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.

Distribution and Functions of Monodehydroascorbate Reductases in Plants: Comprehensive Reverse Genetic Analysis of Arabidopsis thaliana Enzymes.

Monodehydroascorbate reductase (MDAR) is an enzyme involved in ascorbate recycling. Arabidopsis thaliana has five MDAR genes that encode two cytosolic, one cytosolic/peroxisomal, one peroxisomal membrane-attached, and one chloroplastic/mitochondrial isoform. In contrast, tomato plants possess only three enzymes, lacking the cytosol-specific enzymes. Thus, the number and distribution of MDAR isoforms differ according to plant species. Moreover, the physiological significance of MDARs remains poorly understood. In this study, we classify plant MDARs into three classes: class I, chloroplastic/mitochondrial enzymes; class II, peroxisomal membrane-attached enzymes; and class III, cytosolic/peroxisomal enzymes. The cytosol-specific isoforms form a subclass of class III and are conserved only in Brassicaceae plants. With some exceptions, all land plants and a charophyte algae, Klebsormidium flaccidum, contain all three classes. Using reverse genetic analysis of Arabidopsis thaliana mutants lacking one or more isoforms, we provide new insight into the roles of MDARs; for example, (1) the lack of two isoforms in a specific combination results in lethality, and (2) the role of MDARs in ascorbate redox regulation in leaves can be largely compensated by other systems. Based on these findings, we discuss the distribution and function of MDAR isoforms in land plants and their cooperation with other recycling systems.

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.

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.

Chloroplast development activates the expression of ascorbate biosynthesis-associated genes in Arabidopsis roots.

Transcriptional activation of ascorbate biosynthesis-associated genes under illumination is one of the important steps in ascorbate pool size regulation in photosynthetic tissues. Several biological processes within chloroplasts such as photosynthesis are required for this activation, suggesting functional chloroplasts to play a key role. We herein found that when grown on agar plate, ascorbate content in Arabidopsis non-photosynthetic tissues, roots, are unexpectedly almost comparable to that in shoots. The high accumulation of ascorbate was particularly observed in root regions closer to the root-hypocotyl junction, in which chloroplast development occurred because of a direct exposure to light. When chloroplast development in roots were further stimulated by shoot removal, the expression of biosynthetic genes, especially VTC2 gene that encodes GDP-l-galactose phosphorylase, was activated, resulting in an increase in ascorbate pool size. These positive effects were canceled when the roots were treated with a photosynthetic inhibitor. A null mutation in the LONG HYPOCOTYL 5 (HY5) gene almost completely inhibited root greening as well as the VTC2 expression. Overall, these findings show that chloroplast development can trigger the expression of ascorbate biosynthesis-associated genes not only in leaves but also in roots.

A major isoform of mitochondrial trans-2-enoyl-CoA reductase is dispensable for wax ester production in Euglena gracilis under anaerobic conditions.

Under anaerobic conditions, Euglena gracilis produces a large amount of wax ester through mitochondrial fatty acid synthesis from storage polysaccharides termed paramylon, to generate ATP. Trans-2-enoyl-CoA reductases (TERs) in mitochondria have been considered to play a key role in this process, because the enzymes catalyze the reduction of short chain length CoA-substrates (such as crotonyl-CoA). A TER enzyme (EgTER1) has been previously identified and enzymologically characterized; however, its physiological significance remained to be evaluated by genetic analysis. We herein generated EgTER1-knockdown Euglena cells, in which total crotonyl-CoA reductase activity was decreased to 10% of control value. Notably, the knockdown cells showed a severe bleaching phenotype with deficiencies in chlorophylls and glycolipids, but grew normally under heterotrophic conditions (with glucose supplementation). Moreover, the knockdown cells accumulated much greater quantities of wax ester than control cells before and after transfer to anaerobic conditions, which was accompanied by a large metabolomic change. Furthermore, we failed to find any contribution of other potential TER genes in wax ester production. Our findings propose a novel role of EgTER1 in the greening process and demonstrate that this enzyme is dispensable for wax ester production under anaerobic conditions.

Comparative proteomic analysis of mitochondria isolated from Euglena gracilis under aerobic and hypoxic conditions.

The unicellular microalga Euglena gracilis produces wax esters for ATP acquisition under low-oxygen conditions. The regulatory mechanism of wax ester production is not yet understood. Indeed, our previous transcriptomic analysis showed that transcript levels of genes involved in the wax ester synthesis hardly changed under hypoxic conditions, suggesting contribution of post-transcriptional regulation. In this study, we conducted a proteome analysis of E. gracilis mitochondria, as this organelle employs the fatty-acid synthesis pathway under hypoxic conditions. Mitochondria were isolated from E. gracilis SM-ZK strain treated with both aerobic and hypoxic conditions and used for shotgun proteomic analysis. Three independent proteomic analyses succeeded in identifying a total of 714 non-redundant proteins. Of these, 229 were detected in common to all experiments, and 116 were significantly recognized as differentially expressed proteins. GO enrichment analysis suggested dynamic changes in mitochondrial metabolic pathways and redox reactions under aerobic and hypoxic conditions. Protein levels of bifunctional enzymes isocitrate lyase and malate synthase in glyoxylate cycle were 1.35-fold higher under hypoxic conditions. Abundances of the propionyl-CoA synthetic enzymes, succinyl-CoA synthetase and propionyl-CoA carboxylase, were also 1.35- and 1.47-fold higher, respectively, under hypoxic conditions. Protein levels of pyruvate:NADP+ oxidoreductase, a key enzyme for anaerobic synthesis of acetyl-CoA, which serves as a C2 donor for fatty acids, showed a 1.68-fold increase under hypoxic conditions, whereas those of pyruvate dehydrogenase subunits showed a 0.77-0.81-fold decrease. Protein levels of the fatty-acid synthesis enzymes, 3-ketoacyl-CoA thiolase isoforms (KAT1 and KAT2), 3-hydroxyacyl-CoA dehydrogenases, and acyl-CoA dehydrogenase were up-regulated by 1.20- to 1.42-fold in response to hypoxic treatment. Overall, our proteomic analysis revealed that wax ester synthesis-related enzymes are up-regulated at the protein level post-transcriptionally to promote wax ester production in E. gracilis under low-oxygen conditions.

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.

Prediction of arsenic and antimony transporter major intrinsic proteins from the genomes of crop plants.

Major intrinsic proteins (MIPs), commonly known as aquaporins, transport water and non-polar small solutes. Comparing the 3D models and the primary selectivity-related motifs (two Asn-Pro-Ala (NPA) regions, the aromatic/arginine (ar/R) selectivity filter, and Froger's positions (FPs)) of all plant MIPs that have been experimentally proven to transport arsenic (As) and antimony (Sb), some substrate-specific signature sequences (SSSS) or specificity determining sites (SDPs) have been predicted. These SSSS or SDPs were determined in 543 MIPs found in the genomes of 12 crop plants; the As and Sb transporters were predicted to be distributed in noduline-26 like intrinsic proteins (NIPs), and every plant had one or several As and Sb transporter NIPs. Phylogenetic grouping of the NIP subfamily based on the ar/R selectivity filter and FPs were linked to As and Sb transport. We further determined the group-wise substrate selectivity profiles of the NIPs in the 12 crop plants. In addition to two NPA regions, the ar/R filter, and FPs, certain amino acids especially in the pore line, loop D, and termini contribute to the functional distinctiveness of the NIP groups. Expression analysis of transcripts in different organs indicated that most of the As and Sb transporter NIPs were expressed in roots.

Wax Ester Synthase/Diacylglycerol Acyltransferase Isoenzymes Play a Pivotal Role in Wax Ester Biosynthesis in Euglena gracilis.

Wax ester fermentation is a unique energy gaining pathway for a unicellular phytoflagellated protozoan, Euglena gracilis, to survive under anaerobiosis. Wax esters produced in E. gracilis are composed of saturated fatty acids and alcohols, which are the major constituents of myristic acid and myristyl alcohol. Thus, wax esters can be promising alternative biofuels. Here, we report the identification and characterization of wax ester synthase/diacylglycerol acyltrasferase (WSD) isoenzymes as the terminal enzymes of wax ester production in E. gracilis. Among six possible Euglena WSD orthologs predicted by BLASTX search, gene expression analysis and in vivo evaluation for enzyme activity with yeast expressing individual recombinant WSDs indicated that two of them (EgWSD2 and EgWSD5) predominantly function as wax ester synthase. Furthermore, experiments with gene silencing demonstrated a pivotal role of both EgWSD2 and EgWSD5 in wax ester synthesis, as evidenced by remarkably reduced wax ester contents in EgWSD2/5-double knockdown E. gracilis cells treated with anaerobic conditions. Interestingly, the decreased ability to produce wax ester did not affect adaptation of E. gracilis to anaerobiosis. Lipid profile analysis suggested allocation of metabolites to other compounds including triacylglycerol instead of wax esters.

Genome-Wide Characterization of Major Intrinsic Proteins in Four Grass Plants and Their Non-Aqua Transport Selectivity Profiles with Comparative Perspective.

Major intrinsic proteins (MIPs), commonly known as aquaporins, transport not only water in plants but also other substrates of physiological significance and heavy metals. In most of the higher plants, MIPs are divided into five subfamilies (PIPs, TIPs, NIPs, SIPs and XIPs). Herein, we identified 68, 42, 38 and 28 full-length MIPs, respectively in the genomes of four monocot grass plants, specifically Panicum virgatum, Setaria italica, Sorghum bicolor and Brachypodium distachyon. Phylogenetic analysis showed that the grass plants had only four MIP subfamilies including PIPs, TIPs, NIPs and SIPs without XIPs. Based on structural analysis of the homology models and comparing the primary selectivity-related motifs [two NPA regions, aromatic/arginine (ar/R) selectivity filter and Froger's positions (FPs)] of all plant MIPs that have been experimentally proven to transport non-aqua substrates, we predicted the transport profiles of all MIPs in the four grass plants and also in eight other plants. Groups of MIP subfamilies based on ar/R selectivity filter and FPs were linked to the non-aqua transport profiles. We further deciphered the substrate selectivity profiles of the MIPs in the four grass plants and compared them with their counterparts in rice, maize, soybean, poplar, cotton, Arabidopsis thaliana, Physcomitrella patens and Selaginella moellendorffii. In addition to two NPA regions, ar/R filter and FPs, certain residues, especially in loops B and C, contribute to the functional distinctiveness of MIP groups. Expression analysis of transcripts in different organs indicated that non-aqua transport was related to expression of MIPs since most of the unexpressed MIPs were not predicted to facilitate the transport of non-aqua molecules. Among all MIPs in every plant, TIP (BdTIP1;1, SiTIP1;2, SbTIP2;1 and PvTIP1;2) had the overall highest mean expression. Our study generates significant information for understanding the diversity, evolution, non-aqua transport profiles and insight into comparative transport selectivity of plant MIPs, and provides tools for the development of transgenic plants.

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.

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.

Biochemical and physiological analyses of NADPH-dependent thioredoxin reductase isozymes in Euglena gracilis.

At least four peroxiredoxins that are coupled with the thioredoxin (Trx) system have been shown to play a key role in redox metabolism in the unicellular phytoflagellate Euglena gracilis. In order to clarify Trx-mediated redox regulation in this alga, we herein identified three NADPH-dependent thioredoxin reductases (NTRs) using a homologous search and characterized their enzymatic properties and physiological roles. Each Euglena NTR protein belonged to the small, large, and NTRC types, and were named EgNTR1, EgNTR2, and EgNTRC, respectively. EgNTR2 was phylogenetically different from the known NTRs in eukaryotic algae. EgNTR1 was predicted to be localized in mitochondria, EgNTR2 in the cytosol, and EgNTRC in plastids. The catalytic efficiency of EgNTR2 for NADPH was 30-46-fold higher than those of EgNTR1 and truncated form of EgNTRC, suggested that large type EgNTR2 reduced Trx more efficiently. The silencing of EgNTR2 gene expression resulted in significant growth inhibition and cell hypertrophy in Euglena cells. These results suggest that EgNTRs function in each cellular compartment and are physiologically important, particularly in the cytosol.

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.

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.

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).

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.