Low Ascorbate Research Articles

The ascorbate biosynthesis pathway in plants is known, but there is a way to go with understanding control and functions.

Ascorbate (vitamin C) is one of the most abundant primary metabolites in plants. Its complex chemistry enables it to function as an antioxidant, as a free radical scavenger, and as a reductant for iron and copper. Ascorbate biosynthesis occurs via the mannose/l-galactose pathway in green plants, and the evidence for this pathway being the major route is reviewed. Ascorbate accumulation is leaves is responsive to light, reflecting various roles in photoprotection. GDP-l-galactose phosphorylase (GGP) is the first dedicated step in the pathway and is important in controlling ascorbate synthesis. Its expression is determined by a combination of transcription and translation. Translation is controlled by an upstream open reading frame (uORF) which blocks translation of the main GGP-coding sequence, possibly in an ascorbate-dependent manner. GGP associates with a PAS-LOV protein, inhibiting its activity, and dissociation is induced by blue light. While low ascorbate mutants are susceptible to oxidative stress, they grow nearly normally. In contrast, mutants lacking ascorbate do not grow unless rescued by supplementation. Further research should investigate possible basal functions of ascorbate in severely deficient plants involving prevention of iron overoxidation in 2-oxoglutarate-dependent dioxygenases and iron mobilization during seed development and germination.

Non-volatile signals and redox mechanisms are required for the responses of Arabidopsis roots to Pseudomonas oryzihabitans.

Soil bacteria promote plant growth and protect against environmental stresses, but the mechanisms involved remain poorly characterized, particularly when there is no direct contact between the roots and bacteria. Here, we explored the effects of Pseudomonas oryzihabitans PGP01 on the root system architecture (RSA) in Arabidopsis thaliana seedlings. Significant increases in lateral root (LR) density were observed when seedlings were grown in the presence of P. oryzihabitans, as well as an increased abundance of transcripts associated with altered nutrient transport and phytohormone responses. However, no bacterial transcripts were detected on the root samples by RNAseq analysis, demonstrating that the bacteria do not colonize the roots. Separating the agar containing bacteria from the seedlings prevented the bacteria-induced changes in RSA. Bacteria-induced changes in RSA were absent from mutants defective in ethylene response factor (ERF109), glutathione synthesis (pad2-1, cad2-1, and rax1-1) and in strigolactone synthesis (max3-9 and max4-1) or signalling (max2-3). However, the P. oryzihabitans-induced changes in RSA were similar in the low ascorbate mutants (vtc2-1and vtc2-2) to the wild-type controls. Taken together, these results demonstrate the importance of non-volatile signals and redox mechanisms in the root architecture regulation that occurs following long-distance perception of P. oryzihabitans.

Network Theory and the Resilience of Redox Signaling

The redox status inside and around cells is critically important to control, being used to maintain reduced compounds in the correct state and for cell signaling mechanisms. A myriad of compounds and proteins are involved in a vast network system to regulate the redox state of biological systems. These include reactive molecules such reactive oxygen species (ROS), nitric oxide (NO·) and hydrogen sulfide (H2S) along with systems for their removal, such as antioxidants. Redox buffering involves molecules such as glutathione, low molecular weight thiols and ascorbate. Network Theory attempts to give the mechanisms underlying complex networks a mathematical and model-based underpinning and it has been suggested that metabolic systems can be described as scale-free networks, having a power law degree distribution. Such networks are said to be both robust but vulnerable, suggesting a level of resilience. Redox metabolism also has to be robust, being maintained in what has been described as the Goldilocks Zone, while it is also vulnerable to outside influence, often leading to the phenomenon referred to as oxidative stress. Therefore, it is suggested here that a holistic approach to understand redox networks should embrace Network Theory, which may be able to predict characteristics of the redox network that can be targeted for new therapeutics or agricultural treatments.

Ascorbate-mediated regulation of growth, photoprotection, and photoinhibition in Arabidopsis thaliana.

The requirements for ascorbate for growth and photosynthesis were assessed under low (LL; 250 µmol m-2 s-1) or high (HL; 1600 µmol m-2 s-1) irradiance in wild-type Arabidopsis thaliana and two ascorbate synthesis mutants (vtc2-1 and vtc2-4) that have 30% wild-type ascorbate levels. The low ascorbate mutants had the same numbers of leaves but lower rosette area and biomass than the wild type under LL. Wild-type plants experiencing HL had higher leaf ascorbate, anthocyanin, and xanthophyll pigments than under LL. In contrast, leaf ascorbate levels were not increased under HL in the mutant lines. While the degree of oxidation measured using an in vivo redox reporter in the nuclei and cytosol of the leaf epidermal and stomatal cells was similar under both irradiances in all lines, anthocyanin levels were significantly lower in the low ascorbate mutants than in the wild type under HL. Differences in the photosynthetic responses of vtc2-1 and vtc2-4 mutants were observed. Unlike vtc2-1, the vtc2-4 mutants had wild-type zeaxanthin contents. While both low ascorbate mutants had lower levels of non-photochemical quenching of chlorophyll a fluorescence (NPQ) than the wild type under HL, qPd values were greater only in vtc2-1 leaves. Ascorbate is therefore essential for growth but not for photoprotection.

Redox Changes During the Cell Cycle in the Embryonic Root Meristem of Arabidopsis thaliana.

Aims: The aim of this study was to characterize redox changes in the nuclei and cytosol occurring during the mitotic cell cycle in the embryonic roots of germinating Arabidopsis seedlings, and to determine how redox cycling was modified in mutants with a decreased capacity for ascorbate synthesis.Results: Using an in vivo reduction-oxidation (redox) reporter (roGFP2), we show that transient oxidation of the cytosol and the nuclei occurred at G1 in the synchronized dividing cells of the Arabidopsis root apical meristem, with reduction at G2 and mitosis. This redox cycle was absent from low ascorbate mutants in which nuclei were significantly more oxidized than controls. The cell cycle-dependent increase in nuclear size was impaired in the ascorbate-deficient mutants, which had fewer cells per unit area in the root proliferation zone. The transcript profile of the dry seeds and size of the imbibed seeds was strongly influenced by low ascorbate but germination, dormancy release and seed aging characteristics were unaffected.Innovation: These data demonstrate the presence of a redox cycle within the plant cell cycle and that the redox state of the nuclei is an important factor in cell cycle progression.Conclusions: Controlled oxidation is a key feature of the early stages of the plant cell cycle. However, sustained mild oxidation restricts nuclear functions and impairs progression through the cell cycle leading to fewer cells in the root apical meristem. Antioxid. Redox Signal. 27, 1505–1519.

Redox Control of Aphid Resistance through Altered Cell Wall Composition and Nutritional Quality.

The mechanisms underpinning plant perception of phloem-feeding insects, particularly aphids, remain poorly characterized. Therefore, the role of apoplastic redox state in controlling aphid infestation was explored using transgenic tobacco (Nicotiana tabacum) plants that have either high (PAO) or low (TAO) ascorbate oxidase (AO) activities relative to the wild type. Only a small number of leaf transcripts and metabolites were changed in response to genotype, and cell wall composition was largely unaffected. Aphid fecundity was decreased significantly in TAO plants compared with other lines. Leaf sugar levels were increased and maximum extractable AO activities were decreased in response to aphids in all genotypes. Transcripts encoding the Respiratory Burst Oxidase Homolog F, signaling components involved in ethylene and other hormone-mediated pathways, photosynthetic electron transport components, sugar, amino acid, and cell wall metabolism, were increased significantly in the TAO plants in response to aphid perception relative to other lines. The levels of galactosylated xyloglucan were decreased significantly in response to aphid feeding in all the lines, the effect being the least in the TAO plants. Similarly, all lines exhibited increases in tightly bound (1→4)-β-galactan. Taken together, these findings identify AO-dependent mechanisms that limit aphid infestation.

Lipoprotein(a) and vitamin C impair development of breast cancer tumors in Lp(a)+; Gulo-/- mice.

Cancer progression is characterized by loss of extracellular matrix (ECM) integrity, which is a precondition for tumor growth and metastasis. In order to elucidate the precise mechanisms of ECM degradation in cancer we used a genetically modified mouse mimicking two distinct human metabolic features associated with carcinogenesis, the lack of endogenous vitamin C synthesis and the production of human Lp(a). Female Lp(a)+; Gulo(−/−) and control wild-type Balb/c mice without these two metabolic features were orthotopically inoculated with 4T1 breast cancer cells (5×105). The transgenic and control mice were divided into 4 different dietary groups in respect to dietary vitamin C intake: i) low ascorbate intake for 6 weeks; ii) high ascorbate intake for 6 weeks; iii) low ascorbate intake for 3 weeks followed by high ascorbate for 3 weeks; iv) high ascorbate intake for 3 weeks followed by low ascorbate for 3 weeks. After 6 weeks, all wild-type mice developed tumors. In contrast, Lp(a)+; Gulo(−/−) mice developed one third less primary tumors (low ascorbate diet) or no primary tumors at all (high ascorbate diet). Significantly, tumors from Lp(a)+; Gulo(−/−) mice immunostained positively for Lp(a) and their size was inversely proportional to Lp(a) serum levels. The results implicate that Lp(a) may play a role in controlling tumor growth and expansion. The most likely mechanism is the competitive inhibition of plasmin-induced ECM degradation due to the homology of Lp(a) components to plasminogen. The confirmation of this pathomechanism could lead to a universal therapeutic target for the prevention and treatment of cancer.

Amelioration of persistent left ventricular function impairment through increased plasma ascorbate levels following myocardial infarction

Purpose: Percutaneous coronary angioplasty (PCA) has been demonstrated to reduce mortality and morbidity and thereby improve the prognosis of patients undergoing acute myocardial infarctions (AMIs). However, this procedure paradoxically increases the initial damage as the result of a condition known as ‘myocardial reperfusion injury’. Oxidative stress may contribute to the mechanism of this injury. The goal of the present study was to ascertain whether high plasma ascorbate levels could ameliorate the reperfusion injuries that occur after the successful restoration of blood flow. Methods: Patients from three clinical centers of the public health system were included in the study. The groups were formed by either-sex patients with a diagnosis of ST-segment elevation myocardial infarction with an indication for primary PCA. Only the patients who presented with their first myocardial infarction were enrolled. Ascorbate was administered through an infusion given prior to the restoration of the coronary flow, which was then followed by oral treatment with vitamin C (500 mg/12 hours) plus vitamin E (400 IU/day) for 84 days. The left ventricular ejection fraction (LVEF) was determined by using cardiac magnetic resonance on days 6 and 84 following the onset of the reperfusion. In addition, the microvascular function was assessed by an angiographic evaluation using the Thrombolysis In Myocardial Infarction (TIMI) myocardial perfusion grade (TMPG). The results were grouped according to the plasma ascorbate concentration achieved immediately following the onset of reperfusion into either the HA group (high ascorbate, >1 mmol/l) or the LA group (low ascorbate, <1 mmol/l). The biochemical parameters were analyzed throughout the protocol. Results: The LVEF of the HA group was significantly higher than that of the LA group, values on day 84 in the HA group were 33% higher than those of the LA group. The amelioration of the LVEF was accompanied by an improvement in the microvascular dysfunction, after PCA, 95% of the patients in the HA group achieved a TMPG of 2–3, in the LA group only 79% of patients showed a TMPG of 2–3. Conclusions: These data are consistent with the protective effect of high plasma levels of ascorbate against the oxidative challenge caused by reperfusion injury in patients subjected to PCA following an AMI. Further studies are needed to elucidate the mechanism accounting for this beneficial antioxidant effect.

Abstract 2288: Lipoprotein(a) and vitamin C affect the development of breast cancer tumors in Lp(a)+; Gulo-/- mice

Abstract Lipoprotein(a), composed of LDL and an adhesive protein apo(a), is produced in humans and primates, the species which lost an ability to synthesize vitamin C endogenously. We have shown earlier that Lp(a), due to its strong ECM binding properties, may be considered a biological ‘stability’ molecule for the structurally weakened connective tissue in the vascular wall. The development and progression of cancer is characterized by loss of ECM integrity which facilitates tumor growth and metastasis. We developed a unique mouse model lacking endogenous vitamin C production (Gulo-/-) and synthesizing human Lp(a) (lp(a)+), which has been used in this study to investigate the role of Lp(a) and other lipoproteins in cancer. The female Gulo-/-:Lp(a)+ and control wild type Balb/c mice were orthotopically inoculated with 4T1 breast cancer cells (500,000) The transgenic and control mice were divided into 4 different dietary groups in respect to dietary vitamin C intake: A) low ascorbate intake for 6 weeks; B) high ascorbate intake for 6 weeks; C) low ascorbate intake for 3 weeks followed by high ascorbate for 3 weeks; D)high ascorbate intake for 3 weeks followed by low ascorbate for 3 weeks. Control groups of Lp(a)+;Gulo(-/-) mice without tumor inoculation were put on the same Vitamin C regimens. Wild type controls included mice without and with 4T1 inoculation kept on regular mouse chow for 6 weeks. After 6 weeks all (100%) wild type mice developed tumors, while 50% of Lp(a)+;Gulo(-/-) mice kept on high ascorbate diet for 6 weeks did not develop primary tumors and in only a small number of mice some residual tumor cells or inflammatory infiltrates were detected in the lungs by histology. In transgenic mice supplemented with low vitamin C for 6 weeks, the reduction of primary tumors incidence was 33% compared to wild type mice. In addition, primary tumors from wild type mice were on average over 2-fold larger (1.80+/-0.62g) than tumors from Lp(a)+;Gulo(-/-) mice on continual low Vitamin C (0.77+/-0.98g) or continual high Vitamin C (0.63+/-1.09g. Primary tumors from Lp(a)+;Gulo(-/-) mice immunostained positively for Lp(a) and their size was inversely proportional Lp(a) cholesterol serum levels. Lp(a) could not be detected in tumors from wild type mice and the presence of tumors was associated with higher LDL serum levels. The results implicate that Lp(a) may play a role in controlling tumor growth and expansion through its ECM adhesive properties. Citation Format: John Cha, M. Waheed Roomi, Aleksandra Niedzwiecki, Matthias Rath. Lipoprotein(a) and vitamin C affect the development of breast cancer tumors in Lp(a)+; Gulo-/- mice. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2288. doi:10.1158/1538-7445.AM2015-2288

Abstract 2563: Modulation of DNA damage responses and cytotoxicity of bleomycin by vitamin C

Abstract Bleomycin (BL) is an iron-chelating chemical that is used for treatment of several types of cancer. The administration of this drug to patients is also associated with significant side effects, including life-threatening lung fibrosis. Biological factors affecting chemotherapeutic effectiveness of BL and the severity of its major side effects remain poorly understood. Cytotoxic effects of BL are attributed to its ability to cause DNA breaks via oxidative reactions by chelated iron. Ascorbate is a main cellular antioxidant, which can also promote redox cycling of iron by reducing its ferric form. Standard cultures of human cells contain less than 1% of physiological amounts of vitamin C, raising concerns whether common in vitro models adequately recapitulate major mechanistic aspects of BL toxicity in vivo. In this work, we investigated the effects of intracellular ascorbate on cell killing and DNA damage-activated stress responses by BL. We found that replenishment of human lung carcinoma cells with ascorbate increased cytotoxicity and clonogenic lethality of BL. Examination of DNA damage responses in BL-treated cells found that cellular ascorbate enhanced some signaling branches while suppressing others. Ascorbate-restored cells also displayed an increased dependence on nonhomologous end-joining DNA repair for their long-term survival. Overall, our results show that restoration of ascorbate levels increases sensitivity of cancer cells to killing by BL and alters their DNA damage responses. Low ascorbate levels in hypoxic tumors can contribute to their resistance to BL-based therapy. Citation Format: Blazej Rubis, Anatoly Zhitkovich. Modulation of DNA damage responses and cytotoxicity of bleomycin by vitamin C. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2563. doi:10.1158/1538-7445.AM2015-2563

Prolonged exposure to elevated temperature induces floral transition via up-regulation of cytosolic ascorbate peroxidase 1 and subsequent reduction of the ascorbate redox ratio in Oncidium hybrid orchid.

The bolting time of the Oncidium hybrid orchid is not season dependent and so it is a useful year-round model system to study thermal-induced flowering mechanisms in planta. Previously, we reported that a low ascorbate (AsA) content is essential for floral transition in Oncidium; however, the environmental factors governing initiation of the flowering process remained to be elucidated. The current study revealed that a prolonged elevated temperature treatment (30°C over a 14 d period) induces floral transition. This floral induction in response to thermal stress was associated with a significantly increased reactive oxygen species (ROS) level and a lowered AsA redox ratio, as well as prominently up-regulated expression of cytosolic ascorbate peroxidase (cytAPX1). Transcriptome analysis confirmed that increased temperature affected the differential expression of genes involved in antioxidant metabolism. Likewise, transgenic Arabidopsis ectopically overexpressing Oncidium cytAPX1 displayed an early-flowering phenotype and low AsA redox ratio under thermal stress, while cytAPX1 mutants, apx1-1 and apx1-2, exhibited a delayed-flowering phenotype and a high AsA redox ratio. Our present data illustrate that the floral transition response to thermal stress is mediated by the AsA redox ratio, and that CytAPX plays a pivotal role in modulating the AsA redox ratio in Oncidium hybrid orchid. Taken together, the results from this investigation of the thermal-induced flowering mechanism indicated that the AsA redox ratio is a master switch to mediate phase transition from the vegetative to reproductive stage.

Abstract 494: Tumor ascorbate content is associated with extended disease-free survival and decreased hypoxia-inducible factor-1 activation in patients with colorectal cancer

Abstract Ascorbate (vitamin C) is a specific cofactor for a range of dioxygenase enzymes, including those involved in histone and DNA demethylation and also the enzymes that ‘switch off’ hypoxia-inducible factor (HIF)-1. These processes have to potential to influence tumor biology, where inadequate intracellular ascorbate levels may contribute to tumor progression and an over-active HIF-1 survival response. We previously demonstrated an association between low ascorbate levels and high HIF-1 activation in clinical samples from patients with endometrial cancer. In order to determine whether this is a universal phenomenon, we have now proceeded to a second cancer type, colorectal cancer. Clinical, pathological and follow-up data, as well as samples of tumor and adjacent normal tissue, were collected from 50 consenting colorectal cancer patients. Protein levels of HIF-1α and three pro-survival target gene products, Bcl-2/adenovirus E1B 19 kDa interacting protein 3 (BNIP3), glucose transporter 1 (GLUT-1) and VEGF, were measured by Western blot or ELISA and related to the tumor ascorbate content. HIF-1α, BNIP3 and VEGF protein levels were all elevated in tumor compared to adjacent normal tissue (p&amp;lt;0.01), while ascorbate levels were lower in tumor tissue (p&amp;lt;0.001). There was a significant inverse relationship between the tumor ascorbate content and HIF-1 pathway activation (p=0.002) and tumor size (p=0.018). High grade tumors had up to 40% less ascorbate than adjacent normal tissue and also compared to low grade tumors. Patients with tumors containing low ascorbate had significantly shorter disease-free survival than patients with high levels of ascorbate in their tumors (p=0.04). The ability of tumors to accumulate ascorbate, according to the ratio of ascorbate in tumor compared to paired normal tissue, also tended to be associated with disease-free survival (p=0.08). Levels of ascorbate in normal tissue were not associated with disease-free survival (p=0.614). This is the first report demonstrating a relationship between ascorbate levels in human tumors and patient survival. This relationship may rely on the ability of ascorbate to promote dioxygenase activity and dampen the HIF survival response, as this is the second tumor type where low cellular ascorbate levels were associated with increased HIF-1 activation. An intervention clinical trial is now warranted to determine if tumor levels of ascorbate can be manipulated. Citation Format: Caroline Kuiper, Gabi Dachs, Margaret Currie, John Pearson, Delwyn Munn, Margreet Vissers. Tumor ascorbate content is associated with extended disease-free survival and decreased hypoxia-inducible factor-1 activation in patients with colorectal cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 494. doi:10.1158/1538-7445.AM2014-494

Differential Morpho-Agronomic and Physiological Responses of Grass Pea (Lathyrus sativus L.) and Lentil (Lens culinaris Medik.) Genotypes to Arsenic

Tolerance of four improved grass pea and lentil genotypes to arsenic (As) was tested in pot experiment using 30 mg As l -1 up to 60 days of growth after commencement of treatment (DAC). As exposure significantly reduced growth and seed yield of lentil variety VLM1 and VLM 4, but no such effect was observed in grass pea variety BioL-202 and PUSA-90-2. Results revealed normal leaf photosynthesis and antioxidant metabolism in both lentil varieties at 20 DAC. However, high superoxide dismutase activity coupled with low ascorbate and glutathione redox and declining ascorbate peroxidase as well as glutathione reductase (GR) level led to abnormal rise in H2O2 content at reproductive stages (40 and 60 DAC), consequently, resulting in significantly enhanced As-induced oxidative damage and physiological impediment in both varieties. By contrast, H2O2 level in both the grass pea varieties were modulated in fine tune at reproductive stage by balanced action of H2O2-metabolizing defense, powered by effective regeneration of glutathione through normal to high GR activity. This favored plant growth and ensured good grain yield in grass pea varieties even after prolonged As-exposure by effectively preventing oxidative damage. Results revealed criticality of foliar H2O2 level in determining As-tolerance of lentil and grass pea genotypes.

Growth Responses and Leaf Antioxidant Metabolism of Grass Pea (Lathyrus sativus L.) Genotypes under Salinity Stress

Response of six improved grass pea genotypes to prolonged salinity stress was investigated on seedlings grown in pot experiment using 150 mM NaCl up to 60 days of growth after commencement of treatment (DAC). NaCl exposure significantly reduced growth potential of varieties PUSA-90-2 and WBK-CB-14, but no such effect was observed in varieties B1, BioL-212 and in two mutant lines LR3 and LR4. A time-bound measurement at 15, 30 and 60 DAC revealed significant reduction in plant dry matter production, orchestrated through abnormally low capacity of leaf photosynthesis accompanied by low K+/Na+ ratio and onset of oxidative stress in all six genotypes at 15 DAC and the extension of the phenomena in PUSA-90-2 and WBK-CB-14 to 60 DAC. High superoxide dismutase (SOD) activity coupled with low ascorbate redox and declining ascorbate peroxidase (APX) and catalases (CAT) levels led to abnormal rise in H2O2 content at reproductive stage (30 DAC) in the latter two genotypes, consequently, resulting in NaCl-induced oxidative damage. H2O2 level in the rest of the four genotypes was modulated in a controlled way by balanced action of SOD, APX and CAT, preventing oxidative damage even under prolonged NaCl-exposure. Enzyme isoforms were involved in regulation of foliar H2O2-metabolism, which was critical in determining As tolerance of grass pea genotypes.

Higher sensitivity of pad2-1 and vtc2-1 mutants to cadmium is related to lower subcellular glutathione rather than ascorbate contents

Cadmium (Cd) interferes with ascorbate and glutathione metabolism as it induces the production of reactive oxygen species (ROS), binds to glutathione due to its high affinity to thiol groups, and induces the production of phytochelatins (PCs) which use glutathione as a precursor. In this study, changes in the compartment specific distribution of ascorbate and glutathione were monitored over a time period of 14 days in Cd-treated (50 and 100 μM) Arabidopsis Col-0 plants, and two mutant lines deficient in glutathione (pad2-1) and ascorbate (vtc2-1). Both mutants showed higher sensitivity to Cd than Col-0 plants. Strongly reduced compartment specific glutathione, rather than decreased ascorbate contents, could be correlated with the development of symptoms in these mutants suggesting that higher sensitivity to Cd is related to low glutathione contents rather than low ascorbate contents. On the subcellular level it became obvious that long-term treatment of wildtype plants with Cd induced the depletion of glutathione and ascorbate contents in all cell compartments except chloroplasts indicating an important protective role for antioxidants in chloroplasts against Cd. Additionally, we could observe an immediate decrease of glutathione and ascorbate in all cell compartments 12 h after Cd treatment indicating that glutathione and ascorbate are either withdrawn from or not redistributed into other organelles after their production in chloroplasts, cytosol (production centers for glutathione) and mitochondria (production center for ascorbate). The obtained data is discussed in respect to recently proposed stress models involving antioxidants in the protection of plants against environmental stress conditions.Electronic supplementary materialThe online version of this article (doi:10.1007/s00709-013-0576-x) contains supplementary material, which is available to authorized users.

Plant Growth and Leaf Antioxidant Metabolism of Four Elite Grass Pea (Lathyrus sativus) Genotypes, Differing in Arsenic Tolerance

Response of four improved grass pea (Lathyrus sativus L.) genotypes to arsenic was tested in pot experiment using 30 mg As l−1 up to 60 days of growth after commencement of treatment (DAC). Arsenic exposure significantly reduced growth and seed yield potential of vars B1 and BioL-212, but no such effect was observed in bold-seeded mutant and pod indehiscent mutant lines. Results revealed normal leaf photosynthesis and antioxidant metabolism at 20 DAC in both varieties. However, high superoxide dismutase activity coupled with low ascorbate redox and declining ascorbate peroxidase level led to abnormal rise in H2O2 content at reproductive stages (40 and 60 DAC), consequently, resulting in significantly enhanced arsenic-induced oxidative damage and physiological impediment in both varieties. By contrast, H2O2 level in both the mutants was remarkably modulated at reproductive stage (40 DAC) in a highly controlled manner by balanced action of antioxidant defense. This favored normal photosynthesis and ensured good grain yield even under prolonged arsenic exposure by effectively preventing oxidative damage to membrane in both the mutants. Native PAGE analysis revealed that enzyme isoforms were involved in the regulation of foliar H2O2 metabolism, the level of which was found extremely critical in determining arsenic tolerance of grass pea genotypes.

Decreased Plasma Ascorbate Levels in Stage IV Melanoma Patients

It has been reported that cancer patients frequently express low ascorbate (ascorbic acid, vitamin C) blood levels. However, so far this was not shown for melanoma patients. Total ascorbate (TAA) levels were determined in plasma of healthy control individuals (n=31, mean age: 47.3 years, TAA: 64.86 µM) and in 126 melanoma patients (stage I: n=30, mean age: 51 years, TAA: 59.95 µM; stage II: n=30, mean age: 46.8 years, TAA: 58.85 µM; stage III: n=32, mean age: 48.6 years, TAA: 57.27 µM; stage IV: n=34, mean age: 51.1 years, TAA: 47.16 µM). Plasma TAA levels in stage IV patients were significantly reduced by 27.3% when compared to healthy individuals (p=0.0001, t-test). The reduced plasma TAA levels in stage IV patients negatively correlated with increased S100 and lactate dehydrogenase (LDH) levels. Further, plasma TAA levels were determined in additional 9 stage IV patients directly before and 24 h after intravenous polychemotherapy (carboplatin+paclitaxel, n=5) or immunotherapy (ipilimumab, n=4). TAA levels significantly decreased 24 h after therapy (mean TAA before therapy: 48.7 µM; mean TAA after therapy: 43.0 µM; 11.4% reduction, p Ascorbate levels in the plasma of 126 melanoma patients were significantly decreased in the cohort of stage IV patients and were further decreased by polychemo- or immunotherapy in stage IV patients. Considering the importance of adequate ascorbate supply, ascorbate substitution in physiological doses could be considered for late-stage melanoma patients.

Effect of atmospheric CO2 enrichment on the drought response of barley, durum wheat and oat

Phytotron experiments were conducted to examine the impact of elevated atmospheric CO2 level (750 μmol mol−1) on the drought tolerance of winter barley (Petra), durum wheat (Mv Makaroni) and spring oat (Mv Pehely) varieties. Under drought stress conditions, the durum wheat variety was found to be unaffected by CO2 enrichment, as neither the biomass or grain yield nor the antioxidant enzyme activities changed compared to those at ambient CO2. Despite the fact that the spring oat variety had similar grain yield loss due to drought at both CO2 levels, it exhibited reduced antioxidant enzyme activities under less severe drought, indicating a slightly increased tolerance to drought. Winter barley, which exhibited an extremely positive reaction to CO2 enrichment at the control water supply level, also showed increased drought tolerance in response to high CO2. It had low glutathione reductase, glutathione-S-transferase and ascorbate peroxidase activities even at the most severe drought stress levels, while it could also fully compensate for the negative effects of drought on biomass and grain yield parameters when grown at elevated CO2.

BIOREACTIVE COMPOUNDS AND ANTIOXIDANT PROPERTIES OF METHANOLIC EXTRACT OF FENNEL (FOENICULUM VULGARE MILLER)

Methanolic extract of dried Fennel (Foeniculum vulgare Miller) seeds was analyzed for the presence of bioreactive compounds: ascorbate, riboflavin, tocopherol and polyphenols with potential antioxidant properties. The extract had low ascorbate (197.12+1.82μg), riboflavin (11.97+0.35μg) and tocopherol (280.33+5.67μg/g dry seeds) content. However the extract had high polyphenol content (16.506+0.32mg/g dry seeds). Antioxidant activity of the extract was determined by various mechanisms including DPPH free radical scavenging, metal induced protein and lipid oxidation inhibition and protection of DNA against H2O2 induced damage. Fennel had excellent free radical scavenging activity with IC50 2.1mg dry seed weight. IC50 observed for protection of proteins and lipids against metal ion induced oxidation is 2.1 and 2.5mg dry seed weight respectively. Extract equivalent to 0.5μg seeds is enough to protect DNA against H2O2 induced oxidation. The results suggest that polyphenols are the principal components responsible for high antioxidant potential of methanolic extract of fennel.

Balanced Hydrogen peroxide Metabolism is Central in Controlling NaCl-Induced Oxidative Stress in Medicinal Legume, Fenugreek (Trigonella foenum-graecum L.)

Fenugreek, a medicinal and spice-yielding legume, is increasingly facing threats of salinity-induced oxidative stress. The aim of the present study was to test the antioxidant defense response responsible for H2O2-metabolism in two improved fenugreek cultivars to prolonged salinity treatment. A pot experiment was designed in controlled environment using 100 mM NaCl up to 60 days of commencement of treatment (DAS) in a completely randomized block design with four replicates. Foliar ascorbate (AsA)-redox pool along with activities of superoxide dismutase (SOD), ascorbate peroxidase (APX) and catalases (CAT) was assayed at 15, 40 and 60 DAS keeping a control in each set. High SOD activity coupled with low ascorbate redox and declining APX and CAT level in variety Azad led to abnormal rise in H2O2 content at reproductive stage (40 and 60 DAS), consequently, resulting in NaCl-induced oxidative damage due to high level of lipid peroxidation. Despite initial low H2O2 level, variety RM-16 experienced onset of oxidative stress at early vegetative stage but remarkably bounced back to normalcy at re- productive stage of growth exhibiting controlled rise of H2O2 level and decrease in lipid peroxidation as well as electrolyte leakage. Results revealed H2O2 level was modulated in fine tune by balanced action of SOD and APX, preventing oxidative damage in RM-16 even under prolonged NaCl-exposure. Enzyme isoforms were involved in regulation of foliar H2O2-metabolism, the balance of which was found extremely critical in determining NaCl-tolerance of fenugreek genotypes.