Effect of manganese on the resistance of tomato to Pseudocercospora fuligena

Black leaf mold caused by Pseudocercospora fuligena is an important fungal disease of tomato in Southeast Asia. The objective of this study was to evaluate the control of this disease using high manganese (Mn) applied to the root substrate and to evaluate the role of the leaf apoplast in plant response to fungal infection. In a nethouse experiment in Thailand, Mn above the optimum for plant growth but below toxicity increased resistance of tomato plants to black leaf mold. Enhanced resistance caused by Mn was also obtained when tomato plants were grown under controlled conditions in a mist chamber and artificially inoculated with the fungus. Manganese significantly increased plant peroxidases in the leaf apoplast. The highest peroxidase activity was measured when plants were inoculated with Pseudocercospora fuligena. Defense‐related proteins in the leaf apoplast increased when plants were inoculated with Pseudocercospora fuligena but not when treated with high Mn. It is concluded that Mn above the optimum level for plant growth can contribute to the control of Pseudocercospora fuligena in tomato. The Mn effect on disease resistance is associated with the activation of plant peroxidases in the leaf apoplast. A systemic response, possibly mediated by NADH peroxidase activity, also seems to trigger disease resistance in leaves with low Mn concentrations.     

A quick and sensitive method for the quantification of peroxidase activity of organic surface soil from forests

The purpose of the present study was to test the non-mutagenic compound 3,3′,5,5′-tetramethylbenzidine (TMB) as a model substrate for peroxidase in forest topsoil, as an alternative to the conventional substrate l-3,4-dihydroxyphenylalanine (l-DOPA). TMB was highly sensitive; linear absorbance changes of 0.6 were achieved within 20 min for 1000-fold diluted soil. Brief heating (denaturation) of the soil suspension gave a 34-fold reduction of TMB oxidation, indicating that the reaction measured by TMB was indeed an enzymatic reaction. TMB oxidation showed a narrow peak at pH 4.4. A proportional decrease in peroxidase activity, when the soil suspension was diluted, demonstrated that TMB estimates of peroxidase activity are directly comparable when corrected for differences in sample size. Oxidation of TMB was slow in the absence of H₂O₂ suggesting that TMB is a poor substrate for phenol oxidases. TMB oxidation was tested in nine different forest topsoils. The peroxidase activity, when normalised to the amount of soil organic matter, ranged from 1.4±0.1 Δabs₄₅₀ h^?1 mg^?1 to 34.9±4.3 Δabs₄₅₀ h^?1 mg^?1. In comparison, l-DOPA oxidation by soil peroxidases and commercial peroxidases gave inconsistent results, suggesting that one should be cautious when using l-DOPA as a soil peroxidase substrate. The high sensitivity of TMB, compared to l-DOPA, and the low interference from phenol oxidase and humic substances suggest that TMB is a better substrate than l-DOPA for estimation of peroxidase activity of forest topsoil.     

Purification and kinetic characterization of an anionic peroxidase from melon (Cucumis melo L.) cultivated under different salinity conditions

The partial characterization of an anionic peroxidase in melon fruit is described. Four melon peroxidase (MPX) isoenzymes were detected in crude extracts after isoelectric focusing. The major MPX isoenzyme (pI = 3.7) was partially purified by including hydrophobic and anion-exchange chromatography in the purification scheme. The sample obtained was used to characterize MPX. This peroxidase did not show activity on ascorbic acid but oxidized guaiacol at a high rate, showing an optimum pH of 5.5 when acting on this last reducing substrate. Melon fruits grown under highly saline conditions showed slightly increased levels of this anionic isoenzyme. Kinetic studies using 2,2'-azinobis(3-ethylbenzothiazolinesulfonic acid) (ABTS) as reducing substrate showed that increased salinity in the growth medium did not modify the kinetic parameters of melon peroxidase on both hydrogen peroxide and reducing substrate.     

Purification and properties of a neutral peroxidase isozyme from turnip (Brassica napus L. Var. Purple Top White Globe) roots

A neutral peroxidase isozyme (pI 7.2) from turnip roots (TNP) was purified to homogeneity and partially characterized. TNP is a monomeric glycoprotein with 9.1% carbohydrate content and a molecular weight of 36 kDa. Optimum pH values for activity using 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid (ABTS) and guaiacol as H donors were 4.5 and 5.5, whereas the K(m) values were 0.7 and 3.7 mM, respectively. The ABTS K(m) was approximately 7 times higher than that reported for basic commercial horseradish peroxidase (HRP-C). TNP retained approximately 70% activity after 11 min of heating at 65 degrees C, whereas the activation energy for inactivation (132 kJ/mol) was higher than or comparable to that of other peroxidases. The low ABTS K(m) and high specific activity (1930 units/mg) gave a high catalytic efficiency (500 M(-1) s(-1)). These properties make TNP an enzyme with a high potential as an alternative to HRP in various applications.     

Induction of soluble and cell wall peroxidases by aphid infestation in barley

Peroxidase enzymes have been found in soluble, ionically bound, and covalently bound forms and have been implicated in several physiological processes in plants. This paper investigates the effect of aphid infestation on soluble and bound-cell wall peroxidase activity and bound-cell wall isoform changes of barley plants. Peroxidase activity was measured in control plants and plants infested with the aphid Schizaphis graminum (Rondani). The activity of soluble peroxidases increased with time of infestation, older plants being more affected than younger ones. The increase in bound-cell wall peroxidase activity as a function of age was higher in infested than in control plants, being higher in ionically bound than in covalently bound peroxidases. When the aphids were removed from plants, the activities of both types of peroxidases decreased to control levels. Isoelectrofocusing analyses of the ionically bound peroxidases showed changes in the isoform pattern. A new isoform was induced by infestation. The activities of all covalently bound isoforms increased after infestation. The physiological implications of these changes are discussed.     

Purification and characterization of a peroxidase from corn steep water

Three cationic peroxidases have been detected in early, middle, and late corn steep water, with pI values of approximately 8.9, approximately 9.5, and >10.0. The major cationic corn steep water peroxidase (CSWP), with a pI >10, was purified 36400-fold with a 12% recovery from late steep water by a combination of acetone and ammonium sulfate precipitation and sequential chromatography on CM-cellulose, phenyl-Sepharose, and Sephadex G-75. The UV-vis spectrum of purified CSWP is typical of other plant class III peroxidases. The RZ (A(403)/A(280)) of CSWP was between 2.6 and 2.9. It is not glycosylated and exhibited an M(r) of 30662 +/- 7 by MALDI-TOF MS. The pH optimum of CSWP depends on the substrate, and it is active on 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid), guaiacol, ferulic acid, o-dianisidine, o-phenylenediamine, and pyrogallol but is not active on either syringaldazine or ascorbate. At 75 degrees C and pH 4.5, the enzyme has half-lives of 22.7 min (0 mM Ca(2+)) and 248 min (1 mM Ca(2+)). The enzyme is stable at room temperature (22-25 degrees C), losing <3% of the activity at pH 4.5 and <10% at pH 6.2 over 400 h in the presence of 1 mM Ca(2+).     

硼对不同甘蓝型油菜品种细胞壁酶活性的影响

在溶液培养条件下,选用甘蓝型油菜硼高效和硼低效品种,研究硼对细胞壁酶活性的影响。结果表明,不同生育期缺硼均使油菜根系细胞壁过氧化物酶(POD) 活性显著升高;上部叶片细胞壁POD 活性显著下降;下部叶片处理间的差异不显著,但同一生育期细胞壁POD 活性硼高效品种显著高于硼低效品种。油菜根系细胞壁IAA 氧化酶活性,缺硼时两品种薹期和花期显著升高,苗期差异不显著;上部叶片细胞壁IAA 氧化酶活性缺硼时显著降低,硼高效品种花期差异不显著;缺硼对下部叶片细胞壁IAA 氧化酶活性影响不大,但低效品种花期该酶活性显著降低。     

Isolation and thermal characterization of an acidic isoperoxidase from turnip roots

An acidic peroxidase (pI approximately 2.5) was purified from turnip roots (TAP), and its thermal properties were evaluated. TAP is a monomeric protein having a molecular weight (MW) of 49 kDa and a carbohydrate content accounting for 18% of the MW. The yield of pure TAP was relatively high ( approximately 2 mg/kg of fresh roots), with a specific activity of 1810 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) units/mg at pH 6. The activity increased 4-fold at the optimum pH (4.0) to 7250 ABTS units/mg, higher than that of most peroxidases. TAP was heat stable; heat treatment of 25 min at 60 degrees C resulted in 90% initial activity retention, whereas an activity of 20% was retained after 25 min of heating at 80 degrees C. TAP regained 85% of its original activity within 90 min of incubation at 25 degrees C, following heat treatment at 70 degrees C for 25 min. Thermal inactivation caused noticeable changes in the heme environment as evaluated by circular dichroism and visible spectrophotometry. TAP was rapidly denatured by heating in the presence of 1.0 mM ethylene glycol bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid, but the Soret band and activity were fully recovered by adding an excess of Ca(2+). This is further evidence that Ca(2+) plays an important role in the stability of TAP. The high specific activity of TAP, together with its relatively high thermal stability, has high potential for applications in which a thermally stable enzyme is required.     

Broccoli processing wastes as a source of peroxidase

A peroxidase isozyme (BP) was purified to homogeneity from broccoli stems ( Brassica oleraceae var. maraton) discarded from industrial processing wastes. BP specific activity was 1216 ABTS [2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid)] units/mg, representing 466-fold that of crude extract. BP is a monomeric glycoprotein containing 16% carbohydrates, with a molecular mass of 49 kDa and an isoelectric point close to 4.2. From kinetic data it showed a two-substrate ping-pong mechanism, and the catalytic efficiency measured as the rate-limiting step of free BP regeneration was 3.4 x 10(6) M(-1) s(-1). The ABTS K m value was 0.2 mM, which was about 20 times lower than that reported for acidic commercial horseradish peroxidase (HRP). Assessment of BP secondary structure showed 30% helical character, similar to HRP and cytochrome c peroxidase. BP lost only 25% activity after 10 min of heating at 55 degrees C and pH 6; it was stable in the pH range from 4 to 9 and showed an optimum pH of 4.6 using ABTS as substrate. BP was active on substrates normally involved in lignin biosynthesis, such as caffeic and ferulic acids, and also displayed good catechol oxidation activity in the presence of hydrogen peroxide. Reverse micellar extraction was successfully used as potential large-scale prepurification of broccoli peroxidase, achieving a purification factor of 7, with 60% activity yield. Stems from the broccoli processing industry have a high potential as an alternative for peroxidase purification.     

Effect of NPK and biofertilizer on date palm trees grown in Siwa Oasis,Egypt

The effect of applying chemical fertilizers alone or in combination with biofertilizer on the growth, yield and fruit quality and leaf mineral nutrient content of Sewy date palm was investigated in three successive seasons from 2004 to 2006. This study was carried out at the Siwa Oasis in the western desert of Egypt between longitude 25°18′ and 26°06′ E and latitudes 29°05′ and 29°20′ N. Chemical fertilizer applications were 1.2 or 1.5 kg N/tree, 0.065 or 0.044 kg P/tree and 0.250 or 0.420 kg K/tree. Nitrogen and potassium were given as three split applications, whereas P was mixed with organic manure (40 kg/palm tree) and supplied as a single application. Biofertilizer treatments were applied at both rates of N, P and K. The control treatment was the existing nutrient application of just 40 kg of organic sheep manure/tree/year, given as a single application. The results indicated that the application of the larger amounts of N, P and K increased the number of leaves and bunches per palm, fruit yield and total sugar content with a high level of fruit quality and production economics. In addition, this treatment increased the mineral content of leaves and reduced the biennial bearing phenomenon of Sewy date palm.     

Structural motifs of syringyl peroxidases are conserved during angiosperm evolution

The most distinctive variation in the monomer composition of lignins in vascular land plants is that between the two main groups of seed plants. Thus, whereas gymnosperm (softwood) lignins are typically composed of guaiacyl (G) units, angiosperm (hardwood) lignins are largely composed of similar levels of G and syringyl (S) units. However, there are some studies that suggest that certain angiosperm peroxidases are unable to oxidize sinapyl alcohol, and a coniferyl alcohol shuttle has been proposed for oxidizing S units during the biosynthesis of lignins. With this in mind, a screening of the presence of S peroxidases in angiosperms (including woody species and forages) was performed. Contrarily to what might be expected, the intercellular washing fluids from lignifying tissues of 25 woody, herbaceous, and shrub species, belonging to both monocots and dicotyledons, all showed both S peroxidase activities and basic peroxidase isoenzymes analogous, with regard the isoelectric point, to the Zinnia elegans basic peroxidase isoenzyme, the only S peroxidase that has been fully characterized. These results led to the protein database in the search for homologies between angiosperm peroxidases and a true eudicot S peroxidase, the Z. elegans peroxidase. The findings showed that certain structural motifs of S peroxidases are conserved within the first 15 million years of angiosperm history, because they are found in peroxidases from the two major lineages of flowering plants, eumagnoliids and eudicotyledons, of note being the presence of these peroxidases in Amborella and Nymphaeales, which represent the first stages of angiosperm evolution. These phylogenetic studies also suggest that guaiacyl peroxidases apparently constitute the most "evolved state" of the plant peroxidase family evolution.     

Oxido-Reductases and Lipases as Dough-Bleaching Agents

To replace benzoyl peroxide as a bread dough-bleaching agent, pure and commercial oxido-reductases (peroxidases, catalases, glucose oxidases, lipoxygenase, and laccase) were screened based on degradation of β-carotene in a liquid system (5 μg of β-carotene/mL of 0.1M citrate phosphate buffer at pH 5.5 or 6.5) or dough. Peroxidases had the best bleaching activity; some catalases also showed bleaching potential in a liquid system but not in bread dough, suggesting that screening enzymes in liquid media has limited application for dough. In 100 g of flour, combinations of peroxidase (3,000 U), lipase (815–1,630 U), and linoleic acid (0–300 mg) completely bleached bread dough.     

Soybean peroxidase-catalyzed oxidation of luminol by hydrogen peroxide

Anionic soybean peroxidase Glycine max (SbP) is shown to efficiently catalyze luminol oxidation by hydrogen peroxide. Contrary to horseradish peroxidase, the presence of p-iodophenol in the reaction medium affects slightly the efficiency of SbP catalysis. A maximal intensity of chemiluminescence, produced through this enzymatic reaction, was detected at pH 8.4-8.6. Contrary to anionic palm tree peroxidase, in the presence of SbP, chemiluminescence intensity increases with the reaction buffer concentration. The detection limit of SbP in the reaction of luminol oxidation is 0.3 x 10(-12) M. Therefore, high sensitivity in combination with the long-term chemiluminescent signal is indicative of good prospects for application of this enzyme in enzyme immunoassay with chemiluminescent detection.     

Azo dye-mediated regulation of total phenolics and peroxidase activity in thyme (Thymus vulgaris L.) and rosemary (Rosmarinus officinalis L.) clonal lines

Thyme (Thymus vulgaris L.) and rosemary (Rosmarinus officinalis L.) clonal lines, which were previously isolated from a heterogeneous seed population by plant tissue culture techniques, have been targeted as potential plants for phytoremediation of organic pollutants such as azo dyes and related aromatic compounds. Three thyme clonal lines and three rosemary clonal lines were tested for the ability to grow on hormone-free medium containing 0.01% of azo dye Poly S-119. The results showed that dye tolerance was associated with reduced phenolics and enhanced peroxidase activity in these clonal lines. There was a clear inverse correlation between total phenolics and peroxidase activity in these plants in response to Poly S-119. The tolerance of these clonal lines showed variations at different growing stages. These observations suggested that the peroxidase activity was inducible. Because peroxidases are involved in lignification, wound healing, aromatic compound degradation, pathogen defense, and stiffening, the results suggest that azo dye stimulated the defense response of thyme and rosemary clonal plants by increasing the peroxidase activity. Stereomicroscopic observations revealed that the azo dye was sequestered within the growing axis of the plant roots, which may also enhance the polymerization of azo dye onto the cell wall with the help of enhanced peroxidase activity.     

Biochemical and spectroscopic characterization of morning glory peroxidase from an invasive and hallucinogenic plant weed Ipomoea carnea

A novel heme peroxidase MGP from the latex of Ipomoea carnea subsp. fistulosa (morning glory) belonging to the Convolvulaceae family was purified to homogeneity using ammonium sulfate precipitation, anion exchange, hydrophobic interaction, and gel filtration chromatography. The enzyme is glycosylated and has a molecular mass of 42.06 kDa (MALDI-TOF) and an isoelectric point of pH 4.3. The enzyme has high yield, broad substrate specificity, and a high stability toward pH, temperature, chaotrophs, and organic solvents. The extinction coefficient (epsilon 280 (1%)) of the enzyme was estimated as 20.56 and it consists of 13 tryptophan, 9 tyrosine, and 8 cysteine residues forming 4 disulfide bridges. There is significant effect of inhibitors targeting S-S bridges (mercaptoethanol, l-cysteine, glutathione), as well as of inhibitors targeting heme (sodium azide and hydroxylamine) on peroxidase activity, whereas inhibition was not observed with ethylmaleinimide due to the absence of reduced cysteine in the enzyme. Polyclonal antibodies against the enzyme have been raised in rabbit, and immunodiffusion suggests that the antigenic determinants of MGP are unique. The N-terminal sequence of MGP (D-E-A-C-I-F-S-A-V-K-E-V-V-D-A) exhibited considerable similarity to the sequence of other known plant peroxidases. Spectroscopic studies (absorbance, fluorescence, and circular dichroism) reveal that MGP has secondary structural features with alpha/beta type with approximately 20% alpha-helicity.     

酸雨对木芙蓉幼苗光合作用及抗氧化酶活性的影响

以pH 5.6为对照,采用pH 4.0、pH 3.0、pH 2.0强度的酸雨对2年生木芙蓉进行人工模拟胁迫,研究酸雨胁迫对木芙蓉叶片可见伤害、质膜透性(Membrane Permeability,MP)、叶绿素(Chlorophyll,Chl)含量、抗氧化酶系统及气体交换参数的影响.研究结果表明,pH 2.0和pH 3...     

Interactive effect of boron and zinc on growth and metabolism of mustard

Abstract

Mustard (Brassica campestris L.) cv. T9 was grown in refined sand at three levels of boron (B), deficient (0.0033 ppm), normal (0.33 ppm), and excess (3.3 ppm), each at three levels of zinc (Zn), low (0.00065 ppm) adequate (0.065 ppm), and high (6.5 ppm). The B deficiency effects were accentuated by low zinc viz., the decreased biomass, B and Zn concentrations in leaves and seeds and the activity of carbonic anhydrase and accumulation of reducing sugars and stimulated activities of peroxidase, ribonuclease, and acid phosphatase in B deficient leaves were aggravated further. Synergism was also observed between the two nutrients when both B and Zn were in excess together as excess B accelerated the effects of high Zn by lowering further the reduced biomass, economic yield, and carbonic anhydrase activity and raised further the increased concentration of B and Zn in leaves and seeds, reducing sugars and activity of peroxidase obtained in excess Zn. In mustard, additive effects of high Zn and low B was reflected when high Zn increased the reduced biomass, seed yield, leaf B, and decreased the stimulated activities of peroxidase, ribonuclease, acid phosphatase, and high concentration of non‐reducing sugars to some extent in low B.     

Antioxidant phytochemicals in hazelnut kernel (Corylus avellana L.) and hazelnut byproducts

Antioxidant efficacies of ethanol extracts of defatted raw hazelnut kernel and hazelnut byproducts (skin, hard shell, green leafy cover, and tree leaf) were evaluated by monitoring total antioxidant activity (TAA) and free-radical scavenging activity tests [hydrogen peroxide, superoxide radical, and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical], together with antioxidant activity in a beta-carotene-linoleate model system, inhibition of oxidation of human low-density lipoprotein (LDL) cholesterol, and inhibition of strand breaking of supercoiled deoxyribonucleic acid (DNA). In addition, yield, content of phenolics, and phenolic acid profiles (free and esterified fractions) were also examined. Generally, extracts of hazelnut byproducts (skin, hard shell, green leafy cover, and tree leaf) exhibited stronger activities than hazelnut kernel at all concentrations tested. Hazelnut extracts examined showed different antioxidative efficacies, expected to be related to the presence of phenolic compounds. Among samples, extracts of hazelnut skin, in general, showed superior antioxidative efficacy and higher phenolic content as compared to other extracts. Five phenolic acids (gallic acid, caffeic acid, p-coumaric acid, ferulic acid, and sinapic acid) were tentatively identified and quantified (both free and esterified forms). Extracts contained different levels of phenolic acids. These results suggest that hazelnut byproducts could potentially be considered as an excellent and readily available source of natural antioxidants.     

Lipase-catalyzed preparation of human milk fat substitutes from palm stearin in a solvent-free system

Human milk fat substitutes (HMFSs) were synthesized by lipozyme RM IM-catalyzed acidolysis of chemically interesterified palm stearin (mp = 58 °C) with mixed FAs from rapeseed oil, sunflower oil, palm kernel oil, stearic acid, and myristic acid in a solvent-free system. Response surface methodology (RSM) was used to model and optimize the reactions, and the factors chosen were reaction time, temperature, substrate molar ratio, and enzyme load. The optimal conditions generated from the models were as follows: reaction time, 3.4 h; temperature, 57 °C; substrate molar ratio, 14.6 mol/mol; and enzyme load, 10.7 wt % (by the weight of total substrates). Under these conditions, the contents of palmitic acid (PA) and PA at sn-2 position (sn-2 PA) were 29.7 and 62.8%, respectively, and other observed FAs were all within the range of FAs of HMF. The product was evaluated by the cited model, and a high score (85.8) was obtained, which indicated a high degree of similarity of the product to HMF.