Identification of resistance-associated proteins in closely-related maize lines varying in aflatoxin accumulation

Aspergillus flavus infection of maize and subsequent contamination with carcinogenic aflatoxins poses serious health concerns, especially in developing countries. Maize lines resistant to A. flavus infection have been identified; however, the development of commercially-useful aflatoxin-resistant maize lines has been hindered due to a lack of breeding markers. To identify maize resistance-associated proteins (RAPs) as potential markers for breeding, 52 BC1S4 lines developed from crosses between five African maize inbreds and five temperate aflatoxin-resistant lines were screened using the kernel screening assay. Five pairs of closely-related lines that had 75?C94% genetic similarity within each pair and which varied within each pair in aflatoxin accumulation were selected for proteomic investigation. Kernel embryo and endosperm protein profile differences within the pair and across pairs were compared using two-dimensional polyacrylamide gel electrophoresis. Differentially expressed (??1.5-fold) RAPs were sequenced through tandem mass spectrometry and were identified as antifungal, stress-related, storage or regulatory proteins. Sequence homology analysis highlighted several proteins in maize that confer resistance to A. flavus infection and/or aflatoxin production.     

Properties of anionic peroxidases purified from Chinese cabbage roots

Two anionic peroxidases were isolated from Chinese cabbage roots and purified using gel filtration followed by ion-exchange chromatography. Following purification a specific activity of peroxidases was estimated as 50 units.mg^-1 (A1) and 30 units.mg^-1 (A2) compared with that of a crude extract of the peroxidases which was 2.31 units.mg^-1. The pH for its optimum activity was 5.0 and the addition of Ca²⁺ produced a 15 % increase in peroxidase activity. Isoelectric focusing techniques were carried out in order to classify the peroxidases based on their isoelectric point (pI). Two anionic peroxidases, A1 and A2, were found to have pI values of 4.83 and 4.78, respectively. The peroxidases were found to be heat-stable, with 20 % (A1) and 16 % (A2) of the enzymatic activity remaining after heat treatment at 70 °C for 20 min. The heat inactivation rate followed first-order kinetics with the activation energy; Ea, estimated as 38.2 kcal.mol^-1 and 36.4 kcal.mol^-1 for A1 and A2, respectively.     

Possible function of two insect phospholipid-hydroperoxide glutathione peroxidases

We compared the functional properties of two insect members of the phospholipid hydroperoxide glutathione peroxidases (PHGPx) family, VLP1, a major component of virus-like particles from the hymenopteran endoparasitoid Venturia canescens and its closest Drosophila relative, one of the putative PHGPx-proteins predicted from the Berkeley Drosophila genome sequence project. Recombinant Drosophila PHGPx shows enzymatic activity towards a number of PHGPx substrates, while the recombinant PHGPx-like domain of VLP1 lacks a functionally relevant cysteine and enzyme activity. A possible function of a non-enzymatic extracellular PHGPx-like protein is discussed.     

Distribution of the stress-related anionic peroxidases in different cucumber organs

The distribution of the stress-related anionic peroxidase (srPRX) activity was investigated in various organs of cucumber (Cucumis sativus L. cv. Laura) during their development by activity staining and immunoblotting. In shoots, including cotyledon, leaf, stem and tendril, three stress-related peroxidase isoenzymes were present, particularly in old ones. The PRX1 was the only srPRX isoenzyme found in both young and old roots. As fruits became mature, srPRX activity increased dramatically and was particularly enriched in the external parts of the fruit. The PRX1 isoenzyme was highly accumulated in the course of seed germination, while the absence of other two srPRX isoenzymes (PRX2 and PRX3) was recorded. The possible function of the srPRX is discussed, with respect to this spatio-temporal distribution.     

Organ-specific expression of the stress-related anionic peroxidases in cucumber flowers

The study revealed a marked qualitative and quantitative differences in the pattern of expression of three stress-related cucumber (Cucumis sativus L., cv. Laura) isoperoxidases. Activity staining, as well as the protein gel blot (Western blotting) confirmed that the proteins studied are differentially expressed in both male and female sepals, in male but not in female pedicels and in, pistil. By using antibodies specific to three stress-related peroxidases, one serologically related pistil-specific anionic peroxidase was detected. This specific band had never been observed in other flower organs. Differential appearance of the stress-related peroxidase isoenzymes in both male and female flowers demonstrates that these proteins are developmentally regulated, showing an organ-specific expression.     

Redox properties of heme peroxidases

Peroxidases are heme enzymes found in bacteria, fungi, plants and animals, which exploit the reduction of hydrogen peroxide to catalyze a number of oxidative reactions, involving a wide variety of organic and inorganic substrates. The catalytic cycle of heme peroxidases is based on three consecutive redox steps, involving two high-valent intermediates (Compound I and Compound II), which perform the oxidation of the substrates. Therefore, the thermodynamics and the kinetics of the catalytic cycle are influenced by the reduction potentials of three redox couples, namely Compound I/Fe³⁺, Compound I/Compound II and Compound II/Fe³⁺. In particular, the oxidative power of heme peroxidases is controlled by the (high) reduction potential of the latter two couples. Moreover, the rapid H₂O₂-mediated two-electron oxidation of peroxidases to Compound I requires a stable ferric state in physiological conditions, which depends on the reduction potential of the Fe³⁺/Fe²⁺ couple. The understanding of the molecular determinants of the reduction potentials of the above redox couples is crucial for the comprehension of the molecular determinants of the catalytic properties of heme peroxidases.This review provides an overview of the data available on the redox properties of Fe³⁺/Fe²⁺, Compound I/Fe³⁺, Compound I/Compound II and Compound II/Fe³⁺ couples in native and mutated heme peroxidases. The influence of the electron donor properties of the axial histidine and of the polarity of the heme environment is analyzed and the correlation between the redox properties of the heme group with the catalytic activity of this important class of metallo-enzymes is discussed.     

Expression of cucumber stress-related anionic peroxidases during flower development or a cryptic infective process

The striking diversity in the expression pattern of the stress-related anionic peroxidase was observed during development of female cucumber flower. While the isoenzyme Prx3 was accumulated constitutively in the course of flower development, the expression patterns of other two isoenzymes (Prx1 and Prx2) were restricted to the period after flower opening. The virus infection was simulated by careful opening of the intact female flower buds 3 d before anthesis followed by exposition to the glasshouse environment for 3 d. The results obtained in this experiment revealed a marked accumulation of the isoenzyme Prx1 and Prx2 at anthesis. Under normal flower development, the pistils did not accumulate these isoenzymes at this stage. In contrast, the pattern of expression of Prx3 as well as of the pistil-specific peroxidase isoenzyme remained unchanged, confirming a constitutive type of expression. Beside the pistil, a 3-d exposition of the stripped flowers resulted in a marked accumulation of Prx1 and Prx2 isoenzymes also in both adjacent flower organs - the ovary and the pedicel. At the same time of the normal development of female flower these organs did not accumulate these isoenzymes.     

In vitro oxidation of indoleacetic Acid by soluble auxin-oxidases and peroxidases from maize roots

Soluble auxin-oxidases were extracted from Zea mays L. cv LG11 apical root segments and partially separated from peroxidases (EC 1.11.1.7) by size-exclusion chromatography. Auxin-oxidases were resolved into one main peak corresponding to a molecular mass of 32.5 kilodaltons and a minor peak at 54.5 kilodaltons. Peroxidases were separated into at least four peaks, with molecular masses from 32.5 to 78 kilodaltons. In vitro activity of indoleacetic acid-oxidases was dependent on the presence of MnCl₂ and p-coumaric acid. Compound(s) present in the crude extract and several synthetic auxin transport inhibitors (including 2,3,5-triiodobenzoic acid and N-1-naphthylphthalamic acid) inhibited auxin-oxidase activity, but had no effect on peroxidases. The products resulting from the in vitro enzymatic oxidation of [³H] indoleacetic acid were separated by HPLC and the major metabolite was found to cochromatograph with indol-3yl-methanol.     

Purification and some properties of peroxidases of rat bone marrow

Myeloperoxidase and eosinophil peroxidase were separated and purified from rat bone marrow cells using cetyltrimethylammonium bromide as the solubilizer and then with column chromatographies on CM-Sephadex C-50 and Con A-Sepharose. Both purified enzymes were observed to be apparently homogeneous by SDS-polyacrylamide gel electrophoresis. Myeloperoxidase consisted of two subunits of Mr 57,000 and 15,000, and eosinophil peroxidase two of 53,000 and 14,000. On structural analysis of the enzymes, their visual and ESR spectra revealed that the structure surrounding the heme in myeloperoxidase was different from that in eosinophil peroxidase. Moreover, substrate specificity and sensitivity to inhibitors such as azide and cyanide differed between the two enzymes. Rat bone marrow possesses two distinct peroxidases, myeloperoxidase and eosinophil peroxidase, which have different subunits and different heme microenvironments. Therefore, the difference in enzymatic function between the two peroxidases may be due to their structures.     

Identification and characterisation of Ca-pectate binding peroxidases inArabidopsis thaliana

The Arabidopsis genome encodes many secretory guaiacol peroxidases (class III plant peroxidases, EC 1.11.1.7). These higher plant enzymes are found either in the vacuole or in the apoplast, where several functions have been attributed to them. Their localisation within the cell wall matrix is most likely important for their activity. In the present work, a gel consisting of polygalacturonate chains cross-linked by Ca²⁺ and embedded in polyacrylamide was used to separate proteins from Arabidopsis leaves having an affinity for the Ca²⁺-mediated conformation of pectin. This chromatographic technique selected a small number of cationic isoperoxidases able to bind to Ca²⁺-pectate but not to Ca²⁺-alginate, a polyuronate gel similar to Ca²⁺-pectate. This result suggested that some of the Arabidopsis peroxidases have an affinity for pectin in vivo. Such a property could allow them to be properly distributed within the cell wall network. In addition, eleven cDNAs encoding an Arabidopsis peroxidase were expressed in the baculovirus-insect cell system. The capacity of the resulting recombinant peroxidases to bind Ca²⁺-pectate and Ca²⁺-alginate was also assessed. It appeared that 3 of them exhibited a Ca²⁺-pectate binding activity that was resistant to the action of NaCl. The binding of these recombinant peroxidases to Ca²⁺-alginate was much weaker than to Ca²⁺-pectate, confirming the specificity of the interaction with the pectic structure.     

Identification of multiple ear-colonizing insect and disease resistance in CIMMYT maize inbred lines with varying levels of silk maysin

Ninety four corn inbred lines selected from International Center for the Improvement of Maize and Wheat (CIMMYT) in Mexico were evaluated for levels of silk maysin in 2001 and 2002. Damage by major ear-feeding insects [i.e., corn earworm, Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae); maize weevil, Sitophilus zeamais (Motschulsky) (Coleoptera: Curculionidae); brown stink bug, Euschistus servus (Say); southern green stink bugs, Nezara viridula (L.) (Heteroptera: Pentatomidae)], and common smut [Ustilago maydis DC (Corda)] infection on these inbred lines were evaluated in 2005 and 2006 under subtropical conditions at Tifton, GA. Ten inbred lines possessing good agronomic traits were also resistant to the corn earworm. The correlation between ear-feeding insect damage or smut infection and three phenotypic traits (silk maysin level, husk extension, and husk tightness of corn ears) was also examined. Corn earworm and stink bug damage was negatively correlated to husk extension, but not to either silk maysin levels or husk tightness. In combination with the best agronomic trait ratings that show the least corn earworm and stink bug damage, lowest smut infection rate, and good insect-resistant phenotypic traits (i.e., high maysin and good husk coverage and husk tightness), 10 best inbred lines (CML90, CML92, CML94, CML99, CML104, CML108, CML114, CML128, CML137, and CML373) were identified from the 94 lines examined. These selected inbred lines will be used for further examination of their resistance mechanisms and development of new corn germplasm that confers multiple ear-colonizing pest resistance.     

Membrane-bound class III peroxidases: identification, biochemical properties and sequence analysis of isoenzymes purified from maize (Zea mays L.) roots

The occurrence of three plasma membrane-bound class III peroxidases has been demonstrated for maize (Zea mays L.) roots [Mika and Lüthje (2003) Plant Physiol. 132:1489-1498]. In the present work a novel PM-bound peroxidase (pmPOX3) was partially purified. The experimental molecular mass of the heme protein was 38 kDa after size exclusion, and 57 kDa in non-reducing SDS-PAGE stained with the peroxidase substrates tetramethylbenzidine and H(2)O(2). The glycosylation of pmPOX1, pmPOX2b and pmPOX3 was shown by different approaches. The full length sequences of pmPOX1, pmPOX2b and pmPOX3 were identified by ESI-MS/MS and MALDI-TOF MS analysis in combination with in silico and in vivo cloning. Thus, we report the first sequence analysis of membrane-bound class III peroxidases. A partial gene analysis revealed two or three introns. Experimental and theoretical isoelectric points and molecular masses were compared. Targeting signals, the putative protein structures and the localization of the active center of the enzymes on the outside of the plasma membrane were deduced of the amino acid sequences. In contrast to other class III peroxidases, pmPOX1 seems to have a dimeric structure. Predictions of hydrophobic domains in comparison with solubilization experiments suggest an N-terminal transmembrane domain for the isoenzymes.     

Coconut leaf bioactivity toward generalist maize insect pests

Tropical plants are often more resistant to insects than temperate plants due to evolution of robust defenses to cope with a more constant insect threat. Coconut [Cocos nucifera L. (Arecaceae)] has very few chewing‐type leaf feeding insect pests and was tested for feeding suitability against two generalist leaf feeding caterpillar species, corn earworm, Helicoverpa zea (Boddie), and fall armyworm, Spodoptera frugiperda (J.E. Smith) (both Lepidoptera: Noctuidae). Feeding on leaf tissues from the most recently expanded leaves of a coconut variety caused significant mortality and reduced growth rates (as indicated by survivor weights) of S. frugiperda and H. zea compared to when they fed on leaves from a typical host, maize [Zea mays L. (Poaceae)], or the standard artificial diet. Proteins or other polymers did not appear to be responsible for the bioactivity noted against the caterpillars. Components responsible for activity were acetone extractable and separable by thin layer chromatography. Extracts from multiple areas of the thin layer chromatography (TLC) plates caused significant reductions in growth rates of S. frugiperda. The most bioactive TLC‐separated component, identified as pheophytin a, caused oxidative browning of test diets, suggesting that cytotoxicity of reactive oxygen species is a likely mode of action against H. zea and S. frugiperda.     

Predictive inference on cytoplasmic and mitochondrial thioredoxin peroxidases in the highly radioresistant Lepidopteran insect Spodoptera frugiperda

Lepidopteran insects show remarkable resistance to radiation and chemical stress than insects of other orders. Despite this, the antioxidant machinery of insects of this order is poorly understood. Recently we demonstrated the significance of cytoplasmic NOS and a stronger mitochondrial antioxidant enzyme system in the stress-resistance of Lepidopteran insects. In the present study, we hypothesize two thioredoxin peroxidase orthologues (Sf-TPx1 and Sf-TPx2) in Lepidopteran insect Spodoptera frugiperda and demonstrate their structural/functional features important for cellular antioxidant activity and stress resistance. Results show a higher mitochondrial localization score (WoLFPSORT) of Sf-TPx2 (mitochondria-18.0, cytoplasm-7.0, nucleus-4.0) than its Drosophila orthologue Jafrac2 (secretory-30.0; mitochondria/nucleus/cytoplasm-no signal), which is important for antioxidant activity, and a higher cytoplasmic localization score of Sf-TPx1 (mitochondria-no signal; cytoplasm-22.0; nucleus-3.5) than the Drosophila Jafrac1 (mitochondria-17; nucleus- 11; cytoplasm-no signal). Structural modeling data show certain motifs present in Jafrac1 and Jafrac2 that affect active site conformation and separate cysteine residues at distances not suitable for disulphide bridge formation (5.21Å; 5.73Å). These motifs are absent in Sf-TPx1 and Sf-TPx2, yielding shorter distance (2.01Å; 2.05Å) between the cysteine residues suitable for disulphide bridge formation. Taken together, the disulphide bridge as well as mitochondrial and cytoplasmic localization are crucial for peroxidatic activity of TPx''s. Therefore,we hypothesize that the Spodoptera TPx''s offer potentially stronger anti-oxidant activity than that of Drosophila orthologues, and may contribute in the high radioresistance of Lepidopteran insects.     

Purification and properties of cytochrome c and two peroxidases from spinach leaves

1. A simultaneous purification procedure of cytochrome c, peroxidases,ferredoxin, ferredoxin-NADP reductase and sulfite reductasefrom spinach leaves is described. Cytochrome c, ferredoxin andferredoxin-NADP reductase were prepared in crystalline states.The two peroxidases were obtained in homogeneous states as evidencedby their electrophoretic patterns on acrylamide gel and sedimentationanalysis. 2. Crystalline cytochrome c showed a molecular weight of 13,800and an E₀' of 270 mv at pH 7.0. In addition to these properties,its spectral pattern also indicated that this cytochrome c wasderived from mitochondria. 3. Two peroxidases were isolated in high spin forms after treatmentwith HgCl₂. They had a-peaks at 556 mµ in their reducedforms. Although both peroxidases showed small differences inchromatographic behavior on a carboxymethyl cellulose column,' they had similar spectral properties, dissociation constantsof peroxidase-cyanide complex and rate constants for peroxidasereactions. (Received December 24, 1970; )     

Identification of insect parasitic nematodes--a review

Information is presented that can be used by the working parasitologist to identify the type of insect parasitic nematode he has found. The importance of obtaining identifiable adult nematode specimens and using certain fixative solutions is stressed. Tables showing a current classification of the three groups of insect parasitic nematodes—the sphaerulariids, the mermithids, and the neoaplectanids are given, along with numerous photographs and line drawings of those nematodes most commonly found parasitic in insects. Future efforts should be directed toward more field experimentation and the activation of the private sector to mass produce these parasites.