Peanut Cells Research Articles

Elicitor-specific induction of one member of the chitinase gene family in Arachis hypogaea

Chitinases are believed to play an important role in plant defence against bacterial and fungal attack. In peanut (Arachis hypogaea) chitinase genes form a small multigene family. Four chitinase cDNAs (chit 1-4) were isolated from cultured peanut cells. Expression of individual chit genes was assayed by the polymerase chain reaction (PCR) followed by analysis of restriction fragment length polymorphisms (RFLP). UV irradiation, dilution of cell cultures and treatment with Phytophthora megasperma (Pmg) elicitor or yeast extract were used to induce expression of chit genes. The chit 3 gene is constitutively expressed at a low level in untreated as well as in treated cultures; the expression of chit 4 gene is induced by each of the stimuli tested, whereas the chit 1 gene is activated by cell culture dilution and by yeast extract treatment. The chit 2 gene is strongly activated by treatment with cell wall components from the fungus Phytophthora megasperma but not by the other stimuli. These results indicate that chit 2 gene expression may be controlled by pathogen-specific regulatory elements.

Molecular cloning of complementary DNAs encoding two cationic peroxidases from cultivated peanut cells.

We have isolated, cloned, and characterized two cDNAs corresponding to the mRNAs for cationic peroxidases synthesized by cultured peanut cells. The first clone was obtained from a phage lambda gt11 library screened with antibodies directed against the major secreted isozyme. Its predicted amino acid sequence, deduced from the 1228-base-pair (bp) cDNA, revealed a 22-amino acid signal peptide and a 294-amino acid mature protein (Mr, 31,228). The second clone was isolated from a lambda gt10 library screened with oligonucleotides corresponding to the regions for acid/base catalysis and the fifth ligand of heme. This cDNA (1344 bp) encodes a protein (330 amino acids) with a mature peptide of 307 residues (Mr, 32,954). The two peanut peroxidases are 46% homologous. The estimated gene copy numbers of these peroxidases might be close to 1 or 2 per haploid genome. A comparison of the amino acid sequence of these peanut peroxidases with other known isozymes shows two already known regions of homology (the region for acid/base catalysis and the fifth ligand of heme). Moreover, some new characteristics appeared such as a glycosylation site identical in five of the seven isozymes, a putative antigenic determinant common to all the isozymes, and a region of the highest homology. A secondary structure prediction showed that it corresponds to a 16-amino acid helix linked to the next one by a long stretch of beta strands and coils and might represent a critical structural element.

Comparison of anionic with cationic peroxidase from cultured peanut cells

A cationic and an anionic peanut peroxidase were isolated to purity as shown by 2D electrophoresis. Amino acid analysis offered evidence for differences. Variations between the isozymes were also noted in a slight difference in the heme absorption maxima, specific enzyme activity and particularly in the relative amount of each in the suspension medium measured by the heme absorption. In contrast the two isozymes were at least partially similar in their structure as demonstrated by the crossreaction with the antisera. The percent crossreactions were used in turn to amend the calculation for the synthetic rate of each isozyme. In spite of the difference in amount secreted in the suspension medium, the in vivo biosynthetic rate of the two isozyme measured cellularly is much the same.

Immunochemical relatedness of two peroxidase isozymes from peanut cell culture

The immunological relationships between a cationic and an anionic peroxidase from peanut cell suspension culture were probed with polyclonal antibodies raised against each and with a monoclonal antibody raised against the cationic isozyme. Partial identity of the two isozymes was established by cross-reactivities of the polyclonal antibodies directed to one peroxidase with the alternate isozyme. Some peptides from Western blots of the partially hydrolysed peroxidases were recognized by both antisera, even though the peptide maps of the two isozymes differed. In contrast, the monoclonal antibody reacted more specifically to the fragments derived from the cationic isozyme. The anti-anionic peroxidase serum was passed over a cationic peroxidase (C.PRX) – Sepharose affinity column to separate antibodies cross-reacting with C.PRX from those specific to the anionic isozyme. Deglycosylation of the cationic and the anionic peroxidases eliminated binding of the antibody fraction eluted from the C.PRX–Sepharose column to the deglycosylated forms of peroxidases. These data suggest (i) that complex structural relationships exist between the cationic and the anionic peanut peroxidases, (ii) that the carbohydrate moieties of the two isozymes are antigenically similar to each other, and (iii) that epitope mapping will help in characterizing further each isoperoxidase.Key words: peroxidase, isozymes, ELISA, immunoblotting, peptide mapping.

Propachlor metabolism in soybean plants, excised soybean tissues, and soil

Propachlor was rapidly metabolized to a homoglutathione conjugate in the roots and foliage of soybean plants. No other competing reactions were observed. The homoglutathione conjugate was rapidly metabolized to the cysteine conjugate which was slowly converted to a variety of other metabolites, four of which were present up to 72 days after treatment. Those four metabolites were the malonylcysteine, malonylcysteine S-oxide, 3-sulfinyllactic acid, and the O-malonylglucoside conjugates of propachlor. A cysteine S-oxide conjugate was also observed as a transient metabolite. Fast atom bombardment mass spectrometry was used to characterize the polar metabolites. Some metabolites were isolated and identified from both soybean plants and peanut cell suspension cultures. The terminal metabolites in soybean plants were concentrated in the roots and foliage. Less than 1% of the metabolites were present in the beans and pods of the mature plants. In a sandy loam soil, propachlor and the cysteine conjugate of propachlor were both metabolized in approximately the same ratio to the bound residue fraction and three major metabolites: N-isopropyloxanilic acid, 2-sulfo-N-isopropylacetanilide, and 2-(sulfinylmethylenecarboxy)-N-isopropylacetanilide. The methyl sulfoxide and methyl sulfone analogs of propachlor were characterized as minor soil metabolites. The major metabolites present in soybean plants grown in soil treated with propachlor were those produced in the soil and taken up by the plants. It was speculated that propachlor metabolism in the soil involved microbial activity and conjugation with glutathione or cysteine. It was concluded that homoglutathione conjugates are metabolized in soybean in a manner similar to the metabolism of other glutathione conjugates in species such as peanut.

Biosynthesis and Localization of Peanut Peroxidases. A Comparison of the Cationic and the Anionic Isozymes

Summary The biosynthesis and release of the cationic (C.PRX) and the anionic peroxidases (A.PRX) were studied in suspension cultured peanut (Aracbis hypogaea L.) cells by radiolabeling and immunoprecipitation. The incorporation rate of [35S]-methionine into the microsomal fraction was similar for both isozymes over the 2 h labeling period, although the cationic form accumulated four-times as much in the culture medium as the anionic. The mature peroxidases isolated from the medium were 40 kD for the C.PRX and 42 kD for the A.PRX as determined by SDS-PAGE. The polypeptides immunoprecipitated from the microsomal fraction were similar between the two isozymes. They were 58 kD and 31 kD for the cationic, 62 kD and 32 kD for the anionic form, respectively. The polypeptides of 31 kD and 32 kD correlate in size to the trifluoromethane sulfonic acid-deglycosylated C.PRX and A.PRX, respectively, suggesting the absence of carbohydrates. Immunocytochemical labeling demonstrated that both isozymes were associated with Golgi complexes and amyloplasts. The C.PRX was also localized on cell walls, while the A.PRX on plasmalemma.

Purification and yield of a cationic peroxidase from a peanut suspension cell culture

Variability in the purity of peanut ( Arachis hypogaea) peroxidase has necessitated the introduction of further purification steps beyond those used formerly. This appeared to be necessary when the purity of the peroxidase obtained by the established approach was found to be deficient as determined by electrophoresis and by absorption spectra. The addition of lectin affinity and gel filtration steps yielded a homogeneous preparation as indicated by polyacrylamide gel electrophoresis (PAGE) of 20 μg of protein on a mini gel. The RZ (405/280 nm) value was as good as the best former values. A calculation of the capacity of the cultured peanut cells to produce the highly purified peroxidase is presented.

Peptides Released by Cultured Peanut Cells during Growth

Peptides released by cultured peanut ( Arachis hypogaea) cells into their growth medium were examined by electrophoresis. A 2-dimensional separation showed that 27 peptides were detected. The majority of these have a cationic charge and they vary between 14 and 70 kDa. Glycosylation of these peptides, as expressed by staining, was most pronounced for some of the cationic ones. The release of peptides by plant cells is discussed.

Molecular analysis of resveratrol synthase

Resveratrol synthase (RS), a key enzyme in biosynthesis of stilbene-type phytoalexins, catalyzes the formation of resveratrol from coumaroyl-CoA and malonyl-CoA. Two cDNA clones, pGSC1 and pGSC2, have been isolated from cDNA libraries established with poly(A)-rich RNA from peanut (Arachis hypogaea) cell cultures specifically induced for RS. These cDNAs were used to identify two genomic clones (pGSG10 and pGSG11). Sequence analysis shows that the two clones overlap in a large stretch of nearly identical sequences, and that pGSG10 contains the 5' and pGSG11 the 3' end of RS genes. The sequences reveal a single intron, and the size of the predicted protein is 42.7 kDa, in close agreement with that observed in polyacrylamide gels (43 kDa). Chalcone synthase (CHS), a key enzyme of flavonoid biosynthesis, utilizes the same substrates as RS, but the product is different (naringenin chalcone). Comparison of RS with CHS consensus sequences shows that the two genes are related. Homology extends throughout the coding region, and the intron in RS is at the same position as a conserved intron in CHS. However, RS reveals a substantial number of amino acid differences to CHS in positions highly conserved in all CHS enzymes. It is proposed that the two proteins possess a common scaffold necessary for binding of the substrates and the type of enzyme reaction, and that the differences are responsible for the formation of different products.

Control of gene expression during induction of cultured peanut cells: mRNA levels, protein synthesis and enzyme activity of stilbene synthase

Stilbene synthase is an inducible enzyme occurring in a small number of plants. The enzyme is amenable to analysis and biochemical studies only after the cells are subjected to induction. Cell suspension cultures of peanut react very selectively if elicited with biotic inducers. Just as intact peanut plants produce stilbene phytoalexins when attacked by fungi so also do sterile cultured cells when treated with sterilized insoluble fungal cell walls. Both systems react by synthesizing stilbene synthase. The time courses of increase in enzyme activity, protein synthesis and mRNA activity were studied, and their relation to other activities of the cells was elaborated. The results show that, after applying the fungal elicitor, the system responds very quickly and selectively: within 2 hours the synthesis rate of stilbene synthase protein is increased more than 30-fold, the increase being detectable 40 min after induction. The first increase in translatable mRNA for stilbene synthase can be seen 20 min after application of the stimulus. Stilbene synthase synthesized in vivo was compared to stilbene synthase prepared by translation in vitro. There was no difference in size, and limited proteolysis did not indicate significant differences in the peptide structure of the primary translation product and the active enzyme.

A Highly Specific Monoclonal Antibody to a Catonic Peroxidase from Cultured Peanut Cells

Summary A hybridoma cell line (CP-4) producing a monoclonal antibody (IgG2a), against a cationic peroxidase isolated from cultured peanut cells is described. This IgG selectively recognized the peroxidase molecule from cultured peanut cells on western blots, as detected by antimouse- 125 IIgG.

Ca2+ and peroxidase derived from cultured peanut cells

A 5% increase of Ca2+ content of the incubation medium for cultured peanut (Arachis hypogaea L.) cells caused a rise of peroxidase (EC 1.11.1.7) activity in the medium, which could be abolished by the addition of the chelator EGTA [eth‐yleneglycol‐bis‐(β‐aminoethyl ether)‐N,N,N',N'‐tetraacetic acid]. However, the determination of in vivo peroxidase synthesis in these cells showed that Ca2+ had a direct effect on the biosynthesis rather than on transport alone. This concept was re‐enforced by the lack of effect by the ionophore A23187 on the transport. The Ca2+ content was 2 and 5 mol (mol protein−1) for the cationic and anionic peanut peroxidase, respectively. The latter is different from the value reported for the anionic peroxidase from horseradish.

Immunochemical Localization of Peroxidase in Cultured Peanut Cells

Summary Peanut ( Arachis hypogaea L.) cells in suspension culture were incubated with 35 S-methionine. Four groups of organelles (amyloplasts, nuclei, mitochondria and microsomes) were isolated from these cells by differential centrifugation. The homogeneity of the isolated fractions was checked by the assay of marker enzymes for each organelle and by electron microscopic observations. Proteins from these isolated organelles were sequentially extracted with a low ionic (0.05M phosphate buffer pH 7.0) and high ionic (low ionic buffer with 0.8M KCl added) buffer. The peroxidase and total proteins were precipitated using antibodies specific to cationic peroxidase and 10 % trichloro acetic acid, respectively. The results show that most of the newly synthesized peroxidase extracted with the high ionic buffer is associated/derived from the mlcrosomes.

Site of haem synthesis in cultured peanut cells

Cationic peroxidase is the major haemoprotein secreted by cultured peanut cells, into their medium, as determined by immunoprecipitation. The haem moiety of cationic peroxidase has been identified as protohaem, based on results obtained by mass spectrometry. By incubating the cultured cells with [ 14C]-δ-aminolevulinic acid and subsequently isolating the mitochondria and amyloplasts from these cells, it has been shown that mitochondria are the site of synthesis of haem in these cells.

Spectral and kinetic properties of a cationic peroxidase secreted by cultured peanut cells

It is demonstrated that the cationic peroxidase isolated from the growth medium of cultured peanut cells reacts via the same mechanism as other peroxidases, namely conversion of the native enzyme into compound I by reaction with hydrogen peroxide, followed by two reductions by one-electron donors to compound II and then back to the native enzyme. From the pyridine hemochromogen spectrum it is concluded that the prosthetic group of the native enzyme is ferriprotoporphyrin IX. Optical spectra are recorded for (i) the native (ferric) enzyme and its cyanide, azide, fluoride and alkaline forms, (ii) ferrous peroxidase and its cyanide and carbon monoxide complexes, and (iii) compounds I, II, and III. Equilibrium studies show that the ferric cyanide complex has a dissociation constant of 3.0 ± 0.5 μM over the pH range 3–8. The fluoride complex has a dissociation constant which varies from 1.6 μM at pH 4.0 to 28 μM at pH 4.8. Azide has a much lower affinity than fluoride. The alkaline transition occurs with an apparent pKa value of 9.2. Rate constants were recorded for cyanide binding, the alkaline transition, compound I formation, and for the reactions of compound II with a series of substrates. Similarities and differences to horseradish peroxidase are discussed.

Effects of caffeine treatment alone and in combination with gamma exposure on cultured peanut cells

Cell growth, mitotic index, and peroxidase activity were measured, and radioimmunoassay with [35S]methionine for peroxidase synthesis was carried out on peanut suspension cells treated with 10−5 M caffeine with or without 50 kR gamma exposure. Decreases in the amount of peroxidase in the medium from caffeine treatment were thought to be induced by events causing simultaneous mitotic reduction and degradation of peroxidase by gamma exposure. However, the data based on a comparison of the rate of total protein and of peroxidase synthesis suggest that caffeine treatment induces quantitative and qualitative changes in the rate of peroxidase synthesis. The data on qualitative differences were obtained from peroxidase extraction without or with high ionic strength buffer.

Characterization of Peroxidase in Plant Cells

Two peroxidases, one anionic and one cationic, have been purified from the proteins secreted by peanut (Arachis hypogaea L. var Virginia 56R) cells in suspension culture. These two peroxidases apparently have identical catalytic properties.

Comparison of the metabolism of 5,6-dihydro-2-methyl-N-phenyl-1,4-oxathiin-3-carboxamide (carboxin) in peanut plants and peanut cell suspension cultures

Comparison of the metabolism of 5,6-dihydro-2-methyl-N-phenyl-1,4-oxathiin-3-carboxamide (carboxin) in peanut plants and peanut cell suspension cultures

Age-dependent effects of gamma-exposure on form, growth and peroxidase activity of cultured peanut cells

In peanut suspension cells exposed to 500 krad at different ages of cel growth, mitotic index, giant cell frequency and peroxidase activity in the medium were surveyed. The frequency of giant cells significantly increased in the cells exposed at the age just before their maximum cell division. Peroxidase activity sharply decreased after gamma-exposure and then increased in each cell-group. Renewed accumulation of peroxidase in the medium was recognized, particularly in stationary phase cells, suggesting physiological reversion of enzyme synthesis and/or release. Additionally, a reduction in the mitotic index of non-exposed cells was detected after culturing in 500 krad-exposed medium.

Immunoaffinity Studies on Cationic Peanut Peroxidase Fraction

Cationic peroxidase immobilized on sepharose has been used to separate specific antibodies from the antiperoxidase whole rabbit serum immunoglobulins (IgG) raised against it. The IgGs so separated completely pellet the peroxidase activity of the cationic peroxidase at a ratio of 6 : 1 (w/w). This shows the specificity of purified IgGs which is also shown by the single reaction arc obtained by immunoelectrophoresis of the isolated IgGs followed by challenging with crude peroxidase. These specific IgGs when linked to cyanogen bromide activated sepharose have been used to purify the cationic peroxidase from the spent medium proteins and from the cell extract of the peanut cells grown in suspension culture.