Induction of Reactive Oxygen Species and Phytoalexins in Onion (Allium cepa) Cell Culture by Biotic Elicitors Derived from the Fungus Botrytis cinerea

Metabolites of a phytopathogenic fungus Botrytis cinerea Pers. were analyzed for the presence of biotic elicitors. Three groups of elicitors competent in inducing defense responses inAllium cepa cells were identified and partly purified. The recognition of the elicitor signal in onion cells was shown to elevate the concentration of reactive oxygen species (ROS), namely, superoxide anion-radical (O₂^{\overset{-}.}) and hydrogen peroxide (₂₂). The intensity of ROS release depended on chemical identity of elicitor and its concentration. The most active ROS production in onion cells was induced by a protein fraction isolated from the medium for fungus culturing. The carbohydrate elicitors extracted from the fungus cytoplasm and cell walls of mycelia were much less effective. The dynamics of ROS generation comprised two stages. The first stage represented fast and low-amplitude changes that peaked in 15 min after the elicitor treatment. The second stage was more durable and extensive; it occurred in 1.5–6 h after the treatment.     

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.     

Regulation of CDPKs,including identification of PAL kinase,in biotically stressed cells of French bean

Changes in protein kinase activity have been investigated during the early response of suspension cultured cells of French bean to fungal elicitor. One of the kinases activated has a known target, phenylalanine ammonia-lyase (PAL), which has an important role in plant defence responses, and was purified. Kinase acivity during purification was monitored for both the PAL-derived peptide and syntide-2, which it also phosphorylated. The kinase had an M _r of 55000 on the basis of gel migration, ⁴⁵Ca²⁺ binding, autophosphorylation and phosphorylation of various substrates using in-gel assays. The kinase has been characterised with respect to kinetics and other properties in vitro and appears to be a CDPK. In-gel assays were also used to show that this kinase and a number of other CDPKs of similar M _r showed complex changes in elicitor-treated suspension-cultured cells of French bean. An activation was observed within 10 min and was maintained for up to 4 h. The time course of activation was different from MAP kinase and casein kinase assayed in the same extracts. However, at 5 min after addition of elicitor there is a transient inactivation of the CDPKs before activation. This inactivation can be mimicked by adding forskolin to the cells 30 min before elicitation, which brings about changes in the cellular pH. Forskolin potentiates the oxidative burst when elicitor is subsequently added while the CDPK cannot be activated by elicitor upon forskolin treatment. In contrast, intracellular acidification brought about by forskolin brings about slight activation of MAPkinase.     

Rapid induction of ethylene biosynthesis in cultured parsley cells by fungal elicitor and its relationship to the induction of phenylalanine ammonia-lyase

The biosynthesis of ethylene was examined in suspension-cultured cells of parsley (Petroselinum hortense) treated with an elicitor from cell walls of Phytophthora megasperma. Untreated cells contained 50 nmol g^-1 of the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), and produced ethylene at a rate of about 0.5 nmol g^-1 h^-1. Within 2 h after addition of elicitor to the culture medium, the cells started to produce more ethylene and accumulated more ACC. Exogenously added ACC did not increase the rate of ethylene production in control or elicitor-treated cells, indicating that the enzyme converting ACC to ethylene was limiting in both cases. The first enzyme in ethylene biosynthesis, ACC synthase, was very rapidly and transiently induced by the elicitor treatment. Its activity increased more than tenfold within 60 min. Density labelling with ²H₂O showed that this increase was caused by the denovo synthesis of the enzyme protein. Cordycepin and actinomycin D did not affect the induction of ACC synthase, indicating that the synthesis of new mRNA was not required. The peak of ACC-synthase activity preceded the maximal phenylalanine ammonia-lyase (PAL) activity by several hours. Exogenously supplied ethylene or ACC did not induce PAL. However, aminoethoxyvinylglycine, an inhibitor of ACC synthase, suppressed the rise in ethylene production in elicitor-treated cells and partially inhibited the induction of PAL. Exogenously supplied ACC reversed this inhibition. It is concluded that induction of the ethylene biosynthetic pathway is a very early symptom of elicitor action. Although ethylene alone is not a sufficient signal for PAL induction, the enhanced activity of ACC synthase and the ethylene biosynthetic pathway may be important for the subsequent induction of PAL.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglycine - PAL phenylalanine ammonia-lyase     

Elicitation of silymarin in cell cultures of Silybum marianum: effect of subculture and repeated addition of methyl jasmonate

Production of silymarin and the effect of the elicitor, methyl jasmonate (MeJA), was monitored in cell cultures of Silybum marianum over 4 years. Silymarin concentrations gradually declined after prolonged subculture, making the success of elicitor strategy limited in long-term cultures. The continuous presence of MeJA in cultures for an extended period was necessary for induction of silymarin accumulation. A repeated elicitor strategy was not a good option for improving silymarin productivity in batch cultures. Removal of medium from elicited cultures and addition of fresh medium avoided the toxic effects of elicitor accumulation, allowing the system to respond to a repeated MeJA treatment without loss of productivity.     

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     

Induction of pathogenesis-related proteins in sugarcane leaves and cell-cultures by a glycoprotein elicitor isolated from <Emphasis Type="Italic">Colletotrichum falcatum</Emphasis>

The induction of pathogenesis-related (PR) proteins in sugarcane (Saccharum officinarum L.) leaves and suspension-cultured cells in response to treatment with a glycoprotein elicitor isolated from Colletotrichum falcatum (the red rot pathogen) was investigated. Treatment of leaves and cells with the elicitor resulted in a much marked increase in the activities of chitinase and β-1,3-glucanase in red rot resistant (BO 91) than susceptible (CoC 671) sugarcane cultivar. SDS-PAGE analysis revealed that C. falcatum elicitor induced the accumulation of several proteins in suspension-cultured cells of resistant cultivar (BO 91); among them the 35 kDa protein was predominant. Whereas, a 27 kDa protein was induced predominantly in the cells of susceptible cultivar upon treatment with the elicitor. When sugarcane leaves were treated with C. falcatum elicitor, two proteins with apparent molecular masses of 25 and 27 kDa were induced both in the resistant and susceptible cultivars. However, the induction was stronger in the resistant than the susceptible cultivar. Immunoblot analysis for chitinase indicated that a protein with an apparent molecular mass of 37 kDa cross-reacting with barley chitinase antiserum was strongly induced in the suspension cultured cells of both the cultivars. The induction of 37 kDa chitinase was more in the cells of resistant cultivar than in the susceptible cultivar. Western blot analysis revealed that a 25 kDa thaumatin-like protein (TLP) cross-reacting with bean TLP antiserum was strongly induced in leaves and cultured cells of both resistant and susceptible cultivars due to elicitor treatment.     

Marked effect of Cuscuta on puerarin accumulation in cell cultures of Pueraria tuberosa grown in shake flasks and a bioreactor

Isoflavonoid production in cell cultures of Pueraria tuberosa as influenced by an angiospermic parasite, Cuscuta reflexa, was studied. During the time course, maximum isoflavonoid content was recorded when Cuscuta elicitor was added on day 15 of culture. Among various concentrations of elicitor tried, 1 g l⁻¹ of Cuscuta elicitor was found to be the most effective. The optimized elicitation conditions were used in vessels of varying capacity where maximum yield of ~91 mg l⁻¹ of isoflavonoid was recorded in a 2-l bioreactor which was about 19% higher than the control cultures. In this case, puerarin content increased up to 11 mg l⁻¹ which was 580% higher that the value recorded in the control cultures. In the bioreactor, 8 days of elicitation was optimal for the high accumulation of isoflavonoid, giving productivity of ~4 mg l⁻¹ day⁻¹. The study showed persistent high isoflavonoid yield even during scale-up. Use of a preparation of Cuscuta reflexa as an elicitor is reported for the first time. The increase in isoflavonoid content was elicitor dose-dependent and can be explored to trigger high yields of isoflavonoid/secondary metabolites in production.     

Elicitation of 2,3-Dihydroxybenzoic Acid and Ajmalicine in a Catharanthus roseus Suspension Culture*

Addition of an elicitor preparation from cell walls of Phytophthora megasperma f. sp. glycinea (Pmg elicitor) to a newly established cell suspension culture of Catharanthus roseus induced extracellular free 2,3-dihydroxybenzoic acid, suggesting its role in pathogen defense. The same substance also accumulated intracellularly in a bound form. Treatment of the crude Pmg elicitor preparation with trypsin abolished elicitor activity, suggesting that the active fraction is proteinaceous. The cells became more sensitive to low but not to elevated elicitor concentrations when they were pretreated with 2,6-dichloroisonicotinic (DCIA) or 5-chlorosalicylic (5CSA) acid for about 1 day before addition of the elicitor. This indicates that the elicitor reception/transduction system becomes improved by these compounds known to be related to systemic acquired resistance against plant pathogens. The newly established cell line initially accumulated also the indole alkaloid ajmalicine, a process enhanced by Pmg elicitor. This potency was lost during subculturing for about 1 year and was also not restored by preincubation with DCIA or 5CSA. In contrast, elicitation of 2,3-dihydroxybenzoic acid synthesis was undiminished, suggesting that the Pmg elicitor perception system was still functioning and not the cause for the decline in elicited indole alkaloid production.     

Elicitor-stimulated biosynthesis of hydroxycinnamoyltyramines in cell suspension cultures of Solanum tuberosum

Treatment of suspension-cultured potato cells (Solanum tuberosum L. cv. Desirée) with an elicitor from Phytophthora infestans induced increased incorporation of 4-hydroxybenzaldehyde, 4-hydroxybenzoate, and N-4-coumaroyl- and N-feruloyltyramine into the cell␣wall and secretion of N-4-coumaroyl- and N-feruloyltyramine into the culture medium. Induced metabolite accumulation was preceded by rapid and transient increases in activities of phenylalanine ammonia-lyase (EC 4.3.1.5) and tyrosine decarboxylase (TyrDC; EC 4.1.1.25), exhibiting maximal activities 5–10 h after initiation of elicitor treatment. Activities of hydroxycinnamoyl-CoA:tyramine hydroxycinnamoyltransferase (EC 2.3.1.110), catalyzing the formation of N-4-coumaroyl- and N-feruloyltyramine, increased later and remained at high levels. The phenolic defense compounds appear to be involved in cell wall reinforcement and may further directly affect fungal growth in the apoplastic space. Received: 26 July 1997 / Accepted: 9 September 1997     

Oxidative Stress and Taxol Production Induced by Fungal Elicitor in Cell Suspension Cultures of Taxus Chinensis

Treatment of Taxus chinensis cell suspension cultures with fungal elicitor resulted in an oxidative stress characterized by H₂O₂ production, malondiadehyde (MDA) accumulation and cell death. This oxidative stress was dependent on the concentration of elicitor. Cells exposed to elicitor accumulated taxol, however, not proportional to elicitor concentration. High production of taxol occurred in cells treated with the suitable elicitor concentration. We concluded that oxidative stress had the deleterious effect on taxol production. Simultaneous treatment with elicitor and ascorbic acid (ASA) changed the oxidative stress and taxol production. Production of taxol in cells treated with 200 mg dm^–3 elicitor and ASA was enhanced compared with that in cells treated with only 200 mg dm^–3 elicitor, while production of taxol in cells treated with 100 and 50 mg dm^–3 elicitor and ASA was decreased compared with that in cells treated with 100 and 50 mg dm^–3 elicitor.     

Differential induction of peroxidase and polyphenol oxidase isozymes in suspension-cultured cells of blast resistant and susceptible genotypes of rice in response to treatment with Magnaporthe grisea elicitor and toxin

The effect of elicitor from mycelial walls of Magnaporthe grisea, the rice blast fungus and α-picolinic acid, one of the toxins produced by M. grisea on induction of peroxidase (PO), polyphenol oxidase (PPO) in suspension-cultured rice (Oryza sativa L.) cells was studied. Cultured cells of blast resistant (Usen) and susceptible (CO39) rice genotypes were treated with elicitor (50?μg of glucose equivalents per ml) or α-picolinic acid (400?ppm). The cells were harvested at different time intervals and analysed for the induction of PO and PPO. PO isozyme analysis indicated that the elicitor strongly induced the activities of PO-2 and PO-3 in cultured cells of Usen 3?days after treatment. In Usen, toxin also induced the activities of PO-3 and PO-4. However, similar levels of activities corresponding to these isozymes were recorded 7?days after treatment. In CO39, the activities of PO-1 and PO-2 were induced 3?days after elicitor treatment. In contrast, the toxin suppressed the activity of PO-2. The elicitor induced the activities of PPO-1, PPO-2 and PPO-3 in both Usen and CO39. In Usen, steady increase of PPO-3 was observed and higher level of activity was recorded 5?days after treatment. In CO39, higher level of PPO-3 was observed 1?day after treatment and declined thereafter. However, the activities of PPO-1 and PPO-2 increased 3?days after treatment in CO39. In the toxin-treated cells of Usen, higher level of activity of PPO-3 was observed 3?days after treatment.     

The effects of CA2+ during the elicitation of shikonin derivatives inOnosma paniculatum cells

Summary A fungal elicitor extracted fromAspergillus oryzae (Ahlb.) Cobn mycelia promoted the production of shikonin derivatives inOnosma paniculatum Bur et Franch cell suspension cultures. Elicitor treatment also increased Ca²⁺ concentration in RM₉ medium, which could be measured earlier than the elicited increase of shikonin formation. Several reagents known to induce Ca²⁺-influx and increase the intracellular-free Ca²⁺ level, such as the addition of Ca (NO₃)₂·4H₂O, the Ca²⁺ ionophore A₂₃₁₈₇, and abscisic acid (ABA), appreciably suppressed the elicitor-promoted shikonin formation inOnosma cells. In contrast, the decrease of intracellular-free Ca²⁺ level by the specific Ca²⁺-chelator ethylene glycol bis (β-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) or the Ca²⁺—channel blocker, verapamil, enhanced the biosynthesis of shikonin even in the absence of elicitor. Treatment of cells with trifluoperazine (TFP) also stimulated shikonin formation inOnosma cell cultures. A rapid and transient drop of free Ca²⁺ level in one protoplast was directly determined after the addition of elicitor toOnosma cell cultures. The inhibitory effect on shikonin formation by ABA was largely on account of its ability to restore the intracellular Ca²⁺ level lowered by the elicitor. These results suggest that Ca²⁺ play a significant role in an early stage of the elicitation process ofOnosma cells. The rapid drop of cytoplasmic Ca²⁺ carries the elicitor signal and in turn regulates the biosynthesis of shikonin derivatives.     

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.     

Stress responses in alfalfa (Medicago sativa L.) III. Induction of medicarpin and cytochrome P450 enzyme activities in elicitor-treated cell suspension cultures and protoplasts

Summary Cell suspension cultures of alfalfa (Medicago sativa L.) accumulated phenolic secondary metabolites in a pattern similar to that seen in alfalfa roots. Upon treatment with a crude elicitor preparation from the bean pathogen Colletotrichum lindemuthianum, the pterocarpan phytoalexin medicarpin accumulated in cells and culture medium. The extractable activities of six enzymes involved in medicarpin biosynthesis (including three cytochrome P450 activities) were induced by treatment with elicitor, and their induction kinetics correlated with the rate of medicarpin accumulation. However, protoplasts prepared from these cultures accumulated neither medicarpin nor other secondary products after treatment with elicitor. The cytochrome P450 activities were induced during the preparation of the protoplasts, but could be further induced by treatment with fungal elicitor. The results are discussed in relation to the use of alfalfa protoplasts as a system for functional analysis of cloned defense genes.Abbreviations AUFS absorption unit full scale - CHI chalcone isomerase (EC 5.5.1.6) - CHS chalcone synthase (EC 2.3.1.74) - C40H cinnamic acid 4-hydroxylase (EC 1.14.13.11) - CLE elicitor from Colletotrichum lindemuthianum - IFOH isoflavone 2-hydroxylase - IFS isoflavone synthase - PAL L-phenylalanine ammonia-lyase (EC 4.3.1.5)     

Methylation reactions and the phytoalexin response in alfalfa suspension cultures

 In order to determine why the activated methyl cycle is up-regulated in plants undergoing defence responses to fungal pathogens we have monitored the utilisation of methyl groups derived from methionine in cell-suspension cultures of alfalfa (Medicago sativa L.) treated for various times with fungal elicitor, by carrying out a parallel labelling study with [³⁵S]methionine and [methyl-³H]methionine. The distribution of the two radiolabels among the medium, soluble cellular components and cell wall was then determined. In the absence of elicitor the utilisation of the two radiolabels was similar. However, in the presence of the elicitor the total incorporation of radioactivity from [methyl-³H]methionine into metabolites was far greater than from [³⁵S]methionine, indicating that the methyl label had been utilised in methylation reactions. Elicitor treatment resulted in up to a sixfold increase in the use of ³H-methyl groups in the methylation of hydrophobic metabolites. In the period 0–24 h after elicitor treatment, increased methylation was directed largely into the synthesis of the isoflavonoid phytoalexin medicarpin and related metabolites. Newly synthesized phytoalexins were exported into the medium, while a significant proportion of the medicarpin accumulating in the cell in the early stages of elicitation was derived from the hydrolysis of its respective conjugate. Elicitor treatment also modified the incorporation of ³H-methyl groups into the cell wall. Between 0 and 24 h after elicitor treatment the methylation of pectin in the cell wall declined. After 24 h, pectin methylation recovered and was associated with an increase in the methylation of other wall-bound polysaccharide components. Since no other major metabolic sink for the increased methylation was determined we conclude that the increased activity of the activated methyl cycle during defence interactions in alfalfa is required to support phytoalexin synthesis and cell wall modifications. Received: 1 August 1996 / Accepted: 24 October 1996     

Mechanism of resistance induced by plant activators against Colletotrichum falcatum in sugarcane

Abstract

A search for plant activators capable of inducing systemic resistance in sugarcane showed that plants pre-treated with synthetic signal inducers confer a high degree of resistance to Colletotrichum falcatum – the red rot pathogen. Among the various treatments, Acibenzolar S- methyl (ASM) was found to be very effective in restricting the pathogen colonization inside the inoculated cane stalk tissues. The induction of resistance was accompanied by a significant increase in peroxidases and polyphenoloxidases activities. A considerable decrease of pathogen titre in the pre-treated tissues as determined by ELISA, clearly demonstrated the restriction of pathogen colonization and proliferation in the sensitized cane stalks. Specific induction of new isoforms of peroxidases and polyphenoloxidases in C. falcatum elicitor treatment indicates the pathogen elicitor induced specific cellular response of sugarcane suspension-cultured cells.     

Purification and Properties of an Elicitor of Castor Bean Phytoalexin from Culture Filtrates of the Fungus Rhizopus stolonifer

Evidence has been obtained for the presence in filtrates of 3-day-old cultures of the fungus Rhizopus stolonifer grown on potato-dextrose medium of both high molecular weight and low molecular weight elicitors of the production of the phytoalexin casbene in cell-free extracts of castor bean (Ricinus communis L.) seedlings. The high molecular weight elicitor activity was purified by means of gel filtration chromatography. Both protein and carbohydrate are associated with the most purified fraction containing elicitor activity. The elicitor is inactivated by treatments at 60 C or higher temperatures for 15 minutes. The molecular weight of the purified elicitor was estimated from gel filtration chromatography in 10 mm Na-phosphate (pH 7) to be 30,000 ± 5,000. Treatments of the purified elicitor fraction with either sodium periodate or the nonspecific protease preparation, pronase, substantially reduced its activity as an elicitor of casbene production. On the basis of these properties it is concluded that the elicitor is most likely a protein and may be a glycoprotein. It is estimated that 2 × 10⁻⁸ m elicitor gives about a 14-fold increase in casbene synthetase activity in extracts of treated split seedlings in comparison with controls. This corresponds to about 50% of the maximal activity obtainable in this assay system developed to measure elicitor activity.