Effect of biotic elicitors on the accumulation of bilobalide and ginkgolides in Ginkgo biloba cell cultures

The effect of biotic elicitors on the production of bilobalide and ginkgolides in Ginkgo biloba cell suspension cultures was studied. The treatment of cell cultures with Candida albicans and Staphylococcus aureus as elicitors increased the amounts of bilobalide (BB), ginkgolide A (GA) and ginkgolide B (GB), with slight growth inhibition. The native bacterial elicitor was more effective for secondary metabolite accumulations both in cells and culture medium than autoclaved. However, exposure times of the cells to the elicitors strongly influenced the production of BB, GA and GB. This study suggests that biotic elicitors can regulate the production of BB, GA and GB either directly or indirectly. These results also describe the establishment of optimum conditions that determine the effects of biotic elicitors on secondary metabolism of bilobalides.     

Elicitor activity of cerebroside,a sphingolipid elicitor,in cell suspension cultures of rice

Cerebrosides, compounds categorized as glycosphingolipids, were found to occur in a wide range of phytopathogens as novel elicitors and to induce the effective disease resistance for rice plants in our previous study. Here, we showed that cerebroside elicitors lead to the accumulation of phytoalexins and pathogenesis-related (PR) protein in cell suspension cultures of rice with the structural specificity similar to that for the rice whole plants. This elicitor activity of the cerebroside was greater than jasmonic acid (JA) and chitin oligomer (which is known to be an elicitor for cell suspension cultures of rice). Treatment of cell suspension cultures with cerebroside and chitin oligomer resulted in a synergetic induction of phytoalexins, suggesting that cerebroside and carbohydrate elicitors, such as glucan and chitin elicitor, enhance the defense signals of rice in vivo. Induction of phytoalexins by the treatment with cerebroside elicitor was markedly inhibited by LaCl(3) and GdCl(3), Ca(2+ )channel blockers. It is possible that Ca(2+) may be involved in the signaling pathway of elicitor activity of cerebroside.     

Influence of fungal elicitors on production of ajmalicine by cell cultures of Catharanthus roseus

Suspension cultures of Catharanthus roseus (C. roseus) were elicited with fungal cell wall fragments of Aspergillus niger (A. niger), Fusarium moniliforme (F. moniliforme), and Trichoderma viride (T. viride). The effects of elicitor dosage, exposures time, and age of subculture on ajmalicine accumulation were studied. A higher concentration of elicitor extract responded positively to C. roseus suspension cultures. Ajmalicine accumulation increased by about 3-fold when cells were treated with A. niger, F.moniliforme, and T. viride. The maximum ajmalicine production (75 microg g(-1) dry weight (DW)) was observed in cells treated with T. viride. Cell cultures were elicited with 5% preparation of A. niger, F. moniliforme, and T. viride and exposed for 24, 48, 72, and 96 h. for elicitation. Suspension cultures elicited with T. viride for 48 h showed a 3-fold increase (87 microg g(-1) DW) in ajmalicine contents, whereas A. niger and F. moniliforme synthesized a 2-fold increase in alkaloid and yielded 52 and 56 microg g(-1) DW ajmalicine, respectively. C. roseus cells of different age (5,10, 15, 20, and 25 days old) were treated with a 5% elicitor of A. niger, F. moniliforme, and T. viride and investigated elicitors activity at different age of cell cultures. Maximum yield 166 microg g(-1) DW of ajmalicine was synthesized in 20 day old suspension cultures treated with T. viride. A longer period of incubation of cell cultures with elicitors adversely affected the ajmalicine synthesis.     

Coronatine,a more powerful elicitor for inducing taxane biosynthesis in Taxus media cell cultures than methyl jasmonate

Coronatine is a toxin produced by the pathogen Pseudomonas syringae. This compound has received much attention recently for its potential to act as a plant growth regulator and elicitor of plant secondary metabolism. To gain more insight into the mechanism by which elicitors can affect the biosynthesis of paclitaxel (Px) and related taxanes, the effect of coronatine (Cor) and methyl jasmonate (MeJA) on Taxus media cell cultures has been studied. For this study, a two-stage cell culture was established, in which cells were first cultured for 14 days in a medium optimised for growth, after which the cells were transferred to medium optimised for secondary metabolite production. The two elicitors were added to the medium at the beginning of the second stage. Total taxane production in the cell suspension was significantly enhanced by both elicitors, increasing from a maximum level of 8.14 mg/L in control conditions to 21.48 mg/L (day 12) with MeJA and 77.46 mg/L (day 16) with Cor. Expression analysis indicated that the txs, t13oh, t2oh, t7oh, dbat, pam, bata and dbtnbt genes were variably induced by the presence of the elicitors. Genes encoding enzymes involved in the formation of the polihydroxylated hypothetical intermediate (TXS, T13OH, T2OH, T7OH) and the phenylalanoil CoA chain (PAM) were stronger induced than those encoding enzymes catalysing the last steps of the Px biosynthetic pathway (DBAT, BAPT and DBTNBT). Notably, although taxane accumulation differed qualitatively and quantitatively following MeJA- or Cor-elicitation, gene expression induction patterns were similar, inferring that both elicitors may involve distinct but yet uncharacterised regulatory mechanisms.     

Phaseollin production in cell suspensions and whole plants of Phaseolus vulgaris

Production of phaseollin was measured in cell suspension cultures and whole plants of Phaseolus vulgaris. In suspension cultures phaseollin appeared when there was no further increase in cell mass. Cells transferred to a medium without auxins yielded three times higher phaseollin concentrations than cells grown in their presence. Addition of autoclaved fungal mycelia or polysaccharides as elicitors resulted in an increased phaseollin concentration in the cell suspension.In whole plants phaseollin could be detected only after the plants were challenged by a fungus which caused lesions (browning) of the upper root neck region, Rhizoctonia solani. Treatment of non-infected plants with autoclaved fungal mycelia or other elicitors did not induce phaseollin production. However, when they were added before or together with the pathogenic fungus, the elicitors further increased phaseollin concentration in the root neck regions of the plants. This indicated that the pathogenic fungus was important for the penetration of the elicitors to inner plant tissues where phaseollin (and probably other phytoalexins) is produced.     

Synergistic effects of sequential treatment with methyl jasmonate, salicylic acid and yeast extract on benzophenanthridine alkaloid accumulation and protein expression in Eschscholtzia californica suspension cultures

To develop an optimal bioprocess for secondary metabolite production and explain the bioprocess at the molecular level, we examine the synergistic effects of sequential treatment with methyl jasmonate (MJ), salicylic acid (SA) and yeast extract (YE) on benzophenanthridine alkaloid accumulation and protein expression in Eschscholtzia californica suspension cultures. Serial treatment of MJ, SA and YE at 24 h intervals enhanced the accumulation of dihydrosanguinarine (2.5 times) and sanguinarine (5.5 times). This sequential treatment using different signal elicitors was more effective than single elicitor or simultaneous treatment of the elicitors; it induced benzophenanthridine alkaloid accumulation to 917.7 ± 42.0 mg/L. Also, (S)-methylcoclaurine-3′-hydroxylase (CYP80B1) and 3′-hydroxy-(S)-N-methylcoclaurine-4′-O-methyltransferase (4′OMT) expressions among enzymes in sanguinarine biosynthetic pathway explained the synergistic effects by sequential treatment of the elicitors. The sequential treatment strategy using elicitors related to different signal transduction pathways can be used to design better processes to increase accumulation of secondary metabolites in plant cell culture. Analysis of protein expression provides the detailed information about metabolite accumulation through the correlated results.     

Stimulation of betacyanin synthesis through exogenous methyl jasmonate and other elicitors in suspension-cultured cells of Portulaca

Betacyanin production in suspension-cultured cells of Portulaca was significantly enhanced by both abiotic and biotic elicitors. Betacyanin levels increased 1.3 and 1.5-fold over the controls in the presence of two abiotic elicitors (20 mumol/L CuSO4 and 100 mumol/L FeEDTA) and increased 1.8 and 1.6-fold in the presence of two biotic elicitors (0.5 mg/L beta-glucan and 0.5 mg/L chitosan). Maximum betacyanin synthesis with the two most effective elicitors was obtained when cultures were treated on day 1 and day 0 by beta-glucan and FeEDTA, respectively. A concentration-dependent response was exhibited by cultures treated with exogenous methyl jasmonate (MJ). MJ alone at 0.1 mumol/L caused a 2.6-fold increase in betacyanin synthesis when administered to the suspension culture on day 3. However, no additive effect on betacyanin accumulation was observed in treatments, which combined MJ and beta-glucan or FeEDTA. Treatment with ibuprofen (IB), an inhibitor of jasmonate biosynthesis, reduced the level of betacyanin in cells cultured in standard medium at all concentrations tested (25, 50, 100 mumol/L). The effect of IB on betacyanin synthesis in the cells treated with MJ or beta-glucan, however, differed with the IB concentration applied. The two higher concentrations (50 and 100 mumol/L) of IB significantly reduced the betacyanin content while the lower concentration (25 mumol/L) did not show an adverse effect on the betacyanin enhancement triggered by MJ or beta-glucan. Our findings suggest that, in suspension-cultured cells of Portulaca, an MJ-mediated signal transduction pathway prominently exists in betacyanin synthesis. This pathway seems to act antagonistically towards beta-glucan-mediated signaling. As far as we know this is the first report on the elevation of betacyanin level by jasmonate or other elicitors in cell suspension cultures.     

Effects of exogenous methyl jasmonate in elicited anthocyanin-producing cell cultures of ohelo (Vaccinium phalae)

Summary Elicitation of anthocyanin-producing cells of ohelo (Vaccinium pahalae) by both biotic (purified β-glucan and chitosan) and abiotic [sodium ferric ethylenediamine di-(o-hydroxyphenylacetate) FeEDDHA, and CuSO₄] elicitors resulted in significant enhancement of anthocyanin accumulation. Anthocyanin production increased up to 1.8 and 1.5-fold over the control in the presence of abiotic elicitors (90 μM FeEDDHA and 20 μM CuSO₄, respectively), and increased 1.9 and 1.6-fold in the presence of biotic elicitors (10 mg L⁻¹ β-glucan and 100 mg L⁻¹ chitosan). Maximum anthocyanin production with the two most effective elicitors was achieved when cultures were treated on Day 3 (β-glucan) or Day 0 (FeEDDHA) after the initiation of fresh cell cultures. A concentration-dependent response was exhibited by cultures treated with exogenous methyl jasmonate (MJ). The addition of 0.5 μM MJ alone provoked a 2–3-fold increase in anthocyanin production over that of the control; however, no additive effect on anthocyanin production was observed in any treatments which combined MJ and β-glucan or FeEDDHA. Conditioning of the cells with a preculture in either MJ, β-glucan, or FeEDDHA similarly did not enhance anthocyanin production. Inoculation of cultures elicited by MJ or β-glucan with ibuprofen, a reported inhibitor of jasmonate biosynthesis, dramatically stimulated, rather than inhibited, anthocyanin production, resulting in levels of accumulation beyond any of the tested elicitor combinations. Hypotheses for the observed influence of ibuprofen in this system are discussed.     

Jasmonic acid-induced hypericin production in cell suspension cultures of Hypericum perforatum L. (St. John's wort)

Hypericum perforatum L. (St. John's wort) is an herbal remedy widely used in the treatment of mild to moderate depression. Hypericin, a photosensitive napthodianthrone, is believed to be the compound responsible for reversing the depression symptoms. In this study, novel in vitro cell culture systems of H. perforatum were used to monitor the effect of elicitation on cell growth and production of hypericin. A dramatic increase in cell growth and hypericin production was observed after exposure to jasmonic acid (JA). However, other elicitors such as salicylic acid (SA) and fungal cell wall elicitors failed to show any stimulatory effect on either cell growth or hypericin production. Cell cultures treated with JA and incubated in the dark showed increased growth and hypericin production as compared to the cultures grown under light conditions. Jasmonate induction in dark conditions played an important role in growth and hypericin production in cell suspension cultures, to our knowledge an undocumented observation.     

Enhancement of lignan biosynthesis in suspension cultures of <Emphasis Type="Italic">Linum nodiflorum</Emphasis> by coronalon,indanoyl-isoleucine and methyl jasmonate

The effect of the two synthetic elicitors coronalon and indanoyl-isoleucine and of methyl jasmonate (MeJA) on the accumulation and biosynthesis of lignans by cell suspension cultures of Linum nodiflorum (Linaceae) was investigated. The production of 6-methoxypodophyllotoxin (MPTOX) could be increased more than tenfold, the maximal content reaching up to over 2.5% of the cell dry weight. The highest yield was achieved by administering 50 μM of the synthetic elicitors on the fourth day and extracting the products on the tenth day of the culture period. An additional lignan accumulated in elicitor-treated cultures. Its structure was elucidated by extensive 1D and 2D NMR measurements, revealing its identity as 5′-demethoxy-MPTOX (5′-dMPTOX). Its average content amounted up to over 5% of the cell dry weight. Growth was only slightly affected by the addition of the elicitors. Methyl jasmonate exerted a moderate stimulating effect on the L. nodiflorum cells with MPTOX and 5′-dMPTOX contents going up to 1.4 and 2.1% of the cell dry weight, respectively. The activities of deoxypodophyllotoxin 6-hydroxylase and β-peltatin 6-O-methyltransferase, two enzymes involved in MPTOX biosynthesis, were increased up to 21.9-fold and 14.6-fold, respectively, in the treated cultures.     

Polysaccharide elicitors enhance anthocyanin and phenolic acid accumulation in cell suspension cultures of <Emphasis Type="Italic">Vitis vinifera</Emphasis>

The effects of yeast extract and selected polysaccharide elicitors on secondary metabolite production, particularly of anthocyanin and phenolic acid, in cell suspension cultures of Vitis vinifera were investigated. All elicitors either maintained or promoted cell growth in culture. Overall, secondary metabolite production in V. vinifera cell suspension cultures responded differently to different elicitors. Chitosan, pectin, and alginate enhanced production of anthocyanin within 13 days of culture with levels of 2.5-, 2.5-, and 2.6-fold increase, respectively, over that of control. Chitosan, alginate, and gum arabic significantly promoted accumulation of phenolic acids, particularly 3-O-glucosyl-resveratrol, in V. vinifera cultures, as well as in the culture medium. Intracellular phenolic acid production was significantly enhanced by alginate and chitosan, with 1.7- and 1.5-fold levels, respectively, of that of control. Extracellular phenolic acid production was also significantly increased in the presence of chitosan and gum arabic, with levels of 3.3- and 1.7-fold higher, respectively, than those of control. In addition, DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging activity was enhanced in the presence of elicitors, and this was positively correlated with increased accumulation of anthocyanin in V. vinifera cell suspension cultures.     

Enhanced catharanthine production in catharanthus roseus cell cultures by combined elicitor treatment in shake flasks and bioreactors

Chemical and fungal elicitors were added to Catharanthus roseus cell suspension cultures so as to improve the production of indole alkaloids. A synergistic effect on alkaloid accumulation was observed in C. roseus cell cultures when treated with some combined elicitors of fungal preparations and chemicals. Among them, the combination of tetramethyl amminium bromide and Aspergillum niger mycelial homogenate gave the highest ajmalicine yield (63 mg l(-1)) and an improved catharanthine accumulation (17 mg l(-1)). The combined elicitors of malate and sodium alginate resulted in the highest catharanthine yield (26 mg l(-1)) and a high ajmalicine accumulation (41 mg l(-1)) in the cell cultures. Based on the synergistic effect of malate and sodium alginate, a process with enhanced catharanthine production in Catharanthus roseus cell cultures was developed in shake flasks and a bioreactor. After 10 days of culture, 25 mg l(-1), 32 mg l(-1) and 22 mg l(-1) catharanthine yield were obtained in 500-ml flasks, 1000-ml flasks and in a 20-l airlift bioreactor, respectively. Upon malate-alginate combining treatments, peroxidase, catalase and superoxide dismutase activities decreased in elicited cells but phenylalanine ammonia lyase and lipoxygenase activities increased dramatically. That suggests a typical defense responses took place in the combined elicitors-treated cell cultures. Furthermore, the combined elicitors also caused a significant increase of malondialdehyde level in cell cultures, which suggests a serious lipid peroxidation occurred in the elicited cell cultures. Comparison of these results suggests that malate and alginate combining treatment also stimulates defense responses, such as lipid peroxidation, in all C. roseus culture processes and this may mediate the indole alkaloid production via jasmonate pathway.     

Nitric oxide elicitation for secondary metabolite production in cultured plant cells

Nitric oxide (NO) is an important signal molecule in stress responses. Accumulation of secondary metabolites often occurs in plants subjected to stresses including various elicitors or signal molecules. NO has been reported to play important roles in elicitor-induced secondary metabolite production in tissue and cell cultures of medicinal plants. Better understanding of NO role in the biosynthesis of such metabolites is very important for optimizing the commercial production of those pharmaceutically significant secondary metabolites. This paper summarizes progress made on several aspects of NO signal leading to the production of plant secondary metabolites, including various abiotic and biotic elicitors that induce NO production, elicitor-triggered NO generation cascades, the impact of NO on growth development and programmed cell death in medicinal plants, and NO-mediated regulation of the biosynthetic pathways of such metabolites. Cross-talks among NO signaling and reactive oxygen species, salicylic acid, and jasmonic acid are discussed. Some perspectives on the application of NO donors for induction of the secondary metabolite accumulation in plant cultures are also presented.     

Metabolic analysis of elicited cell suspension cultures of Cannabis sativa L. by 1H-NMR spectroscopy

Cannabis sativa L. plants produce a diverse array of secondary metabolites. Cannabis cell cultures were treated with jasmonic acid (JA) and pectin as elicitors to evaluate their effect on metabolism from two cell lines using NMR spectroscopy and multivariate data analysis. According to principal component analysis (PCA) and partial least square-discriminant analysis (PLS-DA), the chloroform extract of the pectin-treated cultures were more different than control and JA-treated cultures; but in the methanol/water extract the metabolome of the JA-treated cells showed clear differences with control and pectin-treated cultures. Tyrosol, an antioxidant metabolite, was detected in cannabis cell cultures. The tyrosol content increased after eliciting with JA.     

Elicitor-enhanced production of gymnemic acid in cell suspension cultures of <Emphasis Type="Italic">Gymnema sylvestre</Emphasis> R. Br.

Cell suspension cultures of Gymnema sylvestre treated with four different elicitors, methyl jasmonate (MJ), yeast extract, chitin and pectin were studied for the production of gymnemic acid as gymnemagenin equivalent, that was analyzed by high performance liquid chromatography (HPLC). All the four tested elicitors induced gymnemic acid production in cell suspension cultures. Highest gymnemic acid content was achieved following treatment with yeast extract (100.47 ± 0.28 mg/l), this was followed by MJ (70.43 ± 0.26 mg/l), pectin (64.19 ± 0.23 mg/l) and chitin (62.72 ± 0.13 mg/l). The addition of elicitors has shown a significant influence on cell growth that affected cell growth compared to respective controls. The highest gymnemic acid production was obtained after 20 days of elicitation in cultures treated with 0.5 g l^−l yeast extract, it was 5.25-folds greater than in control. These results suggest that the addition of an elicitor to Gymnema sylvestre cell suspension cultures could stimulate and enhance gymnemic acid production. In our present study we could able to overproduce gymnemic acid up to 51.97 ± 0.26 mg l^−l (dry weight basis) in yeast extract treated cell suspension cultures.     

Elicitation of benzophenanthridine alkaloid synthesis in Eschscholtzia cell cultures

Quaternary benzophenanthridine alkaloids (sanguinarine, chelerythrine, chelirubine, chelilutine and macarpine) are specifically induced by cell wall components of Penicillium and Saccharomyces in a colorless strain of Eschscholtzia californica cell suspension cultures. Classical elicitors such as the Phytophthora megasperma elicitor are inactive. The alkaloid synthesis is, however, strongly induced by certain polypeptide antibiotics. Out of 190 tested plant species the yeast elicitor provoked benzophenanthridine synthesis in 13 cultures. One of the branch point enzymes, namely the berberine bridge enzyme, catalysing the formation of (S)-scoulerine from (S)-reticuline, is strongly stimulated during the elicitation process. These results clearly demonstrate the induction of the benzophenanthridine biosynthetic pathway by microbial elicitors.Abbreviations ACC 1-Aminocyclopropane-1-carbonic acid - EDTA Ethylenediaminetetraacetic acid - LS-medium Linsmaier and Skoog medium - Pmg Phytophthora megasperma     

前体饲喂、诱导子和光照联合使用对葡萄细胞培养合成花青素的影响

苯丙氨酸前体饲喂分别和环糊精、葡聚糖、茉莉酸甲酯、黑曲霉和直喙镰孢菌提取液五种诱导子联合作用,其中以与茉莉酸甲酯的联合作用对葡萄细胞培养生产花青素的影响最大,可使单位鲜细胞花青素含量提高2.7倍,花青素产量提高3.4倍,实验证明两者在培养后第4天加入效果最好。在30μmol/L苯丙氨酸、218μmol/L茉莉酸甲酯和3000~4000lx光照条件下,不同花青素产量的细胞株都能显著提高花青素产量,但低产株VV06比高产株VV05具有更大的产率提高潜力。该条件下VV05和VV06花青素产量分别达到2975和4090CV/L,是对照组的2.5倍和5.2倍。     

Phenylpropanoid defence responses in transgenic Lotus corniculatus: II. Modelling plant defence responses in transgenic root cultures using thiol and carbohydrate elicitors

Transformed root cultures of Lotus corniculatus L. cv. Leo weretreated with a range of thiol and carbohydrate elicitors. Boththiol reagents and fungal carbohydrate preparations resultedin an increase in the activity of phenylalanine ammonia lyase(PAL) in a concentration-dependent manner. One representativethiol elicitor, glutathione (GSH), and one fungal elicitor,derived from Rhynchosporium orthosporum autoclaved cell walls(Ro), were analysed in more detail. Both elicitors induced thetransient accumulation of vestitol, an isoflavan phytoalexin,in tissue and in culture medium. Treatment of Lotus root cultureswith the Ro elicitor resulted in a more rapid initial accumulationof this end product when compared with GSH, however, sativan(the 2–methoxy ester of vestitol) previously reportedto co-accumulate in Lotus leaves was only detected followingelicitation with high concentrations of GSH. Ro and GSH elicitorsalso induced the accumulation of a number of other phenylpropanoidcompounds putatively identified as chalcones. The addition ofthiol and carbohydrate elicitors to Lotus root cultures alsoresulted in characteristic changes in root morphology. Glutathione,in particular, resulted in the inhibition of root growth dueto differential damage of meristem cells. Key words: Lotus corniculatus, hairy roots, elicitors, phytoalexins.