Peroxidase Isozymes Research Articles

Genetic purity and diversity in isoenzymePeroxidase of pearl millet hybrids [Pennisetum glaucum(L.) Br. R.

The prospective of Peroxidase Isozyme biochemical marker, have been investigation in the present study, ten pearl millet hybrids [ Pennisetum glaucum (L.) R. Br.] Genotypes were used to examine the suitability of peroxidase enzymes for characterization of pearl millet genotypes. Band number 1 (Rm = 0.120) was present in three hybrids i.e. GHB-526, GHB-538 and GHB-719. Band number 2 (0.158) was also present in three hybrids i.e. GHB-538, GHB-577 and GHB-719. Band number 3 (Rm = 0.380) was present in only one hybrid i.e. GHB-719 while band number 4 (Rm = 0.429) was present in five hybrids GHB-538, GHB-719, GHB-732, GHB-235 and GHB-757. All the seven bands were present in only one hybrid GHB-719. 9 DAG stage was found to be more effective as compared to 3 DAG and 6 DAG by showing more number of bands. Among the 10 genotypes studied, two could be differentiated from each other Total 13 bands of peroxidase isozymes were observed at 3, 6 and 9 day after germination (DAG). A total of 15 alleles were generated by isozymes at different DAG (3, 6 and 9 days) with an average of 5 bands per day. Banding pattern at 6 DAG showed that band number1, 2 and 3 (Rm = 0.525, 0.573 and 0.600) were present in all the hybrids except for GHB-744, in band number 1. At 9 DAG seven bands of peroxidase isozymes were observed having Rm value of 0.120, 0.158, 0.380, 0.429, 0.430, 0.478, and 0.760.

Peroxide isozymes and Malondialdehyde content and ascorbate peroxidase activity in Berberis integerrima and Cercis siliquastrum under Cd-induced stress .

ROS detoxification is an essential process in protecting plant cells and their organelles, which is caused by ROS generated in stress condition. Peroxidase (POD) isozyme, Malondialdehyde (MDA) content, and Ascorbate peroxidase (APX) activity are the indicators of plants for measuring pollution effects. The purpose of the present study was to investigate Cd-induced stress on POD isozymes, MDA content, and APX activity in seedlings of Berberis integerrima and Cercis siliquastrum. 100 seedlings were treated with cadmium chloride separately at concentrations of 0, 1000, 2000, 4000, 6000 mg/kg three times at intervals of 15 days. POD activity varied at different levels of Cd stress Cercis siliquastrum, while this was not the case with Berberis integerrima. MDA was slightly increased up to 6000 mg/kg from 5% to 27%, but a sharp increase (41%) was found at 6000 mg/kg compared with 4000 mg/kg Cd treatment in Cercis siliquastrum. The highest MDA content (10.5 µM/ g FW) was recorded at 6000 mg/kg trearment in Berberis integerrima, while the lowest value (2.3 µM/ g FW)was found in control Cercis siliquastrum plants. The maximum and minimum APX activity were found in the treatments of 6000 mg/kg (0.13 unit/mg protein) and control (0.02 unit/mg protein), respectively, for both Cercis siliquastrum and Berberis integerrima. In control Berberis integerrima, APX activity (0.02 unit/mg protein) was higher than that of Cercis siliquastrum (0.006 unit/mg protein) (p ˂ 0.05). Overall, the findings suggest that Berberis integerrima is less tolerant than Cercis siliquastrum under Cd-induced stress.

Toxicological effects, mechanisms, and implied toxicity thresholds in the roots of Vicia faba L. seedlings grown in copper-contaminated soil.

Copper (Cu) contamination has become a global concern because of industrial, agricultural, and other anthropogenic activities. In the present experiments, the toxicological effects, mechanisms, and potential toxicity thresholds were investigated in the roots of Vicia faba L. seedlings that were cultivated in Cu-amended soils (0, 6.25, 12.5, 25, 50, 100, 200, 400, and 600mgkg(-1)) for 20days, based on an analysis of the soil physicochemical properties, native Cu, available Cu, and root-enriched Cu contents. The superoxide dismutase (SOD), ascorbate peroxidase (APX), and guaiacol peroxidase (POD) isozymes and activities, as well as glutathione (GSH) and heat shock protein 70 (HSP70), changed like biphasic dose-response curves, cooperating to control the redox homeostasis. The APX and POD enzymes exhibited enhanced activities and became H2O2 scavengers primarily when the catalase (CAT) activities tended to decrease. Endoprotease (EP) isozymes and activities might be enhanced to degrade carbonylated proteins and alleviate metabolic disturbance in the roots. Additionally, HSP70 may not be suitable as a biomarker for relatively higher soil Cu concentrations and relatively longer exposure times for the roots. As a result, the isozymes and activities of SOD, CAT, and EP, as well as GSH, can be adopted as the most sensitive biomarkers. The toxicity threshold is estimated as 0.76-1.21mgkg(-1) of available Cu in the soils or 25.04-36.65μgCug(-1) dry weights (DW) in the roots.

Slow-growth conservation and clonal fidelity of Tetrastigma hemsleyanum microplants

Storage under slow growth is one of the most successful techniques for medium-term germplasm conservation and could effectively reduce somaclonal variation during subculture. Tetrastigma hemsleyanum is an endangered medicinal herb with anticancer potential. In the present study, an effective protocol was developed for clonal propagation and medium-term conservation of this endangered medicinal plant. Subculture duration was significantly prolonged to at least 10 mo by using 0.2 mg L−1 of maleic hydrazide (MH) at 8°C. Analyses of inter-simple sequence repeat (ISSR) markers, sequence-related amplified polymorphism (SRAP) markers, total proteins, and peroxidase isozymes together demonstrated the genetic stability of the regenerated germplasm. Total flavonoid content of regenerated microplants also remained stable. These results suggest that MH has potential application in slow-growth conservation of plants.

Obtención de semilla biotecnológica de caña de azúcar (Saccharum spp. híbrido) de alta calidad genética y fitosanitaria en el Ecuador

<strong>Título en ingles: Obtainment of sugarcane (<em>Saccharum</em> spp. hybrid) biotechnological seed of high genetic and phytosanitary quality in Ecuador </strong><p><strong>Título corto: Semilla biotecnológica de caña de azúcar</strong></p><p><strong>Resumen:</strong>Los métodos de saneamiento de plantas se basan fundamentalmente en el empleo combinado de cultivo <em>in vitro</em> de meristemos con tratamientos mediante hidrotermoterapia. Este trabajo se realizó con el objetivo de obtener plantas para usar como semilla libre de patógenos sistémicos, con énfasis en la enfermedad raquitismo de los retoños de la caña de azúcar (RSD). El trabajo se llevó a cabo con diferentes cultivares de caña de azúcar, los cuales fueron previamente identificados mediante marcadores bioquímicos basados en los patrones electroforéticos de isoenzimas peroxidasas. El saneamiento se realizó en tres etapas sucesivas mediante tratamiento hidrotérmico a 52 <sup>o</sup>C durante 2h y uso del fungicida vitavax, un segundo tratamiento hidrotérmico a los meristemos a 51 <sup>o</sup>C durante 10 min y una tercera etapa donde las vitroplántulas se propagaron en presencia del antibiótico gentamicina. La detección de RSD se efectuó mediante el empleo de la técnica de tinción de haces vasculares funcionales. Los resultados permitieron la creación de un banco de germoplasma de plantas libres de los principales patógenos bacterianos sistémicos y con alta calidad genética, lo que aportó semilla básica de excelente calidad para establecer semilleros. La conservación incluyó el mantenimiento de plántulas <em>in vitro</em> y la creación de un “banco de ADN” que permite conservar el genofondo en condiciones de laboratorio. Se propone finalmente una metodología para la obtención de plantas saneadas a partir de yemas y meristemos.<strong>Palabras clave:</strong> <em>Saccharum</em> spp.; saneamiento; cultivo de yemas; meristemo.<strong></strong></p><p><strong>Abreviaturas:</strong> BAP: 6-benzyl-aminopurina; LSD: Escaldadura foliar; RSD: Raquitismo de los retoños; VMCA: Virus del mosaico de la caña de azúcar; THV: tinción de los haces vasculares.</p><p><strong>Abstract: </strong>Methods for obtaining healthy plants are mainly based on the combined use of meristem <em>in vitro</em> culture and hydrothermotherapy. This work was carried out in order to obtain free- pathogen plants for using as seeds, emphasizing ratoon stunting disease (RSD) of sugarcane. The work was performed using a group of sugarcane cultivars, which was confirmed the variety identification by means of peroxidases isozymes electrophoretic patterns. Plant sanitation was performed in three successive steps by means of hydrothermal treatment at 52 <sup>o</sup>C during 2h and using the fungicide vitavax, a second hydrothermal treatment at 51 <sup>o</sup>C during 10 min to the meristem and a third step in which vitroplantlets were propagated in presence of the antibiotic gentamicin. RSD detection was done by using the staining transpiration methods (STM) of functional bundles. Results allowed the creation of a germoplasm bank of sugarcane plants free of the most important systemic bacterial pathogens, with high genetic quality, which provided basic seed of excellent quality for establishing seedlings. Conservation included an <em>in vitro</em>-plantlets bank and the creation of a “DNA bank” which allows preserving the genetic fond in laboratory conditions. Finally a methodology for obtaining free-pathogens plants from buds and meristems is included.</p><p><strong>Key words:</strong> <em>Saccharum</em> spp.; plant sanitation; culture of buds; meristem.</p><p><strong>Recibido:</strong> noviembre 28 de 2014<strong> Aprobado: </strong>abril 24 de 2015</p>

Role of defense enzymes, proteins, solutes and Δ1-pyrroline-5-carboxylate synthase in wheat tolerance to drought

Growth parameters of 17-day-old wheat seedlings (Sids4, Sd; Gmiza10, Gm) were similar under normal control conditions; however, water deficit for the following 16 days (up to 33 days after sowing, DAS) led to greater reductions in Gm than in Sd, suggesting that Sd was more tolerant to drought than Gm. At the same time, drought led to significant losses of several proteins in Sd, despite the similarities detected in both cultivars under normal conditions to the extent that 70 % were lost in Gm on the 33rd DAS relative to only 40 % in Sd. In contrast to Sd, drought decreased catalase (CAT) and peroxidase (POX) isozymes and Rubisco as well as the expression of Δ1-pyrroline-5-carboxylate synthase (P5CS) gene in Gm concomitant with slight effects, if any, on soluble sugars, glycine betaine (GB), free amino acids and proline. Three CAT isozymes and ten POX isozymes were detected in both cultivars; nonetheless, drought down expressed some of these isozymes particularly in Gm. The impacts of drought on Rubisco were greater in Gm than in Sd. The accumulation of soluble sugars, GB, free amino acids and proline by drought was higher in Sd than in Gm and the magnitude increased with drought severity. Also, drought led to a significant rise in P5CS in Sd, but caused a decline in Gm. After rewatering, P5CS increased in Gm, but decreased in Sd to reach the level of control. The accumulation of proline in Sd synchronous with increases in P5CS transcription concludes that drought adaptation is efficient only in the tolerant cultivar. In support, rewatering decreased P5CS elevation caused by drought in Sd, concluding that this mechanism of adaptation is effective only under stress conditions.

小叶锦鸡儿抗沙埋生长与抗氧化酶及同工酶变化的关系

PDF HTML阅读 XML下载 导出引用 引用提醒 小叶锦鸡儿抗沙埋生长与抗氧化酶及同工酶变化的关系 DOI: 10.5846/stxb201306131701 作者: 作者单位: 鲁东大学,鲁东大学，生命科学学院,鲁东大学，生命科学学院,鲁东大学，生命科学学院,鲁东大学，生命科学学院,鲁东大学，生命科学学院 作者简介: 通讯作者: 中图分类号: 基金项目: 国家重点基础研究发展计划(2009CB421303); 国家自然科学基金项目(30972422); 中国科学院方向性项目(KZCXZ-YW-431) The relationship between growth of Caraganas stenophylla and the activities and isoforms of protective enzymes under different depths of sand burial Author: Affiliation: College of Life Sciences, Ludong University,,,,, Fund Project: 摘要 | 图/表 | 访问统计 | 参考文献 | 相似文献 | 引证文献 | 资源附件 | 文章评论 摘要:小叶锦鸡儿(Caraganas tenophylla L.)是广泛应用于流动沙丘治理的优良固沙植物。然而关于其抗沙埋生理机理目前尚不清楚。选择生长在科尔沁沙地的小叶锦儿为试验材料,依据株高对其进行不同程度沙埋(轻度、中度、重度沙埋), 并通过测定沙埋过程中植株高度、不同部位叶片丙二醛(MDA)含量、抗氧化酶活力、抗氧化酶同工酶谱变化, 以揭示其抗沙埋生理适应机理和基因调控机理。结果表明:沙埋6d,植株各部位生长加快,尤其是顶部和基部生长更快。叶片MDA含量降低、整株植物叶片平均过氧化氢酶(CAT)、过氧化物酶(POD)、过氧化物歧化酶(SOD)活力增加,但重度沙埋使抗氧化酶活力下降。沙埋12d,植株各部位生长继续加大, 沙下叶片凋落。与对照相比,沙上叶片MDA含量成倍增加,并与叶片POD、SOD和CAT活力的大幅度提高呈正相关,并与对照差异显著(P < 0.01)。同时,不同厚度沙埋6d,叶片CAT同工酶出现两新带CAT III和CATII;POD同工酶谱带(6条酶带)随沙埋厚度增加,叶片PODII区带加宽、色加深,POD I 和POD III酶带消失。但是,不同厚度沙埋下,沙上和沙下叶片CAT、SOD和POD酶谱带数和活力均相同。这表明在沙埋应激适应反应期(6d),叶片抗氧化酶活力的增强与抗氧化酶基因表达增强和基因启动有关。受到沙埋重力胁迫的成熟叶可能将胁迫信号传递给沙上没有沙埋的叶子及生长点,导致整株叶片产生整体适应性反应,激活抗氧化酶系统,以致加速生长。因此,小叶锦鸡儿萌蘖生物学特性和抗氧化酶对沙埋胁迫快速响应在维护氧自由基代谢平衡和植株快速恢复生长中起重要保护作用。 Abstract:The shrub Caraganas stenophylla is widely distributed and used as a sand binding plant on moving sand dunes in China. The physiological mechanisms involved in C. stenophylla responds to coverage with sand are unknown. Therefore we investigated responses to three levels of sand burial based on plant height: light (1/4 plant height), modest (2/4 plant height), and severe (3/4 plant height) as compared with controls (no sand burial). To understand the physiological and molecular regulatory mechanisms for C. stenophylla in adaption to sand burial, changes in length of different parts of plant stems were observed 3 days (d), 6d, 9d, and 12d after sand burial treatment and contents of malondialdehyde (MDA) and activities of antioxidant enzymes peroxidase (POD), catalase (CAT), and superoxide dismutase (SOD) were measured 6d and 12d after treatment. CAT and POD isozymes were also analyzed in leaves 6d after sand burial treatment. Results showed that 6d after sand burial treatment, growth rates of different parts of the stem increased as burial deepened. At the same time, MDA content in the leaves decreased and activities of CAT, POD and SOD in different plant parts increased under light and modest sand burial treatments. Growth rate was higher 12d after treatment than at 6d after treatment but the leaves under sand died. Meanwhile, MDA content and SOD activity were markedly increased by 64% and 121% in leaves above sand under modest and severe sand burial. CAT activity in leaves above sand was also higher under light and modest sand burial. POD activity in the leaves under different depths of sand burial was higher by 308%-104%. SOD activity was higher by 32% and 233% in the leaves with modest and severe sand burial compared with controls. This suggests that the higher growth rate during 7-12d was positively correlated with higher activities in protective enzymes, and increases in antioxidant capacity after sand burial, which might have played an important role in the stem growth and the balance of oxygen free radical metabolism. Analysis of isomers of antioxidant enzymes by native-PAGE and activity staining revealed new isoforms, CAT III and CATII, in the leaves with modest and severe sand burial, and their concentrations were enhanced by sand burial. There are six isoforms of POD; POD II intensity increased with increasing depth of sand burial; a new band, POD IV, was observed while POD I and POD III disappeared. This suggests that during acclimation to sand burial, the activity of protective enzymes is highly related to differential gene expression. Isofo rms of CAT, POD and SOD in the leaves over sand were similar to those under sand 6d after sand burial, which indicates that the signal of sand burial was transduced from leaves under sand to leaves over sand, resulting in the whole plant receiving the signal of sand stress to adapt to sand burial. Therefore, differential gene expression plays an important role in increasing protective enzyme activity to maintain the balance of oxygen free radical metabolism in C. stenophylla. 参考文献 相似文献 引证文献

Evaluation of Genetic Variations in Maize Seedlings Exposed to Electric Field Based on Protein and DNA Markers.

The current study analyzed proteins and nuclear DNA of electric fields (ELF) exposed and nonexposed maize seedlings for different exposure periods using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), isozymes, random amplified polymorphic DNA (RAPD), and comet assay, respectively. SDS-PAGE analysis revealed total of 46 polypeptides bands with different molecular weights ranging from 186.20 to 36.00 KDa. It generated distinctive polymorphism value of 84.62%. Leucine-aminopeptidase, peroxidase, and catalase isozymes showed the highest values of polymorphism (100%) based on zymograms number, relative front (R f), and optical intensity while esterase isozyme generated polymorphism value of 83.33%. Amino acids were analyzed using high-performance liquid chromatography, which revealed the presence of 17 amino acids of variable contents ranging from 22.65% to 28.09%. RAPD revealed that 78 amplified DNA products had highly polymorphism value (95.08%) based on band numbers, with variable sizes ranging from 120 to 992 base pairs and band intensity. Comet assay recorded the highest extent of nuclear DNA damage as percentage of tailed DNA (2.38%) and tail moment unit (5.36) at ELF exposure of maize nuclei for 5 days. The current study concluded that the longer ELF exposing periods had genotoxic stress on macromolecules of maize cells and biomarkers used should be augmented for reliable estimates of genotoxicity after exposure of economic plants to ELF stressors.

Acquisition, identification and analysis of an interspecific Capsicum hybrid (C. annuum × C. chinense)

SummaryUsing an embryo rescue technique, we obtained an F1 plantlet whose female parent was a purple chilli pepper (Capsicum annuum 7033) and whose male parent was a green chilli pepper (C. chinense 7020).We verified the origin of this interspecific hybrid using a peroxidase isozyme assay and random amplified polymorphic DNA (RAPD) markers. Most of the morphological features of the F1 plant, particularly the purple colour of its foliage, flowers and immature fruit, were intermediate between those of its parents. Cytological analysis indicated that the meiotic chromosomes of the pollen mother cells of the F1 plant exhibited abnormal behaviour, resulting in a marked reduction in the proportion of fertile pollen compared to both its parents. Sequence analysis of the internal transcribed spacer of nuclear ribosomal DNA demonstrated that, compared to the parental sequences, the F1 hybrid had 186 polymorphic sites, 139 of which (74.7%) were inherited from the male parent, and 23 (12.4%) were inherited from the female parent, and 24 (12.9%) were transversions or base pair mutations. Therefore, our morphological and cytological observations, as well as isozyme and molecular marker analyses confirmed that the F1 plant was a genuine interspecific hybrid that had inherited more genetic information from its male parent than from its female parent. This work will help locate purple colour-related genes and the breeding of pepper cultivars containing higher concentrations of anthocyanin and capsaicin.

Biochemical Characterization of Ridge Gourd and Sponge Gourd Genotypes

A genetic analysis of ridge gourd and sponge gourd genotypes were performed using protein profiling and isozymes analysis of four enzymes viz., peroxidase, polyphenol oxidase, esterase and super oxide dismutase. Fifteen day old seedlings of the genotypes were tested in Native-PAGE for the study of genetic diversity among them. Protein profiling of the genotypes developed nine bands having Rm values of 0.037, 0.075, 0.154, 0.213, 0.266, 0.326, 0.532, 0.584 and 0.663. Out of them, three bands were monomorphic and others were polymorphic. Seventeen ridge gourd and sponge gourd genotypes were grouped into two main clusters I and II with an average similarity of 47 per cent. Four bands of peroxidase isozymes were observed having Rm values of 0.083, 0.236, 0.515 and 0.803. Out of them two bands were polymorphic in nature. Four bands of polyphenol oxidase were detected with Rm values of 0.318, 0.438, 0.570 and 0.686. Out of them two bands were polymorphic in nature. Total five bands of esterase were observed having Rm values of 0.037, 0.102, 0.170, 0.269 and 0.372. Out of them four bands were polymorphic in nature and a band with Rm value 0.102 was monomorphic. Three bands of Rm values 0.246, 0.339 and 0.486 were observed for super oxide dismutase isozymes. The band of Rm value 0.486 was present in all the 17 genotypes and was monomorphic in nature.

Expression of a manganese peroxidase isozyme 2 transgene in the ethanologenic white rot fungus Phlebia sp. strain MG-60

BackgroundThe white-rot fungus Phlebia sp. strain MG-60 was proposed as a candidate for integrated fungal fermentation process (IFFP), which unifies aerobic delignification and semi-aerobic consolidated biological processing by a single microorganism based on its ability to efficiently degrade lignin and ferment the sugars from cellulose. To improve IFFP, the development of a molecular breeding method for strain MG-60 is necessary. The purpose of this study is to establish the transformation method for the strain MG-60 and to obtain the over-expressing transformants of lignin-degrading enzyme, manganese peroxidase.FindingsIn the present study, the expression vector regulated by Phlebia brevispora glyceraldehyde-3-phosphate dehydrogenase promoter and terminator was constructed. A polyethylene glycol transformation method for the ethanol-fermenting white-rot fungus Phlebia sp. MG-60 was established with high transformation efficiency, and the manganese peroxidase isozyme 2 gene (MGmnp2) transformants were obtained, showing higher MnP activity than control transformants. MGmnp2 transformants showed higher selective lignin degradation on Quercus wood powder.ConclusionsThis first report of MG-60 transformation provides a useful methodology for widely accessible to interested researches. These results indicate the possibility of metabolic engineering of strain MG-60 for improving IFFP.Electronic supplementary materialThe online version of this article (doi:10.1186/2193-1801-3-699) contains supplementary material, which is available to authorized users.

Engineering a horseradish peroxidase C stable to radical attacks by mutating multiple radical coupling sites.

Peroxidases have great potential as industrial biocatalysts. In particular, the oxidative polymerization of phenolic compounds catalyzed by peroxidases has been extensively examined because of the advantage of this method over other conventional chemical methods. However, the industrial application of peroxidases is often limited because of their rapid inactivation by phenoxyl radicals during oxidative polymerization. In this work, we report a novel protein engineering approach to improve the radical stability of horseradish peroxidase isozyme C (HRPC). Phenylalanine residues that are vulnerable to modification by the phenoxyl radicals were identified using mass spectrometry analysis. UV-Vis and CD spectra showed that radical coupling did not change the secondary structure or the active site of HRPC. Four phenylalanine (Phe) residues (F68, F142, F143, and F179) were each mutated to alanine residues to generate single mutants to examine the role of these sites in radical coupling. Despite marginal improvement of radical stability, each single mutant still exhibited rapid radical inactivation. To further reduce inactivation by radical coupling, the four substitution mutations were combined in F68A/F142A/F143A/F179A. This mutant demonstrated dramatic enhancement of radical stability by retaining 41% of its initial activity compared to the wild-type, which was completely inactivated. Structure and sequence alignment revealed that radical-vulnerable Phe residues of HPRC are conserved in homologous peroxidases, which showed the same rapid inactivation tendency as HRPC. Based on our site-directed mutagenesis and biochemical characterization, we have shown that engineering radical-vulnerable residues to eliminate multiple radical coupling can be a good strategy to improve the stability of peroxidases against radical attack.

Effects of calmodulin on expression of lignin-modifying enzymes in Pleurotus ostreatus.

Previously, we suppressed the expression of genes encoding isozymes of lignin peroxidase (LiP) and manganese peroxidase (MnP) using a calmodulin (CaM) inhibitor, W7, in the white-rot fungus Phanerochaete chrysosporium; this suggested that CaM positively regulates their expression. Here, we studied the role of CaM in another white-rot fungus, Pleurotus ostreatus, which produces MnP and versatile peroxidase (VP), but not LiP. W7 upregulated Mn(2+)-dependent oxidation of guaiacol, suggesting that CaM negatively regulates the production of the enzymes. Suppression of CaM in P. ostreatus using RNAi also led to upregulation of enzyme activity, whereas overexpression of CaM in P. ostreatus caused downregulation. Real-time RT-PCR showed that MnP1-6 and VP3 levels in the CaM-knockdown strain were higher than those in the wild-type strain, while MnP-5 and -6 and VP1 and 2 levels in the CaM-overexpressing strain were lower than in the wild type. Moreover, we also found that another ligninolytic enzyme, laccase, which is not produced by P. chrysosporium, was negatively regulated by CaM in P. ostreatus similar to MnP and VP. Although overexpression of CaM did not reduce the ability of P. ostreatus to digest beech wood powder, the percentage of lignin remaining in the digest was slightly higher than in the wild-type strain digest.

Peroxidase inactivation by covalent modification with phenoxyl radical during phenol oxidation

The mechanism-based inactivation of peroxidases limits the usefulness of these versatile enzymes. In this study, we propose a dominant inactivation mechanism for peroxidase during phenol oxidation. Two peroxidases, Coprinus cinereus peroxidase (CiP) and horseradish peroxidase isozyme C (HRPC) showed much higher inactivation rates after simultaneous addition of phenol and hydrogen peroxide, whereas addition of hydrogen peroxide alone or polymeric products had relatively little impact on peroxidase activity. During the oxidation of a phenol substrate, the molecular weights of polypeptides originating from inactivated peroxidases were slightly increased, and a large fraction of CiP and HRPC hemes remained intact even after phenoxyl radical coupling. Our study strongly supports the hypothesis that the inactivation of a peroxidase during the oxidation of phenolic compounds occurs by a covalent modification of the peroxidase polypeptide chain with a phenoxyl radical. These findings will elucidate the method of inactivation for peroxidase and other heme proteins.

PEROXIDASE ISOZYME IDENTIFICATION OF SOME RICE GENOTYPES IN M1 GENERATION UNDER DROUGHT STRESS LEVEL OF -0.03 MPa

The effort to fulfill the need of rice through the improvement of dryland productivity can be viewed as a more environmentally-friendly way. This research used 36 rice genotypes in M 1 generation that were grown hydrophonically under drought stress level of -0.03 MPa. The identifications were conducted based on peroxidase isozyme marker. The isozyme patterns in zymogram were binary-coded by visual scores for each genotype, based on the thickness and the number in the appearance of bands on certain migration distance. The migration distances were measured based on values of Rf. The similarity coefficients were calculated using Dice’s coefficient that were used to construct dendrogram using the UPGMA employing the SAHN from the NTSYSpc 2.02. The results showed that the most resistant genotype under drought stress was R-4, and the genetic relationships among the genotypes were divided into two main groups, aromatic and non-aromatic group, in which some genotypes experienced the reduced levels of aromatic character (R-8 and R-9) and the drought resistance character (IU-2, IU-3, IU-4, IU-5, IU-6, IU-7, and IU-8), but there were some genotypes to able to improve the resistance under drought stress (R-2, R-3, R-5, R-6, R-7, IT-4, IT-5, and IT-7). Keywords: drought resistance, gamma irradiation, peroxidase isozyme, rice, sodium azide

Accelerated degradation of lignin by lignin peroxidase isozyme H8 (LiPH8) from Phanerochaete chrysosporium with engineered 4-O-methyltransferase from Clarkia breweri

Free-hydroxyl phenolic units can decrease or even abort the catalytic activity of lignin peroxidase H8 during oxidation of veratryl alcohol and model lignin dimers, resulting in slow and inefficient lignin degradation. In this study we applied engineered 4-O-methyltransferase from Clarkia breweri to detoxify the inhibiting free-hydroxyl phenolic groups by converting them to methylated phenolic groups. The multistep, enzyme-catalyzed process that combines 4-O-methyltransferase and lignin peroxidase H8 suggested in this work can increase the efficiency of lignin-degradation. This study also suggests approaching the field of multi-enzyme in vitro systems to improve the understanding and development of plant biomass in biorefinery operations.

Antioxidant and Hormone Responses to Heat Stress in Two Kentucky Bluegrass Cultivars Contrasting in Heat Tolerance

Heat stress is a major limiting factor for growth of cool-season perennial grass species, and mechanisms of heat tolerance have not been well understood. This study was designed to investigate antioxidant enzyme and hormone metabolism responses to heat stress in two kentucky bluegrass (Poa pratensis L.) cultivars contrasting in heat tolerance. The plants were subjected to 20/20 °C [day/night (control)] or 38/30 °C [day/night (heat stress)] for 28 days in growth chambers. Heat stress increased leaf electrolyte leakage (EL) and malondialdehyde (MDA) with heat-tolerant cultivar EverGlade exhibiting lower levels of EL and MDA relative to heat-sensitive cultivar Kenblue under heat stress. Superoxide dismutase (SOD) and catalase (CAT) activity increased and then declined during 28 days of heat stress. Peroxidase (POD) and ascorbate peroxidase (APX) activity declined and then increased during heat stress. ‘EverGlade’ had greater activities of SOD, CAT, POD, and APX relative to ‘Kenblue’ under heat stress. In addition, ‘EverGlade’ had two additional SOD isozymes and three additional POD isozymes relative to ‘Kenblue’ under heat stress. Leaf abscisic acid (ABA) increased in response to heat stress. Leaf indole-3-acetic acid (IAA) increased and then declined during heat stress. ‘OverGlade’ had higher ABA and IAA content relative to ‘Kenblue’. At the end of heat stress, leaf IAA and ABA content were 27.8% and 73% higher in ‘EverGlade’ relative to ‘Kenblue’, respectively. The results indicated that antioxidant enzymes and the hormones (ABA and IAA) were associated with kentucky bluegrass heat tolerance. Selection and use of cultivars with higher IAA and ABA content and greater antioxidant enzyme activities may improve kentucky bluegrass growth and quality under heat stress.

Biochemical and molecular genetic characterization of some species of family Malvaceae, Egypt

The aim of the present study intended mainly to investigate the interrelationships between the six studied taxa namely Abutilon theophrasti, Lavatera cretica, Hibiscus trionum, Hibiscus sabdariffa, Malva parviflora and Sida alba collected from ten different accessions in Egypt belonging to family Malvaceae. Biochemical studies include protein profile using SDS-PAGE technique and three isozymes (esterase, peroxidase and acid phosphatase). The electrophoretic analysis revealed the presence of eighteen bands of molecular weight ranging from 11.3 to 115.3 KD. The highest number of bands 15 was observed in H. sabdariffa (Hs1) collected from Menia el-Kamh district and the two accessions of M. parviflora where as the lowest number 11 bands were recorded in H. trionum collected from Talkha district. Four loci of peroxidase isozyme distinguished, three loci of acid phosphatase isozyme and two loci of esterase isozyme. In regarding to random amplified polymorphic DNA technique (RAPD), ten primers were used to differentiate between these accessions. Primer OPA-4 gave the highest percentage of polymorphism (100%), while primer OP-B6 produced the lowest percentage (50%).

Hairy root exudates of allelopathic weed Chenopodium murale L. induce oxidative stress and down-regulate core cell cycle genes in Arabidopsis and wheat seedlings

The effects of Chenopodium murale root exudates, applied as phytotoxic medias (PMs), were tested on Arabidopsis thaliana and Triticum aestivum. The effects of PMs, where wild-type roots (K), hairy roots derived from roots (R clones) or from cotyledons (C clones) were cultured, were different. K medium suppressed Arabidopsis germination, while other PMs reduced root and leaf elongation and the number of rosette leaves. R media were more phytotoxic than C media. Treatment of Arabidopsis with R8 down-regulated expression of core cell cycle genes: cyclin-dependent kinase (CDK) A1;1, four B-class CDKs, and cyclins CYCA3;1, CYCB2;4, CYCD4;2 and CYCH1 in root and shoot tips. Only CYCD2;1 transcript was elevated in treated shoots, but down-regulated in roots. Wheat Ta-CDC2 and Ta-CYCD2 genes showed the same expression profiles as their Arabidopsis counterparts, CDKA1;1 and CYCD2;1. PMs also caused increase of antioxidative enzyme activities in both plants. Exposure of Arabidopsis to PMs induced one catalase isoform, but repressed another, resulting in no net change of catalase activity. Wheat seedlings treated with PMs had catalase activity significantly elevated in all treatments, particularly in shoots. In both plants, PMs induced the activity of different peroxidase isozymes and total peroxidase activity. Both plants responded to phytotoxic treatments by induction of CuZn-superoxide dismutase. Thus, the phytotoxicity of C. murale root exudates is, at least partially, based on down-regulation of the cell cycle regulators and on generation of oxidative stress in the affected plants. We propose that C. murale root exudates should be considered as means of biological weed control.

MOLECULAR AND BIOCHEMICAL MARKERS ASSOCIATED WITH TOLERANCE TO Cassida vittata VILL (COLEOPTERA: CHRYSOMELIDAE) INFESTATIONS IN SUGAR BEET

Insect infestation of sugar beet is one of the most important problems in sugar beet fields at Delta, Egypt. Plant defense against insects represents the most successful element in integrated pest management (IPM). So, the behavior of insect attack, biochemical and molecular analysis were utilized to study protein, peroxidase and esterase isozymes banding patterns under insect attacks to sugar beet varieties. In addition, protein electrophoresis, genomic DNA (ISSR technique) and RAPD-PCR were used to find molecular markers associated with insect attack tolerance in sugar beet plants. These studies were carried out during 2012/2013 and 2013/2014 seasons on two sugar beet varieties (Pyramids and Zinagri). The insect attack behavior study showed that Pyramids variety is resistant to Cassida vittata and Zinagri variety is susceptible to Cassida vittata. From biochemical and genetic studies some peroxidase and esterase enzymes markers were found in the resistant variety. Presence of bands number 3 for peroxidase and number 5 for esterase can be considered as a marker associated with plant defense to Cassida vittata. Also, the bands with Mw 127 and 17.5 KDa can be considered as negative markers associated with plant defense to Cassida vittata, the band with Mw 80 KDa can be considered as a positive marker associated with plant defense to Cassida vittata. A high level of DNA polymorphism was detected by ISSR and RAPD-PCR techniques for the two sugar beet varieties showing some positive and negative markers associated with plant tolerance to insect attack.