes were also induced. High B concentration in the culture medium provokes oxidative damage in tomato leaves and induces a general increase in antioxidant enzyme activity. In particular, B toxicity increased ascorbate pool size. It also increased the activity of l-galactose dehydrogenase, an enzyme involved in ascorbate biosynthesis, and the activity of enzymes of the Halliwell-Asada cycle. This work therefore provides a starting point towards a better understanding of the role of ascorbate in the plant response against B stress.

Light-Dependent Oxidative Stress Determines Physiological Leaf Spot Formation in Barley

ABSTRACT We reported previously that physiological leaf spot (PLS) formation in winter and spring barley is dependent on genotype-related oxidative stress under field conditions. In the present study, we searched for factors inducing PLS symptoms in the greenhouse similar to those observed in the field and investigated its relationship to reactive oxygen species (ROS) metabolism. We found that in the greenhouse, oxidative stress induced spring barley cv. Extract, which is sensitive to PLS, to express symptoms similar to those observed in the field. Leaves severely affected by PLS showed significantly lower activities of key enzymes in the Halliwell-Asada cycle such as ascorbate peroxidase, glutathione reductase, dehy-droascorbate reductase, and monodehydroascorbate reductase. The sensitive cultivar also showed lower levels of total superoxide dismutase (SOD) and Cu/Zn-SOD activity but a higher level of chloroplast-specific Fe-SOD activity than that of the insensitive cultivar. Thus, an unbalanced ROS metabolism in chloroplasts may trigger PLS incidence in sensitive cultivars, which is in agreement with the fact that light is essential for the induction of PLS expression under both field and greenhouse conditions. Accordingly, under greenhouse conditions, continuous light stress (7 days), but not light shock treatments, induced PLS similar to that of field conditions in sensitive cv. Extract, but not in resistant cv. Scarlett. Light with a high proportion of energy in the blue wavelength spectrum (350 to 560 nm) was significantly more PLS inductive than light with a pronounced red (photosynthetically active radiation) spectrum (580 to 650 nm). Exposure to ozone did not produce PLS-like symptoms. Furthermore, similar to earlier observations in the field, PLS symptom expression was closely correlated with the accumulation of superoxide (O(2) (-)) detected by both biochemical and histochemical assays. Taken together, these data suggest that PLS in barley is genotype-dependent but its expression appears to be induced by certain environmental stress factors, among which photosyn-thetically active radiation plays a major role.