Molecular characterization and physiological role of ascorbate peroxidase from halotolerant Chlamydomonas sp. W80 strain

A cDNA clone encoding an ascorbate peroxidase was isolated from the cDNA library from halotolerant Chlamydomonas W80 by a simple screening method based on the bacterial expression system. The cDNA clone contained an open reading frame encoding a mature protein of 282 amino acids with a calculated molecular mass of 30,031 Da, preceded by the chloroplast transit peptide consisting of 37 amino acids. In fact, ascorbate peroxidase was localized in the chloroplasts of Chlamydomonas W80 cells; the activity was detected in the stromal fraction but not in the thylakoid membrane. The deduced amino acid sequence of the cDNA showed 54 and 49% homology to chloroplastic and cytosolic ascorbate peroxidase isoenzymes of spinach leaves, respectively. The enzyme from Chlamydomonas W80 cells was purified to electrophoretic homogeneity. The molecular properties of the purified enzyme were similar to those of the other algal ascorbate peroxidases rather than those of ascorbate peroxidases from higher plants. The enzyme was relatively stable in ascorbate-depleted medium compared with the chloroplastic ascorbate peroxidase isoenzymes of higher plants. The presence of NaCl (3%) as well as of beta-d-thiogalactopyranoside was needed for the expression of Chlamydomonas W80 ascorbate peroxidase in Escherichia coli.     

Functional divergence in the glutathione transferase superfamily in plants. Identification of two classes with putative functions in redox homeostasis in Arabidopsis thaliana

Searches with the human Omega glutathione transferase (GST) identified two outlying groups of the GST superfamily in Arabidopsis thaliana which differed from all other plant GSTs by containing a cysteine in place of a serine at the active site. One group consisted of four genes, three of which encoded active glutathione-dependent dehydroascorbate reductases (DHARs). Two DHARs were predicted to be cytosolic, whereas the other contained a chloroplast targeting peptide. The DHARs were also active as thiol transferases but had no glutathione conjugating activity. Unlike most other GSTs, DHARs were monomeric. The other class of GST comprised two genes termed the Lambda GSTs (GSTLs). The recombinant GSTLs were also monomeric and had glutathione-dependent thiol transferase activity. One GSTL was cytosolic, whereas the other was chloroplast-targeted. When incubated with oxidized glutathione, the putative active site cysteine of the GSTLs and cytosolic DHARs formed mixed disulfides with glutathione, whereas the plastidic DHAR formed an intramolecular disulfide. DHAR S-glutathionylation was consistent with a proposed catalytic mechanism for dehydroascorbate reduction. Roles for the cytosolic DHARs and GSTLs as antioxidant enzymes were also inferred from the induction of the respective genes following exposure to chemicals and oxidative stress.     

Molecular Properties and Functional Divergence of the Dehydroascorbate Reductase Gene Family in Lower and Higher Plants

Dehydroascorbate reductase (DHAR), which reduces oxidized ascorbate, is important for maintaining an appropriate ascorbate redox state in plant cells. To date, genome-wide molecular characterization of DHARs has only been conducted in bryophytes (Physcomitrella patens) and eudicots (e.g. Arabidopsis thaliana). In this study, to gain a general understanding of the molecular properties and functional divergence of the DHARs in land plants, we further conducted a comprehensive analysis of DHARs from the lycophyte Selaginella moellendorffii, gymnosperm Picea abies and monocot Zea mays. DHARs were present as a small gene family in all of the land plants we examined, with gene numbers ranging from two to four. All the plants contained cytosolic and chloroplastic DHARs, indicating dehydroascorbate (DHA) can be directly reduced in the cytoplasm and chloroplast by DHARs in all the plants. A novel vacuolar DHAR was found in Z. mays, indicating DHA may also be reduced in the vacuole by DHARs in Z. mays. The DHARs within each species showed extensive functional divergence in their gene structures, subcellular localizations, and enzymatic characteristics. This study provides new insights into the molecular characteristics and functional divergence of DHARs in land plants.     

Monodehydroascorbate Reductase in Spinach Chloroplasts and Its Participation in Regeneration of Ascorbate for Scavenging Hydrogen Peroxide

The primary reaction product of chloroplast ascorbate peroxidaseactivity was shown to be monodehydroascorbate radical (MDA).MDA reductase (EC 1.6.5.4 [EC] ) was localized in spinach chloroplaststroma. The MDA reductase activity of spinach chloroplasts,using NAD(P)H as electron donor, could account for the regenerationof ascorbate from MDA produced by ascorbate peroxidase activity.In the absence of MDA reductase, MDA disproportionated to ascorbate(AsA) and dehydroascorbate (DHA). The DHA was reduced to AsAby DHA reductase (EC 1.8.5.1 [EC] ) in chloroplasts. Both NADH andNADPH served as the electron donor of partially purified MDAreductase from spinach leaves. (Received September 24, 1983; Accepted January 23, 1984)     

江南卷柏脱氢抗坏血酸还原酶的分子特性

脱氢抗坏血酸还原酶 (DHAR) 在植物抗坏血酸?谷胱甘肽循环中发挥着重要作用。利用同源克隆技术从江南卷柏中克隆到2个脱氢抗坏血酸还原酶基因,分别命名为SmDHAR1和SmDHAR2。SmDHAR1和SmDHAR2分别编码218和241个氨基酸,预测分子量分别是23.97 kDa和27.33 kDa。基因组序列分析显示这2个基因分别含有5和6个内含子。器官表达模式分析发现这2个基因在根、茎、叶中均有表达,是组成型表达基因。在大肠杆菌中表达并纯化了2个基因的重组蛋白。酶活性分析显示SmDHAR1和SmDHAR2蛋白对底物DHA的活性有显著差异,分别是19.76和0.17 μmol/(min·mg)。热力学稳定性分析显示这2个重组蛋白的热力学稳定性具有明显差异。因此,基因结构与酶学性质的差异预示着这2个基因可能存在功能上的分化。     

Antioxidant protection during ageing and senescence in chloroplasts of tobacco with modulated life span

We studied changes in antioxidant protection during ageing and senescence in chloroplasts of tobacco (Nicotiana tabacum L., cv. Wisconsin) with introduced SAG(12) promoter fused with ipt gene for cytokinin synthesis (transgenic plants with increased levels of cytokinins, SAG) or without it (control). Old leaves of SAG plants as well as their chloroplasts maintained higher physiological parameters compared to controls; accordingly, we concluded that their ageing was diverted due to increased cytokinin content. The chloroplast antioxidant protection did not decrease as well. Although antioxidant protection usually decreased in whole leaves of senescing control plants, ascorbate peroxidase (APX) and dehydroascorbate reductase (DHAR) activity, which maintained the high redox state of ascorbate, increased in chloroplasts of old control leaves.     

Light-dependent reduction of dehydroascorbate and uptake of exogenous ascorbate by spinach chloroplasts

A reconstituted spinach chloroplast system containing thylakoids, stroma and 0.1 mM NADPH supported O₂ evolution in the presence of oxidised glutathione (GSSG). The properties of the reaction were consistent with light-coupled GSSG-reductase activity involving H₂O as eventual electron donor. The reconstituted system also supported dehydroascorbate-dependent O₂ evolution in the presence of 0.6 mM reduced glutathione (GSH) and 0.1 mM NADPH with the concomitant production of ascorbate. The GSSG could replace GSH in which case the production of GSH preceded the accumulation of ascorbate. The data are consistent with the light-dependent reduction of dehydroascorbate using H₂O as eventual electron donor via the sequence H₂O→NADP→GSSG→dehydroascorbate. Approximately 30% of the GSH-dehydrogenase activity of spinach leaf protoplasts is localised in chloroplasts: this could not be attributed to contamination of chloroplasts by activity from the extrachloroplast compartment. Washed intact chloroplasts supported the uptake of ascorbate but the uptake mechanism had a very low affinity for ascorbate (K_m approximately 20 mM). The rate of uptake of ascorbate was less than the rate of light-dependent reduction of dehydroascorbate and too slow to account for the rate of H₂O₂ reduction by washed intact chloroplasts.     

Molecular cloning and characterization of a rice dehydroascorbate reductase

Plant dehydroascorbate reductase (DHAR), which re-reduces oxidized ascorbate to maintain an appropriate level of ascorbate, is very important, but no gene or cDNA for plant DHAR has been cloned yet. Here, we describe a cDNA for a rice glutathione-dependent DHAR (designated DHAR1). A recombinant Dhar1p produced in Escherichia coli was functional. The expression sequence tag database suggests that Dhar1p homologs exist in various plants. Furthermore, the rice Dhar1p has a low similarity to rat DHAR, although the rice enzyme has a considerably higher specific activity than the mammalian one. The mRNA level of DHAR1, the protein level of Dhar1p and the DHAR activity in rice seedlings were elevated by high temperature, suggesting the protection role of DHAR at high temperature.     

Purification and characterization of pea cytosolic ascorbate peroxidase

The cytosolic isoform of ascorbate peroxidase was purified to homogeneity from 14-day-old pea (Pisum sativum L.) shoots. The enzyme is a homodimer with molecular weight of 57,500, composed of two subunits with molecular weight of 29,500. Spectral analysis and inhibitor studies were consistent with the presence of a heme moiety. When compared with ascorbate peroxidase activity derived from ruptured intact chloroplasts, the purified enzyme was found to have a higher stability, a broader pH optimum for activity, and the capacity to utilize alternate electron donors. Unlike classical plant peroxidases, the cytosolic ascorbate peroxidase had a very high preference for ascorbate as an electron donor and was specifically inhibited by p-chloromercurisulfonic acid and hydroxyurea. Antibodies raised against the cytosolic ascorbate peroxidase from pea did not cross-react with either protein extracts obtained from intact pea chloroplasts or horseradish peroxidase. The amino acid sequence of the N-terminal region of the purified enzyme was determined. Little homology was observed among pea cytosolic ascorbate peroxidase, the tea chloroplastic ascorbate peroxidase, and horseradish peroxidase; homology was, however, found with chloroplastic ascorbate peroxidase isolated from spinach leaves.     

Enhanced tolerance to oxidative stress in transgenic tobacco plants expressing three antioxidant enzymes in chloroplasts

The effect of simultaneous expression of genes encoding three antioxidant enzymes, copper zinc superoxide dismutase (CuZnSOD, EC 1.15.1.1), ascorbate peroxidase (APX, EC 1.11.1.11), and dehydroascorbate (DHA) reductase (DHAR, EC 1.8.5.1), in the chloroplasts of tobacco plants was investigated under oxidative stress conditions. In previous studies, transgenic tobacco plants expressing both CuZnSOD and APX in chloroplast (CA plants), or DHAR in chloroplast showed enhanced tolerance to oxidative stresses, such as paraquat and salt. In this study, in order to develop transgenic plants that were more resistant to oxidative stress, we introduced the gene encoding DHAR into CA transgenic plants. Mature leaves of transgenic plants expressing all three antioxidant genes (CAD plants) had approximately 1.6–2.1 times higher DHAR activity, and higher ratios of reduced ascorbate (AsA) to DHA, and oxidized glutathione (GSSG) to reduced glutathione (GSH) compared to CA plants. CAD plants were more resistant to paraquat-induced stress, exhibiting only 18.1% reduction in membrane damage relative to CA plants. In addition, seedlings of CAD plants had enhanced tolerance to NaCI (100 mM) compared to CA plants. These results indicate that the simultaneous expression of multiple antioxidant enzymes, such as CuZnSOD, APX, and DHAR, in chloroplasts is more effective than single or double expression for developing transgenic plants with enhanced tolerance to multiple environmental stresses.     

Mechanism of the reaction catalyzed by dehydroascorbate reductase from spinach chloroplasts.

Dehydroascorbate reductase (DHAR) reduces dehydroascorbate (DHA) to ascorbate with glutathione (GSH) as the electron donor. We analyzed the reaction mechanism of spinach chloroplast DHAR, which had a much higher reaction specificity for DHA than animal enzymes, using a recombinant enzyme expressed in Escherichia coli. Kinetic analysis suggested that the reaction proceeded by a bi-uni-uni-uni-ping-pong mechanism, in which binding of DHA to the free, reduced form of the enzyme was followed by binding of GSH. The Km value for DHA and the summed Km value for GSH were determined to be 53 +/- 12 micro m and 2.2 +/- 1.0 mm, respectively, with a turnover rate of 490 +/- 40 s-1. Incubation of 10 microm DHAR with 1 mm DHA and 10 microm GSH resulted in stable binding of GSH to the enzyme. Bound GSH was released upon reduction of the GSH-enzyme adduct by 2-mercaptoethanol, suggesting that the adduct is a reaction intermediate. Site-directed mutagenesis indicated that C23 in DHAR is indispensable for the reduction of DHA. The mechanism of catalysis of spinach chloroplast DHAR is proposed.     

Studies on Fructose—1,6—Diphosphatase (FDP-ASE) in Chloroplasts I. Isolation of Chloroplast FDP-ASE and Comparison of Some Kinetic Properties of Purified Chloroplast FDP-ASE and That of FDP-ASE in Freshly Ruptured Chloroplasts

Chloroplast FDPase was purified from spinach leaves by ammonium sulfate precipitation, Sephadex G-100 chromatography and DEAE-cellulose chromatography. It was found that treatment of the spinach leaves with liquid nitrogen prior to homoge- nization facilitated the subsequent isolation process, the optimal pH for FDPase activity was 8 to 9 and the enzyme was most stable at pH 6, under which it could be stored over several months without appreciable loss of activity. Acrylamide disc electrophoresis of the final enzyme fraction showed only one essential band. The two forms of FDPase, purified spinach chloroplast FDPase and that in fresilly ruptured spinach chloroplast, behaved differently in some of their kinetic properties. Their activities depended throughout on the concentration of Mg++, but the Km (Mg++) were quite different. The Km (Mg++) of the purified enzyme was about 6.0 mM, that of FDPase in freshly ruptured chloroplasts was, however, 1.0 mM, which corresponded to the concentration of Mg+* in the stroma of illuminated chloroplasts. Mg++ concentration was a limiting factor for the activity of purified FDPase. As the amount of Mg++ in the reaction mixture was lowered, the Km and Vmax were both greatly changed. The shortage of Mg++ could not be compensated by increasing the substrate concentration. The purified FDPase was completely inhibited by 15 μ moles EDTA in the teaction mixture, whereas the FDPase in freshly ruptured chloroplasts was inhibited only 70% by 30 to 45 μ moles EDTA, which was 2 to 3 fold of the concentration sufficient to inhibit completely the activity of the purified enzyme. Moreover, the former was more stable. Its activity did not decline even after incubation for over two hours The FDPase activity was higher in chloroplasts ruptured in 0.2% (w/v) Triton X-100 than that ruptured in water. This phenomenon suggests that this enzyme in vivo might be in some way associated, at least partly; with chloroplast lamellae.     

Tobacco chloroplast transformants expressing genes encoding dehydroascorbate reductase, glutathione reductase, and glutathione-S-transferase, exhibit altered anti-oxidant metabolism and improved abiotic stress tolerance

One approach to understanding the Reactive Oxygen Species (ROS)-scavenging systems in plant stress tolerance is to manipulate the levels of antioxidant enzyme activities. In this study, we expressed in the chloroplast three such enzymes: dehydroascorbate reductase (DHAR), glutathione-S-transferase (GST) and glutathione reductase (GR). Homoplasmic chloroplast transformants containing either DHAR or GST, or a combination of DHAR:GR and GST:GR were generated and confirmed by molecular analysis. They exhibited the predicted changes in enzyme activities, and levels or redox state of ascorbate and glutathione. Progeny of these plants were then subjected to environmental stresses including methyl viologen (MV)-induced oxidative stress, salt, cold and heavy metal stresses. Overexpression of these different enzymes enhanced salt and cold tolerance. The simultaneous expression of DHAR:GR and GST:GR conferred MV tolerance while expression of either transgene on its own didn't. This study provides evidence that increasing part of the antioxidant pathway within the chloroplast enhances the plant's ability to tolerate abiotic stress.     

Purification and cDNA cloning of chloroplastic monodehydroascorbate reductase from spinach

The chloroplastic isoform of monodehydroascorbate (MDA) radical reductase was purified from spinach chloroplasts and leaves. The cDNA of chloroplastic MDA reductase was cloned, and its deduced amino acid sequence, consisting of 497 residues, showed high homology with those of putative organellar MDA reductases deduced from cDNAs of several plants. The amino acid sequence of the amino terminal of the purified enzyme suggested that the chloroplastic enzyme has a transit peptide consisting of 53 residues. A southern blot analysis suggested the occurrence of a gene encoding another isoform homologous to the chloroplastic isoform in spinach. The recombinant enzyme was highly expressed in Eschericia coli using the cDNA, and purified to a homogeneous state with high specific activity. The enzyme properties of the chloroplastic isoform are presented in comparison with those of the cytosolic form.     

Purification, subunit structure and immunological comparison of fructose-bisphosphate aldolases from spinach and corn leaves

The cytosol and chloroplast fructose-bisphosphate aldolases from spinach leaves were separated by ion-exchange chromatography on DEAE-cellulose, and were purified by subsequent affinity chromatography on phosphocellulose to apparent homogeneity as judged from polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The two aldolases had specific activities of 7.2 and 7.8 units mg protein-1. Molecular weight determinations by electrophoresis in sodium dodecyl sulfate gels and by sedimentation velocity centrifugation in sucrose gradients showed that the aldolases contained four subunits of Mr 38 000 and 35 000, respectively. Antibodies against the cytosol and chloroplast aldolase from spinach leaves were raised in a guinea pig and in a rabbit, respectively. In the Ouchterlony double-diffusion test, the two aldolases did not cross-react. A small degree of cross-reaction was observed by a test in which immune complexes were adsorbed to a solid-phase support (Staphylococcus aureus Cowan I cells) and nonbound enzyme activity was determined after centrifugation. These results imply major structural differences between the two spinach leaf aldolases. Only one major aldolase could be resolved on DEAE-cellulose from corn leaves. The aldolase was purified and had a specific activity of 6.4 units X mg protein-1. The corn leaf aldolase cross-reacted with the antiserum raised against the chloroplast enzyme from spinach leaves, but not with the other antiserum. Thus, the corn leaf aldolase could be identified as a chloroplast enzyme. Since aldolase activity is mostly restricted to the bundle sheath cells of corn leaf, it was concluded that it is compartmentalized in the chloroplasts of these cells but not in chloroplasts of the mesophyll cells.     

Hydrogen-peroxide-scavenging systems within pea chloroplasts

The subcellular distribution of ascorbate peroxidase and glutathione reductase (EC 1.6.4.2) in pea leaves was compared with that of organelle markers. Enzyme distribution was found to be similar to that of the chloroplast enzyme NADPH-glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13). Isolated chloroplasts showed a close correlation between intactness and the percentage of enzyme activity recovered. Chloroplasts of 85% intactness were found to contain a high proportion of leaf dehydroascorbate reductase activity (EC 1.8.5.1), 10% of leaf glutathione and 30% of leaf ascorbate. These results are discussed in relation to the potential role of chloroplast antioxidant systems in plant resistance to environmental and other stress conditions.Abbreviations GSH reduced glutathione - GSSG oxidized glutathione - NADPH-GPD glyceraldehyde-3-phosphate dehydrogenase - SOD superoxide dismutase     

The Tropical Fig Ficus microcarpa L. f. cv. Golden Leaves Lacks Heat-Stable Dehydroascorbate Reductase Activity

Dehydroascorbate reductase (DHAR) is an enzyme which mediatesa regeneration of ascorbate from dehydroascorbate using GSHas the electron donor. Here we report a tropical fig lackinga heat-stable DHAR activity in leaves. The leaves of Ficus microcarpaL. f. cv. Golden Leaves are normally green under dim light butare yellow under full sunlight in the field. We compared DHARactivity from the leaves of Golden Leaves to that of the wildtype using two distinct assay methods. Total leaf DHAR activityof Golden Leaves, determined in the presence of 2-mercaptoethanoland EDTA, was 0.3 µmol min^–1 (mg protein)^–1which was comparable to that of the wild type or spinach leaves.This activity was completely eliminated by heat treatment at80°C, whereas 40% of the activity was resistant to thistreatment in the wild type leaves. Similar results were obtainedwith another assay method employing dithiothreitol and gel filtration.We conclude that the absence of heat-stable DHAR activity isa significant biochemical marker that distinguishes Golden Leavesfrom the wild type. A physiological role for DHAR in adaptationto high light stress is proposed. (Received December 4, 1998; Accepted April 15, 1999)     

Feedback inhibition of spinach L-galactose dehydrogenase by L-ascorbate

We have studied the enzymological properties of L-galactose dehydrogenase (l-GalDH), a key enzyme in the biosynthetic pathway of l-ascorbate (AsA) in plants. L-GalDH was purified approximately 560-fold from spinach leaves. The enzyme was a homodimer with a subunit mass of 36 kDa. We also cloned the full-length cDNA of spinach L-GalDH, which contained an open reading frame encoding 322 amino acid residues with a calculated molecular mass of 35,261 Da. The deduced amino acid sequence of the cDNA showed 82, 79 and 75% homology to L-GalDH from kiwifruit, apple and Arabidopsis, respectively. Recombinant enzyme expressed from the cDNA in Escherichia coli showed L-GalDH activity. Southern blot analysis revealed that the spinach L-GalDH gene occurs in a single copy. Northern blot analysis suggests that L-GalDH is expressed in different organs of spinach. The purified native L-GalDH showed high specificity for L-galactose with a Km of 116.2+/-3.2 microM. Interestingly, spinach L-GalDH exhibited reversible inhibition by AsA, the end-product of the biosynthetic pathway. The inhibition kinetics indicated a linear-competitive inhibition with a Ki of 133.2+/-7.2 microM, suggesting feedback regulation in AsA synthesis in the plant.     

Ascorbic acid contents in transgenic potato plants overexpressing two dehydroascorbate reductase genes

Ascorbic acid (AsA, vitamin C) is one of the most important nutritional quality factors in many horticultural crops and has many biological activities in the human body. Dehydroascorbate reductase (EC 1.8.5.1; DHAR) plays an important role in maintaining the normal level of ascorbic acid (AsA) by recycling oxidized ascorbic acid. To increase AsA content of potato, we isolated and characterized the cDNAs encoding two isoform DHARs localized in cytosol and chloroplast from potato, and developed two types of transgenic potato plants overexpressing cytosolic DHAR gene and chloroplastic DHAR, respectively. Incorporation of the transgene in the genome of potato was confirmed by PCR and real time RT-PCR. The overexpression of cytosolic DHAR significantly increased DHAR activities and AsA contents in potato leaves and tubers, whereas chloroplastic DHAR overexpression only increased DHAR activities and AsA contents in leaves, and did not change them in tubers. These results indicated that AsA content of potato can be elevated by enhancing recycling ascorbate via DHAR overexpression, moreover, cytosolic DHAR might play main important roles in improving the AsA contents of potato tubers.