Dimorphic DNA variation in the anionic peroxidase gene AtPrx53 of Arabidopsis thaliana

The DNA polymorphism in the AtPrx53 gene which encodes anionic peroxidase was analyzed in 20 Arabidopsis thaliana accessions. There are two divergent sequence types (Col and Dj-like haplotypes) in the AtPrx53 gene that differ by 2 indel and 16 non-singleton nucleotide polymorphisms including 5 nucleotide polymorphic sites responsible for 4 deduced amino acid replacements. Two of the amino acid substitutions (Phe/Ser180and Asp/Asn270) could be responsible for the difference in electrophoretic mobility of AtPrx53 allozymes. One of them (Phe/Ser180) lies within the hypervariable region, indicating that this amino acid polymorphism is subjected to balancing selection. The revealed difference between deduced allozymes is related to the dimorphism in mobility of three major anionic peroxidase isoforms which according to previously established data encoded by AtPrx53 gene. The haplotype Col which included 12 accessions from three different continents is characterized by faster mobility of three isoforms in comparison with the Dj haplotype represented by eight accessions. There is a significant association between the haplotype and several developmental traits: leaf number, flowering time, main stem height etc. Lines of the Dj haplotype have shorter duration of vegetative stages and flower earlier than most of Col haplotype accessions. The reasons of this association are discussed.     

Genes encoding plant-specific class III peroxidases are responsible for increased cold tolerance of the brassinosteroid-insensitive 1 mutant

We previously reported that one of the brassinosteroidinsensitive mutants, bri1-9, showed increased cold tolerance compared with both wild type and BRI1-overexpressing transgenic plants, despite its severe growth retardation. This increased tolerance in bri1-9 resulted from the constitutively high expression of stress-inducible genes under normal conditions. In this report, we focused on the genes encoding class III plant peroxidases (AtPrxs) because we found that, compared with wild type, bri1-9 plants contain higher levels of reactive oxygen species (ROS) that are not involved with the activation of NADPH oxidase and show an increased level of expression of a subset of genes encoding class III plant peroxidases. Treatment with a peroxidase inhibitor, salicylhydroxamic acid (SHAM), led to the reduction of cold resistance in bri1-9. Among 73 genes that encode AtPrxs in Arabidopsis, we selected four (AtPrx1, AtPrx22, AtPrx39, and AtPrx69) for further functional analyses in response to cold temperatures. T-DNA insertional knockout mutants showed increased sensitivity to cold stress as measured by leaf damage and ion leakage. In contrast, the overexpression of AtPrx22, AtPrx39, and AtPrx69 increased cold tolerance in the BRI1-GFP plants. Taken together, these results indicate that the appropriate expression of a particular subset of AtPrx genes and the resulting higher levels of ROS production are required for the cold tolerance.     

Catalytic Profile of Arabidopsis Peroxidases,AtPrx-2, 25 and 71, Contributing to Stem Lignification

Lignins are aromatic heteropolymers that arise from oxidative coupling of lignin precursors, including lignin monomers (p-coumaryl, coniferyl, and sinapyl alcohols), oligomers, and polymers. Whereas plant peroxidases have been shown to catalyze oxidative coupling of monolignols, the oxidation activity of well-studied plant peroxidases, such as horseradish peroxidase C (HRP-C) and AtPrx53, are quite low for sinapyl alcohol. This characteristic difference has led to controversy regarding the oxidation mechanism of sinapyl alcohol and lignin oligomers and polymers by plant peroxidases. The present study explored the oxidation activities of three plant peroxidases, AtPrx2, AtPrx25, and AtPrx71, which have been already shown to be involved in lignification in the Arabidopsis stem. Recombinant proteins of these peroxidases (rAtPrxs) were produced in Escherichia coli as inclusion bodies and successfully refolded to yield their active forms. rAtPrx2, rAtPrx25, and rAtPrx71 were found to oxidize two syringyl compounds (2,6-dimethoxyphenol and syringaldazine), which were employed here as model monolignol compounds, with higher specific activities than HRP-C and rAtPrx53. Interestingly, rAtPrx2 and rAtPrx71 oxidized syringyl compounds more efficiently than guaiacol. Moreover, assays with ferrocytochrome c as a substrate showed that AtPrx2, AtPrx25, and AtPrx71 possessed the ability to oxidize large molecules. This characteristic may originate in a protein radical. These results suggest that the plant peroxidases responsible for lignin polymerization are able to directly oxidize all lignin precursors.     

Multiple β-defensin isoforms identified in early developmental stages of the teleost Paralichthys olivaceus

The β-defensin-like gene and its cloned isoforms (fBDI-1 to -5) were identified in an expressed sequence tag (EST) library from the early developmental stages of the olive flounder, Paralichthys olivaceus. The fBDI cDNA clones show identical amino acid sequences in 24 residues of the signal peptide and 38 residues of the mature peptide; however, the propiece region varies in sequence and length, from 5 to 15 amino acid residues. The predicted molecular weight of the mature peptide is 3.83 kDa, and its predicted isoelectric point is 4.1, showing anionic properties. The genomic organisation of the isoforms was analysed using bacterial artificial chromosome (BAC) DNA containing the fBDI gene. Southern blotting and sequence analyses of fBDI BAC DNA confirmed that the fBDI isoforms cluster at the same locus and exhibit the conserved gene organisation reported for other fish defensin genes. The fBDI mRNA was expressed constitutively in early developmental stages after hatching, and pathogen challenge induced fBDI expression in the head kidney of juvenile fish. We also produced a recombinant fBDI peptide (smfBD) using the expression plasmid pET32 and examined its bioactivity toward Escherichia coli.     

Lateral Gene Transfer Acts As an Evolutionary Shortcut to Efficient C4 Biochemistry

The adaptation of proteins for novel functions often requires changes in their kinetics via amino acid replacement. This process can require multiple mutations, and therefore extended periods of selection. The transfer of genes among distinct species might speed up the process, by providing proteins already adapted for the novel function. However, this hypothesis remains untested in multicellular eukaryotes. The grass Alloteropsis is an ideal system to test this hypothesis due to its diversity of genes encoding phosphoenolpyruvate carboxylase, an enzyme that catalyzes one of the key reactions in the C₄ pathway. Different accessions of Alloteropsis either use native isoforms relatively recently co-opted from other functions or isoforms that were laterally acquired from distantly related species that evolved the C₄ trait much earlier. By comparing the enzyme kinetics, we show that native isoforms with few amino acid replacements have substrate K_M values similar to the non-C₄ ancestral form, but exhibit marked increases in catalytic efficiency. The co-option of native isoforms was therefore followed by rapid catalytic improvements, which appear to rely on standing genetic variation observed within one species. Native C₄ isoforms with more amino acid replacements exhibit additional changes in affinities, suggesting that the initial catalytic improvements are followed by gradual modifications. Finally, laterally acquired genes show both strong increases in catalytic efficiency and important changes in substrate handling. We conclude that the transfer of genes among distant species sharing the same physiological novelty creates an evolutionary shortcut toward more efficient enzymes, effectively accelerating evolution.     

Purification and cloning of a Chinese red radish peroxidase that metabolise pelargonidin and forms a gene family in Brassicaceae

An anionic peroxidase RsPrx1 was purified (RZ=3.0) and characterized from roots of Chinese red radish (Raphanus sativus var. niger, Brassicaceae). The specific activity of RsPrx1 (micromol mg(-1) min(-1)) is 413.5 (ferulic acid); 258.7 (ABTS); 177.3 (caffeic acid) and 10.0 (guaiacol acid). The optimum pH is 4.0 (citrate buffer) using ABTS as substrate. RsPrx1 can utilise the red pigment present in the root, pelargonidin, as substrate and the specific activity is 93.6 micromol mg(-1) min(-1). The molecular mass of RsPrx1 is 45 kDa (denatured) and 46 kDa (native) as determined by SDS-PAGE and gel filtration, respectively. The isoelectric point (pI) determined by native IEF is 4.7 and by chromatofocusing (Mono P) is 5.1. Analysis of tryptic peptides by nanoscale liquid chromatography-tandem mass spectrometry (LC-MS/MS) covered 27% of the RsPrx1 sequence and confirmed its identity. The gene encoding RsPrx1 was cloned by PCR and the amino acid sequence showed the highest identity (82%) to peroxidase AtPrx22 and AtPrx23 from Arabidopsis thaliana and to HRPC3 and HRPE5 from horseradish, respectively. Activity-stained IEF gels show that RsPrx1 is primarily expressed in the roots in agreement with the expression profile of the orthologous genes. These five orthologous peroxidases have three introns of variable length and sequence at conserved locations between the distal and proximal histidine. The results suggest that RsPrx1 orthologs are widespread in the Brassicaceae plant family with a 15-residue-long C-terminal propeptide in common. Based on the results, we propose that RsPrx1 and orthologs are targeted to the vacuoles to modify stored anthocyanins like pelargonidin.     

Intraspecific variation of the crotamine and crotasin genes in Crotalus durissus rattlesnakes

Crotamine is a small basic myotoxin peptide of Crotalus durissus venom, with β-defensin scafold and variable concentration in individual venoms. The crotamine gene was mapped to the end of chromosome 2 and the signal intensity differed significantly between the two homologues. In contrast to crotamine, the paralogous crotasin gene is scarcely expressed in the venom glands. In this study, we analyzed the crotamine concentrations in the venoms of a total of 23 rattlesnakes from diverse Brazilian localities by ELISA as well as the copy number of both crotamine and crotasin genes by real-time PCR. Crotamine was found to constitute 5–29% of venom proteins varying greatly among individual animals. The crotamine gene exists from 1 to 32 copies per haploid genome, whereas the crotasin gene is present from 1 to 7 copies. Furthermore, we observed that the crotamine concentration and crotamine gene copy number are positively correlated (r² = 0.68), implying the variation of crotamine in venom results from the variation of the gene copy number. Sequencing of 50 independent copies of crotamine and crotasin genes from four different rattlesnakes revealed the presence of six crotasin isoforms with a single amino acid difference from the original crotasin sequence, whereas only two additional crotamine isoforms were observed. Taken together, our results suggested that after duplication from a common ancestor gene, crotamine and crotasin may have diverged in such a way that the crotamine gene underwent repetitive duplication to increase its copy number, whereas the crotasin gene diversified its sequence.     

Bioinformatic and functional characterization of the basic peroxidase 72 from Arabidopsis thaliana involved in lignin biosynthesis

Lignins result from the oxidative polymerization of three hydroxycinnamyl (p-coumaryl, coniferyl, and sinapyl) alcohols in a reaction mediated by peroxidases. The most important of these is the cationic peroxidase from Zinnia elegans (ZePrx), an enzyme considered to be responsible for the last step of lignification in this plant. Bibliographical evidence indicates that the arabidopsis peroxidase 72 (AtPrx72), which is homolog to ZePrx, could have an important role in lignification. For this reason, we performed a bioinformatic, histochemical, photosynthetic, and phenotypical and lignin composition analysis of an arabidopsis knock-out mutant of AtPrx72 with the aim of characterizing the effects that occurred due to the absence of expression of this peroxidase from the aspects of plant physiology such as vascular development, lignification, and photosynthesis. In silico analyses indicated a high homology between AtPrx72 and ZePrx, cell wall localization and probably optimal levels of translation of AtPrx72. The histochemical study revealed a low content in syringyl units and a decrease in the amount of lignin in the atprx72 mutant plants compared to WT. The atprx72 mutant plants grew more slowly than WT plants, with both smaller rosette and principal stem, and with fewer branches and siliques than the WT plants. Lastly, chlorophyll a fluorescence revealed a significant decrease in Φ_PSII and q _L in atprx72 mutant plants that could be related to changes in carbon partitioning and/or utilization of redox equivalents in arabidopsis metabolism. The results suggest an important role of AtPrx72 in lignin biosynthesis. In addition, knock-out plants were able to respond and adapt to an insufficiency of lignification.     

Overexpression of a peroxidase gene (<Emphasis Type="Italic">AtPrx64</Emphasis>) of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> in tobacco improves plant’s tolerance to aluminum stress

Key message

AtPrx64 is one of the peroxidases gene up-regulated in Al stress and has some functions in the formation of plant second cell wall. Its overexpression may improve plant tolerance to Al by some ways. Studies on its function under Al stress may help us to understand the mechanism of plant tolerance to Al stress.

Abstract

In Arabidopsis thaliana, the expressions of some genes (AtPrxs) encoding class III plant peroxidases have been found to be either up-regulated or down-regulated under aluminum (Al) stress. Among 73 genes that encode AtPrxs in Arabidopsis, AtPrx64 is always up-regulated by Al stress, suggesting this gene plays protective roles in response to such stress. In this study, transgenic tobacco plants were generated to examine the effects of overexpressing of AtPrx64 gene on the tolerance to Al stress. The results showed that overexpression of AtPrx64 gene increased the root growth and reduced the accumulation of Al and ROS in the roots. Compared with wild type controls, transgenic tobaccos had much less soluble proteins and malondialdehyde in roots and much more root citrate exudation. The activity of plasma membrane (PM) H⁺-ATPase, the phosphorylation of PM H⁺-ATPase and its interaction with 14-3-3 proteins increased in transgenic tobaccos; moreover, the content of lignin in root tips also increased. Taken together, these results showed that overexpression of AtPrx64 gene might enhance the tolerance of tobacco to Al stress.

     

Allele-Level Haplotype Frequencies and Pairwise Linkage Disequilibrium for 14 KIR Loci in 506 European-American Individuals

The immune responses of natural killer cells are regulated, in part, by killer cell immunoglobulin-like receptors (KIR). The 16 closely-related genes in the KIR gene system have been diversified by gene duplication and unequal crossing over, thereby generating haplotypes with variation in gene copy number. Allelic variation also contributes to diversity within the complex. In this study, we estimated allele-level haplotype frequencies and pairwise linkage disequilibrium statistics for 14 KIR loci. The typing utilized multiple methodologies by four laboratories to provide at least 2x coverage for each allele. The computational methods generated maximum-likelihood estimates of allele-level haplotypes. Our results indicate the most extensive allele diversity was observed for the KIR framework genes and for the genes localized to the telomeric region of the KIR A haplotype. Particular alleles of the stimulatory loci appear to be nearly fixed on specific, common haplotypes while many of the less frequent alleles of the inhibitory loci appeared on multiple haplotypes, some with common haplotype structures. Haplotype structures cA01 and/or tA01 predominate in this cohort, as has been observed in most populations worldwide. Linkage disequilibrium is high within the centromeric and telomeric haplotype regions but not between them and is particularly strong between centromeric gene pairs KIR2DL5∼KIR2DS3S5 and KIR2DS3S5∼KIR2DL1, and telomeric KIR3DL1∼KIR2DS4. Although 93% of the individuals have unique pairs of full-length allelic haplotypes, large genomic blocks sharing specific sets of alleles are seen in the most frequent haplotypes. These high-resolution, high-quality haplotypes extend our basic knowledge of the KIR gene system and may be used to support clinical studies beyond single gene analysis.     

Purification and identification of a Ca(2+)-pectate binding peroxidase from Arabidopsis leaves

A protein fraction was obtained from Arabidopsis (Arabidopsis thaliana, L.) leaf extract by affinity chromatography through a Ca(2+)-pectate/polyacrylamide gel. Further purification by preparative isoelectric focusing and SDS PAGE allowed the separation of a peroxidase that was identified as being peroxidase AtPrx34 (AtprxCb, accession number X71794) by N-terminal amino acid microsequencing. AtPrx34 belongs to a group of five Arabidopsis sequences encoding putative pectin-binding peroxidases. An expression study showed that it is expressed in root, stem, flower and leaf. It was produced by Escherichia coli and tested for its ability to bind to Ca(2+)-pectate. The identity of the amino acids involved in the interaction between the peroxidase and the Ca(2+)-pectate structure is discussed.     

Association of inherited variation in Toll-like receptor genes with malignant melanoma susceptibility and survival

The family of Toll-like receptors (TLRs) is critical in linking innate and acquired immunity. Polymorphisms in the genes encoding TLRs have been associated with autoimmune diseases and cancer. We investigated the genetic variation of TLR genes and its potential impact on melanoma susceptibility and patient survival. The study included 763 cutaneous melanoma cases recruited in Germany and 736 matched controls that were genotyped for 47 single nucleotide polymorphisms (SNPs) in 8 TLR genes. The relationship between genotype, disease status and survival was investigated taking into account patient and tumor characteristics, and melanoma treatment. Analysis of 7 SNPs in TLR2, 7 SNPs in TLR3 and 8 SNPs in TLR4 showed statistically significant differences in distribution of inferred haplotypes between cases and controls. No individual polymorphism was associated with disease susceptibility except for the observed tendency for TLR2-rs3804099 (odds ratio OR  = 1.15, 95% CI 0.99–1.34, p = 0.07) and TLR4-rs2149356 (OR = 0.85, 95% CI 0.73–1.00, p = 0.06). Both polymorphisms were part of the haplotypes associated with risk modulation. An improved overall survival (Hazard ratio HR 0.53, 95% CI 0.32–0.88) and survival following metastasis (HR 0.55, 95% CI 0.34–0.91) were observed in carriers of the variant allele (D299G) of TLR4-rs4986790. In addition various TLR2, TLR4 and TLR5 haplotypes were associated with increased overall survival. Our results point to a novel association between TLR gene variants and haplotypes with melanoma survival. Our data suggest a role for the D299G polymorphism in the TLR4 gene in overall survival and a potential link with systemic treatment at stage IV of the disease. The polymorphic amino acid residue, located in the ectodomain of TLR4, can have functional consequences.     

Analysis of expression profiles of three peroxidase genes associated with lignification in Arabidopsis thaliana

We have investigated the mechanism of lignification during tracheary element (TE) differentiation using a Zinnia elegans xylogenic culture. In the process, we isolated ZPO-C , a peroxidase gene of Z. elegans that is expressed specifically in differentiating TEs. ZPO-C is suggested to be involved in lignification of Z. elegans TEs in vivo and in vitro. Furthermore, a peroxidase gene of Arabidopsis thaliana ( AtPrx66 ), which is homologous to ZPO-C , was identified. The expression profile and functions of the gene in planta remain to be investigated. In this study, we performed promoter :: β-glucuronidase (GUS) assays to investigate the expression profiles and functions of the ZPO-C -like peroxidases in A. thaliana . We generated transgenic A. thaliana lines carrying AtPrx66, AtPrx47 or AtPrx64 (peroxidases showing high sequence similarity to AtPrx66 ) promoter :: GUS reporter gene fusions. The GUS activities of AtPrx66, AtPrx47 and AtPrx64 promoter :: GUS lines were arranged concentrically from the center to the periphery in the roots of seedlings. Furthermore, histochemical GUS assays using inflorescence stems showed that AtPrx66, AtPrx47 and AtPrx64 promoter-driven GUS were mainly expressed in the differentiating vessels, xylem parenchyma and sclerenchyma, respectively. These results suggest that the gene expressions of these three peroxidases, which showed high sequence similarity to one another, are differentially regulated in various tissues and organs. In addition, our results suggest that while AtPrx66 and AtPrx47 are associated with lignification of vessels, AtPrx64 is associated with lignification of sclerenchyma.     

CodY of Streptococcus pneumoniae: link between nutritional gene regulation and colonization

CodY is a nutritional regulator mainly involved in amino acid metabolism. It has been extensively studied in Bacillus subtilis and Lactococcus lactis. We investigated the role of CodY in gene regulation and virulence of the human pathogen Streptococcus pneumoniae. We constructed a codY mutant and examined the effect on gene and protein expression by microarray and two-dimensional differential gel electrophoresis analysis. The pneumococcal CodY regulon was found to consist predominantly of genes involved in amino acid metabolism but also several other cellular processes, such as carbon metabolism and iron uptake. By means of electrophoretic mobility shift assays and DNA footprinting, we showed that most of the targets identified are under the direct control of CodY. By mutating DNA predicted to represent the CodY box based on the L. lactis consensus, we demonstrated that this sequence is indeed required for in vitro DNA binding to target promoters. Similar to L. lactis, DNA binding of CodY was enhanced in the presence of branched-chain amino acids, but not by GTP. We observed in experimental mouse models that codY is transcribed in the murine nasopharynx and lungs and is specifically required for colonization. This finding was underscored by the diminished ability of the codY mutant to adhere to nasopharyngeal cells in vitro. Furthermore, we found that pcpA, activated by CodY, is required for adherence to nasopharyngeal cells, suggesting a direct link between nutritional regulation and adherence. In conclusion, pneumococcal CodY predominantly regulates genes involved in amino acid metabolism and contributes to the early stages of infection, i.e., colonization of the nasopharynx.     

The activity of peroxidase in various cell fractions of wheat plants infected with Septoria nodorum berk.

The activities of peroxidase isoforms and hydrogen peroxide content in leaf cuttings of wheat (Triticum aestivum L., cv. Diamant) resistant to Septoria blotch were studied during aging and following the infection with Septoria nodorum Berk. The differential activation of peroxidase isoforms was regulated by hydrogen peroxide level in the tissue. At early stages of fungus development in plant tissues, the decrease in the activities of soluble, membrane and ion-bound fractions of peroxidase elevated the level of hydrogen peroxide in infected tissues and rapidly activated peroxidase isoforms in infected tissues as compared to the aging ones even before disease symptoms appeared. The anionic peroxidases, which were first to respond to the pathogen, seem to stand for wheat resistance to fungal infections and the protection of leaf tissues from oxidative stress.     

Profile of peroxidase isoforms influenced by spongy tissue disorder in Alphonso mango (<Emphasis Type="Italic">Mangifera indica</Emphasis> L.) fruits

This paper describes the profile of peroxidase (POX) isoenzymes induced due to the natural infection of Staphylococcus xylosus in spongy Alphonso mango fruits. Very low levels of protein and POX activity was observed in non-spongy unripe fruits, and when these fruits turned table-ripe, the levels of both the protein content and POX activity increased several fold. The spongy fruits, however, showed further 2-fold increase in POX activity; although drastic decrease in protein content was observed. Anionic and cationic PAGE, and isoelectric focusing (IEF), resulted in separation of various isoenzymes of POX. Both, anionic and cationic PAGE indicated that, at unripe stage, only basic isoforms were present in trace amounts. In non-spongy ripe fruits, increased levels of both anionic and cationic isoforms were observed after staining the gel with o-dianisidine, the POX substrate. In spongy fruits, however, an anionic PAGE showed appearance of four acidic isoforms with relative electrophoretic mobility (REM) of 0.52, 0.73, 0.78, and 0.84 and an isoenzyme (REM 0.52), showed further activation, as indicated by the intense dark color formation. Cationic PAGE also indicated higher levels of two basic isoforms (REM 0.56 and 0.62), in the spongy fruits. Isoelectric focusing resolved these isoenzymes in acidic, neutral, and basic isoforms. Two acidic isoforms in the pI range of 2–3.5 were detected toward the anode region and two cationic isoforms of pI 7.8 and 8.7, toward the cathode, giving visible indication of increased levels of these isoforms. The increased intensities of the POX bands observed in anionic and cationic PAGE, and IEF, gave confirmatory evidence for the up regulation of anionic and cationic isoforms in spongy fruits. These isoenzymes could have been overexpressed as a defense response of the spongy fruits against the Staphylococcus infection.     

The suppression of AtPrx52 affects fibers but not xylem lignification in Arabidopsis by altering the proportion of syringyl units

Lignins result from the oxidative polymerization of three hydroxycinnamyl (p‐coumaryl, coniferyl and sinapyl) alcohols in a reaction mediated by peroxidases (EC 1.11.1.7) and laccases (EC 1.10.3.2), yielding H, G and S units, respectively. Although both acidic and basic peroxidases can oxidize p‐coumaryl and coniferyl alcohol, only basic peroxidases are able to oxidize sinapyl alcohol. The AtPrx52 from Arabidopsis is a basic peroxidase that has been reported to be highly homologous to the basic peroxidase of Zinnia elegans, the only peroxidase which has been unequivocally linked to lignin formation. Here, we show how the suppression of AtPrx52 causes a change in lignin composition, mainly at the level of stem interfascicular fibers. Quantification of lignins in two different atprx52 knock‐out mutants revealed a decrease of lignin amount compared with wild type. The S/G ratio, obtained by both nitrobenzene oxidation and thioacidolysis, indicated a decrease in S units in the atprx52 mutants. As deduced from Wiesner and mainly Mäule staining, this reduction in S unit content appears to be restricted to the interfascicular fibers. Moreover, quantitative polymerase chain reaction analysis in atprx52 plants showed a general downregulation of genes involved in lignin biosynthetic pathway, as well as genes related to secondary cell wall. On the other hand, other routes from phenylpropanoid metabolism were induced. Taken together, our results indicate that AtPrx52 is involved in the synthesis of S units in interfascicular fibers at late stages of the lignification process.