Peroxidase Isoforms Research Articles

Peroxidase gene-based estimation of genetic relationships and population structure among Citrus spp. and their relatives

Many stress-related interactions such as pathogen infection, insect tolerance, salt tolerance, auxin degradation, cell wall lignification, tissue suberization, and plant senescence involve various isoforms of peroxidases. Peroxidase gene polymorphism (POGP) markers have been used to estimate diversity, relationships and population structure among 80 Citrus and their relatives in Aurantioideae by using unweighted pair group method arithmetic average (UPGMA) and bayesian substructuring analysis. Fourteen primers produced a total of 148 fragments and 147 of them were polymorphic. The UPGMA analysis demonstrated that the accessions had a similarity range from 0.27 to 0.98 and were distinguished. The results of this study were mostly consistent with previous reports of different marker systems, but few different findings were also detected. The subtribe Clauseninae (tribe Clauseneae) did not clearly separate from the subtribes of the tribe Citreae. Substructuring analysis indicated that there were six subpopulations among the accessions studied. This study revealed that the POGP markers can be utilized to estimate genetic diversity, relationships and population structure in Citrus and related species in the Aurantioideae subfamily.

Production of Ligninolytic Enzymes by Cultures of White Rot Fungi

Some Basidiomycota were chosen for studies of key ligninases synthesis (25°C, 30 days) in modified medium (shaken or not cultures) with added wheat straw. Liquid Czapek medium with straw yielded a higher amount of laccase than peroxidase, ground straw induced enzyme worse than chopped straw. With peroxidase the reverse dependencies were observed. Laccase of Lentinus edodes synthesized two enzyme isoforms (ca 30 and 16 kDa). In T. versicolor culture active laccase protein with highest molecular mass ca 65 kDa was found. P. sajor-caju yielded three different peroxidase isoforms. Ligninase biosynthesis depended on strain, straw fragmentation extent, culture method and growth medium.

Biochemical and molecular evidence for the role of class III peroxidases in the resistance of carnation (Dianthus caryophyllus L) to Fusarium oxysporum f. sp. dianthi

Rooted cuttings of two cultivars of carnation (Dianthus caryophyllus L) with differential responses to vascular wilt caused by Fusarium oxysporum f. sp. dianthi (FOD) were inoculated with the pathogen and some parameters associated to guaiacol peroxidase activity (GPX) (E.C. 1.11.1.7) were evaluated. The resistant cultivar (L.P. Candy) showed a significant increase in GPX activity levels at 12, 24, and 48 h post-inoculation (hpi), whereas the susceptible cultivar (Tasman) exhibited only a slight increase at a longer time (96 hpi). The H2O2 content in the resistant cultivar was increased at 12 and 24 hpi, with corresponding increase in GPX activity level (α = 0.05). However, the susceptible cultivar showed an increase in H2O2 content at 12 hpi that did not correlate with GPX activity. Using zymograms, the increased GPX activity in the resistant cultivar was determined to be due to increased levels of constitutive isoforms of peroxidase. Quantification of transcriptional levels indicated that in the resistant cultivar, the mRNA level of the class III peroxidase Dcprx02 increased almost 8 fold compared with the control at 6 hpi, whereas the mRNA level in the susceptible cultivar only increased approximately 2 fold at 12 hpi. The increased levels of H2O2 correlated with the increased levels of Dcprx02 transcription. Differences in the temporal regulation of the variables assessed during infection support the idea that the generation of reactive oxygen species (ROS) and subsequent regulation mediated by the action of antioxidant enzymes such as GPX are relevant to plant resistance to the vascular wilt causal agent.

Tyrosinase and superoxide dismutase activities of peroxidase in the vacuoles of beet roots

In the absence of H2O2, isoforms of vacuolar phenol-dependent peroxidase (PO) in beet (Beta vulgaris) roots oxidized phenolic compounds like tyrosinases. Tyrosinase activity of PO manifested a clearly expressed pH-dependence with the optimum at pH of 8.0–9.0; peroxidase activity was the highest at pH 5.0–7.0. The inhibitory analysis confirmed a specificity of observed reactions. Along with tyrosinase activity, PO manifested SOD-like activity, which was also expressed in the absence of H2O2 and depended on some factors, for example, on the way of analyzed sample preparation. This activity appeared at a long-term dialysis and low temperature. SOD-like PO activity was observed in the presence of such substrates as 3,3′-diaminobenzidine and IAA. The results obtained allow a conclusion that vacuolar PO, as well as PO of other localization and origin is a polyfunctional enzyme, which, under definite conditions, can catalyze reactions of oxidase type.

Dose-Dependent Reactive Species Accumulation and Preferential Double-Strand Breaks Repair are Featured in the γ-ray Response in Medicago truncatula Cells

In plants, there are issues related to the effects of gamma (γ)-rays that are still poorly explored, particularly as concerns the biological response to irradiation delivered at a low dose rate. In the present work, the effects of exposure to increasing γ-ray total doses (6, 12, 25, and 50 Gy) delivered at 0.29 Gy min−1, were evaluated in terms of DNA damage sensing and repair in Medicago truncatula proliferating cell suspension cultures. The profiles of reactive oxygen species (ROS) and nitric oxide (NO) production, monitored by fluorescent staining, were in agreement with the transcriptional response of genes encoding the respiratory burst oxidase-like protein C (MtRBOHC), nitrite reductase (MtNR), the cytosolic isoform of ascorbate peroxidase (MtAPX), and a type 2 metallothionein (MtMT2). The genotoxic effects were assessed using the alkaline and neutral version of single cell gel electrophoresis (SCGE), detecting the occurrence of SSBs and DSBs. Independent on the irradiation dose, M. truncatula cells always revealed the preferential repair of DSBs, while the SSB repair was less effective. The DNA repair response was investigated focusing on genes representative of different DNA repair pathways. The overall picture derived from gene profiling analysis highlights that M. truncatula cells can produce an active response to IR-mediated genotoxic stress, as indicated by the up-regulation of the DSB sensor MtMRE11 gene, the MtTop2 gene, and the MtOGG1gene, an essential component of the base excision repair pathway. In contrast, the MtTop1, MtTdp1 and MtTFIIS genes, believed to be part of the nucleotide excision repair pathway, were significantly down-regulated.

Estimation of peroxidase activity in red cabbage by artificial neural network

Enzymes in plant tissues can have undesirable or desirable effects on the quality of fruits and vegetables such as the post-harvest senescence, oxidation of phenolic substances, starch-sugar conversion and post-harvest demethylation of pectic substances leading to softening of plant tissues during ripening. Peroxidase (POD) is a commonly enzyme in vegetable, with bad effects on quality of their products. Structure of POD and POD isoform differ in each vegetable. Hence, activity of POD is unique to each vegetable. The aim of this study was evaluation efficiency of different essential oils as natural antioxidant in POD inactivation and estimation of POD activity by artificial neural network (ANN) modeling. In this study we used natural antioxidant (cumin, fennel, clove) in red cabbage. An ANN was developed by using a multilayer perceptron model, three input neurons (type and concentration of antioxidant and duration of enzyme activity), one hidden layer and 21 hidden neurons. The ANN model predicted POD activity with a mean square error of 0.0002629 and a good correlation between predicted and experimental data (R2=0.9974). These results show the ability of ANN technology for predicting POD activity of red cabbage under natural antioxidants.

Accumulation and scavenging of reactive oxygen species and nitric oxide correlate with stigma maturation and pollen–stigma interaction in sunflower

During the course of stigma development in sunflower (bud, staminate and pistillate stages), correlation is evident among the accumulation of reactive oxygen species (ROS), nitric oxide (NO), and the activities of ROS scavenging enzymes [superoxide dismutase (SOD) and peroxidase (POD)]. Confocal image analysis shows a gradual increase in ROS and NO accumulation in the stigmatic papillae, which may provide immunity to the developing stigma and function as signalling molecules. A novel, NO-specific probe (MNIP-Cu) has been employed for the detection and quantification of intracellular NO. Mn-SOD (mitochondrial) and Cu/Zn-SOD (cytoplasmic) exhibit differential expression during the staminate stage of stigma development. An increase in total SOD activity at the staminate stage is followed by a peak of POD activity during pistillate stage, thereby indicating the sequential action of the two enzymes in scavenging ROS. An increase in the number of POD isoforms is observed with the passage of stigma development (from three at bud stage to seven at pistillate stage), and two POD isoforms are unique to pistillate stage, thereby highlighting their role in ROS scavenging mechanism. ROS and NO accumulation exhibit reverse trends during pollen–stigma interaction.

Biochemical characterisation of grain mould resistant and susceptible genotypes and PGPR-induced resistance in the host to Curvularia lunata and Fusarium proliferatum

Resistance to biotic stresses in plants is either due to the presence of preformed biochemical compounds or induced in response to external stimulus. In this study, 13 grain mould resistant and seven susceptible lines of sorghum were analysed for biochemical defence mechanism. The levels of total phenols and phenylalanine ammonia lyase were almost same in the resistant and susceptible genotypes. However, two additional isoforms of peroxidase were found in the three of the 13 resistant genotypes. The isoform peroxidase corresponding to the R f value of 0.25 was found in the resistant genotypes IS 13969, ICSB 377 and IS 8219-1, and two genotypes IS 13969 and ICSB 377 had an additional isoform corresponding to the R f value of 0.32. The results indicated the genotype specific association of peroxidases with grain mould resistance in sorghum. Nine bacterial strains (Bacillus pumilus SB 21, Bacillus megaterium HiB 9, Bacillus subtilis BCB 19, Pseudomonas plecoglossicida SRI 156, Brevibacterium antiquum SRI 158, B. pumilus INR 7, P. fluorescens UOM SAR 80, P. fluorescens UOM SAR 14, B. pumilus SE 34) were tested to induce systemic resistance in sorghum cultivars 296B and Bulk Y against the highly pathogenic grain mould pathogens Curvularia lunata and Fusarium proliferatum, respectively. The bacterial isolates were effective in inducing resistance in sorghum. Among the strains tested, SRI 158 was found highly effective in reducing grain mould severity in both the genotypes.

Abstract 1876: Oxidative metabolism by human peritoneal cancer cells stimulated with death ligands.

Abstract Anatomic environments harboring cancer are known to influence tumor sensitivity to most forms of therapy. In general, the effects are negative with respect to clinical response. Thus, manipulation of tumor environments to favor treatment outcome is justified. The human peritoneal cavity harbors multiple types of primary and metastatic cancer whose presence elicits a substantial inflammatory response with consequent diverse and conflicting effects on tumor growth and treatment response. The present study used peritoneal tumors (PT) from patients to investigate expression of oxidative metabolism (OM) genes relevant to inflammation within the peritoneal cavity. Briefly, PT were used to establish cell lines which were stimulated with pathophysiologic concentrations of the inflammatory death ligands, rTNF and rTRAIL followed by gene expression analysis with real time pcr. Lines from a metastatic lesion on the stomach of a colorectal cancer patient (CR) as well as tissue from a patient with extensive peritoneal carcinomatosis (PC) were used. Stimulation (expressed as fold increase above media controls) of the PC line with rTNF elicited significantly increased expression of multiple OM gene families and multiple isoforms within each family including the gluathione peroxidase (isoforms 1-4, range = 2.1-4.8 fold); peroxiredoxin (isoforms 1-6, range = 2.6-5.1 fold); superoxide dismutase (isoforms 1-3, range = 2.4-2.6 fold); and, arachidonate metabolism (Cox1, Cox2 and 12-Lipox, range =2.1-16 fold) families. Significant increases were also seen for ApoE (3.8 fold); gluathione synthetase, transferase, and reductase (3.1, 2.6, and 7.1 fold respectively); and scavenger receptor A (4.1 fold). The results with rTRAIL also showed significant increases in expression of selected isoforms of the same OM families, but were of lesser magnitude and involved only 1 or 2 of the isoforms from each family. OM gene expression in the CR line was not significantly increased in most gene families by inflammatory death ligands, with statistically significant increases of <3 fold observed for glutathione peroxidases 5, 6 and 7; lactoperoxidase; myeloperoxidase; and Cox1. Incubation of the CR and PC line with rTNF and rTRAIL produced only modest inhibition of proliferation in an MTS assay. These results demonstrate that innflammatory death ligands shown to be increased in the peritoneal cavity of cancer patients can elicit increased expression of OM genes in primary PT cell lines. The types of genes stimulated suggest that PT cells can create a highly anti-oxidant local microenvironment within an inflammed peritoneal cavity. This effect may substantially inhibit the tumoricidal effects of chemotherapy, radiation, and antitumor immunity while promoting tumor angiogenesis and progression. Tumor-targeted inhibition of OM may improve clinical treatment outcomes in peritoneal cancers. Citation Format: Donald P. Braun, Derek M. Johnson, Adriana Rosales, C. Komen Brown. Oxidative metabolism by human peritoneal cancer cells stimulated with death ligands. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1876. doi:10.1158/1538-7445.AM2013-1876

Gamma irradiation with different dose rates induces different DNA damage responses in Petunia x hybrida cells

In plants, there is evidence that different dose rate exposures to gamma (γ) rays can cause different biological effects. The dynamics of DNA damage accumulation and molecular mechanisms that regulate recovery from radiation injury as a function of dose rate are poorly explored. To highlight dose-rate dependent differences in DNA damage, single cell gel electrophoresis was carried out on regenerating Petunia x hybrida leaf discs exposed to LDR (total dose 50Gy, delivered at 0.33Gymin−1) and HDR (total doses 50 and 100Gy, delivered at 5.15Gymin−1) γ-ray in the 0–24h time period after treatments. Significant fluctuations of double strand breaks and different repair capacities were observed between treatments in the 0–4h time period following irradiation. Dose-rate-dependent changes in the expression of the PhMT2 and PhAPX genes encoding a type 2 metallothionein and the cytosolic isoform of ascorbate peroxidase, respectively, were detected by Quantitative RealTime-Polymerase Chain Reaction. The PhMT2 and PhAPX genes were significantly up-regulated (3.0- and 0.7-fold) in response to HDR. The results are discussed in light of the potential practical applications of LDR-based treatments in mutation breeding.

Ultrastructural and antioxidative changes in lupine embryo axes in response to salt stress

Embryo axes of lupine (<em>Lupinus luteus</em> L. ‘Mister’) were subjected to 0.1 M NaCl salt stress for 24 and 48 h. The ultrastructure modification and adjustment of antioxidant enzymes activities and izoenzymes profiles were observed. In cells of lupine embryo axes grown for 48 hours in medium with 0.1 M NaCl mitochondria took the forked shape and bulges of the outer mitochondrial membranes appeared. Moreover, the inflating and swelling of rough endoplasmic reticulum (RER) lumen and fragmentation of RER were noticed. The level of H<sub>2</sub>O<sub>2</sub> was higher in salt treated embryo axes after 24 hours and increase of thiobarbituric acid reactive substances was observed after both 24 and 48 h of salt treatment. Native gel electrophoresis showed increased intensities of bands for catalase isozymes in response to salt stress, whereas activity of catalase was higher only in embryo axes grown for 48 h in control conditions. Appearance of two new isoforms of ascorbate peroxidase was observed after 48 h only under control condition, however increased activities were stated for both control and salt-stress condition after 48 h. No changes in isozymes pattern for superoxide dismutase were observed, but significant decrease in superoxide dismutase activity was noticed in relation to time and salt stress. Possible role of these enzymes in salt stress tolerance is discussed. The 0.1 M salt stress is regarded as a middle stress for lupine embryo axes and the efficiency of stress prevention mechanisms is proposed.

Thiol-based redox signaling in the nitrogen-fixing symbiosis

In nitrogen poor soils legumes establish a symbiotic interaction with rhizobia that results in the formation of root nodules. These are unique plant organs where bacteria differentiate into bacteroids, which express the nitrogenase enzyme complex that reduces atmospheric N 2 to ammonia. Nodule metabolism requires a tight control of the concentrations of reactive oxygen and nitrogen species (RONS) so that they can perform useful signaling roles while avoiding nitro-oxidative damage. In nodules a thiol-dependent regulatory network that senses, transmits and responds to redox changes is starting to be elucidated. A combination of enzymatic, immunological, pharmacological and molecular analyses has allowed us to conclude that glutathione and its legume-specific homolog, homoglutathione, are abundant in meristematic and infected cells, that their spatio-temporally distribution is correlated with the corresponding (homo)glutathione synthetase activities, and that they are crucial for nodule development and function. Glutathione is at high concentrations in the bacteroids and at moderate amounts in the mitochondria, cytosol and nuclei. Less information is available on other components of the network. The expression of multiple isoforms of glutathione peroxidases, peroxiredoxins, thioredoxins, glutaredoxins and NADPH-thioredoxin reductases has been detected in nodule cells using antibodies and proteomics. Peroxiredoxins and thioredoxins are essential to regulate and in some cases to detoxify RONS in nodules. Further research is necessary to clarify the regulation of the expression and activity of thiol redox-active proteins in response to abiotic, biotic and developmental cues, their interactions with downstream targets by disulfide-exchange reactions, and their participation in signaling cascades. The availability of mutants and transgenic lines will be crucial to facilitate systematic investigations into the function of the various proteins in the legume-rhizobial symbiosis.

Tyrosinase activity of peroxidase of red beet root vacuoles and plastids

[1–4]. The greatest activity and diversity of peroxidase isoforms are characteristic for vacuoles andcell walls. Polyphenol oxidase (tyrosinase) is a coppercontaining enzyme that uses molecular oxygen to catalyze phenol oxidation, usually accompanied by thegeneration of , which dismutates to form Н

Ascorbate as seen through plant evolution: the rise of a successful molecule?

Ascorbate is a widespread and efficient antioxidant that has multiple functions in plants, traditionally associated with the reactions of photosynthesis. This review aims to look at ascorbate from an evolutionary perspective. Cyanobacteria, algae, and bryophytes contain lower concentrations of ascorbate than higher plants, where the molecule accumulates in high concentrations in both photosynthetic and non-photosynthetic organs and tissues. This increase in ascorbate concentration is paralleled by an increase in the number of isoforms of ascorbate peroxidase and the ascorbate regenerating enzymes mono- and dehydroascorbate reductase. One way of understanding the rise in ascorbate concentrations is to consider ascorbate as a molecule among others that has been subject to selection pressures during evolution, due to its cost or benefit for the cell and the organism. Ascorbate has a low cost in terms of synthesis and toxicity, and its benefits include protection of the glutathione pool and proper functioning of a range of enzymes. The hypothesis presented here is that these features would have favoured increasing roles for the molecule in the development and growth of multicellular organisms. This review then focuses on this diversity of roles for ascorbate in both photosynthetic and non-photosynthetic tissues of higher plants, including fruits and seeds, as well as further functions the molecule may possess by looking at other species. The review also highlights one of the trade-offs of domestication, which has often reduced or diluted ascorbate content in the quest for increased fruit growth and yield, with unknown consequences for the corresponding functional diversity, particularly in terms of stress resistance and adaptive responses to the environment.

Water extraction under terminal drought explains the genotypic differences in yield, not the anti-oxidant changes in leaves of pearl millet (Pennisetum glaucum).

Pearl millet (Pennisetum glaucum (L.) R.Br.) is a resilient crop suiting the harshest conditions of the semi-arid tropics, in which we assessed possible relationships between crop tolerance, anti-oxidative enzyme activity and plant/soil water status. Biochemical acclimation and cell homeostasis traits have been proposed as critical for the drought tolerance of crops, but their limited practical application in breeding so far suggests that the role of biochemical acclimation for drought tolerance is still unclear. Previous research may have been of limited value because it has not approached biochemical acclimation from the angle of plant water relations. Four pearl millet genotypes, contrasting for terminal drought tolerance, were evaluated (sensitive H77/833-2, tolerant PRLT2/89-33 and two near isogenic lines carrying a terminal drought tolerance quantitative trait locus) under water-stress (WS) and well-watered (WW) conditions in a lysimetric system that simulates field-like conditions. We assessed the genotypic variation and relationship between photosynthetic pigments (chlorophylls a and b and carotenoids), antioxidative isoenzymatic spectrum (superoxide dismutase, ascorbate peroxidase and catalase), physiological traits (soil moisture available, normalised transpiration, stay-green score and water extraction) and biomass and yield. Biochemical traits investigated were tightly related among each other under WS conditions but not under WW conditions. Two major ascorbate peroxidase isoforms (APX6&7), whose variation in both water regimes reflected the presence/absence of the drought tolerance quantitative trait locus, were identified, but these did not relate to yield. Both, yield and biochemical traits under terminal drought stress were closely related to the traits linked to plant/soil water status (soil moisture available, normalised transpiration, stay-green score and water extraction), whereas yield and the biochemical indicators were not correlated, except for one. It is concluded that there is no direct effect of biochemical traits on yield parameters since both are consequences of soil-plant water status and their putative relation appear to be secondary - through plant/soil water status.

Cloning, expression and functional validation of drought inducible ascorbate peroxidase (Ec-apx1) from Eleusine coracana

Eleusine coracana (finger millet) is a stress-hardy but under-utilized cereal crop that possesses an efficient antioxidant defense system. The plant is capable of enduring long durations of water deficit stress. Experiments were conducted to clone a potent stress responsive isoform of ascorbate peroxidase and validate its role under drought stress. Reverse transcriptase PCR was used to obtain the partial cDNA of apx1 gene, from a meticulously screened drought tolerant genotype of E. coracana (PR202). Using RACE strategy, the full length apx1 cDNA was cloned and sequenced. The cDNA length of the E. coracana apx1 (Ec-apx1) gene is 1,047bp with a 750bp ORF, encoding a 250 amino acid protein having a molecular weight of 28.5kDa. The identity of the amino acid sequence, deduced from the cDNA, with the APX family homologs was about 74-97%. The full-length apx1 ORF was sub-cloned in a prokaryotic expression vector pET23b. The recombinant fusion protein, Ec-apx1, had high expression level in BL21 strain of E. coli and exhibited APX enzyme activity. The structure-function relationship of the protein was deduced by modelling a three-dimensional structure of Ec-apx1, on the basis of comparative homology using SWISS-MODEL. Real time PCR analysis of Ec-apx1 expression at mRNA level showed that the transcript increased under drought stress, with maximum levels attained 5-days after imposition of stress. Our results suggest that Ec-apx1 has a distinct pattern of expression and plays a pivotal role in drought stress tolerance. Therefore, the cloned isoform of ascorbate peroxidase can be used for developing stress tolerant genotypes of important crops, through transgenic approach.

Characterisation of an acidic peroxidase from papaya (Carica papaya L. cv Tainung No. 2) latex and its application in the determination of micromolar hydrogen peroxide in milk

An acidic peroxidase isoform, POD-A, with a molecular mass of 69.4kDa and an isoelectric point of 3.5 was purified from papaya latex. Using o-phenylenediamine (OPD) as a hydrogen donor (citrate–phosphate as pH buffer), the optimum pH for the function of POD-A was 4.6, and the optimum temperature was 50°C. The peroxidase activity of POD-A toward hydrogen donors was both pH- and concentration-dependent. Under optimal conditions, POD-A catalysed the oxidation of OPD at higher rates than pyrogallol, catechol, quercetin and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). The chemical modification reagents N-bromosuccinimide and sodium azide significantly inhibited POD-A activity. The results of kinetic studies indicated that POD-A followed a ping-pong mechanism and had a Km value of 2.8mM for OPD. Using CPC silica-immobilised POD-A for the determination of micromolar H2O2 in milk, the lower limit of determination was 0.1μM, and the recoveries of added H2O2 were 96–109%.

Cell wall-bound cationic and anionic class III isoperoxidases of pea root: biochemical characterization and function in root growth.

Cell wall isolated from pea roots was used to separate and characterize two fractions possessing class III peroxidase activity: (i) ionically bound proteins and (ii) covalently bound proteins. Modified SDS–PAGE separated peroxidase isoforms by their apparent molecular weights: four bands of 56, 46, 44, and 41kDa were found in the ionically bound fraction (iPOD) and one band (70kDa) was resolved after treatment of the cell wall with cellulase and pectinase (cPOD). Isoelectric focusing (IEF) patterns for iPODs and cPODs were significantly different: five iPODs with highly cationic pI (9.5–9.2) were detected, whereas the nine cPODs were anionic with pI values between pH 3.7 and 5. iPODs and cPODs showed rather specific substrate affinity and different sensitivity to inhibitors, heat, and deglycosylation treatments. Peroxidase and oxidase activities and their IEF patterns for both fractions were determined in different zones along the root and in roots of different ages. New iPODs with pI 9.34 and 9.5 were induced with root growth, while the activity of cPODs was more related to the formation of the cell wall in non-elongating tissue. Treatment with auxin that inhibits root growth led to suppression of iPOD and induction of cPOD. A similar effect was obtained with the widely used elicitor, chitosan, which also induced cPODs with pI 5.3 and 5.7, which may be specifically related to pathogen defence. The differences reported here between biochemical properties of cPOD and iPOD and their differential induction during development and under specific treatments implicate that they are involved in specific and different physiological processes. Abbreviations:cPODcovalently bound peroxidaseDAB3,3'-diaminobenzidineDEPMPOspin-trap (5-diethoxy-phosphoryl-5-methyl-1-pyrroline-n-oxide)EPRelectron paramagnetic resonanceHRPhorseradish peroxidaseIAAindole-3-acetic acidHRPhorseradish peroxidaseIEFisoelectric focusingiPODionically bound peroxidaseNAAnaphthalene acetic acidPNGase Fpeptide N-glycosidase FPRpathogen-relatedSDS–PAGEsodium dodecyl sulphate–polyacrylamide gel electrophoresisSHAMsalicylhydroxamic acidTMBtetramethyl benzidineWGAwheat germ agglutinin

Effect of cadmium stress on antioxidative enzymes during the germination of Serbian spruce ([Picea omorika (Panč.) Purkynĕe)

When considering the effect of heavy metals on trees generally and on forest ecosystems especially, importance is given to their influence on seed germination in metal polluted soil. There is insufficient data on this subject, especially when conifers are concerned. In this work, the influence of high cadmium concentrations on percentage germination, specific activities and isoenzyme patterns of catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD) during the germination of Serbian spruce [Picea omorika (Pan..) Purkynĕ] was studied. Cadmium chloride concentrations of up to 0.1 mM did not cause an inhibition of germination, while 1 mM concentration inhibited germination and the activities of catalase, superoxide dismutase and peroxidase. The isoenzyme profile of catalase and superoxide dismutase did not change at high cadmium concentrations, while peroxidase expression of basic peroxidase (B5) with pI value of 9.1 increased. This isoform of POD can play an important role in the early development of Serbian spruce and its defense mechanism against heavy metals. Key words : Seed germination, catalase, peroxidase, superoxide dismutase, heavy metals.

Effect of sulfur dioxide on ROS production, gene expression and antioxidant enzyme activity in Arabidopsis plants

Sulfur dioxide (SO(2)) is one of the most common and harmful air pollutants. To analyze antioxidant response of plants to SO(2) stress, we investigated the reactive oxygen species (ROS) levels, transcript alterations and antioxidant enzyme activities in Arabidopsis thaliana (Col-0) exposed to 0, 2.5, 10 and 30mgm(-3) of SO(2). The results showed that both superoxide radical (O(2)(-⋅)) generation rate and hydrogen peroxide (H(2)O(2)) content increased in SO(2)-treated Arabidopsis shoots. GeneChip and RT-PCR analysis revealed that transcript levels of peroxidase (POD), glutathione peroxidase (GPX) and superoxide dismutase (SOD) genes enhanced after exposure to 30mgm(-3) SO(2) for 72h. The content of glutathione and activities of SOD, POD and GPX increased significantly during 72h of SO(2) exposure. However, catalases (CAT) activity changed slightly under SO(2) stress. Furthermore, the results of in-gel enzyme assays indicated that SOD (FeSOD and Cu/ZnSOD) and POD isoforms increased after exposure to SO(2) for 72h, whereas two CAT isoforms (CAT2 and CAT3) declined. Malondialdehyde content kept at a low level within 72h of SO(2) exposure, but increased significantly after exposure to 30mgm(-3) SO(2) for 120h along with decrease in the level of ROS and activities of SOD and GPX. Our results indicated that increased ROS may act as a signal to induce defense response to SO(2) stress. Antioxidant status plays an important role in plant protection against SO(2)-caused oxidative stress, though the defense capacity cannot sufficiently alleviate oxidative damage occurring under prolonged exposure to higher concentrations of SO(2).