Differential induction of enzyme in soybean cell cultures by elicitor or osmotic stress

Soybean cell cultures were challenged either by glucan elicitor from Phytophthora megasperma f.sp. glycinea or by osmotic stress (0.4 M glucose). Osmotic stress induced production of a microsomal NADPH-dependent flavone synthase (flavone synthase II) which catalyses conversion of (2S)-naringenin to apigenin. In one of our cell-lines this enzyme activity was not detected either in unchallenged cells or in cells treated with glucan elicitor. Inducibility of flavone synthase II by 0.4 M glucose was highest at the end of the linear growth phase. Changes in the activities of a number of other enzymes were determined after treatment of the cells with elicitor or 0.4 M glucose. The activities of phenylalanine ammonialyase, cinnamate 4-hydroxylase, chalcone synthase and dihydroxypterocarpan 6a-hydroxylase all increased with elicitor and with osmoticum, albeit to a different degree. The rise in enzyme activity occurred later with osmoticum than with elicitor. The prenyltransferase involved in glyceollin synthesis was induced strongly by elicitor but only very weakly by osmoticum, whereas isoflavone synthase and NADPH: cytochrome-c reductase were only induced by elicitor. The activity of glucose-6-phosphate dehydrogenase did not change with elicitor or with osmoticum. Different product patterns were also obtained: whereas with elicitor, glyceollin I was the major product, intermediates of the glyceollin pathway (7,4-dihydroxyflavanone, trihydroxypterocarpan) accumulated with osmoticum.     

Effects of sodium orthovanadate on benzophenanthridine alkaloid formation and distribution in cell suspension cultures of Eschscholtzia californica

Cell suspension cultures of Eschscholtzia californica produce relatively large amounts of benzophenanthridine alkaloids upon elicitation. Sodium orthovanadate is used as an abiotic elicitor to induce alkaloid biosynthesis in cultures of E. californica. The response of the cell culture to this abiotic elicitor is very similar to that observed after elicitation with a biotic elicitor (a carbohydrate fraction from yeast extract). Treatment with orthovanadate leads to alkalinization of the growth medium, a 20-fold induction of the key enzyme tyrosine decarboxylase and increased alkaloid formation (up to 40 mg.L^–1). Cells treated with the yeast elicitor excrete a large portion of alkaloids produced into the growth medium (up to 50 % of total alkaloids) while cells treated with orthovanadate release very small amounts of alkaloids into the medium (less than 10 % of total alkaloids). These results suggest that an active transport system, possibly specific for benzophenanthridine alkaloids, is present in the plasma membrane of E. californica cells. The nature of this putative vanadate-sensitive transporter is not known at present.     

Host-Pathogen Interactions: IX. Quantitative Assays of Elicitor Activity and Characterization of the Elicitor Present in the Extracellular Medium of Cultures of Phytophthora megasperma var. sojae

Resistance of soybean (Glycine max L.) seedlings to Phytophthora megasperma var. sojae (Pms) is in part due to the accumulation in infected tissue of a compound which is toxic to Pms. The accumulation of this compound, a phytoalexin called glyceollin, is triggered by infection, but it can also be triggered by molecules, “elicitors,” present in cultures of Pms. The ability of the Pms elicitor to stimulate phytoalexin accumulation in soybean tissues has been used as the basis for biological assays of elicitor activity. Two bioassays were developed and characterized in this study of the Pms elicitor. These bioassays use the cotyledons and the hypocotyls of soybean seedlings. The cotyledon assay was used to characterize the extracellular Pms elicitor. This elicitor was isolated from Pms cultures and purified by ion exchange and molecular sieving chromatography. The extracellular Pms elicitor was determined to be a predominantly 3-linked glucan, which is similar in composition and structure to a polysaccharide component of Pms mycelial walls.     

Release of a Soluble Phytoalexin Elicitor from Mycelial Walls of Phytophthora megasperma var. sojae by Soybean Tissues

A soluble elicitor of glyceollin accumulation was released from insoluble mycelial walls of Phytophthora megasperma var. sojae after incubation with soybean cotyledon tissue for as little as 2 minutes. Various enzymic and chemical treatments of the released elicitor indicated that the activity resided in a carbohydrate moiety, and gel filtration disclosed the presence of at least two active molecular species. Cell-free extracts from soybean cotyledons or hypocotyls also released soluble elicitors from fungal cell walls that were similar to those released by living cotyledon tissue. These results may suggest that contact of fungal pathogens with host tissues is required to release fungal wall elicitors which then initiate phytoalexin accumulation in the plant.     

Phytoalexin Elicitor Activity of Carbohydrates from Phytophthora megasperma f.sp. glycinea and Other Sources

Three unique classes of carbohydrates were isolated from the hyphal cell walls of Phytophthora megasperma f.sp. glycinea (Pmg) and compared with other substances for their activity as elicitors of the phytoalexin glyceollin in soybean tissues. Glucomannans extracted from cell walls with soybean β-1,3-endoglucanase were purified and proved to be the most active elicitors yet reported. They were approximately 10 times more active in soybean cotyledons than the heterogeneous β-glucan elicitor fraction extracted from Pmg walls. In addition, the glucomannan fraction gave race-specific elicitor activity in soybean hypocotyls. Pronase was found to be a suitable reagent for the mild extraction of glycopeptides from Pmg cell walls. All of the carbohydrates isolated from Pmg cell walls possessed significant elicitor activity, but other glucans, a glucomannan and mannan from other sources, were much less active. Chitin and chitosan, reported to function as elicitors in other plants, had low activity in soybean cotyledons. Arachidonic acid was inactive, despite its previously observed elicitor activity in potato tubers. The results indicated that, for Pmg, the carbohydrate elicitor most probably involved in the initiation of phytoalexinmediated defense during fungus infection of soybean plants is the glucomannan fraction liberated by endoglucanase.     

Host-Pathogen Interactions: XIV. Isolation and Partial Characterization of an Elicitor from Yeast Extract

An elicitor of glyceollin accumulation in soybeans (Glycine max L.) has been isolated from a commercially available extract of brewers' yeast. Yeast is not a known pathogen of plants. The elicitor was isolated by precipitation in 80% (v/v) ethanol followed by column chromatography on DEAE-cellulose, sulfopropyl-Sephadex, and concanavalin A-Sepharose. Compositional and structural analysis showed the elicitor to be a glucan containing terminal, 3-, 6-, and 3,6-linked glucosyl residues. The yeast elicitor stimulates the accumulation of glyceollin in the cotyledons and hypocotyls of soybeans when as little as 15 nanograms or 100 nanograms of the elicitor is applied to the respective tissues. The yeast elicitor is very similar in both structure and absolute elicitor activity to an elicitor isolated from the mycelial walls of Phytophthora megasperma var. sojae, a pathogen of soybeans. These and other results of this laboratory suggest that plants are able to respond to the presence of a wide range of fungi by recognizing, as foreign to the plant, structural polysaccharides of the mycelial walls of the fungi.     

l-Phenylalanine ammonia-lyase in suspension culture cells of spruce (Picea abies)

The activity of l-phenylalanine ammonia-lyase (PAL) (EC 4.3.1.5) was determined in seedlings, callus cells, cell suspension cultures and in young needles of spruce (Picea abies) (L.) (Karst). PAL activity increased up to 10 fold in response to transferring suspension cultured cells into new cultivation medium. PAL was also induced about 10 fold when callus cells were transferrd into liquid medium. The increase was transient and it required the presence of a carbohydrate.In cell suspension cultures, grown in the dark (white cells), but not in light-grown cultures (green cells), PAL activity was induced up to 30 fold by UV-light.With a cell wall preparation of Rhizosphaera kalkhoffii, a forest pathogenic fungus, used as elicitor, the activity of PAL could be induced more than 10 fold. The degree of induction depended on the elicitor concentration. Induction was prevented by cycloheximide but not by actinomycin D.     

Rapid Accumulation of Anionic Peroxidases and Phenolic Polymers in Soybean Cotyledon Tissues following Treatment with Phytophthora megasperma f. sp. Glycinea Wall Glucan

Phytophthora megasperma Drechs. f. sp. glycinea Kuan & Erwin (PMG) cell wall glucan has been extensively characterized as an elicitor of the pterocarpan phytoalexins, the glyceollins in soybean (Glycine max L.). Just recently, this glucan was shown to be a potent elicitor of conjugates of the isoflavones, daidzein and genistein as well. Here we report that PMG wall glucan also induces a rapid and massive accumulation of phenolic polymers in soybean cotyledon cells proximal to the point of elicitor application. Deposition of phenolic polymers is over then times that in wounded controls within just 4 hours of elicitor treatment and reaches a maximum by 24 hours. In the same tissues, isoflavone conjugates begin to accumulate at 8 hours and glyceollin at 12 hours. By 24 hours, the total deposition of wall bound phenolics in elicitor-treated tissues is several times greater than the peak glyceollin and isoflavone responses combined. Histochemical stains and quantitation of phenolic residues released after saponification and nitrobenzene or copper oxide oxidation suggest that the covalently linked phenolics include both lignin- and suberin-like polymers as well as simple esterified coumaric and ferulic acid monomers. Accumulations of phenolic polymers are accompanied by equally rapid and massive increases in activity of a specific group of anionic peroxidases. Although increases in peroxidase activity are not strictly limited to cells immediately adjacent to the area of elicitor treatment, the deposition of phenolic polymers is significantly less extensive in distal cells.     

Inhibition of elicitor-induced phytoalexin formation in cotton and soybean cells by citrate

Addition of an elicitor preparation from Verticillium dahliae to soybean or cotton cell suspension cultures induces the formation of the phytoalexins, glycelollin or sesquiterpene aldehydes, respectively. Recent work (PS Low, PF Heinstein 1986 Arch Biochem Biophys 249: 472-479) has shown that the induction of phytoalexin biosynthesis in these cells is preceded by rapid changes in the plant cell membrane which can be conveniently monitored by membrane associated fluorescent probes. Using this elicitation assay, we have found that citrate, a common metabolite of higher plants, acts as a potent inhibitor of elicitation when added prior to treatment with elicitor. The citrate concentration required to obtain a 50% inhibition of the elicitor-induced fluorescence transition in cultured cotton cells was found to be about 2 millimolar, while the concentration of citrate observed to inhibit elicitor-induced sesquiterpene aldehyde formation in the same cell suspensions was also 2 millimolar. Curiously, in the presence of elicitor, citrate at less than ID₅₀ concentrations increased cell mass accumulation significantly above control incubations without elicitor. A similar inhibition of glyceollin formation with an increase in cell mass accumulation was also observed upon addition of 1 to 5 millimolar citrate to soybean cell suspension cultures. The physiological significance of the inhibition by citrate of phytoalexin formation in plant cell suspensions was supported by the observation that a similar inhibition of sesquiterpene aldehyde formation occurs in cotton plantlets elicited by cold shock or V. dahliae stress. The specificity of citrate as an inhibitor of phytoalexin formation was demonstrated by data showing that other di- and tricarboxylic-hydroxy acids did not inhibit, with the exception of malate which inhibited phytoalexin formation in soybean cells with roughly half the potency of citrate. These experiments not only demonstrate that citrate can act as a specific inhibitor of elicitation, but they further confirm the validity of monitoring elicitation and its modulation with fluorescent probes.     

Quantification of glyceollins in non-elicited seedlings of Glycine max by gas chromatography-mass spectrometry

A sensitive gas chromatography-mass spectrometry method is described for detection and quantification of small amounts of phytoalexins, especially glyceollins. This method allowed identification of canescacarpin (glyceollin V) besides appreciable amounts of glyceollin isomers I–III and glyceofuran in unchallenged parts of soybean seedlings. Based on this quantification method the glyceollin content of soybean roots, either untreated, wounded or incubated with a glucan elicitor from Phytophthora sojae, was compared. The proportions of glyceollin isomers in the mixture of compounds were determined in different soybean tissues. Single-ion monitoring revealed the presence of four additional glyceollin isomers with benzofuranoid structure.     

Effects of Ca2+ on phytoalexin induction by fungal elicitor in soybean cells

A glucan elicitor from the cell walls of the fungus Phytophthora megasperma f.sp. glycinea caused increases in the activities of the phytoalexin biosynthetic enzymes, phenylalanine ammonia-lyase and chalcone synthase, and induced the production of the phytoalexin, glyceollin, in soybean (Glycine max) cell suspension cultures when tested in culture medium containing 1.2 mmol/liter Ca2+. Removal of extracellular Ca2+ by treatment with ethylene glycol bis(beta-aminoethyl ether)-N, N'-tetraacetic acid followed by washing the cells with Ca2+-free culture medium abolished the elicitor-mediated phytoalexin response. This suppression was largely reversed on readdition of Ca2+. Elicitor-mediated enhancement of biosynthetic enzyme activities and accumulation of glyceollin was strongly inhibited by La3+; effective concentrations for 50% inhibition were (mumol/liter) 40 for phenylalanine ammonia-lyase, 100 for chalcone synthase, and 30 for glyceollin. Verapamil caused similar effects only at concentrations higher than 0.1 mmol/liter, whereas trifluoperazine and 8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate did not affect enzyme induction by the elicitor in the concentration range tested. Uptake of alpha-amino isobutyric acid into soybean cells, which was rapidly inhibited in the presence of the glucan elicitor, was not affected by La3+ nor was uptake inhibition by the elicitor relieved by La3+. The Ca2+ ionophore, A23187, enhanced phytoalexin biosynthetic enzyme activities and glyceollin accumulation in a dose-dependent manner, with 50% stimulation (relative to the elicitor) occurring at about 5 mumol/liter. The results suggest that the glucan elicitor causes changes in metabolite fluxes across the plasma membrane of soybean cells, among which changes in Ca2+ fluxes appear to be important for the stimulation of the phytoalexin response.     

Dose responses for Colletotrichum lindemuthianum elicitor-mediated enzyme induction in French bean cell suspension cultures

The induction of L-phenylalanine ammonialyase (PAL, EC 4.3.1.5) and flavanone synthase in French bean cell suspension cultures in response to heat-released elicitor from cell walls of the phytopathogenic fungus Colletotrichum lindemuthianum is highly dependent upon elicitor concentration. The elicitor dose-response curve for PAL induction shows two maxima at around 17.5 and 50 g elicitor carbohydrate per ml culture, whereas the flavanone synthase response shows one maximum at around 100 g ml^-1. The PAL response is independent of the elicitor concentration present during the lag phase of enzyme induction; if the initial elicitor concentration is increased after 2 h by addition of extra elicitor, or decreased by dilution of the cultures, the dose response curves obtained reflect the concentration of elicitor present at the time of harvest. PAL induction is not prevented by addition of methyl sugar derivatives to the cultures; -methyl-D-glucoside, itself a weak elicitor of PAL activity, elicits a multiphasic PAL response when increasing concentrations are added in the presence of Colletotrichum elicitor. Eight fractions with different monosaccharide compositions, obtained from the crude elicitor by gel-filtration, each elicit different dose-responses for PAL induction; the response to unfractionated elicitor is not the sum of the response to the isolated fractions. There is no correlation between the ability of the fractions to induce PAL in the cultures and their ability to act as elicitors of isoflavonoid phytoalexin accumulation in bean hypocotyls.Abbreviations PAL phenylalanine ammonia-lyase - PMS Phytophthora megasperma var sojae     

Elicitor-Induced l-Tyrosine Decarboxylase from Plant Cell Suspension Cultures : II. Partial Characterization

l-Tyrosine decarboxylase (EC 4.1.1.25) activity was induced in cell suspension cultures of Thalictrum rugosum Ait. and Eschscholtzia californica Cham. with a yeast polysaccharide preparation (elicitor). The highest l-tyrosine decarboxylase activity in extracts from 7-day-old cell cultures of E. californica was observed 5 hours after addition of 30 to 40 micrograms elicitor per gram cell fresh weight. The enzyme extracted from cells of E. californica was purified 1540-fold to a specific activity of 2.6 micromoles CO₂ produced per minute per milligram protein at pH 8.4 and 30°C. Purified enzyme from T. rugosum showed a specific activity of 0.18 micromoles per minute per milligram protein. The purification procedure involved ammonium sulfate fractionation, anion-exchange fast protein liquid chromatography, ultrafiltration, and hydrophobic interaction chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the enzyme from the two plant cell cultures had subunits of identical molecular weight (56,300 ± 300 daltons.     

Host-Pathogen Interactions: X. Fractionation and Biological Activity of an Elicitor Isolated from the Mycelial Walls of Phytophthora megasperma var. sojae

An elicitor of phytoalexin production in soybean (Glycine max L.) tissues was isolated from purified Phytophthora megasperma var. sojae mycelial walls by a heat treatment similar to that used to solubilize the surface antigens from the cell walls of Saccharomyces cerevisiae. The wall-released elicitor is a discrete, minor portion of the P. megasperma var. sojae mycelial walls. The elicitor released from the mycelial walls was divided by diethylaminoethylcellulose and concanavalin A-Sepharose chromatography into four fractions, each having different chemical characteristics. The four fractions were obtained from each of the three races of P. megasperma var. sojae. The corresponding fractions from each of the three races are very similar in composition and elicitor activity. The results suggest that the elicitor activity of each fraction resides in the glucan component of the fraction. Evidence is presented to demonstrate that the elicitors are not race-specific and that the accumulation of glyceollin is not sufficient to account for race-specific resistance.     

Metabolic Regulation during Glyceollin Biosynthesis in Green Soybean Hypocotyls

The accumulation of the isoflavonoid phytoalexin, glyceollin, occurs in hypocotyls of green soybean seedlings (Glycine max L. Merr. cv Harosoy 63) in response to the injection of a glucan elicitor isolated from the mycelial walls of the fungus, Phytophthora megasperma f. sp. glycinea. This accumulation, which levels off after 24 hours, is preceded by a dramatic, transient rise in extractable activities of two early enzymes in the biosynthetic pathway, phenylalanine ammonia-lyase (PAL) and p-coumaryl CoA ligase (pCL). The maximum amount of extractable activity occurs 12 to 16 hours after elicitor treatment and is coincident with the most rapid period of glyceollin accumulation. These results suggest a regulatory role for these early enzymes in the biosynthesis of this secondary metabolite. High performance liquid chromatography analysis of the early intermediates in the pathway further corroborates this hypothesis. The relative pool size and rate of turnover of p-coumaric acid, an early intermediate in glyceollin production, increase during the period of rapid increases in enzyme activities. Removal of cotyledons from elicitor-treated seedlings reduces glyceollin accumulation approximately 70%. This limitation of phytoalexin accumulation by cotyledon removal is correlated with a similar cotyledon effect on reduction of extractable activities of both PAL and pCL as well as a decrease in the flux of carbon through the p-coumaric acid pool. This research further supports the hypothesis that early enzymic steps in a biosynthetic pathway diverting carbon from primary to secondary metabolites function as regulatory control points.     

Elicitor enhanced production of protoberberine alkaloids from in vitro cell suspension cultures of <Emphasis Type="Italic">Tinospora cordifolia</Emphasis> (Willd.) Miers ex Hook. F. &amp; Thoms

The present research investigates the effect of Piriformospora indica, an endophytic fungus, on production of protoberberine alkaloids in in vitro cell suspension cultures of Tinospora cordifolia. Although T. cordifolia produces a number of protoberberine alkaloids, the simultaneous production of jatrorrhizine and palmatine in cell suspension cultures of T. cordifolia was observed for the first time with the use of P. indica as biotic elicitor. The cells in suspension cultures were elicitated with P. indica on 14th day of culture initiation and the production of the alkaloids on 16th day was monitored. The autoclaved as well as filter sterilized cultures of P. indica were used in addition to the use of fungal cell extract. The elicitor effect of P. indica was analyzed and compared with other abiotic elicitor (methyl jasmonate) and biotic elicitors (chitin and chitosan). The culture filtrate of P. indica in the filter sterilized (5.0% v/v) form gave better response with enhanced 4.2-fold production of jatrorrhizine (10.72 mg/g DW) and 4.0-fold production of palmatine (4.39 mg/g DW). The production of these compounds was at par with that achieved in methyl jasmonate (at 250 µM) treated cell suspension cultures.     

Induction of enzymes of phytoalexin synthesis in cultured soybean cells by an elicitor from Phytophthora megasperma f. sp. glycinea

The glucan elicitor from cell walls of the fungal pathogen, Phytophthora megasperma f. sp. glycinea, induced rapid but transient increases in enzyme activities of general phenylpropanoid metabolism (phenylalanine ammonia-lyase and 4-coumarate: CoA ligase) and of the flavonoid pathway (chalcone synthase) in cell suspension cultures of soybean (Glycine max). After transferring cells into fresh medium, two peaks of inducibility for the enzymes by elicitor were observed, one shortly after transfer (stage I), and one at the end of the linear growth phase (stage II). Only one of the two isoenzymes of 4-coumarate: CoA ligase (isoenzyme 2), for which a specific involvement in flavonoid biosynthesis has been postulated, was affected by the elicitor. For two of the induced enzymes, phenylalanine ammonia-lyase and chalcone synthase, the changes in activity at stage I were shown to be preceded by large changes in their rates of synthesis, as determined by in vivo labelling with [³⁵S] methionine and immunoprecipitation.Abbreviations Pmg Phytophthora megasperma f. sp. glycinea - glyceollin is a term used to designate the 3 isomers which accumulate in challenged soybean tissue (Moesta and Grisebach 1981b)     

Elicitor-induced accumulation of glyceollin and callose in soybean roots and localized resistance against Phytophthora megasperma f.sp. glycinea

Immersion of roots of 2-day-old soybean seedlings (Glycine max cv. Harosoy 63) into solutions of several glucan elicitors caused the accumulation to various degrees of the soybean phytoalexin glyceollin. Laminarin and polytran proved to be more effective elicitors in this system than the glucan elicitor from Phytophthora megasperma f.sp. glycinea (Pmg). Digitonin and tomatin caused, in addition to glyceollin accumulation, the deposition of callose in the rhizodermis. Pretreatment of the soybean roots with laminarin effected an increase in resistance of the seedlings against a compatible race of Pmg.     

Induction of sesquiterpene cyclase and suppression of squalene synthetase activities in plant cell cultures treated with fungal elicitor

Addition of elicitor, cell wall fragments of the fungus Phytophthora parasitica, to tobacco cell suspension cultures (Nicotiana tabacum) resulted in the rapid synthesis and secretion of large amounts of antibiotic sesquiterpenoids. Pulse-labeling experiments with [¹⁴C]acetate and [³H] mevalonate demonstrated that the induction of sesquiterpenoid biosynthesis, maximal by 6 to 9 hours after elicitor addition to the cell cultures, was paralleled by a rapid and large decline in the incorporation rate of radioactivity into sterols. Consequently, sterol accumulation was also inhibited upon addition of elicitor to the cell cultures. Sesquiterpene cyclase activity was absent from control cell cultures but induced to a maximum within 10 hours of elicitor addition to the cell cultures. The cyclase activity remained elevated for an additional 30 hours before declining. In contrast, squalene synthetase activity was suppressed to less than 15% of that found in control cells within 7 hours of elicitor addition. Our results suggest that the channeling of isoprenoid intermediates, and especially farnesyl diphosphate, into sesquiterpenoids occurred by a coordinated increase in the sesquiterpene cyclase and a decrease in the squalene synthetase enzyme activities. A reexamination of the data pertaining to the transient induction of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity (EC 1.1.1.34) in elicitor-treated cells suggested that, while the reductase activity was necessary for sesquiterpenoid biosynthesis, it functioned more to maintain a sufficient level of intermediates between mevalonate and farnesyl diphosphate rather than as a rate limiting step controlling the synthesis rate of any one class of isoprenoids.     

Involvement of lipoxygenase in elicitor-stimulated sanguinarine accumulation in Papaver somniferum suspension cultures

The involvement of lipoxygenase (LOX, EC 1.13.11.12) in elicitor-induced opium poppy defense response was investigated. Papaver somniferum L. suspension cultures were treated with abiotic elicitor methyl jasmonate (MJ), fungal elicitor (Botrytis cinerea homogenate) and phenidone (specific inhibitor of LOX) to determine the involvement of this enzyme in production of sanguinarine, the major secondary metabolite of opium poppy cultures. P. somniferum suspension cultures responded to elicitor treatment with strong and transient increase of LOX activity followed by sanguinarine accumulation. LOX activity increased in elicited cultures, reaching 9.8 times of the initial value at 10 h after MJ application and 2.9 times after B. cinerea application. Sanguinarine accumulated to maximal levels of 169.5 ± 12.5 μg g^?1 dry cell weight in MJ-elicited cultures and 288.0 ± 10.0 μg g^?1 dry cell weight in B. cinerea-elicited cultures. The treatment of cells with phenidone before elicitor addition, significantly reduced sanguinarine production. The relative molecular weight of P. somniferum LOX (83 kDa) was estimated by using immunobloting and its pH optimum was shown to be pH 6.5.