Drought stress induces oxidative stress and the antioxidant defense system in ascorbate-deficient vtc1 mutants of Arabidopsis thaliana

Drought stress has a negative impact on plant cells and results in the generation of reactive oxygen species (ROS). To increase our understanding of the effects of drought stress on antioxidant processes, we investigated the response of the ascorbate-deficient Arabidopsis thaliana vtc1 mutant to drought stress. After drought stress, vtc1 mutants exhibited increases in several oxidative parameters, including H₂O₂ content and the production of thiobarbituric acid reactive substances. Decreases in chlorophyll content and chlorophyll fluorescence parameters were also observed. The vtc1 mutants had higher total glutathione than did wild-type (WT) plants after 48 h of drought stress. A reduced ratio of glutathione/total glutathione and an increased ratio of dehydroascorbate/total ascorbate were observed in the vtc1 mutants compared with the WT plants. In addition, the activities of enzymes that are responsible for ROS scavenging, including superoxide dismutase, catalase, and ascorbate peroxidase, were decreased in the vtc1 mutants compared with the WT plants. Similar reductions in activity in the vtc1 mutant were observed for the enzymes that are responsible for the regeneration of ascorbate and glutathione, including monodehydroascorbate reductase, dehydroascorbate reductase, and glutathione reductase. These results suggest that low intrinsic ascorbate and impaired ascorbate–glutathione cycling in the vtc1 mutant induced a decrease in the reduced form of ascorbate, which enhanced sensitivity to drought stress.     

Ascorbate plays a key role in alleviating low temperature-induced oxidative stress in Arabidopsis

Low temperature has a negative impact on plant cells and results in the generation of reactive oxygen species (ROS). In order to study the role of ascorbate under chilling stress, the response of an ascorbate-deficient Arabidopsis thaliana mutant vtc2-1 to low temperature (2°C) was investigated. After chilling stress, vtc2-1 mutants exhibited oxidative damage. An increase in the H₂O₂ generation and the production of thiobarbituric acid reactive substances (TBARS), and a decrease in chlorophyll content, the maximal photochemical efficiency of PSII (F_v/F_m) and oxidizable P₇₀₀ were also noted. The ratio of ascorbate/dehydroascorbate and reduced glutathione/oxidzed glutathione in the vtc2-1 mutants were reduced, compared with the wild type (WT) plants. The activities of antioxidant enzymes, such as catalase (CAT) and ascorbate peroxidase (APX), and soluble antioxidants were lower in the vtc2-1 mutants than those in WT plants. These results suggested that the ascorbate-deficient mutant vtc2-1 was more sensitive to chilling treatment than WT plants. The low temperature-induced oxidative stress was the major cause of the decrease of PSII and PSI function in the vtc2-1 mutants. Ascorbate plays a critical role of defense without which the rest of the ROS defense network is unable to react effectively.     

Ascorbate-deficient mutants of Arabidopsis grow in high light despite chronic photooxidative stress

Acclimation to changing environments, such as increases in light intensity, is necessary, especially for the survival of sedentary organisms like plants. To learn more about the importance of ascorbate in the acclimation of plants to high light (HL), vtc2, an ascorbate-deficient mutant of Arabidopsis, and the double mutants vtc2npq4 and vtc2npq1 were tested for growth in low light and HL and compared with the wild type. The vtc2 mutant has only 10% to 30% of wild-type levels of ascorbate, vtc2npq4 has lower ascorbate levels and lacks non-photochemical quenching of chlorophyll fluorescence (NPQ) because of the absence of the photosystem II protein PsbS, and vtc2npq1 is NPQ deficient and also lacks zeaxanthin in HL but has PsbS. All three genotypes were able to grow in HL and had wild-type levels of Lhcb1, cytochrome f, PsaF, and 2-cysteine peroxiredoxin. However, the mutants had lower electron transport and oxygen evolution rates and lower quantum efficiency of PSII compared with the wild type, implying that they experienced chronic photooxidative stress. The mutants lacking NPQ in addition to ascorbate were only slightly more affected than vtc2. All three mutants had higher glutathione levels than the wild type in HL, suggesting a possible compensation for the lower ascorbate content. These results demonstrate the importance of ascorbate for the long-term acclimation of plants to HL.     

Increased sensitivity to salt stress in an ascorbate-deficient Arabidopsis mutant

The Arabidopsis thaliana ascorbate-deficient vtc-1 mutant has only 30% ascorbate contents of the wild type (WT). This ascorbate-deficient mutant was used here to study the physiological roles of ascorbate under salt stress in vivo. Salt stress resulted in a more significant decrease in CO2 assimilatory capacity in the vtc-1 mutant than in the WT. Photosystem II function in the Arabidopsis vtc-1 mutant also showed an increased sensitivity to salt stress. Oxidative stress, indicated by the hydrogen peroxide content, increased more dramatically in the vtc-1 mutant than in the WT under salt stress. To clarify the reason for the increased oxidative stress in the vtc-1 mutant, the contents of small antioxidant compounds and the activities of several antioxidant enzymes in the ascorbate-glutathione cycle were measured. Despite an elevated glutathione pool in the vtc-1 mutant, the ascorbate contents and the reduced form of ascorbate decreased very rapidly under salt stress. These results showed that the activities of MDAR and DHAR were lower in the vtc-1 mutant than in the WT under salt stress. Thus, low intrinsic ascorbate and an impaired ascorbate-glutathione cycle in the vtc-1 mutant under salt stress probably induced a dramatic decrease in the reduced form of ascorbate, which resulted in both enhanced ROS contents and decreased NPQ in the vtc-1 mutant.     

Alterations in tocopherol cyclase activity in transgenic and mutant plants of Arabidopsis affect tocopherol content, tocopherol composition, and oxidative stress

Tocopherol belongs to the Vitamin E class of lipid soluble antioxidants that are essential for human nutrition. In plants, tocopherol is synthesized in plastids where it protects membranes from oxidative degradation by reactive oxygen species. Tocopherol cyclase (VTE1) catalyzes the penultimate step of tocopherol synthesis, and an Arabidopsis (Arabidopsis thaliana) mutant deficient in VTE1 (vte1) is totally devoid of tocopherol. Overexpression of VTE1 resulted in an increase in total tocopherol of at least 7-fold in leaves, and a dramatic shift from alpha-tocopherol to gamma-tocopherol. Expression studies demonstrated that indeed VTE1 is a major limiting factor of tocopherol synthesis in leaves. Tocopherol deficiency in vte1 resulted in the increase in ascorbate and glutathione, whereas accumulation of tocopherol in VTE1 overexpressing plants led to a decrease in ascorbate and glutathione. Deficiency in one antioxidant in vte1, vtc1 (ascorbate deficient), or cad2 (glutathione deficient) led to increased oxidative stress and to the concomitant increase in alternative antioxidants. Double mutants of vte1 were generated with vtc1 and cad2. Whereas growth, chlorophyll content, and photosynthetic quantum yield were very similar to wild type in vte1, vtc1, cad2, or vte1vtc1, they were reduced in vte1cad2, indicating that the simultaneous loss of tocopherol and glutathione results in moderate oxidative stress that affects the stability and the efficiency of the photosynthetic apparatus.     

Zeaxanthin deficiency enhances the high light sensitivity of an ascorbate-deficient mutant of Arabidopsis

The ascorbate content of plants is usually increased in high light (HL), implying a function for ascorbate in the acclimation of plants to HL. Nevertheless, the importance of ascorbate in HL acclimation has not yet been tested directly. Here, we report on the acclimation process of an ascorbate-deficient Arabidopsis mutant to HL. The mutant vtc2 has only 10% to 30% of wild-type levels of ascorbate, and it is also slightly deficient in feedback de-excitation (qE), a photoprotective mechanism that causes the dissipation of excess light as heat. The vtc2 mutant was unable to acclimate to HL, when transferred from low light to HL. Its mature leaves bleached, and it showed an increased degree of lipid peroxidation and photoinhibition. In parallel, we tested the photosensitivity of an ascorbate-deficient xanthophyll cycle mutant, vtc2npq1, which also lacks zeaxanthin and nearly all qE. The double mutant bleached sooner and had higher degrees of lipid peroxidation and photoinhibition than the vtc2 mutant. This was in contrast to the npq1 single mutant that showed only slight deviations from the wild-type phenotype under the conditions used. These results demonstrate the antioxidant role of ascorbate in the acclimation process to HL and point to the relative importance of ascorbate in comparison with other photoprotective processes, such as specific xanthophylls or feedback de-excitation. The results also provide further support for the proposed role of zeaxanthin as an antioxidant and lipid stabilizer.     

Role of salicylic acid in regulating ultraviolet radiation-induced oxidative stress in pepper leaves

The effect of salicylic acid (SA) counteracting the UV-A, UV-B, and UV-C-induced action on pepper (Capsicum annuum L.) plants was studied. For this purpose, the activities of antioxidant enzymes (peroxidase, polyphenol oxidase, ascorbate peroxidase, catalase, and glutathione reductase) were measured. Plants were sprayed with SA and treated with UV-A (320–390 nm), UV-B (312 nm), and UV-C (254 nm) radiation with a density of 6.1, 5.8, and 5.7 W/m². The activities of antioxidant enzymes were enhanced in leaves in response to UV-B and UV-C radiation. SA treatment moderated an increase in the activities of some antioxidant enzymes (peroxidase, ascorbate peroxidase, catalase, and glutathione reductase) in plants that were treated with UV radiation. The activity of antioxidant enzyme polyphenol oxidase in plants that were treated with UV-B, UV-C, and SA was significantly increased. The aim of the present study was to investigate the possible protective effect of SA treatment on UV-A, UV-B, and UV-C stress.     

Ascorbate deficient semi-dwarf asfL1 mutant of Lathyrus sativus exhibits alterations in antioxidant defense

An ascorbate-deficient semi-dwarf mutant asfL-1 was detected in 250 Gy γ-ray treated grass pea (Lathyrus sativus L.) cv. BioR-231. The mutant contained only 42 % of leaf and 20 % of root ascorbate content of mother control (MC). I investigated the possible causes of ascorbate deficiency and its effect on growth and antioxidant defense in control and 150 mM NaCl-treated seedling after 60 d growth period. Ascorbate deficiency was due to significant reduction in activities of monodehydroascorbate reductase and dehydroascorbate reductase as well as increase in ascorbate oxidase, leading to considerable decrease in redox state. Despite low ascorbate pool and decrease in ascorbate peroxidase activity, shoot and root biomass production in asfL-1 mutant were similar to MC plants, even at NaCl treatment. High accumulation of glutathione (GSH) coupled with high activities of GSH reductase, catalase, GSH peroxidase and peroxidase in both tissues of the mutant permitted efficient recycling of GSH and scavenging of H₂O₂ through well integrated catalase/peroxidase system, despite high superoxide dismutase activity under NaCl treatment. The collapse of this system led to inhibition of growth in NaCl-treated mother plants. Together, the results suggested that asfL-1 plants undertook a major reshuffle in its antioxidant defense machinery, which effectively counterbalanced the negative impact of ascorbate deficiency and remained unperturbed by NaCl treatment to maintain normal growth and biomass production.     

Exogenously applied salicylic acid maintains redox homeostasis in salt-stressed <Emphasis Type="Italic">Arabidopsis gr1</Emphasis> mutants expressing cytosolic roGFP1

Exogenous salicylic acid (SA) can be used for chemical hardening to alleviate oxidative stress in plants exposed to salinity. The treatment of 5-week-old Arabidopsis thaliana plants with increasing doses of SA alters the ascorbate (ASC) and glutathione (GSH) pools, and modulates their redox status and the activity of several antioxidant enzymes, such as ascorbate peroxidase (APX) and glutathione reductase (GR). To investigate the role of GR in the maintenance of cytoplasmic redox homeostasis after hardening by SA, wild type (WT) and gr1 mutant plants, expressing the cytoplasmic redox-sensitive green fluorescent protein (c-roGFP1), were pre-treated with 10^?7 and 10^?5 M SA for 2 weeks and subsequently exposed to 100 mM NaCl. The redox status of the salt-stressed WT plants became more oxidized, which was prevented by pretreatment with 10^?5 M SA. The gr1 mutants showed more positive redox potential than WT plants, which could be reversed by treatment with 10^?5 M SA. In mutants, the increased GSH levels may have compensated for the deleterious effect of GR deficiency and stabilized the redox potential in plants exposed to salinity. The ASC regeneration in WT plants shifted from the GSH-dependent dehydroascorbate reductase (DHAR) reaction to the NAD(P)H-dependent monodehydroascorbate reductase (MDHAR) activity during chemical hardening, which contributed to the preservation of the GSH pool in plants under salt stress. Our results suggest that the maintenance of GSH levels and redox homeostasis by SA-mediated hardening play a major role in priming and defending against salt stress.     

Ultraviolet-B- and ozone-induced biochemical changes in antioxidant enzymes of Arabidopsis thaliana.

Earlier studies with Arabidopsis thaliana exposed to ultraviolet B (UV-B) and ozone (O3) have indicated the differential responses of superoxide dismutase and glutathione reductase. In this study, we have investigated whether A. thaliana genotype Landsberg erecta and its flavonoid-deficient mutant transparent testa (tt5) is capable of metabolizing UV-B- and O3-induced activated oxygen species by invoking similar antioxidant enzymes. UV-B exposure preferentially enhanced guaiacol-peroxidases, ascorbate peroxidase, and peroxidases specific to coniferyl alcohol and modified the substrate affinity of ascorbate peroxidase. O3 exposure enhanced superoxide dismutase, peroxidases, glutathione reductase, and ascorbate peroxidase to a similar degree and modified the substrate affinity of both glutathione reductase and ascorbate peroxidase. Both UV-B and O3 exposure enhanced similar Cu,Zn-superoxide dismutase isoforms. New isoforms of peroxidases and ascorbate peroxidase were synthesized in tt5 plants irradiated with UV-B. UV-B radiation, in contrast to O3, enhanced the activated oxygen species by increasing membrane-localized NADPH-oxidase activity and decreasing catalase activities. These results collectively suggest that (a) UV-B exposure preferentially induces peroxidase-related enzymes, whereas O3 exposure invokes the enzymes of superoxide dismutase/ascorbate-glutathione cycle, and (b) in contrast to O3, UV-B exposure generated activated oxygen species by increasing NADPH-oxidase activity.     

Fungal pathogen-induced changes in the antioxidant systems of leaf peroxisomes from infected tomato plants

Peroxisomes, being one of the main organelles where reactive oxygen species (ROS) are both generated and detoxified, have been suggested to be instrumental in redox-mediated plant cell defence against oxidative stress. We studied the involvement of tomato (Lycopersicon esculentum Mill.) leaf peroxisomes in defence response to oxidative stress generated upon Botrytis cinerea Pers. infection. The peroxisomal antioxidant potential expressed as superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6) and glutathione peroxidase (GSH-Px, EC 1.11.1.19) as well as the ascorbate-glutathione (AA-GSH) cycle activities was monitored. The initial infection-induced increase in SOD, CAT and GSH-Px indicating antioxidant defence activation was followed by a progressive inhibition concomitant with disease symptom development. Likewise, the activities of AA-GSH cycle enzymes: ascorbate peroxidase (APX, EC 1.11.1.11), monodehydroascorbate reductase (MDHAR, EC 1.6.5.4), dehydroascorbate reductase (DHAR, EC 1.8.5.1) and glutathione reductase (GR, EC 1.6.4.2) as well as ascorbate and glutathione concentrations and redox ratios were significantly decreased. However, the rate and timing of these events differed. Our results indicate that B. cinerea triggers significant changes in the peroxisomal antioxidant system leading to a collapse of the protective mechanism at advanced stage of infection. These changes appear to be partly the effect of pathogen-promoted leaf senescence.     

Effect of sodium nitroprusside and S-nitrosoglutathione on pigment content and antioxidant system of tocopherol-deficient plants of Arabidopsis thaliana

Sodium nitroprusside (SNP) and S-nitrosoglutathione (GSNO) were used as a source of exogenous nitric oxide (NO) to investigate their effects on biochemical parameters and antioxidant enzyme response in leaves of wild type Columbia and tocopherol-deficient vte4 and vte1 mutant lines of Arabidopsis thaliana plants and possible tocopherol involvement in regulation of antioxidant response under NO-induced stress. SNP enhanced the activity of the enzymes, that scavenge hydrogen peroxide in leaves of all studied lines, and increased glutathione reductase and glutathione-S-transferase activity there. In addition, it decreased the intensity of lipid peroxidation in vte1 mutant line leaves. At the same time, GSNO increased the levels of protein carbonyls and inactivated enzymes ascorbate peroxidase, guaiacol peroxidase and dehydroascorbate reductase in almost all investigated plant lines. In contrast to wild type, GSNO increased superoxide dismutase activity and decreased catalase activity and chlorophyll a/b ratio in the leaves of two mutant lines. It can be assumed that tocopherols in some way are responsible for plant protection against NO-induced stress. However the mechanisms of this protection remain unknown.     

Ascorbic acid in plants: biosynthesis and function

Ascorbic acid (vitamin C) is an abundant component of plants. It reaches a concentration of over 20 mM in chloroplasts and occurs in all cell compartments, including the cell wall. It has proposed functions in photosynthesis as an enzyme cofactor (including synthesis of ethylene, gibberellins and anthocyanins) and in control of cell growth. A biosynthetic pathway via GDP-mannose, GDP-L-galactose, L-galactose, and L-galactono-1,4-lactone has been proposed only recently and is supported by molecular genetic evidence from the ascorbate-deficient vtc 1 mutant of Arabidopsis thaliana. Other pathways via uronic acids could provide minor sources of ascorbate. Ascorbate, at least in some species, is a precursor of tartrate and oxalate. It has a major role in photosynthesis, acting in the Mehler peroxidase reaction with ascorbate peroxidase to regulate the redox state of photosynthetic electron carriers and as a cofactor for violaxanthin de-epoxidase, an enzyme involved in xanthophyll cycle-mediated photoprotection. The hypersensitivity of some of the vtc mutants to ozone and UV-B radiation, the rapid response of ascorbate peroxidase expression to (photo)-oxidative stress, and the properties of transgenic plants with altered ascorbate peroxidase activity all support an important antioxidative role for ascorbate. In relation to cell growth, ascorbate is a cofactor for prolyl hydroxylase that posttranslationally hydroxylates proline residues in cell wall hydroxyproline-rich glycoproteins required for cell division and expansion. Additionally, high ascorbate oxidase activity in the cell wall is correlated with areas of rapid cell expansion. It remains to be determined if this is a causal relationship and, if so, what is the mechanism. Identification of the biosynthetic pathway now opens the way to manipulating ascorbate biosynthesis in plants, and, along with the vtc mutants, this should contribute to a deeper understanding of the proposed functions of this multifaceted molecule.     

Antioxidant defences of the apoplast

Summary The apoplast of barley and oat leaves contained superoxide dismutase (SOD), catalase, ascorbate peroxidase, dehydroascorbate reductase, monodehydroascorbate reductase, and glutathione reductase activities. The activities of these enzymes in the apoplastic extracts were greatly modified 24 h after inoculation with the biotrophic fungal pathogenBlumeria graminis. The quantum efficiency of photosystem II, which is related to photosynthetic electron transport flux, was comparable in inoculated and healthy leaves during this period. Apoplastic soluble acid invertase activity was also modified in inoculated leaves. Inoculation-dependent increases in apoplastic SOD activity were observed in all lines. Major bands of SOD activity, observed in apoplastic protein extracts by activity staining of gels following isoelectric focusing, were similar to those observed in whole leaves but two additional minor bands were found in the apoplastic fraction. The apoplastic extracts contained substantial amounts of dehydroascorbate (DHA) but little or no glutathione (GSH). Biotic stress decreased apoplastic ascorbate and DHA but increased apoplastic GSH in resistant lines. The antioxidant cycle enzymes may function to remove apoplastic H₂O₂ with ascorbate and GSH derived from the cytoplasm. DHA and oxidized glutathione may be reduced in the apoplast or returned to the cytosol for rereduction.Abbreviations AA reduced ascorbate - APX ascorbate peroxidase - DHA dehydroascorbate (oxidised ascorbate) - DHAR dehydroascorbate reductase - G6PDH glucose-6-phosphate dehydrogenase - GSH reduced glutathione - GSSG glutathione disulphide - GR glutathione reductase - MDHA monodehydroascorbate - MDHAR monodehydroascorbate reductase - SOD superoxide dismutase     

Redox regulation of ascorbate and glutathione by a chloroplastic dehydroascorbate reductase is required for high-light stress tolerance in Arabidopsis

Chloroplasts are a significant site for reactive oxygen species production under illumination and, thus, possess a well-organized antioxidant system involving ascorbate. Ascorbate recycling occurs in different manners in this system, including a dehydroascorbate reductase (DHAR) reaction. We herein investigated the physiological significance of DHAR3 in photo-oxidative stress tolerance in Arabidopsis. GFP-fused DHAR3 protein was targeted to chloroplasts in Arabidopsis leaves. A DHAR3 knockout mutant exhibited sensitivity to high light (HL). Under HL, the ascorbate redox states were similar in mutant and wild-type plants, while total ascorbate content was significantly lower in the mutant, suggesting that DHAR3 contributes, at least to some extent, to ascorbate recycling. Activation of monodehydroascorbate reductase occurred in dhar3 mutant, which might compensate for the lack of DHAR3. Interestingly, glutathione oxidation was consistently inhibited in dhar3 mutant. These findings indicate that DHAR3 regulates both ascorbate and glutathione redox states to acclimate to HL.     

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.