Cytosolic ascorbate peroxidase from Arabidopsis thaliana L. is encoded by a small multigene family

A second cytosolic ascorbate peroxidase (cAPX; EC 1.11.1.11) gene from Arabidopsis thaliana has been characterised. This second gene (designated APX1b) maps to linkage group 3 and potentially encodes a cAPX as closely related to that from other dicotyledonous species as to the other member of this gene family (Kubo et al, 1993, FEBS Lett 315: 313–317; here designated APX1a), which maps to linkage group 1. In contrast, the lack of sequence similarity in non-coding regions of the genes implies that they are differentially regulated. Under non-stressed conditions only APX1a is expressed. APX1b was identified during low-stringency probing using a cDNA coding for pea cAPX which, in turn, was recovered from a cDNA library by immunoscreening with an antiserum raised against tea plastidial APX (pAPX). No pAPX cDNAs were recovered, despite the antiserum displaying specificity for pAPX in Western blots.Abbreviations ATG methionine translation initiation codon - bp base pair - cAPX cytosolic ascorbate peroxidase - pAPX plastidial ascorbate peroxidase - RFLP restriction fragment length polymorphism Accession numbers: The APX1b sequence is in the EMBL database under accession number X80036M.S. gratefully acknowledges the support from the Junta Nacional de Investigaçâo Cientifica e Tecnológia, Portugal (grant number BD/394/90-IE). This work was supported by the Biotechnological and Biological Sciences Research Council through a grant-in-aid to the John Innes Centre.     

Changes in expression of the cytosolic ascorbate peroxidase gene,ca-capx1, during germination and development of hot pepper seedlings

Ascorbate peroxidase (APX), an antioxidant enzyme, scavenges H₂O₂ that is produced by normal metabolism and cellular oxidative stresses. To investigate its role during germination and seedling growth, we isolated a cDNA encoding cytosolic APX (cAPX) in hot pepper(Capsicum annuum L). The full-length clone,Ca- cAPX1, is 1011 bp long and has an ORF encoding 249 amino acid residues. During seedling development, cAPX activity and expression levels were higher at Days 5 and 6 post-imbibition, respectively, whereas those of catalase (CAT) increased at Days 8 and 10. The increased amount of H₂O₂ in that early developmental stage (Day 5) may have been counteracted mainly by APX, and further removed by CAT in cooperation with APX. To determine whether the accumulation of H₂O₂ via suppression ofcAPX expression might be a factor in stimulating germination, we constructed a transformant ofCaAPX1. Compared with the wild type, the germination rate for the antisense-suppressedArabidopsis increased by 26%, while its H₂O₂ content rose by 50%. Therefore, we propose that the pre-germination suppression ofcAPX expression stimulates seed germination by promoting the accumulation of H₂O₂.     

Stable Form of Ascorbate Peroxidase from the Red Alga Galdieria partita Similar to Both Chloroplastic and Cytosolic Isoforms of Higher Plants

Depletion of the electron donor ascorbate causes rapid inactivation of chloroplastic ascorbate peroxidase (APX) of higher plants, while cytosolic APX is stable under such conditions. Here we report the cloning of cDNA from Galdieria partita, a unicellular red alga, encoding a novel type of APX (APX-B). The electrophoretic mobility, K _m values, k _cat and absorption spectra of recombinant APX-B produced in Escherichia coli were measured. Recombinant APX-B remained active for at least 180 min after depletion of ascorbate. The amino-terminal half of APX-B, which forms the distal pocket of the active site, was richer in amino acid residues conserved in chloroplastic APXs of higher plants rather than cytosolic APXs. In contrast, the sequence of the carboxyl-terminal half, which forms the proximal pocket, was similar to that of the cytosolic isoform. The stability of APX-B might be due to its cytosolic isoform-like structure of the carboxyl-terminal half.     

Alleviation of ultraviolet-C-induced oxidative damage through overexpression of cytosolic ascorbate peroxidase

Experiments were conducted to investigate the relationship between ultraviolet (UV) C-induced oxidative damage and the activity of ascorbate peroxidase (APX), using transgenic tobacco (Nicotiana tabacum L. cv. Petit Havana) plants overexpressing cytosolic APX gene (apx1). Transgenic plants having 2.3 fold higher total APX activity, as compared to the wild type plants, showed normal morphological characters. Exposure of 70-day-old plants to fixed intensity UV-C radiation caused an increase in the malondialdehyde (MDA) content in wild type as well as transgenic plants. However, the wild type plants showed significantly higher (p < 0.05) lipid peroxidation as compared to the transgenic plants. Higher proline accumulation was recorded in transgenic plants as compared to the wild type plants, after 24 hours of UV-C exposure. Although the ascorbate content decreased continuously with increasing exposure to UV-C radiation, yet the wild type plants exhibited higher ascorbate levels than the transgenic plants. A marked difference in H₂O₂ content, between the wild type and transgenic plants, was consistently observed up to 20 hours of UV-C exposure. A direct correlation of ascorbate, MDA and H₂O₂ levels was recorded with the extent of oxidative stress, signifying that these could be used as potential bio-marker molecules for oxidative stress. The results clearly demonstrate that overexpression of cytosolic APX can protect tobacco plants from UV-C-induced oxidative damage.     

Cloning of a cytosolic ascorbate peroxidase gene from Lycium chinense Mill. and enhanced salt tolerance by overexpressing in tobacco

To evaluate the physiological importance of cytosolic ascorbate peroxidase (APX) in the reactive oxygen species (ROS)-scavenging system, a full-length cDNA clone, named LmAPX, encoding a cytosolic ascorbate peroxidase was isolated from Lycium chinense Mill. using homologous cloning, then the expression of LmAPX under salt stress was investigated. After sequencing and related analysis, the LmAPX cDNA sequence was 965 bp in length and had an open reading frame (ORF) of 750 bp coding for 250 amino acids. Furthermore, the LmAPX sequence was sub-cloned into prokaryotic expression vector pET28a and the recombinant proteins had a high expression level in Escherichia coli. Results from a southern blot analysis indicated that three inserts of this gene existed in the tobacco genome encoding LmAPX. Compared with the control plants (wild-type and empty vector control), the transgenic plants expressing the LmAPX gene exhibited lower amount of hydrogen peroxide (H₂O₂) and relatively higher values of ascorbate peroxidase activity, proline content, and net photosynthetic rate (P_n) under the same salt stress. These results suggested that overexpression of the LmAPX gene could decrease ROS production caused by salt stress and protect plants from oxidative stress.     

Overexpression of SsCHLAPXs confers protection against oxidative stress induced by high light in transgenic Arabidopsis thaliana

To evaluate the physiological importance of chloroplastic ascorbate peroxidase (CHLAPX) in the reactive oxygen species (ROS)‐scavenging system of a euhalophyte, we cloned the CHLAPX of Suaeda salsa (SsCHLAPX) encoding stromal APX (sAPX) and thylakoid‐bound APX. The stromal APX of S. salsa (Ss.sAPX) cDNA consists of 1726 nucleotides including an 1137‐bp open reading frame (ORF) and encodes 378 amino acids. The thylakoid‐bound APX of S. salsa (Ss.tAPX) cDNA consists of 1561 nucleotides, including a 1284‐bp ORF, and encodes 427 amino acids. The N‐terminal 378 amino acids of Ss.sAPX are identical with those of Ss.tAPX, whereas the C‐terminal 49 amino acids differ. Arabidopsis thaliana lines overexpressing Ss.sAPX and Ss.tAPX were constructed using Agrobacterium tumefaciens transformation methods. Under high light (1000 µmol m^?2 s^?1), malondialdehyde (MDA) content was lower in transgenic plants than in the wild type. Under high light, Fv/Fm and chlorophyll contents of both overexpressing lines and the wild type declined but were significantly higher in the overexpressing lines than in the wild type. The activities of APX (EC 1.11.1.11), catalase (CAT 1.11.1.6) and superoxide dismutase (SOD EC 1.15.1.1) were higher in the overexpressing lines than in the wild type. The transgenic plants showed increased tolerance to oxidative stress caused by high light. These results suggest that SsCHLAPX plays an important role in scavenging ROS in chloroplasts under stress conditions such as high light.     

Genotype-Dependent Effect of Exogenous Nitric Oxide on Cd-induced Changes in Antioxidative Metabolism, Ultrastructure, and Photosynthetic Performance in Barley Seedlings (Hordeum vulgare)

A greenhouse hydroponic experiment was performed using Cd-sensitive (cv. Dong 17) and Cd-tolerant (Weisuobuzhi) barley seedlings to evaluate how different genotypes responded to cadmium (Cd) toxicity in the presence of sodium nitroprusside (SNP), a nitric oxide (NO) donor. Results showed that 5 μM Cd increased the accumulation of O₂^•−, H₂O₂, and malondialdehyde (MDA) but reduced plant height, chlorophyll content, net photosynthetic rate (P _n), and biomass, with a much more severe response in the Cd-sensitive genotype. Antioxidant enzyme activities increased significantly under Cd stress in the roots of the tolerant genotype, whereas in leaves of the sensitive genotype, superoxide dismutase (SOD) and ascorbate peroxide (APX), especially cytosol ascorbate peroxidase (cAPX), decreased after 5–15 days Cd exposure. Moreover, Cd induces NO synthesis by stimulating nitrate reductase and nitric oxide synthetase-like enzymes in roots/leaves. A Cd-induced NO transient increase in roots of the Cd-tolerant genotype might partly contribute to its Cd tolerance. Exogenous NO dramatically alleviated Cd toxicity, markedly diminished Cd-induced reactive oxygen species (ROS) and MDA accumulation, ameliorated Cd-induced damage to leaf/root ultrastructure, and increased chlorophyll content and P _n. External NO counteracted the pattern of alterations in certain antioxidant enzymes induced by Cd; for example, it significantly elevated the depressed SOD, APX, and catalase (CAT) activities in the Cd-sensitive genotype after 10- and 15-day treatments. Furthermore, NO significantly increased stromal APX and Mn-SOD activities in both genotypes and upregulated Cd-induced decrease in cAPX activity and gene expression of root/leaf cAPX and leaf CAT1 in the Cd-sensitive genotype. These data suggest that under Cd stress, NO, as a potent antioxidant, protects barley seedlings against oxidative damage by directly and indirectly scavenging ROS and helps to maintain stability and integrity of the subcellular structure.     

Crystal structure of chloroplastic ascorbate peroxidase from tobacco plants and structural insights into its instability

Ascorbate peroxidase (APX) is a heme-containing protein that plays a central role in scavenging H(2)O(2) in higher plants. The structure of stromal APX (sAPX) was determined at 1.6 A to an R-factor of 19.1% and an R-free-factor of 22.3%. The electrostatic potential of the gamma-channel that connects the molecular surface of sAPX to the gamma-edge of heme was more positive than that of cytosolic APX (cAPX) from pea, so sAPX might bind more easily with ascorbate than cAPX. The overall structure of sAPX was similar to those of cAPX from pea and cytochrome c peroxidase (CCP) from yeast, with a substantial difference in a loop structure located in the vicinity of the heme. The side chain of Arg169 in sAPX corresponding to His169 in cAPX and His181 in CCP extended in the opposite direction from the heme, forming two hydrogen bonds with carbonyl groups in the loop structure. The rapid inactivation of sAPX might be due to the characteristic conformation of Arg169 owing to the loop structure of sAPX.     

Role of sulfate in detoxification of arsenate-induced toxicity in Zea mays L. (SRHM 445): nutrient status and antioxidants

The study highlights the role of sulfur (S) in detoxification of arsenate-induced toxicity and the shift in essential element homeostasis in Zea mays L (SRHM 445). Overall growth of arsenate-treated plants under sulfur starvation (?S) was lower than that in the presence of excess sulfur (+S). Translocation of arsenate from roots to shoots, increased under As(?S) and decreased with As(+S). The level of micronutrients (Cu, Zn, Fe) increased in As(?S) plants. Whereas, the level of K and PO₄ was higher in As(?S) plants than in As(+S) plants. Higher malondialdehyde, protein carbonyl, and H₂O₂ levels in As(?S) plants are indicative of higher oxidative stress. Higher superoxide dismutase (SOD) and ascorbate peroxidase (APX) activities, in As(?S) plants coincided with higher H₂O₂ levels showing the activity of these enzymes are independent of S availability. Absence of reduced glutathione/oxidized glutathione pool in (?S) plants manifested into failure of ascorbate–glutathione detoxification pathway. Hence, S has dual role of protecting the plant against arsenate-induced toxicity (1) by restricting arsenic (As) translocation to the upper parts and (2) by increasing the activity SOD and APX.     

Two rice cytosolic ascorbate peroxidases differentially improve salt tolerance in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

In order to determine the different roles of rice (Oryza sativa L.) cytosolic ascorbate peroxidases (OsAPXa and OsAPXb, GenBank accession nos. D45423 and AB053297, respectively) under salt stress, transgenic Arabidopsis plants over-expressing OsAPXa or OsAPXb were generated, and they all exhibited increased tolerance to salt stress compared to wild-type plants. Moreover, transgenic lines over-expressing OsAPXb showed higher salt tolerance than OsAPXa transgenic lines as indicated by root length and total chlorophyll content. In addition to ascorbate peroxidase (APX) activity, antioxidant enzyme activities of catalase (CAT), superoxide dismutase (SOD) and glutathione reductase (GR), which are also involved in the salt tolerance process, and the content of H₂O₂ were also assayed in both transgenic and wild-type plants. The results showed that the overproduction of OsAPXb enhanced and maintained APX activity to a much higher degree than OsAPXa in transgenic Arabidopsis during treatment with different concentrations of NaCl, enhanced the active oxygen scavenging system, and protected plants from salt stress by equilibrating H₂O₂ metabolism. Our findings suggest that the rice cytosolic OsAPXb gene has a more functional role than OsAPXa in the improvement of salt tolerance in transgenic plants. Zhenqiang Lu and Dali Liu contributed equally.     

Peroxisomal APX knockdown triggers antioxidant mechanisms favourable for coping with high photorespiratory H2O2 induced by CAT deficiency in rice

The physiological role of peroxisomal ascorbate peroxidases (pAPX) is unknown; therefore, we utilized pAPX4 knockdown rice and catalase (CAT) inhibition to assess its role in CAT compensation under high photorespiration. pAPX4 knockdown induced co‐suppression in the expression of pAPX3. The rice mutants exhibited metabolic changes such as lower CAT and glycolate oxidase (GO) activities and reduced glyoxylate content; however, APX activity was not altered. CAT inhibition triggered different changes in the expression of CAT, APX and glutathione peroxidase (GPX) isoforms between non‐transformed (NT) and silenced plants. These responses were associated with alterations in APX, GPX and GO activities, suggesting redox homeostasis differences. The glutathione oxidation‐reduction states were modulated differently in mutants, and the ascorbate redox state was greatly affected in both genotypes. The pAPX suffered less oxidative stress and photosystem II (PSII) damage and displayed higher photosynthesis than the NT plants. The improved acclimation exhibited by the pAPX plants was indicated by lower H₂O₂ accumulation, which was associated with lower GO activity and glyoxylate content. The suppression of both pAPXs and/or its downstream metabolic and molecular effects may trigger favourable antioxidant and compensatory mechanisms to cope with CAT deficiency. This physiological acclimation may involve signalling by peroxisomal H₂O₂, which minimized the photorespiration.     

Activity of Enzymes Related to H2O2 Generation and Metabolism in Leaf Apoplastic Fraction of Tomato Leaves Infected with Botrytis cinerea

Hydrogen peroxide generation rates of uninfected and infected leaves of two tomato (Lycopersicon esculentum) cultivars showing differential susceptibility to Botrytis cinerea were determined. The superoxide anion, hydroxyl radical, ascorbate contents and changes in NADH peroxidase, superoxide dismutase (SOD), ascorbate peroxidase (APX) and catalase (CAT) activities in the apoplast fraction were analysed. Infected leaves had an increased hydrogen peroxide level. It was greater and generally occurred earlier in plants of the less susceptible cv. Perkoz than in those of the more susceptible cv. Corindo. Induction of nitrotetrazolium blue reducing activity and SOD levels in apoplast were higher in cv. Perkoz 24 h after inoculation. In the controls, NADH peroxidase activity in apoplast was higher in the more susceptible cv. Corindo, but after infection it increased faster and to a higher level in the less susceptible cv. Perkoz. NADH oxidation was inhibited by only 15% by a specific inhibitor DPI (diphenylene‐iodonium) but was completely inhibited by KCN and NaN₃. Similar increases in APX activity after 48 h and a small increase in catalase activities were observed in both cultivars soon after infection. These results indicate that resistance of tomato plants to infection by the necrotrophic fungus B. cinerea may result from early stimulation of hydrogen peroxide and superoxide radical generations by NADH peroxidase and SOD in apoplastic space, and they confirm the important role of their enhanced production in apoplastic spaces of plants.     

Expression of the <Emphasis Type="Italic">Apx</Emphasis> gene family during leaf senescence of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Oxygen-free radicals are thought to play an essential role in senescence. Therefore, the expression patterns of the small gene family encoding the H₂O₂ scavenging enzymes ascorbate peroxidase (APX; EC 1.11.1.11) were analyzed during senescence of Arabidopsis thaliana (L.) Heinh. Applying real-time RT-PCR, the mRNA levels were quantified for three cytosolic (APX1, APX2, APX6), two chloroplastic types (stromal sAPX, thylakoid tAPX), and three microsomal (APX3, APX4, APX5) isoforms identified in the genome of Arabidopsis. The genes of chloroplastic thylakoid-bound tAPX and the microsomal APX4 exhibit a strong age-related decrease of mRNA level in leaves derived from one rosette as well as in leaves derived from plants of different ages. In contrast to the tAPX, the mRNA of sAPX was only reduced in old leaves of old plants. The microsomal APX3 and APX5, and the cytosolic APX1, APX2, and APX6 did not show remarkable age-related changes in mRNA levels. The data show that expression of the individual APX genes is differentially regulated during senescence indicating possible functional specialization of respective isoenzymes. The hydrogen peroxide levels seem to be controlled very precisely in different cell compartments during plant development.     

Plant shading increases lipid peroxidation and intensifies senescence-induced changes in photosynthesis and activities of ascorbate peroxidase and glutathione reductase in wheat

Plants of spring wheat (Triticum aestivum L. cv. Saxana) were grown during the autumn. Over the growth phase of three leaves (37 d after sowing), some of the plants were shaded and the plants were grown at 100 (control without shading), 70, and 40 % photosynthetically active radiation. Over 12 d, chlorophyll (Chl) and total protein (TP) contents, rate of CO₂ assimilation (P _N), maximal efficiency of photosystem 2 photochemistry (F_V/F_P), level of lipid peroxidation, and activities of antioxidative enzymes ascorbate peroxidase (APX) and glutathione reductase (GR) were followed in the 1_st, 2_nd, and 3_rd leaves (counted according to their emergence). In un-shaded plants, the Chl and TP contents, P _N, and F_V/F_P decreased during plant ageing. Further, lipid peroxidation increased, while the APX and GR activities related to the fresh mass (FM) decreased. The APX activity related to the TP content increased in the 3_rd leaves. The plant shading accelerated senescence including the increase in lipid peroxidation especially in the 1^st leaves and intensified the changes in APX and GR activities. We suggest that in the 2_nd and 3_rd leaves a degradation of APX was slowed down, which could reflect a tendency to maintain the antioxidant protection in chloroplasts of these leaves.     

Response of xanthophyll cycle and chloroplastic antioxidant enzymes to chilling stress in tomato over-expressing glycerol-3-phosphate acyltransferase gene

Over-expression of chloroplastic glycerol-3-phosphate acyltransferase gene (LeGPAT) increased unsaturated fatty acid contents in phosphatidylglycerol (PG) of thylakoid membrane in tomato. The effect of this increase on the xanthophyll cycle and chloroplast antioxidant enzymes was examined by comparing wild type (WT) tomato with the transgenic (TG) lines at chilling temperature (4 °C) under low irradiance (100 μmol m⁻² s⁻¹). Net photosynthetic rate and the maximal photochemical efficiency of photosystem (PS) 2 (F_v/F_m) in TG plants decreased more slowly during chilling stress and F_v/F_m recovered faster than that in WT plants under optimal conditions. The oxidizable P700 in both WT and TG plants decreased during chilling stress under low irradiance, but recovered faster in TG plants than in the WT ones. During chilling stress, non-photochemical quenching (NPQ) and the de-epoxidized ratio of xanthophyll cycle in WT plants were lower than those of TG tomatoes. The higher activities of superoxide dismutase (SOD) and ascorbate peroxidase (APX) in TG plants resulted in the reduction of O₂ ^−· and H₂O₂ contents during chilling stress. Hence the increase in content of unsaturated fatty acids in PG by the over-expression of LeGPAT could alleviate photoinhibition of PS2 and PS1 by improving the de-epoxidized ratio of xanthophyll cycle and activities of SOD and APX in chloroplast.     

Drought Induces Oxidative Stress and Enhances the Activities of Antioxidant Enzymes in Growing Rice Seedlings

When rice seedlings grown for 10 and 20 days were subjected to in vitro drought stress of −0.5 and −2.0 MPa for 24 h, an increase in the concentration of superoxide anion (O₂^.−), increased level of lipid peroxidation and a decrease in the concentration of total soluble protein and thiols was observed in stressed seedlings compared to controls. The concentration of H₂O₂ as well as ascorbic acid declined with imposition of drought stress, however glutathione (GSH) concentration declined only under severe drought stress. The activities of total superoxide dismutases (SODs) as well as ascorbate peroxidase (APX) showed consistent increases with increasing levels of drought stress, however catalase activity declined. Mild drought stressed plants had higher guaiacol peroxidase (GPX) and chloroplastic ascorbate peroxidase (c-APX) activity than control grown plants but the activity declined at the higher level of drought stress. The activities of enzymes involved in regeneration of ascorbate i.e. monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR) and glutathione reductase (GR) were higher in drought stressed plants compared to controls. Results suggest that drought stress induces oxidative stress in rice plants and that besides SOD, the enzymes of ascorbate-glutathione cycle, which have not been studied in detail earlier under stressful conditions, appear to function as important component of antioxidative defense system under drought stress.     

Regulation of ascorbate peroxidase activity in dark-grown radish cotyledons by a catalase inhibitor, 3-Amino-1,2,4-Triazole

The present study was performed to see the physiological role of cytosolic ascorbate peroxidase (APX) and its relationship to other enzymes involved in the H₂O₂ scavenging metabolism, and also to elucidate the regulation of APX expression in dark-grown radish (Raphanus sativus L. cv Taiwang) cotyledons. To do so, 3-amino-l,2,4-triazole (aminotriazole), a known specific inhibitor of catalase, was used to simulate a catalase-deficient phenomenon in cotyledons. Aminotriazole, in very low concetration (10^-4 M), inhibited remarkably the development of catalase activity in cotyledons during dark germination. This inhibition of catalase by aminotriazole, however, did not result in any significant changes in the growth response and the H₂O₂ level of developing cotyledons. In addition, the development of guaiacol peroxidase (GPX) activity was also not significantly affected. Unlike GPX, cytosolic APX activity was induced rapidly and reached a 1.7-fold increase in aminotriazole treated cotyledons at day 7 after germination. However,in vitro incubation of cytosolic APX preparation from cotyledons with aminotriazole did not result in any significant change in activity. One cytosolic APX isozyme (APXa) band involved in this APX activation was predominantly intensified in a native polyacrylamide gel by activity staining assay. This means that this APXa isozyme seems to play a key role in the expression of cytosolic APX activity. On the other hand, 2-day-old control seedlings treated with exogenous 1 mM H₂O₂ for 1 h showed a significant increase of cytosolic APX acitivity even in the absence of aminotriazole. Also, 2 μM cycloheximide treatment substantially inhibited the increase of APX activity due to aminotriazole. Based on these results, we suggest that a radish cytosolic APX could probably be substituted for catalase in H₂O₂ removal and that the expression of APX seems to be regulated by a change of endogenous H₂O₂ level which couples to APX protein synthesis in a translation stage in cotyledons.