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.     

Influence of methyl jasmonate on podophyllotoxin and 6-methoxypodophyllotoxin accumulation in Linum album cell suspension cultures

The accumulation of podophyllotoxin (PTOX) and 6-methoxypodophyllotoxin (6MPTOX) was enhanced about twofold in the suspension culture of Linum album line 2-5 aH following the addition of methyl jasmonate (MeJas) to the cultivation medium, reaching 7.69±1.45 mg/g dry weight and 1.11±0.09 mg/g dry weight, respectively. There was no increase in 6MPTOX accumulation following the addition of MeJas to suspension cells of L. album line X4SF, whereas PTOX accumulation was enhanced about tenfold to 0.49±0.10 mg/g dry weight. Phenylalanine ammonia-lyase activity increased immediately after the addition of MeJas to a cell suspension culture of line X4SF, reaching a maximum between 4 h and 1 day after elicitation, while cinnamyl alcohol dehydrogenase activity and the lignin content of the cells were not affected.     

The kinetics of taxoid accumulation in cell suspension cultures of Taxus following elicitation with methyl jasmonate

Cell suspension cultures of Taxus canadensis and Taxus cuspidata rapidly produced paclitaxel (Taxol) and other taxoids in response to elicitation with methyl jasmonate. By optimizing the concentration of the elicitor, and the timing of elicitation, we have achieved the most rapid accumulation of paclitaxel in a plant cell culture, yet reported. The greatest accumulation of paclitaxel occurred when methyl jasmonate was added to cultures at a final concentration of 200 microM on day 7 of the culture cycle. The concentration of paclitaxel increased in the extracellular (cell-free) medium to 117 mg/day within 5 days following elicitation, equivalent to a rate of 23.4 mg/L per day. Paclitaxel was only one of many taxoids whose concentrations increased significantly in response to elicitation. Despite the rapid accumulation and high concentration of paclitaxel, its concentration never exceeded 20% of the total taxoids produced in the elicited culture. Two other taxoids, 13-acetyl-9-dihydrobaccatin III and baccatin VI, accounted for 39% to 62% of the total taxoids in elicited cultures. The accumulation of baccatin III did not parallel the pattern of accumulation for paclitaxel. Baccatin III continued to accumulate until the end of the culture cycle, at which point most of the cells in the culture were dead, implying a possible role as a degradation product of taxoid biosynthesis, rather than as a precursor.     

Transglutaminase activity in testicular homogenates and serum-free Sertoli cell cultures

Transglutaminase (EC 2.3.2.13) (TGase) activity has been localized in homogenates of rat Leydig cells and seminiferous tubules and is present in cytosol and membrane fractions. The enzyme has a requirement for Ca2+ and when the acceptor substrate casein was deleted from the assay mixture, incorporation of [14C]putrescine into cytosolic and membrane fractions occurred. Transglutaminase was also detected in Sertoli cells cultured in serum-free medium. Sertoli cells reside within the seminiferous tubule and are involved in normal spermatogenesis. Sertoli cell TGase has a strict requirement for Ca2+ and is not activated by Mg2+. Activation of the enzyme occurs with as little as 0.3 microM Ca2+; however, consistent with intracellular calcium levels, maximum stimulation occurred at 1.9 mM Ca2+. Sertoli cell TGase activity is markedly stimulated if the cells are cultured in 10% fetal bovine serum rather than in serum-free medium. Inhibition of Sertoli cell TGase by monodansylcadaverine concomitantly decreased the response of the cells to follicle-stimulating hormone (FSH)-induced secretion of cAMP but did not change basal cAMP levels. These data suggest that TGase may play a facilitative rather than an absolute role in activation of Sertoli cells by FSH and the resultant secretion of cellular products. This may occur through modulation of activities of membrane and cytosolic components by TGase.     

Ligand accumulation in autocrine cell cultures

Cell-culture assays are routinely used to analyze autocrine signaling systems, but quantitative experiments are rarely possible. To enable the quantitative design and analysis of experiments with autocrine cells, we develop a biophysical theory of ligand accumulation in cell-culture assays. Our theory predicts the ligand concentration as a function of time and measurable parameters of autocrine cells and cell-culture experiments. The key step of our analysis is the derivation of the survival probability of a single ligand released from the surface of an autocrine cell. An expression for this probability is derived using the boundary homogenization approach and tested by stochastic simulations. We use this expression in the integral balance equations, from which we find the Laplace transform of the ligand concentration. We demonstrate how the theory works by analyzing the autocrine epidermal growth factor receptor system and discuss the extension of our methods to other experiments with cultured autocrine cells.     

Adventitious root growth and ginsenoside accumulation in Panax ginseng cultures as affected by methyl jasmonate

Adventitious roots of ginseng were treated with methyl jasmonate (MJ) up to 150 microM and cultured for 40 days. Up to 100 microM MJ inhibited the root growth but increase ginsenoside accumulation. In a two-stage bioreactor culture, total ginsenosides, after elicitation with 100 microM MJ peaked after 10 days at 48 mg g(-1) dry wt and then dropped sharply. Of the two groups of ginsenosides (Rb and Rg), higher amounts of Rb accumulated in the adventitious roots.     

Novel oligosaccharides isolated from Fusarium oxysporum L. rapidly induce PAL activity in Rubus cells

Activation of the phenolic pathway is known to be part of a defense response against cell wall-derived elicitors from pathogens. Many examples of a defense response by increasing the synthesis of phenolic compound against the elicitor were demonstrated in the past, but the elicitor structure has so far been poorly characterized. Our results indicate that a disaccharide fraction containing the following structure: alpha-D-mannopyranosyl (1-->2)alpha/beta-D-glucopyranosyl and alpha-D-mannopyranosyl (1-->x) inositol, isolated from Fusarium oxysporum L., promotes rapid and transient phenylalanine ammonia lyase activity in Rubus fructicosus cells at nanomolar concentration. The disaccharides were isolated by size-exclusion chromatography directly from extracts obtained by alkaline treatment of F. oxysporum mycelium. Their structure was determined by 500-MHz-1H-NMR spectroscopy combined with methylation analysis and fast atom bombardment mass spectrometry.     

Fungal elicitor preparations and methyl jasmonate enhance rosmarinic acid accumulation in suspension cultures of Coleus blumei

bstract Suspension cultures of Coleus blumei (Lamiaceae) treated with either an elicitor preparation from the culture medium of the phytopathogenic oomycete Pythium aphanidermatum or with methyl jasmonate enhanced accumulation of rosmarinic acid approximately threefold. The specific activities of phenylalanine ammonia lyase and rosmarinic acid synthase were also enhanced after addition of the fungal elicitor. The addition of methyl jasmonate transiently increased activities of phenylalanine ammonia lyase and hydroxyphenylpyruvate reductase, whereas the activity of rosmarinic acid synthase was not stimulated and the activity of tyrosine aminotransferase was slightly and constantly enhanced. Methyl jasmonate stimulated rosmarinic acid accumulation not only when added directly to the culture medium, but also when it could reach the cells only via the gas phase. Received: 2 April 1997 / Revision received:16 June 1997 / Accepted: 15 September 1997     

Sterol and Fatty Acid Metabolism in Fusarium oxysporum

The effects of temperatures ranging from 10°C to 35°C on sterol and fatty acid production and hydroxymethylglutaryl CoA reductase (EC 1.1,1.34, HMGCoA reductase) activity have been examined. Growth, based on dry weight, was maximal at 25°C to 30°C. Sterol production and the reductase activity were highest at 15°C after 28~32 hr incubation when the total fatty acids were minimal. Fatty acid unsaturation generally increased with decrease in temperature.     

Adventitious root growth and ginsenoside accumulation in Panax ginseng cultures as affected by methyl jasmonate

Adventitious roots of ginseng were treated with methyl jasmonate (MJ) up to 150m and cultured for 40days. Up to 100m MJ inhibited the root growth but increase ginsenoside accumulation. In a two-stage bioreactor culture, total ginsenosides, after elicitation with 100 m MJ peaked after 10days at 48mgg^–1 dry wt and then dropped sharply. Of the two groups of ginsenosides (Rb and Rg), higher amounts of Rb accumulated in the adventitious roots.Revisions requested; 2 July 2004; Revisions received 30 June 2004; 3 September 2004     

Ability of nonpathogenic Fusarium oxysporum strain Fo47 to induce resistance against Pythium ultimum infection in cucumber

The influence exerted by nonpathogenic Fusarium oxysporum strain Fo47 in triggering cucumber protection against infection by Pythium ultimum was investigated ultrastructurally. Macroscopic and microscopic observations of the pathogen colony in dual cultures revealed that reduction of Pythium growth was associated with marked disorders, including generalized disorganization of the host cytoplasm, retraction of the plasmalemma, and complete loss of the protoplasm. Cytochemical labeling of cellulose with an exoglucanase-gold complex showed that the cellulose component of the host cell walls was structurally preserved at a time when the host cytoplasm had undergone complete disorganization. A similar antagonistic process was observed at the root cell surface. Most striking and interesting was the finding that mycoparasitism, as evidenced by the frequent occurrence of Fo47 hyphae within nearly empty cells of the pathogen, occurred not only at the root surface but also within the invaded root tissues. The specific labeling pattern obtained with the exoglucanase-gold complex confirmed that Fo47 successfully penetrated cells of the pathogen, both in the rhizosphere and inside the root tissues. Pythium cells that could evade the first defensive line in the rhizosphere could penetrate the root epidermis, but their growth was restricted to the outermost tissues. Positive correlations between Fo47 treatment and induced resistance to infection by P. ultimum in cucumber were confirmed by (i) the reduction of pathogen viability; (ii) the elaboration of newly formed barriers, a phenomenon which was not seen in Fo47-free plants, where the pathogen proliferated in all root tissues within a few days; and (iii) the occlusion of intercellular spaces with a dense material likely enriched in phenolics. Taken together, our observations provide the first convincing evidence that Fo47 exerts a direct inhibitory effect on P. ultimum through a combination of antibiosis and mycoparasitism, in addition to being a strong inducer of plant defense reactions.     

Ultrastructural changes associated with the accumulation and secretion of sanguinarine in Papaver bracteatum suspension cultures treated with fungal elicitor

Suspension cultures of Papaver bracteatum Arya II Lindl., grown without hormone in the presence of conidial extracts of Verticillium dahliae Kleb., accumulate millimolar quantities of the benzophenanthridine alkaloid, sanguinarine. Under the fluorescence microscope, the elicitor-treated cells display an orange-yellow fluorescence characteristic of sanguinarine, primarily near the periphery of the cells. Electron-microscopic inspection showed the presence of slightly dilated endoplasmic reticulum and of electron-dense protuberances on the tonoplast of large central vacuoles. These osmiophilic aggregates lining the tonoplast bud into spherical bodies, appear to become detached from the membrane and are released into the vacuole. Upon subcellular fractionation of elicited cells on Renografin step gradients, sanguinarine was found to be distributed in all bands but with 86% concentrated in the gradient pellet. Analysis of the pellet by electron microscopy showed that it contained electron-dense fragments similar to the osmiophilic bodies observed on the tonoplast of intact elicited cells. In elicited cell cultures, most of the sanguinarine was recovered from medium in a 100·g sedimenting, cell-free, particulate fraction accounting for as much as 85% of the media sanguinarine and 62% of the total sanguinarine. The sanguinarine-rich 100·g media pellet was determined to be two-thirds protein, one-third RNA and was essentially devoid of phenolics, phospholipid and DNA. The pellet consisted of electrondense material and cytoplasmic remnants resembling those found in the Renografin pellet and tonoplast aggregates of intact cells. When placed under hypotonic conditions or extracted with aqueous buffer, pH 3–11, the pellet did not release sanguinarine. These observations provide evidence for storage of sanguinarine at electron-dense deposits which occur on the tonoplast and as freely floating bodies in vacuoles.Abbreviations BA N⁶-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - EM electron microscopy - ER endoplasmic reticulum - HPLC high-pressure liquid chromatography - MRST Murashige and Skoog's revised tobacco medium     

Solid-state cultures of Fusarium oxysporum transform aromatic components of olive-mill dry residue and reduce its phytotoxicity

The present study mainly investigated the ability of solid-state cultures of the non-pathogenic Fusarium oxysporum strain BAFC 738 to transform aromatic components to reduce the phytotoxicity in olive-mill dry residue (DOR), the waste from the two-phase manufacturing process. Lignin, hemicellulose, fats and water-soluble extractives contents of DOR colonized by the fungus for 20 weeks were reduced by 16%, 25%, 71% and 13%, respectively, while the cellulose content increased by 25%. In addition, the ethyl acetate-extractable phenolic fraction of the waste was reduced by 65%. However, mass-balance ultra-filtration and size-exclusion chromatography experiments suggested that the apparent removal of that fraction, mainly including 2-(3,4-dihydroxyphenyl)ethyl alcohol and 2-(4-hydroxyphenyl)ethyl alcohol, was due to polymerization. Mn-peroxidase and Mn-independent peroxidase activities were found in F. oxysporum solid-state cultures, while laccase and aryl alcohol oxidase activities were not detected. Tests performed with seedlings of tomato (Lycopersicum esculentum L.), soybean (Glycine maximum Merr.), and alfalfa (Medicago sativa L.) grown on soils containing 6% (w/w) of bioconverted DOR (kg soil)(-1) showed that the waste's phytotoxicity was removed by 20 weeks-old fungal cultures. By contrast, the same material exhibited a high residual toxicity towards lettuce (Lactuca sativa L.).     

Effect of methyl jasmonate in combination with carbohydrates on gene expression of PR proteins, stilbene and anthocyanin accumulation in grapevine cell cultures.

Grapevine (Vitis vinifera L.) is subject to a number of diseases which affect yield and wine quality. After veraison, berries become strongly susceptible to pathogens due to different physiological changes including the accumulation of glucose and fructose, on the one hand, and to the decrease of anti-microbial compounds called stilbenes, on the other. To obtain berry protection, pesticides are excessively used leading to important cost to the grower and to undesirable environmental impact of the residues, especially in grape, soil and water. As a consequence, alternative strategies have to be developed. Exogenously applied biotic elicitors induce defense responses. We studied the effects of methyl jasmonate in combination with sucrose on defense-related gene expression, stilbene and anthocyanin production in grapevine cell suspensions. The methyl jasmonate/sucrose treatment was effective in stimulating phenylalanine ammonia lyase, chalcone synthase, stilbene synthase, UDP-glucose: flavonoid-O-glucosyltransferase, proteinase inhibitor and chitinase gene expression, and triggered accumulation of both piceids and anthocyanins in cells, and trans-resveratrol and piceids in the extracellular medium. Methyl jasmonate treatment might be an efficient natural strategy to protect grapevine berries in vineyard.     

Production of reactive oxygen species in Arabidopsis thaliana cell suspension cultures in response to an elicitor from Fusarium oxysporum: implications for basal resistance

The present understanding of ROS generation in the defence responseof Arabidopsis thaliana is reviewed. Evidence suggests thatthe apoplastic oxidative burst generated during basal resistanceis peroxidase-dependent. The ROS generated during this basalresistance may serve to activate NADPH oxidase during the R-gene-mediatedhypersensitive response. The processes involved in the productionof reactive oxygen species in A. thaliana cell suspension culturesin response to an elicitor from Fusarium oxysporum are investigatedin the present work. This system appears analogous to the productionof ROS during the basal resistance response in French bean,which is peroxidase-dependent. A panel of modulators effectivein other pathogen elicitor and plant cell systems has been usedto investigate the Arabidopsis signalling pathways and the plantcell responses involved. Thus as in other systems, an earlycalcium influx into the cytosolic compartment, a rapid effluxof K⁺ and Cl^–, and extracellular alkalinization of elicitedcell cultures has been found. However the alkalinization isnot sufficient to stimulate the apoplastic oxidative burst byitself, unlike in French bean, although vectorial ion fluxesare needed. A secretory component which is sensitive to monensinand N-ethylmaleimide and insensitive to brefeldin A may alsobe necessary for the release and provision of substrates forperoxidase-dependent generation of H₂O₂. Key words: Arabidopsis thaliana, calcium, elicitation, hydrogen peroxide, oxidative burst, secretion     

Tricyclic antidepressants induce sphingomyelinase deficiency in fibroblast and neuroblastoma cell cultures

Tricyclic antidepressants (imipramine and desipramine) gave rise to an important decrease of sphingomyelinase activity in murine neuroblastoma and human fibroblast cell cultures. It occurred within 1 to 2 hours at a final concentration of 1 or 2 X 10(-5) M in cell culture medium. Other lysosomal enzymes such as acid lipase, arylsulfatases A and B and hexosaminidases were not modified. Low level of sphingomyelinase activity may be related to the amphiphilic characteristics of the drugs: iminodibenzyle which has the same tricyclic core but is devoid of the side chain necessary for amphiphilic properties had no effect. As iminodibenzyle has no therapeutic action, amphiphilic may be requisite to antidepressant properties of tricyclic drugs.