Peroxidase genes differentially respond to auxin during the formation of adventitious roots in soybean hypocotyl

In our investigation, auxins (IAA, IBA and NAA) effectively promoted rooting in soybean hypocotyls. The activity of anionic peroxidase (POX) (pI 3.7) and cationic POX (pI 8.5) was significantly suppressed by exogenous auxins on day 2 (the inductive phase). Some particular anionic POXs (pI 4.0 and pI 5.3) significantly increased in IBA-treated tissues as compared with the control when the incubation time was prolonged to day 3 and day 4 (the initiation phase). We sequenced 5′-flanking region of pI 8.5 and pI 5.3 POX genes using the PLACE and PlantCARE databases to identify several potential cis-regulatory elements. The pI 8.5 POX gene promoter contained two sites that were homologous to sequences commonly found in auxin response elements; motifs ARF/AuxRE and CATATGGMSAUR. During the inductive phase, the activity of pI 8.5 POX was significantly suppressed by the exogenously applied auxins. The pI 8.5 POX gene promoter contained both ARF/AuxRE and CATATGGMSAUR motifs that responded to auxins earlier than the pI 5.3 POX gene. Hence, the pI 8.5 POX gene might belong to primary auxin response genes. The pI 5.3 POX gene, which responded to auxins a day or two later, contained only ARF/AuxRE motif. Moreover, unlike pI 8.5 and pI 3.7 POXs that were suppressed by auxins, the pI 5.3 POX was induced or enhanced by the applied auxins, especially IBA. The pI 5.3 POX might generate H₂O₂ which caused the auxin-induced growth at the initiation phase during the formation of adventitious root in soybean hypocotyls.     

Characterisation of phenol oxidase and peroxidase from maize silk

Silk of some maize genotypes contains a high level of phenolics that undergo enzymatic oxidation to form quinones, which condense among themselves or with proteins to form brown pigments. Two phenolic oxidizing enzymes, peroxidase (POD; EC 1.11.1.7) and polyphenol oxidase (PPO; EC 1.10.3.1), from maize (Zea mays L.) silk were characterised with respect to their preferred substrate, different isoforms and specific effectors. One browning silk sample with high, and two non‐browning samples with low phenolic content were investigated. Although POD oxidizes a wide range of phenolic substrates in vitro, its activity rate was independent of silk phenolic content. PPO activity, detected with o‐diphenolic substrates, was abundant only in browning silk, and low or absent in non‐browning silk. Pollination increased POD but not PPO activity. Isoelectric‐focusing (IEF) and specific staining for POD and PPO showed a high degree of polymorphism that varied with silk origin. The IEF pattern of POD revealed a number of anionic and several cationic isoenzymes, with the most pronounced having neutral pI 7 and a basic isoform with pI 10. Detected isoforms of PPO were anionic, except for one neutral form found only in browning silk, and occupied positions different from those of POD. Different inhibitory effects of NaN₃, EDTA, KCN, and L‐cysteine, as well as different impacts of a variety of cations on the oxidation of chlorogenic acid, mediated by PPO or POD, were detected. The findings are discussed in terms of a possible roles of these enzymes in defence and pollination.     

Identification of glucose tolerant acid active β-glucosidases from thermophilic and thermotolerant fungi

Thirteen thermophilic and thermotolerant fungal cultures isolated from composting soils produced diverse β-glucosidases as indicated by zymograms of PAGE developed using 4-methylumbelliferyl-β-d-glucoside. IEF profiling revealed the presence of 28 β-glucosidases separated on the basis of their pI. Eleven of the β-glucosidases were active under acidic conditions. Two β-glucosidase isoforms, ASCβG-II of Aspergillus caespitosus and HIβG-I of Humicola insolens were resistant to inhibition by glucose and were active in the presence of 300 and 100 (mM) glucose, respectively.     

Gene pacA+ codes for the multiple active forms of Pi-repressible acid phosphatase in the mould Aspergillus nidulans

The acid phosphatase secreted by the biA1 strain of the mould Aspergillus nidulans was separated into at least nine isoforms by isoelectric focusing (IEF). The components visualized by activity were predominantly acidic proteins with isoelectric points ranging from pH 4.0 to 6.5. Almost the same isoforms were secreted by strains pabaA1 and palD8 biA1. Furthermore, the isoforms secreted by strain pacA1 biA1 were not visualized by staining after IEF, indicating that these isoforms are encoded by gene pacA. Treatment of the secreted enzyme with endoglycosidase H also reduced the number of isoforms visualized by staining after IEF and enhanced the R_f (electrophoretic mobility) value of this enzyme visualized after PAGE.     

Purification,characterization and catalytic activity of anionic zucchini peroxidase

The isolation and purification, by preparative electrofocusing, of the major anionic (ZPOA) and cationic (ZPOC) isoenzymes, collected from young zucchini squash, are reported. The M _r and sugar content are similar to those found previously for the major isoenzymes from the ripe fruits and in the range commonly observed for plant peroxidases. The amount of the two cationic enzymes was very low compared with that of anionic ZPOA. The anionic enzyme has been characterized by electronic, circular dichroism, proton NMR and electron paramagnetic resonance spectroscopy. The spectra are qualitatively similar to those of the corresponding anionic horseradish peroxidase (HRPA) derivatives, with minor differences attributable to the particular protein environment around the heme. The kinetics of the enzymatic oxidation of a series of phenols by H₂O₂ have been studied. ZPOA shows a parallel behavior to HRPA, but it is systematically more active than HRPA, indicating that the zucchini enzymes have a marked tendency to carry out oxidation of this type of compounds.     

菜心中高等电点高活性的乙醇酸氧化酶同工酶的纯化和特性

乙醇酸氧化酶 (EC 1 1 3 15 ,GO)被认为只含4 0kD一种碱性亚基 ,是因为从多种植物中获得了具GO活性的蛋白 ,SDS PAGE后呈约 4 0kD单带[1] .菠菜GOcDNA编码约 370个氨基酸 ,即 4 0kD多肽 ,其碱 酸性氨基酸的比例高达 0 96 ,富含碱性氨基酸[2 ] .已克隆的GOcDNA在E .coli中表达     

Peroxidase and IAA oxidase activities and peroxidase isoenzymes in the pericarp of seeded and seedless “Redhaven” peach fruit

Parthenocarpic peach fruit (Prunus persica L. Batsch., cv. Redhaven) were induced with 1-(3-chlorophthalimide)-cyclohexane carboxamide (AC 94377). The activities of soluble, and ionically and covalently bound peroxidase and indole-3-acetic acid (IAA) oxidase in the pericarp of both seeded and parthenocarpic fruit were determined from 21–43 days after anthesis. Seedless fruit grew faster during early stage I and ceased growth earlier than seeded fruit. Total peroxidase and IAA oxidase activities increased with development on both types of fruit, but higher values were found in seedless fruit. The ionic fraction showed the greatest increase for both enzyme activities. Isoperoxidase profile showed new cationic isoenzymes and higher levels of the less anionic isoenzymes in the pericarp of seedless fruit, whereas the seeded fruit contained higher levels of the more acidic isoperoxidases.     

Activity and isoenzyme composition of vacuolar peroxidase in the roots of red beet at different stages of development and upon changes in storage conditions

Activity and isoenzyme composition of phenol-dependent vacuolar peroxidase (PO) were examined at different stages of development of red beet (Beta vulgaris L.) roots. The enzyme activity was found to increase during growth and decrease during the period of root dormancy. The isoenzyme composition of PO was also altered; additional cationic and anionic isoforms were revealed at certain stages of growth and dormancy. The dormant roots are often subjected to stresses, such as water deficiency and pathogenesis. For this reason, the enzyme activity in beet roots showing obvious signs of shrivel and infection with pathogenic microorganisms was investigated. The activation of PO was observed in infected roots, whereas the increase in the number of cationic PO isoforms was noted in water-stressed roots. The pH optima of the enzyme were found to shift depending on the developmental stage and the nature of stress factors: during dormancy and under water stress the pH optima shifted towards the acidic values. The shift in pH optima occurred concurrently with the appearance of cationic inducible isoforms. The pH dependences of cationic PO isoforms (these POs were mainly associated with the tonoplast) showed that their activity was optimal at pH 4.0 and 5.0. The observed changes in activity and composition of isoenzymes suggest the active involvement of the vacuolar peroxidase in metabolic processes and cell defense responses in beet roots.     

Influence of light conditions on development, apoplastic peroxidase activities and peroxidase isoenzymes in chicory root explants

Cichorium intybus L. (cv. Bea) root explants grown in continuous light had a higher fresh weight, lignin and chlorophyll content than explants grown in darkness. Intermediary values were found when the light conditions were switched after 6 days. Peroxidase activities (EC 1.11.1.7) were measured in the apoplastic fluid with guaiacol, indoleacetic acid (IAA) and syringaldazine as substrates. There was an inverse relationship between specific IAA oxidase activity and explant growth. Specific syringaldazine oxidase activity (SSO) correlated with the lignin content. Analysis by isoelectric focusing (IEF) showed that the apoplastic fluid contained both cationic and anionic peroxidase isoforms, whose expression differed according to culture conditions. Isoform isoelectric point (pI) 7.0 was only detectable when the explants were cultured for 12 days in darkness. When explants were grown for the first 6 days in light, SSO and lignin content were high and the isoforms pI 4.0, 6.6, 7.6 and 8.1 were highly expressed. Conversely, when the first 6 days were in darkness, specific IAA oxidase activity was high and the isoforms pI 4.5, 6.7 and 6.8. were most strongly expressed. The isoperoxidases pI 7.8 and 7.9 were strongly expressed when the explants were cultured for at least 6 days in darkness.     

Effect of various synthetic dyes on the production of manganese-dependent peroxidase isoenzymes by immobilized <Emphasis Type="Italic">Irpex lacteus</Emphasis>

The effect of manganese and selected synthetic dyes on the production of manganese-dependent peroxidase (MnP) by Irpex lacteus immobilized on polyurethane foam was studied. In the cultures grown in a medium containing 65 μM Mn (II), up to three various isoenzymes of MnP were resolved by isolectrofocusing, with pI values within the range of 3.50–6.04. In the cultures grown in a medium containing 2.9 mM Mn (II), two new MnP isoforms (pI 3.28, 3.75) were produced. The addition of structurally different synthetic dyes, an azo dye Reactive Orange 16 (RO16), an anthraquinonic dye Remazol Brilliant Blue R (RBBR), and a triphenylmethane dye Bromophenol Blue (BPB), to the fungal cultures grown in the presence of high manganese inhibited the production of low pI MnP isoforms. However, in the presence of BPB a new MnP isoform with pI 5.67 was detected. BPB was found to induce MnP isoforms which are more effective in RBBR decolorization in vitro than the low pI isoforms present in the control cultures.     

The isomerization of glutamate dehydrogenase in response to lead toxicity in maize

Maize (Zea mays) was cultivated on lead-adultrated soil up to 600 mg(Pb) kg-1. At maturity, the maize seeds were harvested. The glutamate dehydrogenase (GDH) was fractionated to its isoenzyme population by Rotofor isoelectric focusing (IEF). The increasing Pb concentration progressively enhanced the more acidic isoenzymes (pI 6.3 - 6.5), and at the same time suppressed the less acidic isoenzymes (pI 7.3 - 7.8) and at the 600 mg(Pb) kg-1(soil) only the most acidic couple of isoenzymes (pI 6.3, and 6.5) were detectable. The NH4+ Km values of the GDH increased progressively from 6.2 in the control to 100 mM and the total glutathione content of maize seeds from 60 to 240 nmol g-1 in the 600 mg(Pb) kg-1(soil) treated maize. The orderly, and sequential isomerization of GDH in response to Pb suggests that the enzyme functions as a sensor in the monitoring of environmentally induced stress. This revised version was published online in August 2006 with corrections to the Cover Date.     

Multimodal analysis of metals in copper–zinc superoxide dismutase isoforms separated on electrophoresis gels

It has been suggested that copper–zinc superoxide dismutase (CuZnSOD) isoforms of distinct isoelectric point (pI) could result from differences in their metallation state. Our aim was then to develop and validate analytical methods for the determination and understanding of metallation states in human CuZnSOD isoforms. To avoid metal losses during sample preparation steps, CuZnSOD isoforms were separated according to their pI using non-denaturing isoelectric focusing (IEF) gel electrophoresis. Metal quantification was directly performed in-gel. Cu/Zn ratios of CuZnSOD isoforms were quantified by Particle-Induced X-ray Emission (PIXE) and Laser Ablation-Inductively Coupled Plasma-Mass Spectrometry (LA-ICP-MS). Cu/Zn ratios were measured close to the value of 1 as expected from the known stoichiometry of CuZnSOD with slight, but statistically significant, differences between acidic and basic isoforms. Overall, this study demonstrates that metal quantification can be performed directly on metalloproteins separated on electrophoresis gels.     

Resolution of a complex ionic mixture of an apparently homogenous protein preparation by preparative electrophoresis

Preparations of recombinant envelope glycoprotein E2 of hepatitis C virus (r-HCV E2), found to be homogeneous by N-terminal amino acid sequencing and mass spectrometry, resolved into multiple ionic species (isoforms) when analysed by isoelectric focusing (IEF) gel electrophoresis in the p1 range of 3-10. These isoforms possessed pI values in the range of 4.5-8.2. The major isoform with p1 value of approximately 7.1 was separated from the rest of them by employing a method developed on Gradiflow BF 200, a device based on preparative electrophoresis. This isoform was adjudged to be homogenous by IEF and by native polyacrylamide gel electrophoresis (PAGE).     

An examination of the peroxidases from Lupinus albus L. hypocotyls

Peroxidases (EC 1.11.1.7) from hypocotyls of Lupinus albus L. cv. Rio Maior have been characterised using one- and two-dimensional, native electrophoretic techniques. Data are presented showing the complexity in charge and molecular size or shape of these peroxidases. We report the finding of a new acidic peroxidase and several new basic peroxidases in these hypocotyls, and of their stability to treatments considered to break ligand-induced variants and conformational variants derived from differences in polypeptide folding. Densitometric data demonstrate that these new peroxidases contribute up to 60 of the total peroxidase activity in hypocotyls. Studies of intercellular fluid, cell-wall and soluble fractions, with assays of purity were conducted in an attempt to define the subcellular locations of these additional peroxidases. The acidic form (pI 4.1) is greatly enriched in soluble fractions, three of the basic peroxidases (pIs 9.5, 9.7 and >9.7) are strongly associated to the cell wall, ad a minor, basic component (pI 9.7) is enriched in the intercellular fluid. Individual peroxidase activities with the substrates coniferyl alcohol, ferulic acid or indole acetic acid were compared by densitometric analysis of zymograms with those for guaiacol, and notable differences between these peroxidases in their capacity to oxidise indole acetic acid in vitro were identified. The possible functions of these peroxidases in vivo and their implications to current understanding of peroxidases in L. albus are discussed.Abbreviations APAGE anionic polyacrylamide gel electrophoresis - CA coniferyl alcohol - CPAGE cationic polyacrylamide gel electrophoresis - IEF isoelectric focusin - NEIEF non-equilibrated isoelectric focusing - 2D two dimensional - pI isoelectric point - RCPAGE reversed current polyacrylamide gel electrophoresis     

Isoenzyme Multiplicity and Characterization of Recombinant Manganese Peroxidases from Ceriporiopsis subvermispora and Phanerochaete chrysosporium

We expressed cDNAs coding for manganese peroxidases (MnPs) from the basidiomycetes Ceriporiopsis subvermispora (MnP1) and Phanerochaete chrysosporium (H4) under control of the α-amylase promoter from Aspergillus oryzae in Aspergillus nidulans. The recombinant proteins (rMnP1 and rH4) were expressed at similar levels and had molecular masses, both before and after deglycosylation, that were the same as those described for the MnPs isolated from the corresponding parental strains. Isoelectric focusing (IEF) analysis of rH4 revealed several isoforms with pIs between 4.83 and 4.06, and one of these pIs coincided with the pI described for H4 isolated from P. chrysosporium (pI 4.6). IEF of rMnP1 resolved four isoenzymes with pIs between 3.45 and 3.15, and the pattern closely resembled the pattern observed with MnPs isolated from C. subvermispora grown in solid-state cultures. We compared the abilities of recombinant MnPs to use various substrates and found that rH4 could oxidize o-dianisidine and p-anisidine without externally added manganese, a property not previously reported for this MnP isoenzyme from P. chrysosporium.     

Isoenzyme multiplicity and characterization of recombinant manganese peroxidases from Ceriporiopsis subvermispora and Phanerochaete chrysosporium

We expressed cDNAs coding for manganese peroxidases (MnPs) from the basidiomycetes Ceriporiopsis subvermispora (MnP1) and Phanerochaete chrysosporium (H4) under control of the alpha-amylase promoter from Aspergillus oryzae in Aspergillus nidulans. The recombinant proteins (rMnP1 and rH4) were expressed at similar levels and had molecular masses, both before and after deglycosylation, that were the same as those described for the MnPs isolated from the corresponding parental strains. Isoelectric focusing (IEF) analysis of rH4 revealed several isoforms with pIs between 4.83 and 4.06, and one of these pIs coincided with the pI described for H4 isolated from P. chrysosporium (pI 4.6). IEF of rMnP1 resolved four isoenzymes with pIs between 3.45 and 3.15, and the pattern closely resembled the pattern observed with MnPs isolated from C. subvermispora grown in solid-state cultures. We compared the abilities of recombinant MnPs to use various substrates and found that rH4 could oxidize o-dianisidine and p-anisidine without externally added manganese, a property not previously reported for this MnP isoenzyme from P. chrysosporium.     

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.     

Bacteriolytic activities of the free-living soil amoebae,Acanthamoeba castellanii,Acanthamoeba polyphaga andHartmannella vermiformis

Bacteriolytic activities of axenically grown free-living soil amoebaeAcanthamoeba castellanii, Acanthamoeba polyphaga andHartmannella vermiformis towards various Gram-positive and Gram-negative bacteria were determined. A spectrophotometric assay revealed that the specific bacteriolytic activities of bothAcanthamoeba species were higher as those of the threeHartmannella strains.Bacillus megaterium, Bacillus subtilis, Chromatium vinosum, Micrococcus luteus andPseudomonas fluorescens were more easily lysed than the other bacteria tested.Agrobacterium tumefaciens, Klebsiella aerogenes andSerratia marcescens were hardly affected at all by the amoebal bacteriolytic activities. Among the Gram-negative bacteria we observed differences in lysis sensitivity while the Gram-positive bacteria tested were sensitive to lysis. Isoelectric focusing (IEF) gel-electrophoresis in the pH range 3–10 was performed to separate the bacteriolytic isoenzymes of amoebae. Bacteriolytic patterns were shown by using an activity assay in which lysis bands were formed in the agar/bacteria gel-overlay. The activity assay revealed remarkable differences in typical banding patterns for bacteriolytic activities among amoebae. Distinct differences between typical pI points of bacteriolytic activities inAcanthamoeba andHartmannella were shown. Bacteriolytic activities ofHartmannella were more pronounced and observed in the isoelectric points (pI) range of 4.0–9.3 while forAcanthamoeba the range was pI 4.5–8.9.Abbreviations IEF isoelectric focusing - PAA-IEF polyacrylamide-isoelectric focusing - CCAP culture collection of algae and protozoa - AS amoeba saline medium - pI isoelectric points     

Superoxide dismutase, peroxidase, and germin-like protein activity in plasma membranes and apoplast of maize roots

Summary. The analysis of plasma membranes from maize roots by native gel electrophoresis revealed the existence of Mn-containing 120 kDa and CuZn-containing 70, 40, and 15 kDa superoxide dismutase (SOD) isoform activities. Isoelectric focusing of the plasma membranes differentiated anionic SOD isoforms with a pI of about 5 and cationic SOD isoforms at pI 8.6. Solubilization of the plasma membrane proteins further separated the cationic SOD into pI 8.6, 8.2, 8.4, and 7.2 isoforms. Double staining for both SOD and peroxidase activities showed an overlap of these activities only in the case of the high-molecular-mass (ca. 120 kDa) isoforms. High-temperature treatments demonstrated that the 120 kDa isoform was active even at 100 °C, indicating that it was a germin-like protein with superoxide-dismutating activity, different from the peroxidase with a similar molecular mass and the lower-molecular-mass CuZn-containing superoxide dismutases. These results are compared to those obtained from whole-tissue extract and apoplastic fluid. Correspondence and reprints: Maize Research Institute, POB 89-Zemun, 11081 Belgrade, Serbia and Montenegro.