The d-mannose/l-galactose pathway is the dominant ascorbate biosynthetic route in the moss Physcomitrium patens.

Ascorbate is an abundant and indispensable redox compound in plants. Genetic and biochemical studies have established the d-mannose/l-galactose (d-Man/l-Gal) pathway as the predominant ascorbate biosynthetic pathway in streptophytes, while the d-galacturonate (d-GalUA) pathway is found in prasinophytes and euglenoids. Based on the presence of the complete set of genes encoding enzymes involved in the d-Man/l-Gal pathway and an orthologous gene encoding aldonolactonase (ALase) - a key enzyme for the d-GalUA pathway - Physcomitrium patens may possess both pathways. Here, we have characterized the moss ALase as a functional lactonase and evaluated the ascorbate biosynthesis capability of the two pathways using knockout mutants. Physcomitrium patens expresses two ALase paralogs, namely PpALase1 and PpALase2. Kinetic analyses with recombinant enzymes indicated that PpALase1 is a functional enzyme catalyzing the conversion of l-galactonic acid to the final precursor l-galactono-1,4-lactone and that it also reacts with dehydroascorbate as a substrate. Interestingly, mutants lacking PpALase1 (Δal1) showed 1.2-fold higher total ascorbate content than the wild type, and their dehydroascorbate content was increased by 50% compared with that of the wild type. In contrast, the total ascorbate content of mutants lacking PpVTC2-1 (Δvtc2-1) or PpVTC2-2 (Δvtc2-2), which encode the rate-limiting enzyme GDP-l-Gal phosphorylase in the d-Man/l-Gal pathway, was markedly decreased to 46 and 17%, respectively, compared with that of the wild type. Taken together, the dominant ascorbate biosynthetic pathway in P. patens is the d-Man/l-Gal pathway, not the d-GalUA pathway, and PpALase1 may play a significant role in ascorbate metabolism by facilitating dehydroascorbate degradation rather than ascorbate biosynthesis.

Cooperation of chloroplast ascorbate peroxidases and proton gradient regulation 5 is critical for protecting Arabidopsis plants from photo-oxidative stress.

High-light (HL) stress enhances the production of H2 O2 from the photosynthetic electron transport chain in chloroplasts, potentially causing photo-oxidative damage. Although stromal and thylakoid membrane-bound ascorbate peroxidases (sAPX and tAPX, respectively) are major H2 O2 -scavenging enzymes in chloroplasts, their knockout mutants do not exhibit a visible phenotype under HL stress. Trans-thylakoid proton gradient (∆pH)-dependent mechanisms exist for controlling H2 O2 production from photosynthesis, such as thermal dissipation of light energy and downregulation of electron transfer between photosystems II and I, and these may compensate for the lack of APXs. To test this hypothesis, we focused on a proton gradient regulation 5 (pgr5) mutant, wherein both ∆pH-dependent mechanisms are impaired, and an Arabidopsis sapx tapx double mutant was crossed with the pgr5 single mutant. The sapx tapx pgr5 triple mutant exhibited extreme sensitivity to HL compared with its parental lines. This phenotype was consistent with cellular redox perturbations and enhanced expression of many oxidative stress-responsive genes. These findings demonstrate that the PGR5-dependent mechanisms compensate for chloroplast APXs, and vice versa. An intriguing finding was that the failure of induction of non-photochemical quenching in pgr5 (because of the limitation in ∆pH formation) was partially recovered in sapx tapx pgr5. Further genetic studies suggested that this recovery was dependent on the NADH dehydrogenase-like complex-dependent pathway for cyclic electron flow around photosystem I. Together with data from the sapx tapx npq4 mutant, we discuss the interrelationship between APXs and ∆pH-dependent mechanisms under HL stress.

Dehydroascorbate Reductases and Glutathione Set a Threshold for High-Light-Induced Ascorbate Accumulation.

Plants require a high concentration of ascorbate as a redox buffer for survival under stress conditions, such as high light. Dehydroascorbate reductases (DHARs) are enzymes that catalyze the reduction of DHA to ascorbate using reduced glutathione (GSH) as an electron donor, allowing rapid ascorbate recycling. However, a recent study using an Arabidopsis (Arabidopsis thaliana) triple mutant lacking all three DHAR genes (herein called ∆dhar) did not find evidence for their role in ascorbate recycling under oxidative stress. To further study the function of DHARs, we generated ∆dhar Arabidopsis plants as well as a quadruple mutant line combining ∆dhar with an additional vtc2 mutation that causes ascorbate deficiency. Measurements of ascorbate in these mutants under low- or high-light conditions indicated that DHARs have a nonnegligible impact on full ascorbate accumulation under high light, but that they are dispensable when ascorbate concentrations are low to moderate. Because GSH itself can reduce DHA nonenzymatically, we used the pad2 mutant that contains ∼30% of the wild-type GSH level. The pad2 mutant accumulated ascorbate at a wild-type level under high light; however, when the pad2 mutation was combined with ∆dhar, there was near-complete inhibition of high-light-dependent ascorbate accumulation. The lack of ascorbate accumulation was consistent with a marked increase in the ascorbate degradation product threonate. These findings indicate that ascorbate recycling capacity is limited in ∆dhar pad2 plants, and that both DHAR activity and GSH content set a threshold for high-light-induced ascorbate accumulation.

Enzymatic determination of d-alanine with l-alanine dehydrogenase and alanine racemase.

An enzymatic assay system of d-Ala, which is reported to affect the taste, was constructed using alanine racemase and l-alanine dehydrogenase. d-Ala is converted to l-Ala by alanine racemase and then deaminated by l-alanine dehydrogenase with the reduction of NAD+ to NADH, which is determined with water-soluble tetrazolium. Using the assay system, the d-Ala contents of 7 crustaceans were determined.

Affinity resins as new tools for identifying target proteins of ascorbic acid.

l-Ascorbic acid (AA) has diverse physiological functions, but little is known about the functional mechanisms of AA. In this study, we synthesized two types of affinity resin on which AA is immobilized in a stable form to identify new AA-targeted proteins, which can provide important clues for elucidating unknown functional mechanisms of AA. To our knowledge, an affinity resin on which AA as a ligand is immobilized has not been prepared, because AA is very unstable and rapidly degraded in an aqueous solution. By using the affinity resins, cytochrome c (cyt c) was identified as an AA-targeted protein, and we showed that oxidized cyt c exhibits specific affinity for AA. These results suggest that two kinds of AA-affinity resin can be powerful tools to identify new target proteins of AA.

Anabaena sp. DyP-type peroxidase is a tetramer consisting of two asymmetric dimers.

DyP-type peroxidases are a newly discovered family of heme peroxidases distributed from prokaryotes to eukaryotes. Recently, using a structure-based sequence alignment, we proposed the new classes, P, I and V, as substitutes for classes A, B, C, and D [Arch Biochem Biophys 2015;574:49-55]. Although many class V enzymes from eukaryotes have been characterized, only two from prokaryotes have been reported. Here, we show the crystal structure of one of these two enzymes, Anabaena sp. DyP-type peroxidase (AnaPX). AnaPX is tetramer formed from Cys224-Cys224 disulfide-linked dimers. The tetramer of wild-type AnaPX was stable at all salt concentrations tested. In contrast, the C224A mutant showed salt concentration-dependent oligomeric states: in 600 mM NaCl, it maintained a tetrameric structure, whereas in the absence of salt, it dissociated into monomers, leading to a reduction in thermostability. Although the tetramer exhibits non-crystallographic, 2-fold symmetry in the asymmetric unit, two subunits forming the Cys224-Cys224 disulfide-linked dimer are related by 165° rotation. This asymmetry creates an opening to cavities facing the inside of the tetramer, providing a pathway for hydrogen peroxide access. Finally, a phylogenetic analysis using structure-based sequence alignments showed that class V enzymes from prokaryotes, including AnaPX, are phylogenetically closely related to class V enzymes from eukaryotes.

Diversity and Evolution of Ascorbate Peroxidase Functions in Chloroplasts: More Than Just a Classical Antioxidant Enzyme?

Reactive oxygen species (ROS) have dual functions in plant cells as cytotoxic molecules and emergency signals. The balance between the production and scavenging of these molecules in chloroplasts, major sites for the production of ROS, is one of the key determinants for plant acclimation to stress conditions. The water-water cycle is a crucial regulator of ROS levels in chloroplasts. In this cycle, the stromal and thylakoid membrane-attached isoforms of ascorbate peroxidase (sAPX and tAPX, respectively) are involved in the metabolism of H2O2 Current genome and phylogenetic analyses suggest that the first monofunctional APX was generated as sAPX in unicellular green algae, and that tAPX occurred in multicellular charophytes during plant evolution. Chloroplastic APXs, especially tAPX, have been considered to be the source of a bottleneck in the water-water cycle, at least in higher plants, because of their high susceptibility to H2O2 A number of studies have succeeded in improving plant stress resistance by reinforcing the fragile characteristics of the enzymes. However, researchers have unexpectedly failed to find a 'stress-sensitive phenotype' among loss-of-function mutants, at least in laboratory conditions. Interestingly, the susceptibility of enzymes to H2O2 may have been acquired during plant evolution, thereby allowing for the flexible use of H2O2 as a signaling molecule in plants, and this is supported by growing lines of evidence for the physiological significance of chloroplastic H2O2 as a retrograde signal in plant stress responses. By overviewing historical, biochemical, physiological and genetic studies, we herein discuss the diverse functions of chloroplastic APXs as antioxidant enzymes and signaling modulators.

Identification and functional analysis of peroxiredoxin isoforms in Euglena gracilis.

Euglena gracilis lacks catalase and contains ascorbate peroxidase (APX) which is localized exclusively in the cytosol. Other enzymes that scavenge reactive oxygen species (ROS) in Euglena have not yet been identified; therefore, ROS metabolism, especially in organelles, remains unclear in Euglena. The full-length cDNAs of four Euglena peroxiredoxins (EgPrxs) were isolated in this study. EgPrx1 and -4 were predicted to be localized in the cytosol, and EgPrx2 and -3 in plastids and mitochondria, respectively. The catalytic efficiencies of recombinant EgPrxs were similar to those of plant thiol-peroxidases, but were markedly lower than those of APX from Euglena. However, transcript levels of EgPrx1, -2, and -3 were markedly higher than those of APX. The growth rate of Euglena cells, in which the expression of EgPrx1 and -4 was suppressed by gene silencing, was markedly reduced under normal conditions, indicating physiological significance of Prx proteins.

Substitution of a single amino acid residue in the aromatic/arginine selectivity filter alters the transport profiles of tonoplast aquaporin homologs.

Aquaporins are integral membrane proteins that facilitate the transport of water and some small solutes across cellular membranes. X-ray crystallography of aquaporins indicates that four amino acids constitute an aromatic/arginine (ar/R) pore constriction known as the selectivity filter. On the basis of these four amino acids, tonoplast aquaporins called tonoplast intrinsic proteins (TIPs) are divided into three groups in Arabidopsis. Herein, we describe the characterization of two group I TIP1s (TgTIP1;1 and TgTIP1;2) from tulip (Tulipa gesneriana). TgTIP1;1 and TgTIP1;2 have a novel isoleucine in loop E (LE2 position) of the ar/R filter; the residue at LE2 is a valine in all group I TIPs from model plants. The homologs showed mercury-sensitive water channel activity in a fast kinetics swelling assay upon heterologous expression in Pichia pastoris. Heterologous expression of both homologs promoted the growth of P. pastoris on ammonium or urea as sole sources of nitrogen and decreased growth and survival in the presence of H(2)O(2). TgTIP1;1- and TgTIP1;2-mediated H(2)O(2) conductance was demonstrated further by a fluorescence assay. Substitutions in the ar/R selectivity filter of TgTIP1;1 showed that mutants that mimicked the ar/R constriction of group I TIPs could conduct the same substrates that were transported by wild-type TgTIP1;1. In contrast, mutants that mimicked group II TIPs showed no evidence of urea or H(2)O(2) conductance. These results suggest that the amino acid residue at LE2 position is critical for the transport selectivity of the TIP homologs and group I TIPs might have a broader spectrum of substrate selectivity than group II TIPs.

Expression profiles of aquaporin homologues and petal movement during petal development in Tulipa gesneriana.

Previously, we have characterized two tonoplast intrinsic proteins (TIPs) and four plasma membrane intrinsic proteins (PIPs) from the 2-day-old petals of tulip (Tulipa gesneriana). In this study, we analyzed the development of tulip petals and stems, temperature-dependent petal movement, the amount of ³H2O transported into petals and stems during petal movement, and the transcript levels of two TIP (TgTIP1;1 and TgTIP1;2) and four TgPIP genes in petals and stems, from the first day of petal opening to day 12. The development of the petals and stems was completed by days 6 and 9, respectively, after the first day of petal opening. Temperature-dependent petal movement and the amount of ³H2O that was transported into petals could be detected at significant levels up to day 6 with petal movement reaching a peak at day 3. Real-time reverse transcription-polymerase chain reaction analysis revealed that TgTIP1;1 and TgTIP1;2 were expressed ubiquitously in petals, stems, leaves, bulbs and roots. However, the expression level of TgTIP1;2 was very low in bulbs. The expression of both TgTIP1 genes was upregulated in close association with the development of petals but not with that of the stem. The four TgPIP genes were expressed at almost the same level during the development of the petals and the stem. However, the levels of the TgTIP1 and TgPIP transcripts in petals decreased during the course of petal wilting from day 9 onwards. These results suggest that TgTIP1;1 and TgTIP1;2 may contribute to petal development.

Analysis of two L-Galactono-1,4-lactone-responsive genes with complementary expression during the development of Arabidopsis thaliana.

Unraveling the role of genes annotated as protein of unknown function is of importance in progression of plant science. l-Galactono-1,4-lactone (l-GalL) is the terminal precursor for ascorbic acid (AsA) biosynthesis in Arabidopsis thaliana, and a previous study showed two DUF (domains of unknown function) 642 family genes (At1g80240 and At5g25460, designated as DGR1 and DGR2, respectively) to be sensitive to it. In this work, leaves from wild-type Arabidopsis were fed with d-glucose, l-galactose, l-GalL and AsA, and the expression level of the At1g80240 and At5g25460 genes showed a specific response to l-GalL, but not to the other supplements despite the increases of the tissue AsA contents. Analysis of promoter-ß-glucuronidase (GUS) transgenic plants showed the two genes to be complementarily expressed at the root apex and in the rest of the root excluding the apex, respectively, in both young and old seedlings, and to be expressed at the leaf primordia. The GUS activity under the control of the At5g25460 promoter was high in the cotyledon and leaf veins of young seedlings. These findings were consistent with the results of quantitative real-time PCR. Interestingly, the T-DNA insertion mutant of At5g25460 (SALK_125079) displayed shorter roots and smaller rosettes than Col-0; however, no phenotypic difference was observed between the T-DNA insertion mutant of At1g80240 and the wild type. This is the first report on the expression and functional analysis of these two DUF642 family genes, with the results revealing the contribution of DGR genes to the development of Arabidopsis.

Amino acid residues of both the extracellular and transmembrane domains influence binding of the antiparasitic agent milbemycin to Haemonchus contortus AVR-14B glutamate-gated chloride channels.

Glutamate-gated chloride (GluCl) channels are pentameric receptors for the inhibitory neurotransmitter glutamate in invertebrates and are a major target for macrolide anthelmintics. Three amino acids in GluCl channels are reported to render macrolide resistance in nematodes and insects. To examine whether these three amino acids are involved in binding of the antiparasitic agent milbemycin (MLM) to the GluCl channels of the nematode parasite Haemonchus contortus, the equivalent amino acids (L256, P316, and G329) of the Hco-AVR-14B subunit were substituted with various amino acids. cDNAs encoding the wild type and mutants of this subunit were transfected into COS-1 cells for transient expression and analysis of GluCl channels. The abilities of these mutant channels to bind [(3)H]MLM A(4) were remarkably decreased when compared with the wild-type channel. In patch clamp analysis, L256F and P316S mutant channels were 37- and 100-fold less sensitive to MLM A(4) when compared with the wild-type channel, respectively. These findings indicate that amino acid changes in the ß10 strand, the M2-M3 linker, and the M3 region influence MLM A(4) binding to the channel. Homology modeling and ligand docking studies suggest the presence of two potential binding sites for MLM A(4).

Translocation and the alternative D-galacturonate pathway contribute to increasing the ascorbate level in ripening tomato fruits together with the D-mannose/L-galactose pathway.

The D-mannose/L-galactose pathway for the biosynthesis of vitamin C (L-ascorbic acid; AsA) has greatly improved the understanding of this indispensable compound in plants, where it plays multifunctional roles. However, it is yet to be proven whether the same pathway holds for all the different organs of plants, especially the fruit-bearing plants, at different stages of development. Micro-Tom was used here to elucidate the mechanisms of AsA accumulation and regulation in tomato fruits. The mRNA expression of the genes in the D-mannose/L-galactose pathway were inversely correlated with increasing AsA content of Micro-Tom fruits during ripening. Feeding L-[6-(14)C]AsA to Micro-Tom plants revealed that the bulk of the label from AsA accumulated in the source leaf was transported to the immature green fruits, and the rate of translocation decreased as ripening progressed. L-Galactose feeding, but neither D-galacturonate nor L-gulono-1,4-lactone, enhanced the content of AsA in immature green fruit. On the other hand, L-galactose and D-galacturonate, but not L-gulono-1,4-lactone, resulted in an increase in the AsA content of red ripened fruits. Crude extract prepared from insoluble fractions of green and red fruits showed D-galacturonate reductase- and aldonolactonase-specific activities, the antepenultimate and penultimate enzymes, respectively, in the D-galacturonate pathway, in both fruits. Taken together, the present findings demonstrated that tomato fruits could switch between different sources for AsA supply depending on their ripening stages. The translocation from source leaves and biosynthesis via the D-mannose/L-galactose pathway are dominant sources in immature fruits, while the alternative D-galacturonate pathway contributes to AsA accumulation in ripened Micro-Tom fruits.

L-aspartate dehydrogenase: features and applications.

L-amino acid dehydrogenases are a group of enzymes that catalyze the reversible oxidative deamination of L-amino acids to their corresponding 2-oxoacids, using either nicotinamide adenine dinucleotide (NAD(+)) or nicotinamide adenine dinucleotide phosphate (NADP(+)) as cofactors. These enzymes have been studied widely because of their potential applications in the synthesis of amino acids for use in production of pharmaceutical peptides, herbicides and insecticides, in biosensors or diagnostic kits, and development of coenzyme regeneration systems for industrial processes. This article presents a review of the currently available data about the recently discovered amino acid dehydrogenase superfamily member L-aspartate dehydrogenase (L-AspDH), their relevant catalytic properties and speculated physiological roles, and potential for biotechnological applications. The proposed classification of L-AspDH on the basis of bioinformatic information and potential role in vivo into NadB (NAD biosynthesis-related) and non-NadB type is unique. In particular, the mesophilic non-NadB type L-AspDH is a novel group of amino acid dehydrogenases with great promise as potential industrial biocatalysts owing to their relatively high catalytic properties at room temperature. Considering that only a few L-AspDH homologs have been characterized so far, identification and prodigious enzymological research of the new members will be necessary to shed light on the gray areas pertaining to these enzymes.

Evolution and properties of alanine racemase from Synechocystis sp. PCC6803.

Alanine racemase (EC 5.1.1.1) depends on pyridoxal 5'-phosphate and catalyses the interconversion between L- and D-Ala. The enzyme is responsible for the biosynthesis of D-Ala, which is an essential component of the peptidoglycan layer of bacterial cell walls. Phylogenetic analysis of alanine racemases demonstrated that the cyanobacterial enzyme diverged before the separation of gram-positive and gram-negative enzymes. This result is interesting considering that the peptidoglycans observed in cyanobacteria seem to combine the properties of those in both gram-negative and gram-positive bacteria. We cloned the putative alanine racemase gene (slr0823) of Synechocystis sp. PCC6803 in Escherichia coli cells, expressed and purified the enzyme protein and studied its enzymological properties. The enzymatic properties of the Synechocystis enzyme were similar to those of other gram-positive and gram-negative bacterial enzymes. Alignment of the amino acid sequences of alanine racemase enzymes revealed that the conserved tyrosine residue in the active centre of most of the gram-positive and gram-negative bacterial enzymes has been replaced with tryptophan in most of the cyanobacterial enzymes. We carried out the site-directed mutagenesis involving the corresponding residue of Synechocystis enzyme (W385) and revealed that the residue is involved in the substrate recognition by the enzyme.

Expression of aspartyl protease and C3HC4-type RING zinc finger genes are responsive to ascorbic acid in Arabidopsis thaliana.

Ascorbate (AsA) is a redox buffer and enzyme cofactor with various proposed functions in stress responses and growth. The aim was to identify genes whose transcript levels respond to changes in leaf AsA. The AsA-deficient Arabidopsis mutant vtc2-1 was incubated with the AsA precursor L-galactono-1,4-lactone (L-GalL) to increase leaf AsA concentration. Differentially expressed genes screened by DNA microarray were further characterized for AsA responsiveness in wild-type plants. The analysis of 14 candidates by real-time PCR identified an aspartyl protease gene (ASP, At1g66180) and a C3HC4-type RING zinc finger gene (AtATL15, At1g22500) whose transcripts were rapidly responsive to increases in AsA pool size caused by L-GalL and AsA supplementation and light. Transgenic Arabidopsis plants expressing an AtATL15 promoter::luciferase reporter confirmed that the promoter is L-GalL, AsA, and light responsive. The expression patterns of ASP and AtATL15 suggest they have roles in growth regulation. The promoter of AtATL15 is responsive to AsA status and will provide a tool to investigate the functions of AsA in plants further.

A novel L-aspartate dehydrogenase from the mesophilic bacterium Pseudomonas aeruginosa PAO1: molecular characterization and application for L-aspartate production.

L-aspartate dehydrogenase (EC 1.4.1.21; L: -AspDH) is a rare member of amino acid dehydrogenase superfamily and so far, two thermophilic enzymes have been reported. In our study, an ORF PA3505 encoding for a putative L-AspDH in the mesophilic bacterium Pseudomonas aeruginosa PAO1 was identified, cloned, and overexpressed in Escherichia coli. The homogeneously purified enzyme (PaeAspDH) was a dimeric protein with a molecular mass of about 28 kDa exhibiting a very high specific activity for L-aspartate (L-Asp) and oxaloacetate (OAA) of 127 and 147 U mg(-1), respectively. The enzyme was capable of utilizing both nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) as coenzyme. PaeAspDH showed a T (m) value of 48°C for 20 min that was improved to approximately 60°C by the addition of 0.4 M NaCl or 30% glycerol. The apparent K (m) values for OAA, NADH, and ammonia were 2.12, 0.045, and 10.1 mM, respectively; comparable results were observed with NADPH. The L-Asp production system B consisting of PaeAspDH, Bacillus subtilis malate dehydrogenase and E. coli fumarase, achieved a high level of L-Asp production (625 mM) from fumarate in fed-batch process with a molar conversion yield of 89.4%. Furthermore, the fermentative production system C released 33 mM of L-Asp after 50 h by using succinate as carbon source. This study represented an extensive characterization of the mesophilic AspDH and its potential applicability for efficient and attractive production of L-Asp. Our novel production systems are also hopeful for developing the new processes for other compounds production.

A non-NadB type L-aspartate dehydrogenase from Ralstonia eutropha strain JMP134: molecular characterization and physiological functions.

We report the molecular characterization and physiological function of a novel L-aspartate dehydrogenase (AspDH). The purified enzyme was a 28-kDa dimeric protein, exhibiting high catalytic activity for L-aspartate (L-Asp) oxidation using NAD and/or NADP as cofactors. Quantitative real-time PCR analysis indicated that the genes involved in the AspDH gene cluster, poly-3-hydroxyalkanoate (PHA) biosynthesis, and the TCA cycle were substantially induced by L-Asp in wild-type cells. In contrast, expression of the aspartase and aspartate aminotransferase genes was substantially induced in the AspDH gene knockout mutant (ΔB3576) but not in the wild type. GC-MS analyses revealed that the wild-type strain synthesized poly-3-hydroxybutyrate from fructose or L-Asp, whereas the ΔB3576 mutant did not synthesize PHA from L-Asp. AspDH gene cluster products might be involved in the biosynthesis of the PHA precursor, revealing that AspDH was a non-NadB type enzyme, and thus entirely different from the previously reported NadB type enzymes working in NAD biosynthesis.

Expression analysis of the VTC2 and VTC5 genes encoding GDP-L-galactose phosphorylase, an enzyme involved in ascorbate biosynthesis, in Arabidopsis thaliana.

Arabidopsis thaliana contains two GDP-L-galactose phosphorylase genes, VTC2 and VTC5, which are critical for ascorbate (AsA) biosynthesis. We investigated the expression levels of both VTC2 and VTC5 genes in wild-type A. thaliana and the AsA deficient mutants during early seedling growth. Ascorbate accumulated to an equal extent in all genotypes up to 5 d post-germination (DPG). The transcript level of VTC2 was dominant, and increased in parallel with AsA accumulation in the wild type. On the other hand, the expression of VTC5 compensated for the reduced VTC2 transcription levels in the AsA deficient mutant vtc2-1 in young seedlings. A luciferase activity assay indicated that the VTC5 promoter was more active in young (2 DPG) cotyledons and that the VTC2 and VTC5 promoters drove a day-to-night variation in expression. The present work provides clues to the precise roles of VTC2 and VTC5 in AsA biosynthesis in A. thaliana at the young seedling stage.

Euglena gracilis ascorbate peroxidase forms an intramolecular dimeric structure: its unique molecular characterization.

Euglena gracilis lacks a catalase and contains a single APX (ascorbate peroxidase) and enzymes related to the redox cycle of ascorbate in the cytosol. In the present study, a full-length cDNA clone encoding the Euglena APX was isolated and found to contain an open reading frame encoding a protein of 649 amino acids with a calculated molecular mass of 70.5 kDa. Interestingly, the enzyme consisted of two entirely homologous catalytic domains, designated APX-N and APX-C, and an 102 amino acid extension in the N-terminal region, which had a typical class II signal proposed for plastid targeting in Euglena. A computer-assisted analysis indicated a novel protein structure with an intramolecular dimeric structure. The analysis of cell fractionation showed that the APX protein is distributed in the cytosol, but not the plastids, suggesting that Euglena APX becomes mature in the cytosol after processing of the precursor. The kinetics of the recombinant mature FL (full-length)-APX and the APX-N and APX-C domains with ascorbate and H2O2 were almost the same as that of the native enzyme. However, the substrate specificity of the mature FL-APX and the native enzyme was different from that of APX-N and APX-C. The mature FL-APX, but not the truncated forms, could reduce alkyl hydroperoxides, suggesting that the dimeric structure is correlated with substrate recognition. In Euglena cells transfected with double-stranded RNA, the silencing of APX expression resulted in a significant increase in the cellular level of H2O2, indicating the physiological importance of APX to the metabolism of H2O2.