Evolution and expression of class III peroxidases

Class III peroxidases are members of a large multigenic family, only detected in the plant kingdom and absent from green algae sensu stricto (chlorophyte algae or Chlorophyta). Their evolution is thought to be related to the emergence of the land plants. However class III peroxidases are present in a lower copy number in some basal Streptophytes (Charapyceae), which predate land colonization. Gene structures are variable among organisms and within species with respect to the number of introns, but their positions are highly conserved. Their high copy number, as well as their conservation could be related to plant complexity and adaptation to increasing stresses. No specific function has been assigned to respective isoforms, but in large multigenic families, particular structure-function relations can be expected. Plant peroxidase sequences contain highly conserved residues and motifs, variable domains surrounded by conserved residues and present a low identity level among their promoter regions, further suggesting the existence of sub-functionalization of the different isoforms.     

A large family of class III plant peroxidases

Class III plant peroxidase (POX), a plant-specific oxidoreductase, is one of the many types of peroxidases that are widely distributed in animals, plants and microorganisms. POXs exist as isoenzymes in individual plant species, and each isoenzyme has variable amino acid sequences and shows diverse expression profiles, suggesting their involvement in various physiological processes. Indeed, studies have provided evidence that POXs participate in lignification, suberization, auxin catabolism, wound healing and defense against pathogen infection. Little, however, is known about the signal transduction for inducing expression of the pox genes. Recent studies have provided information on the regulatory mechanisms of wound- and pathogen-induced expression of some pox genes. These studies suggest that pox genes are induced via different signal transduction pathways from those of other known defense-related genes.     

Enzyme-catalyzed mechanism of isoniazid activation in class I and class III peroxidases

There is an urgent need to understand the mechanism of activation of the frontline anti-tuberculosis drug isoniazid by the Mycobacterium tuberculosis catalase-peroxidase. To address this, a combination of NMR spectroscopic, biochemical, and computational methods have been used to obtain a model of the frontline anti-tuberculosis drug isoniazid bound to the active site of the class III peroxidase, horseradish peroxidase C. This information has been used in combination with the new crystal structure of the M. tuberculosis catalase-peroxidase to predict the mode of INH binding across the class I heme peroxidase family. An enzyme-catalyzed mechanism for INH activation is proposed that brings together structural, functional, and spectroscopic data from a variety of sources. Collectively, the information not only provides a molecular basis for understanding INH activation by the M. tuberculosis catalase-peroxidase but also establishes a new conceptual framework for testing hypotheses regarding the enzyme-catalyzed turnover of this compound in a number of heme peroxidases.     

Quantitative analysis of resistance in cotton to three new isolates of the bacterial blight pathogen

Summary Genetic variability for virulence of the bacterial blight pathogen [Xanthomonas campestris pv malvacearum (Smith) Dye] on cotton (Gossypium hirsutum L.) has been shown by the identification of 19 races of the pathogen based on disease reactions of a set of ten host differentials. This study was conducted to determine the inheritance of host resistance to three recently identified isolates of X. campestris pv malvacearum, which are virulent on the entire set of differentials. True leaves of Tamcot CAMD-E, LEBOCAS-3-80, Stoneville 825, and their f₁, F₂, and backcross progenies were wound-inoculated in the field with separate bacterial suspensions of the virulent HV3, HV7, and Sudan isolates of the pathogen. LEBOCAS-3-80 was replaced with S295, a new immune cultivar, for a greenhouse study in which both cotyledons and true leaves were inoculated. Disease reactions were rated on a scale of 1–10, and genetic models were proposed utilizing generation means analysis. Dominance, when significant, was in the direction of resistance in all but one cross-isolate combination. Digenic interaction components indicated a duplicate type. Narrow-sense heritability for resistance ranged from 0.59 to 0.68; therefore, primarily additive-genetic variability among the selected cutlivars was detected, indicating that breeding for improved resistance to these isolates is a practical goal.Contribution of the Department of Soil and Crop Sciences and the Texas Agricultural Experiment Station     

Surface layers ofXanthomonas malvacearum,the cause of bacterial blight of cotton

Mureins were isolated from two strains ofXanthomonas malvacearum, a phytopathogenic bacterium causing bacterial blight of cotton. The purity of murein was 70–95 % and the amino acid and amino sugar components (glutamic acid, alanine, meso-diaminopimelic acid, muramio acid and glucosamine) were present at the molar ratio of 1: 1.9: 1: 1.12: 0.85. The bacterium secreted a copious amount of slime which masked its surface structure. The slime was composed of densely interwoven network of filamentous material originating from the cell surface and extended into the medium without any discernable boundary. The slime was secreted through surface layers pores by force, giving the effect of a spray or jet. Slime also played a role in chain formation of bacterial cells.     

Resistance to bacterial blight of cotton varieties homozygous for combinations of B resistance genes

In the Sudan, inoculation experiments were done on a wide range of Egyptian cotton lines homozygous for single genes and for digenic and trigenic combinations of Knight's B genes for resistance to bacterial blight. Although leaf inoculation was successful, stem inoculation was only partially so and boll inoculation, using two different techniques, failed to produce measurable disease symptoms. There was a good general relationship between leaf and stem resistance, and a close association between resistance to natural attack in the field and leaf resistance to artificial inoculation. Confirmation was obtained of the strong resistance conferred by B₂ B_9K, which was as effective as B₂B₆ or B₂B₃B₆. No other combination was as effective when inoculated artificially. Nevertheless, in a natural field attack, only mild symptoms were found in lines homozygous for B₁ B_9K and for B₄B₆. Under the same conditions lines with B₂B₆ showed no symptoms but those with B₂B₃ were severely attacked. Reasons for continuing to use major genes in the breeding of blight-resistant cotton in the Sudan are discussed.     

Antisense suppression of a (+)-delta-cadinene synthase gene in cotton prevents the induction of this defense response gene during bacterial blight infection but not its constitutive expression

In cotton (Gossypium hirsutum) the enzyme (+)-delta-cadinene synthase (CDNS) catalyzes the first committed step in the biosynthesis of cadinane-type sesquiterpenes, such as gossypol, that provide constitutive and inducible protection against pests and diseases. A cotton cDNA clone encoding CDNS (cdn1-C4) was isolated from developing embryos and functionally characterized. Southern analysis showed that CDNS genes belong to a large multigene family, of which five genomic clones were studied, including three pseudogenes and one gene that may represent another subfamily of CDNS. CDNS expression was shown to be induced in cotton infected with either the bacterial blight or verticillium wilt pathogens. Constructs for the constitutive or seed-specific antisense suppression of cdn1-C4 were introduced into cotton by Agrobacterium-mediated transformation. Gossypol levels were not reduced in the seeds of transformants with either construct, nor was the induction of CDNS expression affected in stems of the constitutive antisense plants infected with Verticillium dahliae Kleb. However, the induction of CDNS mRNA and protein in response to bacterial blight infection of cotyledons was completely blocked in the constitutive antisense plants. These results suggest that cdn1-C4 may be involved specifically in the bacterial blight response and that the CDNS multigene family comprises a complex set of genes differing in their temporal and spatial regulation and responsible for different branches of the cotton sesquiterpene pathway.     

A second class of peroxidases linked to the trypanothione metabolism

Trypanosoma brucei, the causative agent of African sleeping sickness, has three nearly identical genes encoding cysteine homologues of classical selenocysteine-containing glutathione peroxidases. The proteins are expressed in the mammalian and insect stages of the parasite. One of the genes, which contains a mitochondrial as well as a glycosomal targeting signal has been overexpressed. The recombinant T. brucei peroxidase has a high preference for the trypanothione/tryparedoxin couple as electron donor for the reduction of different hydroperoxides but accepts also T. brucei thioredoxin. The apparent rate constants k(2)' for the regeneration of the reduced enzyme are 2 x 10(5) m(-1) s(-1) with tryparedoxin and 5 x 10(3) m(-1) s(-1) with thioredoxin. No saturation kinetics was observed and the rate-limiting step of the overall reaction is reduction of the hydroperoxide. With glutathione, the peroxidase has marginal activity and reduction of the enzymes becomes limiting with a k(2)' value of 3 m (-1) s(-1). The T. brucei peroxidase, in contrast to the related Trypanosoma cruzi enzyme, also accepts hydrogen peroxide as substrate. The catalytic efficiency of the peroxidase studied here is comparable with that of the peroxiredoxin-like tryparedoxin peroxidases, which shows that trypanosomes possess two distinct peroxidase systems both dependent on the unique dithiol trypanothione.     

Stable resistance to bacterial blight disease in rice

Ten rice cultivars were evaluated for stable resistance to 18 isolates of Xanthomonas campestris pv. oryzae. Stable performance of the host cultivar was evaluated by regressing the cultivar means on the mean level of pathogenicity of the isolates averaged over all cultivars. A cultivar with low disease incidence, a regression coefficient near or equal to zero (b= 0) and the deviation from regression as small as possible (S²_d= 0) was considered highly stable while that with low disease incidence, b= 1 -0 and S²_d= 0 was considered to possess average stability. Accordingly, cvs TKM-6, CB II, DV 85 and Ramakrishna were considered to possess average stability while IR 20 and Indira were the most stable. Cvs Cemposelak, IR 1545, BJ 1 and IR 8 showed high response to disease incidence with relatively small changes in level of pathogenicity of the isolates and hence were considered most unstable. The regression technique was considered as a valuable tool for identification of stable resistance to bacterial blight disease of rice.     

Phylogenetic relationships in class I of the superfamily of bacterial, fungal, and plant peroxidases.

Molecular phylogeny among catalase-peroxidases, cytochrome c peroxidases, and ascorbate peroxidases was analysed. Sixty representative sequences covering all known subgroups of class I of the superfamily of bacterial, fungal, and plant heme peroxidases were selected. Each sequence analysed contained the typical peroxidase motifs evolved to bind effectively the prosthetic heme group, enabling peroxidatic activity. The N-terminal and C-terminal domains of catalase-peroxidases matching the ancestral tandem gene duplication event were treated separately in the phylogenetic analysis to reveal their specific evolutionary history. The inferred unrooted phylogenetic tree obtained by three different methods revealed the existence of four clearly separated clades (C-terminal and N-terminal domains of catalase-peroxidases, ascorbate peroxidases, and cytochrome c peroxidases) which were segregated early in the evolution of this superfamily. From the results, it is obvious that the duplication event in the gene for catalase-peroxidase occurred in the later phase of evolution, in which the individual specificities of the peroxidase families distinguished were already formed. Evidence is presented that class I of the heme peroxidase superfamily is spread among prokaryotes and eukaryotes, obeying the birth-and-death process of multigene family evolution.     

Inheritance of resistance to bacterial blight in common bean

Summary Inheritance of resistance to common bacterial blight in the trifoliate leaf, plant canopy, and pods was controlled by a single major gene. Additive followed by dominance effects were more important than epistatic interactions. Narrow-sense heritability values ranged from 0.18 to 0.87 for trifoliate leaf, from 0.26 to 0.76 for canopy, and from 0.11 to 0.36 for pods. Observed gains from selection for resistance were higher than expected gains. Implications of these results in breeding for resistance are discussed.     

Phylogeny, topology, structure and functions of membrane-bound class III peroxidases in vascular plants

Peroxidases are key player in the detoxification of reactive oxygen species during cellular metabolism and oxidative stress. Membrane-bound isoenzymes have been described for peroxidase superfamilies in plants and animals. Recent studies demonstrated a location of peroxidases of the secretory pathway (class III peroxidases) at the tonoplast and the plasma membrane. Proteomic approaches using highly enriched plasma membrane preparations suggest organisation of these peroxidases in microdomains, a developmentally regulation and an induction of isoenzymes by oxidative stress. Phylogenetic relations, topology, putative structures, and physiological function of membrane-bound class III peroxidases will be discussed.     

Contribution of bacterial outer membrane vesicles to innate bacterial defense

Background

Microorganisms produce cell-wall-degrading enzymes as part of their strategies for plant invasion/nutrition. Among these, pectin lyases (PNLs) catalyze the depolymerization of esterified pectin by a β-elimination mechanism. PNLs are grouped together with pectate lyases (PL) in Family 1 of the polysaccharide lyases, as they share a conserved structure in a parallel β-helix. The best-characterized fungal pectin lyases are obtained from saprophytic/opportunistic fungi in the genera Aspergillus and Penicillium and from some pathogens such as Colletotrichum gloeosporioides. The organism used in the present study, Colletotrichum lindemuthianum, is a phytopathogenic fungus that can be subdivided into different physiological races with different capacities to infect its host, Phaseolus vulgaris. These include the non-pathogenic and pathogenic strains known as races 0 and 1472, respectively.

Results

Here we report the isolation and sequence analysis of the Clpnl2 gene, which encodes the pectin lyase 2 of C. lindemuthianum, and its expression in pathogenic and non-pathogenic races of C. lindemuthianum grown on different carbon sources. In addition, we performed a phylogenetic analysis of the deduced amino acid sequence of Clpnl2 based on reported sequences of PNLs from other sources and compared the three-dimensional structure of Clpnl2, as predicted by homology modeling, with those of other organisms. Both analyses revealed an early separation of bacterial pectin lyases from those found in fungi and oomycetes. Furthermore, two groups could be distinguished among the enzymes from fungi and oomycetes: one comprising enzymes from mostly saprophytic/opportunistic fungi and the other formed mainly by enzymes from pathogenic fungi and oomycetes. Clpnl2 was found in the latter group and was grouped together with the pectin lyase from C. gloeosporioides.

Conclusions

The Clpnl2 gene of C. lindemuthianum shares the characteristic elements of genes coding for pectin lyases. A time-course analysis revealed significant differences between the two fungal races in terms of the expression of Clpnl2 encoding for pectin lyase 2. According to the results, pectin lyases from bacteria and fungi separated early during evolution. Likewise, the enzymes from fungi and oomycetes diverged in accordance with their differing lifestyles. It is possible that the diversity and nature of the assimilatory carbon substrates processed by these organisms played a determinant role in this phenomenon.     

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.     

Divergent evolutionary lines of fungal cytochrome c peroxidases belonging to the superfamily of bacterial, fungal and plant heme peroxidases

Novel open reading frames coding for cytochrome c peroxidase (CcP) belonging to the superfamily of bacterial, fungal, and plant heme peroxidases were analyzed in the available fungal genomes. Multiple sequence alignment of 71 selected peroxidase genes revealed the presence of three conserved regions essential for their function: one on the distal and two on the proximal side of the prosthetic heme group. Conserved sequence motifs on the proximal heme side are peculiar for CcPs and are responsible for their reactivity. Phylogenetic analysis performed with the distance method as well as with the maximum likelihood method revealed the existence of three distinct subfamilies of fungal CcP and their relationship to other members of the peroxidase superfamily. These divergent CcP evolutionary lines apparently evolved from a single primordial heme peroxidase gene in parallel with the evolution of ascorbate peroxidase genes. Analyzed CcPs differ significantly in their N-terminal sequences. Only subfamily I did not exhibit a presence of any signal sequence. Subfamily II members possess a well defined signal sequence allowing processing and release into mitochondrion and also in subfamily III a signal sequence was detected. Several here analyzed peroxidase genes mainly from Candida albicans and from Rhizopus oryzae can be considered interesting for the investigation of the structure-function relationship of novel CcPs revealing differences to the well documented properties of cytochrome c peroxidase from Saccharomyces cerevisiae.     

Structure and mechanism in the bacterial dihaem cytochrome c peroxidases

The bacterial cytochrome c peroxidases contain an electron-transferring haem c (E) and a peroxidatic haem c (P). Many are isolated in an inactive oxidised state. Reduction of the E haem promotes Ca(2+)-dependent spin state and coordination changes at the P haem rendering it accessible to ligand. Recent crystallographic work on the oxidised and mixed valence enzymes has suggested a mechanism by which an electron entering the E haem remotely triggers this activation of the P haem. Binding of hydrogen peroxide at the activated P haem leads to an intermediate catalytic form containing two oxidising equivalents, one of which is a ferryl oxene. This form of the enzyme is then reduced by two single electron transfers to the E haem delivered by small redox proteins such as cytochromes or cupredoxins. The binding of these small redox proteins is dominated by global electrostatic forces but the interfaces of the electron transfer complexes that are formed are largely hydrophobic and relatively non-specific. These features allow very high electron transfer rates in the steady state.     

Class III peroxidases and ascorbate metabolism in plants

Class III peroxidases (PODs) have many functions in plant metabolism mainly dependent on their various physiological reducing substrates. Their involvement in plant differentiation and in the response against environmental stress is well known. Several evidences underline that ascorbate (ASC) levels affect POD reactions and, as a consequence, interfere with the metabolic pathways controlled by these isoenzymes. Ascorbate peroxidases (APXs), enzymes belonging to a different class of peroxidases (class I), are often present in the same cellular compartments in which PODs are also active. Since both APXs and PODs specifically utilise hydrogen peroxide as oxidising substrate they can compete, when co-present, for the same substrate. In this review, attention focuses on some of the physiological processes in which both ASC metabolism and PODs are involved. In particular, the scavenging of reactive oxygen species (ROS) during photosynthesis, cell elongation and wall stiffening as well as programmed cell death have been considered thoroughly. The relations between PODs and ASC metabolism have been discussed also in the attempt to outline their relevance for the correct plant development as well as for the perception/response of external stimuli allowing plants to cope with unfavourable conditions.     

The quantum mixed-spin heme state of barley peroxidase: A paradigm for class III peroxidases.

Electronic absorption and resonance Raman (RR) spectra of the ferric form of barley grain peroxidase (BP 1) at various pH values, at both room temperature and 20 K, are reported, together with electron paramagnetic resonance spectra at 10 K. The ferrous forms and the ferric complex with fluoride have also been studied. A quantum mechanically mixed-spin (QS) state has been identified. The QS heme species coexists with 6- and 5-cHS hemes; the relative populations of these three spin states are found to be dependent on pH and temperature. However, the QS species remains in all cases the dominant heme spin species. Barley peroxidase appears to be further characterized by a splitting of the two vinyl stretching modes, indicating that the vinyl groups are differently conjugated with the porphyrin. An analysis of the currently available spectroscopic data for proteins from all three peroxidase classes suggests that the simultaneous occurrence of the QS heme state as well as the splitting of the two vinyl stretching modes is confined to class III enzymes. The former point is discussed in terms of the possible influences of heme deformations on heme spin state. It is found that moderate saddling alone is probably not enough to cause the QS state, although some saddling may be necessary for the QS state.     

Overexpression of defense response genes in transgenic wheat enhances resistance to Fusarium head blight

Fusarium head blight (FHB) of wheat, caused by Fusarium graminearum and other Fusarium species, is a major disease problem for wheat production worldwide. To combat this problem, large-scale breeding efforts have been established. Although progress has been made through standard breeding approaches, the level of resistance attained is insufficient to withstand epidemic conditions. Genetic engineering provides an alternative approach to enhance the level of resistance. Many defense response genes are induced in wheat during F. graminearum infection and may play a role in reducing FHB. The objectives of this study were (1) to develop transgenic wheat overexpressing the defense response genes α-1-purothionin, thaumatin-like protein 1 (tlp-1), and β-1,3-glucanase; and (2) to test the resultant transgenic wheat lines against F. graminearum infection under greenhouse and field conditions. Using the wheat cultivar Bobwhite, we developed one, two, and four lines carrying the α-1-purothionin, tlp-1, and β-1,3-glucanase transgenes, respectively, that had statistically significant reductions in FHB severity in greenhouse evaluations. We tested these seven transgenic lines under field conditions for percent FHB disease severity, deoxynivalenol (DON) mycotoxin accumulation, and percent visually scabby kernels (VSK). Six of the seven lines differed from the nontransgenic parental Bobwhite line for at least one of the disease traits. A β-1,3-glucanase transgenic line had enhanced resistance, showing lower FHB severity, DON concentration, and percent VSK compared to Bobwhite. Taken together, the results showed that overexpression of defense response genes in wheat could enhance the FHB resistance in both greenhouse and field conditions.