Proteome Changes Reveal the Protective Roles of Exogenous Citric Acid in Alleviating Cu Toxicity in Brassica napus L.

Citric acid (CA), as an organic chelator, plays a vital role in alleviating copper (Cu) stress-mediated oxidative damage, wherein a number of molecular mechanisms alter in plants. However, it remains largely unknown how CA regulates differentially abundant proteins (DAPs) in response to Cu stress in Brassica napus L. In the present study, we aimed to investigate the proteome changes in the leaves of B. L. seedlings in response to CA-mediated alleviation of Cu stress. Exposure of 21-day-old seedlings to Cu (25 and 50 µM) and CA (1.0 mM) for 7 days exhibited a dramatic inhibition of overall growth and considerable increase in the enzymatic activities (POD, SOD, CAT). Using a label-free proteome approach, a total of 6345 proteins were identified in differentially treated leaves, from which 426 proteins were differentially expressed among the treatment groups. Gene ontology (GO) and KEGG pathways analysis revealed that most of the differential abundance proteins were found to be involved in energy and carbohydrate metabolism, photosynthesis, protein metabolism, stress and defense, metal detoxification, and cell wall reorganization. Our results suggest that the downregulation of chlorophyll biosynthetic proteins involved in photosynthesis were consistent with reduced chlorophyll content. The increased abundance of proteins involved in stress and defense indicates that these DAPs might provide significant insights into the adaptation of Brassica seedlings to Cu stress. The abundances of key proteins were further verified by monitoring the mRNA expression level of the respective transcripts. Taken together, these findings provide a potential molecular mechanism towards Cu stress tolerance and open a new route in accelerating the phytoextraction of Cu through exogenous application of CA in B. napus.

Class III peroxidase: an indispensable enzyme for biotic/abiotic stress tolerance and a potent candidate for crop improvement.

Class III peroxidases are secretory enzymes which belong to a ubiquitous multigene family in higher plants and have been identified to play role in a broad range of physiological and developmental processes. Potentially, it is involved in generation and detoxification of hydrogen peroxide (H2O2), and their subcellular localization reflects through three different cycles, namely peroxidative cycle, oxidative and hydroxylic cycles to maintain the ROS level inside the cell. Being an antioxidant, class III peroxidases are an important initial defence adapted by plants to cope with biotic and abiotic stresses. Both these stresses have become a major concern in the field of agriculture due to their devastating effect on plant growth and development. Despite numerous studies on plant defence against both the stresses, only a handful role of class III peroxidases have been uncovered by its functional characterization. This review will cover our current understanding on class III peroxidases and the signalling involved in their regulation under both types of stresses. The review will give a view of class III peroxidases and highlights their indispensable role under stress conditions. Its future application will be discussed to showcase their importance in crop improvement by genetic manipulation and by transcriptome analysis.

Antifungal Proteins from Plant Latex.

Latex, a milky fluid found in several plants, is widely used for many purposes, and its proteins have been investigated by researchers. Many studies have shown that latex produced by some plant species is a natural source of biologically active compounds, and many of the hydrolytic enzymes are related to health benefits. Research on the characterization and industrial and pharmaceutical utility of latex has progressed in recent years. Latex proteins are associated with plants' defense mechanisms, against attacks by fungi. In this respect, there are several biotechnological applications of antifungal proteins. Some findings reveal that antifungal proteins inhibit fungi by interrupting the synthesis of fungal cell walls or rupturing the membrane. Moreover, both phytopathogenic and clinical fungal strains are susceptible to latex proteins. The present review describes some important features of proteins isolated from plant latex which presented in vitro antifungal activities: protein classification, function, molecular weight, isoelectric point, as well as the fungal species that are inhibited by them. We also discuss their mechanisms of action.

Peroxidase Activity of Human Hemoproteins: Keeping the Fire under Control.

The heme in the active center of peroxidases reacts with hydrogen peroxide to form highly reactive intermediates, which then oxidize simple substances called peroxidase substrates. Human peroxidases can be divided into two groups: (1) True peroxidases are enzymes whose main function is to generate free radicals in the peroxidase cycle and (pseudo)hypohalous acids in the halogenation cycle. The major true peroxidases are myeloperoxidase, eosinophil peroxidase and lactoperoxidase. (2) Pseudo-peroxidases perform various important functions in the body, but under the influence of external conditions they can display peroxidase-like activity. As oxidative intermediates, these peroxidases produce not only active heme compounds, but also protein-based tyrosyl radicals. Hemoglobin, myoglobin, cytochrome c/cardiolipin complexes and cytoglobin are considered as pseudo-peroxidases. Рeroxidases play an important role in innate immunity and in a number of physiologically important processes like apoptosis and cell signaling. Unfavorable excessive peroxidase activity is implicated in oxidative damage of cells and tissues, thereby initiating the variety of human diseases. Hence, regulation of peroxidase activity is of considerable importance. Since peroxidases differ in structure, properties and location, the mechanisms controlling peroxidase activity and the biological effects of peroxidase products are specific for each hemoprotein. This review summarizes the knowledge about the properties, activities, regulations and biological effects of true and pseudo-peroxidases in order to better understand the mechanisms underlying beneficial and adverse effects of this class of enzymes.

Anthropogenic N Deposition Alters the Composition of Expressed Class II Fungal Peroxidases.

Here, we present evidence that ca. 20 years of experimental N deposition altered the composition of lignin-decaying class II peroxidases expressed by forest floor fungi, a response which has occurred concurrently with reductions in plant litter decomposition and a rapid accumulation of soil organic matter. This finding suggests that anthropogenic N deposition has induced changes in the biological mediation of lignin decay, the rate limiting step in plant litter decomposition. Thus, an altered composition of transcripts for a critical gene that is associated with terrestrial C cycling may explain the increased soil C storage under long-term increases in anthropogenic N deposition.IMPORTANCE Fungal class II peroxidases are enzymes that mediate the rate-limiting step in the decomposition of plant material, which involves the oxidation of lignin and other polyphenols. In field experiments, anthropogenic N deposition has increased soil C storage in forests, a result which could potentially arise from anthropogenic N-induced changes in the composition of class II peroxidases expressed by the fungal community. In this study, we have gained unique insight into how anthropogenic N deposition, a widespread agent of global change, affects the expression of a functional gene encoding an enzyme that plays a critical role in a biologically mediated ecosystem process.

Lignin-degrading peroxidases in white-rot fungus Trametes hirsuta 072. Absolute expression quantification of full multigene family.

Ligninolytic heme peroxidases comprise an extensive family of enzymes, which production is characteristic for white-rot Basidiomycota. The majority of fungal heme peroxidases are encoded by multigene families that differentially express closely related proteins. Currently, there were very few attempts to characterize the complete multigene family of heme peroxidases in a single fungus. Here we are focusing on identification and characterization of peroxidase genes, which are transcribed and secreted by basidiomycete Trametes hirsuta 072, an efficient lignin degrader. The T. hirsuta genome contains 18 ligninolytic peroxidase genes encoding 9 putative lignin peroxidases (LiP), 7 putative short manganese peroxidases (MnP) and 2 putative versatile peroxidases (VP). Using ddPCR method we have quantified the absolute expression of the 18 peroxidase genes under different culture conditions and on different growth stages of basidiomycete. It was shown that only two genes (one MnP and one VP) were prevalently expressed as well as secreted into cultural broth under all conditions investigated. However their transcriptome and protein profiles differed in time depending on the effector used. The expression of other peroxidase genes revealed a significant variability, so one can propose the specific roles of these enzymes in fungal development and lifestyle.

Revisiting the Non-Animal Peroxidase Superfamily.

Peroxidases reduce peroxide through substrate oxidation in order to alleviate oxidative stress in aerobic organisms. Since the initial description of the non-animal peroxidase superfamily, great effort has been made to characterize this large and heterogeneous group of proteins. Next generation sequencing data have permitted an in-depth study of the molecular evolution of this superfamily and allowed us to perform a phylogenetic reconstruction. Through this analysis, we identified two additional class I members and, here, we discuss the similarities and differences among members of this class. Our results provide new insights into the organization of these antioxidant enzymes, allowing us to propose a new model for the emergence and evolution of this superfamily.

Characterisation of Dyp-type peroxidases from Pseudomonas fluorescens Pf-5: Oxidation of Mn(II) and polymeric lignin by Dyp1B.

Members of the DyP family of peroxidases in Gram-positive bacteria have recently been shown to oxidise Mn(II) and lignin model compounds. Gram-negative pseudomonads, which also show activity for lignin oxidation, also contain dyp-type peroxidase genes. Pseudomonas fluorescens Pf-5 contains three dyp-type peroxidases (35, 40 and 55kDa), each of which has been overexpressed in Escherichia coli, purified, and characterised. Each of the three enzymes shows activity for oxidation of phenol substrates, but the 35kDa Dyp1B enzyme also shows activity for oxidation of Mn(II) and Kraft lignin. Treatment of powdered lignocellulose with Dyp1B in the presence of Mn(II) and hydrogen peroxide leads to the release of a low molecular weight lignin fragment, which has been identified by mass spectrometry as a ß-aryl ether lignin dimer containing one G unit and one H unit bearing a benzylic ketone. A mechanism for release of this fragment from lignin oxidation is proposed.

Application of a novel alkali-tolerant thermostable DyP-type peroxidase from Saccharomonospora viridis DSM 43017 in biobleaching of eucalyptus kraft pulp.

Saccharomonospora viridis is a thermophilic actinomycete that may have biotechnological applications because of its dye decolorizing activity, though the enzymatic oxidative system responsible for this activity remains elusive. Bioinformatic analysis revealed a DyP-type peroxidase gene in the genome of S. viridis DSM 43017 with sequence similarity to peroxidase from dye-decolorizing microbes. This gene, svidyp, consists of 1,215 bp encoding a polypeptide of 404 amino acids. The gene encoding SviDyP was cloned, heterologously expressed in Escherichia coli, and then purified. The recombinant protein could efficiently decolorize several triarylmethane dyes, anthraquinonic and azo dyes under neutral to alkaline conditions. The optimum pH and temperature for SviDyP was pH 7.0 and 70°C, respectively. Compared with other DyP-type peroxidases, SviDyP was more active at high temperatures, retaining>63% of its maximum activity at 50-80°C. It also showed broad pH adaptability (>35% activity at pH 4.0-9.0) and alkali-tolerance (>80% activity after incubation at pH 5-10 for 1 h at 37°C), and was highly thermostable (>60% activity after incubation at 70°C for 2 h at pH 7.0). SviDyP had an accelerated action during the biobleaching of eucalyptus kraft pulp, resulting in a 21.8% reduction in kappa number and an increase of 2.98% (ISO) in brightness. These favorable properties make SviDyP peroxidase a promising enzyme for use in the pulp and paper industries.

Roles of peroxinectin in PGE2-mediated cellular immunity in Spodoptera exigua.

BACKGROUND: Prostaglandins (PGs) mediate insect immune responses to infections and invasions. Although the presence of PGs has been confirmed in several insect species, their biosynthesis in insects remains a conundrum because orthologs of the mammalian cyclooxygenases (COXs) have not been found in the known insect genomes. PG-mediated immune reactions have been documented in the beet armyworm, Spodoptera exigua. The purpose of this research is to identify the source of PGs in S. exigua. PRINCIPAL FINDINGS: Peroxidases (POXs) are a sister group of COX genes. Ten putative POXs (SePOX-A ∼ SePOX-J) were expressed in S. exigua. Expressions of SePOX-F and -H were induced by bacterial challenge and expressed in the hemocytes and the fat body. RNAi of each POX was performed by hemocoelic injection of their specific double-stranded RNAs. dsPOX-F or, separately, dsPOX-H, but not the other eight dsRNA constructs, specifically suppressed hemocyte-spreading behavior and nodule formation; these two reactions were also inhibited by aspirin, a COX inhibitor. PGE2, but not arachidonic acid, treatment rescued the immunosuppression. Sequence analysis indicated that both POX genes were clustered with peroxinectin (Pxt) and their cognate proteins shared some conserved domains corresponding to the Pxt of Drosophila melanogaster. CONCLUSIONS: SePOX-F and -H are Pxt-like genes associated with PG biosynthesis in S. exigua.

Evidence that the fosfomycin-producing epoxidase, HppE, is a non-heme-iron peroxidase.

The iron-dependent epoxidase HppE converts (S)-2-hydroxypropyl-1-phosphonate (S-HPP) to the antibiotic fosfomycin [(1R,2S)-epoxypropylphosphonate] in an unusual 1,3-dehydrogenation of a secondary alcohol to an epoxide. HppE has been classified as an oxidase, with proposed mechanisms differing primarily in the identity of the O2-derived iron complex that abstracts hydrogen (H•) from C1 of S-HPP to initiate epoxide ring closure. We show here that the preferred cosubstrate is actually H2O2 and that HppE therefore almost certainly uses an iron(IV)-oxo complex as the H• abstractor. Reaction with H2O2 is accelerated by bound substrate and produces fosfomycin catalytically with a stoichiometry of unity. The ability of catalase to suppress the HppE activity previously attributed to its direct utilization of O2 implies that reduction of O2 and utilization of the resultant H2O2 were actually operant.

Ciona intestinalis peroxinectin is a novel component of the peroxidase-cyclooxygenase gene superfamily upregulated by LPS.

Peroxinectins function as hemoperoxidase and cell adhesion factor involved in invertebrate immune reaction. In this study, the ascidian (Ciona intestinalis) peroxinectin gene (CiPxt) and its expression during the inflammatory response have been examined. CiPxt is a new member of the peroxidase-cyclooxygenase gene superfamily that contains both the peroxidase domain and the integrin KGD (Lys-Gly-Asp) binding motif. A phylogenetic tree showed that CiPxt is very close to the chordate group and appears to be the outgroup of mammalian MPO, EPO and TPO clades. The CiPxt molecular structure model resulted superimposable to the human myeloperoxidase. The CiPxt mRNA expression is upregulated by LPS inoculation suggesting it is involved in C. intestinalis inflammatory response. The CiPxt was expressed in hemocytes (compartment/morula cells), vessel epithelium, and unilocular refractile granulocytes populating the inflamed tunic matrix and in the zones 7, 8 and 9 of the endostyle, a special pharynx organs homolog to the vertebrate thyroid gland.

In silico comparative analysis and expression profile of antioxidant proteins in plants.

The antioxidant system in plants is a very important defensive mechanism to overcome stress conditions. We examined the expression profile of antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX) using a bioinformatics approach. We explored secondary structure prediction and made detailed studies of signature pattern of antioxidant proteins in four plant species (Triticum aestivum, Arabidopsis thaliana, Oryza sativa, and Brassica juncea). Fingerprinting analysis was done with ScanProsite, which includes a large collection of biologically meaningful signatures. Multiple sequence alignment of antioxidant proteins of the different plant species revealed a conserved secondary structure region, indicating homology at the sequence and structural levels. The secondary structure prediction showed that these proteins have maximum tendency for α helical structure. The sequence level similarities were also analyzed with a phylogenetic tree using neighbor-joining method. In the antioxidant enzymes SOD, CAT and APX, three major families of signature were predominant and common; these were PKC_PHOSPHO_SITE, CK2_PHOSPHO_SITE and N-myristoylation site, which are functionally related to various plant signaling pathways. This study provides new strategies for screening of biomodulators involved in plant stress metabolism that will be useful for designing degenerate primers or probes specific for antioxidant. These enzymes could be the first line of defence in the cellular antioxidant defence pathway, activated due to exposure to abiotic stresses.

PeroxiBase: a database for large-scale evolutionary analysis of peroxidases.

The PeroxiBase (http://peroxibase.toulouse.inra.fr/) is a specialized database devoted to peroxidases' families, which are major actors of stress responses. In addition to the increasing number of sequences and the complete modification of the Web interface, new analysis tools and functionalities have been developed since the previous publication in the NAR database issue. Nucleotide sequences and graphical representation of the gene structure can now be included for entries containing genomic cross-references. An expert semi-automatic annotation strategy is being developed to generate new entries from genomic sequences and from EST libraries. Plus, new internal and automatic controls have been included to improve the quality of the entries. To compare gene structure organization among families' members, two new tools are available, CIWOG to detect common introns and GECA to visualize gene structure overlaid with sequence conservation. The multicriteria search tool was greatly improved to allow simple and combined queries. After such requests or a BLAST search, different analysis processes are suggested, such as multiple alignments with ClustalW or MAFFT, a platform for phylogenetic analysis and GECA's display in association with a phylogenetic tree. Finally, we updated our family specific profiles implemented in the PeroxiScan tool and made new profiles to consider new sub-families.

Enzymatic activity and proteomic profile of class III peroxidases during sugarcane stem development.

Class III peroxidases are present as large multigene families in all land plants. This large number of genes together with the diversity of processes catalyzed by peroxidases suggests possible functional specialization of each isoform. However, assigning a precise role for each individual peroxidase gene has continued to be a major bottleneck. Here we investigated the enzyme activity and translational profile of class III peroxidases during stem development of sugarcane as a first step in the estimation of physiological functions of individual isoenzymes. Internodes at three different developmental stages (young, developing and mature) were divided into pith (inner tissue) and rind (outer tissue) fractions. The rind of mature internodes presented the highest enzymatic activity and thus could be considered the ideal tissue for the discovery of peroxidase gene function. In addition, activity staining of 2DE gels revealed different isoperoxidase profiles and protein expression regulation among different tissue fractions. In-gel tryptic digestion of excised spots followed by peptide sequencing by LC-MS/MS positively matched uncharacterized peroxidases in the sugarcane database SUCEST. Multiple spots matching the same peroxidase gene were found, which reflects the generation of more than one isoform from a particular gene by post-translational modifications. The identified sugarcane peroxidases appear to be monocot-specific sequences with no clear ortholog in dicot model plant Arabidopsis thaliana.

Eukaryotic extracellular catalase-peroxidase from Magnaporthe grisea - Biophysical/chemical characterization of the first representative from a novel phytopathogenic KatG group.

All phytopathogenic fungi have two catalase-peroxidase paralogues located either intracellularly (KatG1) or extracellularly (KatG2). Here, for the first time a secreted bifunctional, homodimeric catalase-peroxidase (KatG2 from the rice blast fungus Magnaporthe grisea) has been produced heterologously with almost 100% heme occupancy and comprehensively investigated by using a broad set of methods including UV-Vis, ECD and resonance Raman spectroscopy (RR), thin-layer spectroelectrochemistry, mass spectrometry, steady-state & presteady-state spectroscopy. RR spectroscopy reveals that MagKatG2 shows a unique mixed-spin state, non-planar heme b, and a proximal histidine with pronounced imidazolate character. At pH 7.0 and 25 °C, the standard reduction potential E°' of the Fe(III)/Fe(II) couple for the high-spin native protein was found to fall in the range typical for the KatG family. Binding of cyanide was relatively slow at pH 7.0 and 25 °C and with a K(d) value significantly higher than for the intracellular counterpart. Demonstrated by mass spectrometry MagKatG2 has the typical Trp118-Tyr251-Met277 adduct that is essential for its predominantly catalase activity at the unique acidic pH optimum. In addition, MagKatG2 acts as a versatile peroxidase using both one- and two-electron donors. Based on these data, structure-function relationships of extracellular eukaryotic KatGs are discussed with respect to intracellular KatGs and possible role(s) in host-pathogen interaction.

Directed evolution of a temperature-, peroxide- and alkaline pH-tolerant versatile peroxidase.

The VPs (versatile peroxidases) secreted by white-rot fungi are involved in the natural decay of lignin. In the present study, a fusion gene containing the VP from Pleurotus eryngii was subjected to six rounds of directed evolution, achieving a level of secretion in Saccharomyces cerevisiae (21 mg/l) as yet unseen for any ligninolytic peroxidase. The evolved variant for expression harboured four mutations and increased its total VP activity 129-fold. The signal leader processing by the STE13 protease at the Golgi compartment changed as a consequence of overexpression, retaining the additional N-terminal sequence Glu-Ala-Glu-Ala that enhanced secretion. The engineered N-terminally truncated variant displayed similar biochemical properties to those of the non-truncated counterpart in terms of kinetics, stability and spectroscopic features. Additional cycles of evolution raised the T50 8°C and significantly increased the enzyme's stability at alkaline pHs. In addition, the Km for H2O2 was enhanced up to 15-fold while the catalytic efficiency was maintained, and there was an improvement in peroxide stability (with half-lives for H2O2 of 43 min at a H2O2/enzyme molar ratio of 4000:1). Overall, the directed evolution approach described provides a set of strategies for selecting VPs with improvements in secretion, activity and stability.

Structural-functional features of plant isoperoxidases.

Current data on structural--functional features of plant peroxidases and their involvement in functioning of the pro-/antioxidant system responding to stress factors, especially those of biotic origin, are analyzed. The collection of specific features of individual isoforms allows a plant to withstand an aggressive influence of the environment. Expression of some genes encoding different isoperoxidases is regulated by pathogens (and their metabolites), elicitors, and hormone-like compounds; specific features of this regulation are considered in detail. It is suggested that isoperoxidases interacting with polysaccharides are responsible for a directed deposition of lignin on the cell walls, and this lignin in turn is concurrently an efficient strengthening material and protects the plants against pathogens.

Molecular diversity of katG genes in the soil bacteria Comamonas.

UNLABELLED: Three complete katG genes coding for bifunctional catalase-peroxidases (KatGs) from the beta-proteobacterium Comamonas terrigena and two related strains of Comamonas testosteroni have been cloned and sequenced. Catalase-peroxidases are unique bifunctional enzymes known to be expressed in these soil bacteria in response to environmental and/or oxidative stress. The evolutionary and structural diversity of these enzymes is investigated based on multiple sequence alignment and comprehensive phylogenetic analysis. The reconstructed phylogenetic tree and well-known structure-function relationships were applied to inspect the conservation of essential residues. Observed diversity is discussed with respect to the fact that KatGs are distinctive gene-duplicated peroxidases comprising a N-terminal (enzymatically active) and a C-terminal (heme-less) domain. The unique promoter motifs regulating katG transcription in four strains of Comamonas were detected and compared with E. coli katG promoter. The relationship between the promoter sequences and the corresponding expression levels was analyzed. A significant difference in heat shock-inducible catalatic and peroxidatic activities between E. coli K12 and Comamonas terrigena & testosteroni strains was observed. The peculiar variability in gene-coding sequences appears to be more significant for such activity output among Comamonas strains than differences in their promoter regions. The functional role of observed increased diversity in the C-terminal domain is discussed with respect to potential modification of catalytic features at the N-terminal domain that could be relevant for these soil bacteria to cope with stressors. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00203-009-0541-4) contains supplementary material, which is available to authorized users.

Two distinct groups of fungal catalase/peroxidases.

Catalase/peroxidases (KatGs) are bifunctional haem b-containing (Class I) peroxidases with overwhelming catalase activity and substantial peroxidase activity with various one-electron donors. These unique oxidoreductases evolved in ancestral bacteria revealing a complex gene-duplicated structure. Besides being found in numerous bacteria of all phyla, katG genes were also detected in genomes of lower eukaryotes, most prominently of sac and club fungi. Phylogenetic analysis demonstrates the occurrence of two distinct groups of fungal KatGs that differ in localization, structural and functional properties. Analysis of lateral gene transfer of bacterial katGs into fungal genomes reveals that the most probable progenitor was a katG from a bacteroidetes predecessor. The putative physiological role(s) of both fungal KatG groups is discussed with respect to known structure-function relationships in bacterial KatGs and is related with the acquisition of (phyto)pathogenicity in fungi.