Effect of elicitors on the production of gossypol and methylated gossypol in cotton hairy roots

The effect of two chemical elicitors, salicylic acid and methyl jasmonate, on the production of gossypol, 6-methoxygossypol, and 6,6′-dimethoxygossypol in Gossypium barbadense hairy roots was examined. Methyl jasmonate, but not salicylic acid, was found to increase the production of gossypol and its methylated forms, but with a concomitant reduction in culture growth. The optimal methyl jasmonate dose was between 100 and 300 μM for hairy roots harvested 7 days after elicitation. After 20 d of induction with 100 μM methyl jasmonate, an eightfold increase in the level of gossypol was observed in elicited cultures compared with control cultures, double the highest gossypol levels previously reported for any cotton tissue. A two to threefold increase in the level of 6-methoxygossypol and a slight increase in the levels of 6,6′-dimethoxygossypol were also observed. Although methyl jasmonate stimulated the production of both optical forms of gossypol, the distribution of the enantiomers was different between elicited and control cultures.     

The effects of elicitation on the expression of key enzyme genes and on production of tropane alkaloids in <Emphasis Type="Italic">Anisodus acutangulus</Emphasis> plant

Tropane alkaloids (TAs) including hyoscyamine, anisodamine, anisodine and scopolamine are widely used as anticholinergic agents, which act on the parasympathetic nervous system, and their clinical demands are increasing rapidly. Anisodus acutangulus is a solanaceous perennial plant that is endemic to China with good potential for TA production and classified as an endangered species in China. Therefore there is a need to improve the TA production in this resource plant by biology methods. In our research, three different important elicitors including methyl jasmonate (MJ), yeast elicitor (YE) and abscisic acid (ABA) were used to study their effects on TA production, and genes expressions in A. acutangulus plant under each treatment were examined. The results revealed that TA production was induced by all the three treatments. However, its quantity varies with inducers used. The effect induced by MJ and YE were significant, while the effect induced by ABA was not obviously. To our knowledge, this is the first report on the relationship between TA production and genes expression profile induced by different elicitors in cultivated A. acutangulus plant, which should provide some useful information on how to improve TA production with the cultivated A. acutangulus plant in the near future.     

Improved isoflavonoid production in Pueraria candollei hairy root cultures using elicitation

The effect of abiotic and biotic elicitors (methyl jasmonate, chitosan, salicylic acid, Agrobacterium, and yeast extract) at various concentrations on total isoflavonoid accumulation was studied in the hairy root cultures of Pueraria candollei. All elicitors stimulated isoflavonoid production. Yeast extract (0.5 mg/ml) was the most efficient giving total isoflavonoids at 60 ± 1 mg/g dry wt, which was 4.5-fold higher than control hairy roots on day 3 of elicitation.     

Enhanced paclitaxel production induced by the combination of elicitors in cell suspension cultures of Taxus chinensis

Taxus chinensis suspension cells were cultured in the modified Gamborg's B5 medium. Addition of 50 mg chitosan l^–1, 60 M methyl jasmonate and 30 M Ag⁺ resulted in the greatest paclitaxel production, at 25 mg l^–1 in the cultures, being almost 40 times higher than that of the control culture, 10 times higher than that of the culture exposed to Ag⁺, 6 times higher than that of the culture elicited by chitosan and almost double that of the culture elicited by methyl jasmonate.     

Molecular cloning and mRNA expression profiling of the first specific jasmonate biosynthetic pathway gene allene oxide synthase from <Emphasis Type="Italic">Hyoscyamus niger</Emphasis>

In the endeavor to enhance the production of pharmaceutically valuable tropane alkaloids including hyoscyamine and scopolamine in Hyoscyamus niger, methyl jasmonate (MeJA) showed significant stimulation both in tropane biosynthetic pathway enzymes activities and tropane alkaloids yields. Therefore it was speculated that genetic engineering of jasmonate biosynthetic pathway might enhance the endogenous jasmonates concentration, followed by stimulating the production of tropane alkaloids. Herein a full-length cDNA encoding allene oxide synthase (AOS, EC 4.2.1.92), the first committed step enzyme in jasmonate biosynthetic pathway was reported (named HnAOS, GenBank accession: EF532599). HnAOS was a novel member of the cytochrome P450 (CYP74A) subfamily. Real-time quantitative PCR analysis showed that HnAOS mRNA accumulated mainly in stems, and responded significantly to wounding or methyl jasmonate. The article is published in the original.     

Optimization of induction and culture conditions and tropane alkaloid production in hairy roots of <Emphasis Type="Italic">Anisodus acutangulus</Emphasis>

In this study, an efficient transformation system for the medicinal plant Anisodus acutangulus was successfully developed and optimized using Agrobacterium rhizogenes. Three bacterial strains, A4, R1601, and modified C58C1 and three explant types, leaf blade, petiole, and stem, were examined. The highest transformation efficiency of 94.44% was achieved using strain C58C1 with stem explants. Over 20 independent hairy root lines were successfully established with strain C58C1 using stem explants, all of which contained the ro/B and ro/C genes as confirmed by polymerase chain reaction (PCR). Out of four media compositions, the liquid 1/2 MS medium was found the most suitable for hairy root growth. The maximum biomass of one hairy root line increased up to 80 times in liquid 1/2 MS medium after a 30 day culture period. Different hairy root lines displayed a varied capacity for tropane alkaloid production and the best hairy root line (T4) from the C58C1-stem combination produced up to 10.21 mg/g (dw) of hyoscyamine, which was about 1.5-fold higher than in the wild type plants. To our knowledge, this is the first report to demonstrate the production of tropane alkaloids in hairy roots of A. acutangulus.     

Effects of biotic and abiotic elicitors on cell growth and tanshinone accumulation in Salvia miltiorrhiza cell cultures

This study examined the effects of biotic and abiotic elicitors on the production of diterpenoid tanshinones in Salvia miltiorrhiza cell culture. Four classes of elicitors were tested, heavy metal ions (Co²⁺, Ag⁺, Cd²⁺), polysaccharides (yeast extract and chitosan), plant response-signaling compounds (salicylic acid and methyl jasmonate), and hyperosmotic stress (with sorbitol). Of these, Ag (silver nitrate), Cd (cadmium chloride), and polysaccharide from yeast extract (YE) were most effective to stimulate the tanshinone production, increasing the total tanshinone content of cell by more than ten-fold (2.3 mg g^-1 versus 0.2 mg g^-1 in control). The stimulating effect was concentration-dependent, most significant at 25 μM of Ag and Cd and 100 mg l^-1 (carbohydrate content) of YE. Of the three tanshinones detected, cryptotanshinone was stimulated most dramatically by about 30-fold and tanshinones I and IIA by no more than 5-fold. Meanwhile, most of the elicitors suppressed cell growth, decreasing the biomass yield by about 50% (5.1–5.5 g l^-1 versus 8.9 g l^-1 in control). The elicitors also stimulated the phenylalanine ammonia lyase activity of cells and transient increases in the medium pH and conductivity. The results suggest that the elicitor-stimulated tanshinone accumulation was a stress response of the cells.     

Influence of feeding precursors on tropane alkaloid production during an abiotic stress in Datura innoxia transformed roots

The effects of feeding tropane alkaloid precursors in transformed root culture of Datura innoxia Mill. were studied during a stress treatment. The permeabilizing effect of Tween 20 on tropane alkaloid production by hairy root cultures was studied in flasks with different feeding of precursors (L-ornithine, L-arginine, L-phenylalanine, DL-β-phenyllactic acid, and tropinone). It has been shown that the addition of various precursors alone (0.5 m mol l ^-1) was ineffective in stimulating hyoscyamine production. In contrast, a short treatment with Tween 20, combined with L-phenylalanine feeding, amplified the level of hyoscyamine released into the medium compared with the Tween treatment alone. Thus, the total hyoscyamine content per flask was increased (+ 40%) compared with the control. When DL-β-phenyllactic acid (0.5 m mol l ^-1) was used, this last effect became more pronounced (+ 60%). These results show that permeabilization with Tween modulates tropane alkaloid accumulation by a release of alkaloids into the medium and a restoration of hyoscyamine root content. The simultaneous feeding of DL-β-phenyllactic acid and tropinone during the Tween treatment gave a similar effect to that obtained with DL-β-phenyllactic acid and Tween, suggesting that the synthesis of the tropate moiety determines the flux at the level of the esterification of tropine. This revised version was published online in June 2006 with corrections to the Cover Date.     

The effects of jasmonic acid and methyl jasmonate on rosmarinic acid production in <Emphasis Type="Italic">Mentha</Emphasis> × <Emphasis Type="Italic">piperita</Emphasis> cell suspension cultures

The effects of two elicitors: jasmonic acid and methyl jasmonate on cell growth as well as on rosmarinic acid accumulation in cell suspension cultures of Mentha × piperita were investigated. The highest rosmarinic acid accumulation 117.95 mg g⁻¹ DW (12% DW) was measured 24 h after addition of 100 μM methyl jasmonate. A similar concentration 110.12 mg g⁻¹ DW was detected 48 h after application of 200 μM jasmonic acid. Those values were nearly 1.5 times higher compared to the control sample, without elicitation. There was no substantial influence of elicitors on rosmarinic acid secretion into the culture media. Extracellular concentrations of rosmarinic acid were similar to the values from the control variants. It was documented that suspension cultures of M. piperita treated with elicitors showed a decrease in biomass accumulation when compared to the control.     

Tropane alkaloids production in transgenic Hyoscyamus niger hairy root cultures over-expressing Putrescine N-methyltransferase is methyl jasmonate-dependent

The cDNA from Nicotiana tabacum encoding Putrescine N-methyltransferase (PMT), which catalyzes the first committed step in the biosynthesis of tropane alkaloids, has been introduced into the genome of a scopolamine-producing Hyoscyamus niger mediated by the disarmed Agrobacterium tumefaciens strain C58C1, which also carries Agrobacterium rhizogenes Ri plasmid pRiA4, and expressed under the control of the CaMV 35S promoter. Hairy root lines transformed with pmt presented fivefold higher PMT activity than the control, and the methylputrescine (MPUT) levels of the resulting engineered hairy roots increased four to fivefold compared to the control and wild-type roots, but there was no significant increase in tropane alkaloids. However, after methyl jasmonate (MeJA) treatment, a considerable increase of PMTase and endogenous H6Hase as well as an increase in scopolamine content was found either in the transgenic hairy roots or the control. The results indicate that hairy root lines over-expressing pmt have a high capacity to synthesize MPUT, whereas their ability to convert hyoscyamine into scopolamine is very limited. Exposure to MeJA strongly stimulated both polyamine and tropane biosynthesis pathways and elicitation led to more or less enhanced production simultaneously.     

Improved cardenolide production in Calotropis gigantea hairy roots using mechanical wounding and elicitation

A hairy root culture system of Calotropis gigantea was established and effects of mechanical wounding (MW) and elicitors [methyl jasmonate (MJ), yeast extract (YE) and chitosan (CS)] on cardenolide production were investigated. All treatments stimulated the production of cardenolide in hairy root cultures of C. gigantea. CS was the most effective elicitor, followed by MJ. YE and MW also improved cardenolide yield in individual treatments. The highest cardenolide yield (1,050 ± 55 mg/l) was obtained after adding 50 mg CS/l for 20 days, which was 2.7-fold higher than the control.     

Enhanced production of scopolamine by bacterial elicitors in adventitious hairy root cultures of Scopolia parviflora

In an attempt to increase productivity, the effect of elicitation on tropane alkaloids (TA) biosynthesis was studied in adventitious hairy root cultures of Scopolia parviflora. Two Gram-positive strains and one Gram-negative strain of bacteria were used as biotic elicitors. The raw bacterial elicitors affected the tropane alkaloid profile by increasing the scopolamine concentration, while the autoclaved bacterial elicitors produced similar effects on the control. The conversion ratio of hyoscyamine to scopolamine was increased following elicitation using raw bacterial elicitors. The bacterial elicitor inhibited the expression of H6H (hyoscyamine 6β-hydoxylase) whereas the expression of PMT (putrescine N-methyltransferase) was raised by elicitation. These results have important implications for the large-scale production of tropane alkaloids.     

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.     

Norlittorine and norhyoscyamine identified as products of littorine and hyoscyamine metabolism by 13C-labeling in Datura innoxia hairy roots

The presence of two compounds, norlittorine and norhyoscyamine, has been reported in leaves and roots of Datura innoxia; however their metabolic origin in the tropane alkaloid pathway has remained unknown. Precise knowledge of this pathway is a necessary pre-requisite to optimize the production of hyoscyamine and scopolamine in D. innoxia hairy root cultures. The exact structure of norlittorine and norhyoscyamine was confirmed by LC–MS/MS and NMR analyses. Isotopic labeling experiments, using [1-¹³C]-phenylalanine, [1′-¹³C]-littorine and [1′-¹³C]-hyoscyamine, combined with elicitor treatments, using methyl jasmonate, coronalon and 1-aminocyclopropane-1-carboxylic acid, were used to investigate the metabolic origin of the N-demethylated tropane alkaloids. The results suggest that norlittorine and norhyoscyamine are induced under stress conditions by conversion of littorine and hyoscyamine. We propose the N-demethylation of tropane alkaloids as a mechanism to detoxify cells in overproducing conditions.     

CO2 enrichment and supporting material impact the primary metabolism and 20-hydroxyecdysone levels in Brazilian ginseng grown under photoautotrophy

Plant cell and organ cultures via the implementation of effective elicitation strategies can offer attractive biotechnological platforms for the enhanced production of phytochemicals of pharmaceutical interest. For the first time, the elicitation of exogenous signal molecules was conducted to enhance the production of pharmacologically active alkaloids and flavonoids in Isatis tinctoria L. hairy root cultures (ITHRCs). ITHRCs III and V correspondingly possessing high alkaloid and flavonoid productivity were adopted for elicitation treatments. The maximum accumulation of alkaloids in ITHRCs III elicited by 142.61 µM salicylic acid for 28.18 h and flavonoids in ITHRCs V elicited by 179.54 µM methyl jasmonate for 41.87 h increased 5.89- and 11.21-folds as compared with controls, respectively. Moreover, expressions of 11 genes involved in alkaloid and flavonoid biosynthetic pathways were significantly up-regulated following elicitation, among which YUCCA, CHI and F3′H genes might play a crucial role in the target phytochemical augmentation. Overall, two effective elicitation protocols were provided here to improve the yields of bioactive alkaloids and flavonoids in ITHRCs, which was useful for the scale-up production of these valuable compounds to meet the demands for natural bioactive ingredients by pharmaceutical industries.

     

A differential response to chemical elicitors in Catharanthus roseus in vitro cultures

The effects of methyl jasmonate, salicylic acid and ethylene on alkaloid accumulation in in vitro cell suspension, hairy roots and rootless shoot cultures of Catharanthus roseus were analyzed. Ajmalicine, but not catharanthine, accumulation was promoted by jasmonate and ethylene treatments in cell suspensions. In hairy roots, jasmonate induced the accumulation of both alkaloids, whereas ethylene only induced catharanthine accumulation. In shoot cultures, positive effects of jasmonate and ethylene were recorded only in vindoline accumulation. Ethylene diminished catharanthine accumulation in these cultures. No effect of salicylic acid was observed in any of the studied in vitro culture systems. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Characterization and expression profiling of 4-hydroxyphenylpyruvate dioxygenase gene (<Emphasis Type="Italic">Smhppd</Emphasis>) from <Emphasis Type="Italic">Salvia</Emphasis> <Emphasis Type="Italic">miltiorrhiza</Emphasis> hairy root cultures

A novel 4-hydroxyphenylpyruvate dioxygenase gene (designated as Smhppd) was cloned from hairy roots of Salvia miltiorrhiza Bung. The full-length cDNA of Smhppd was 1,736 bp long with an ORF (open reading frame) that putatively encoded a polypeptide of 481 amino acids, with a predicted molecular mass of 52.54 kDa. The deduced amino acid sequence of the Smhppd gene shared high homology with other known HPPDs. Analysis of Smhppd genomic DNA revealed that it contained two exons and one intron. The analysis of Smhppd promoter region was also presented. Southern-blot analysis revealed that the Smhppd was a low-copy gene in S. miltiorrhiza. Real-time quantitative PCR analysis indicated that Smhppd was constitutively expressed in roots, stems and leaves of S. miltiorrhiza, with the high expression in roots. In addition, Smhppd expreession level under different stress condition was also analyzed during the hairy root culture period, including signaling components for plant defence responses, such as methyl jasmonate and salicylic acid, as well as an abiotic elicitor, Ag⁺ and a biotic elicitor, yeast extract. This study will enable us to further understand the role Smhppd plays in the synthesis of active pharmaceutical compounds in S. miltiorrhiza at molecular level.     

Influence of nano‐zinc oxide on tropane alkaloid production,h6h gene transcription and antioxidant enzyme activity in Hyoscyamus reticulatus L. hairy roots

The use of nanotechnology and biotechnology to improve the production of plant bioactive compounds is growing. Hyoscyamus reticulatus L. is a major source of tropane alkaloids with a wide therapeutic use, including treatment of Parkinson's disease and to calm schizoid patients. In the present study, hairy roots were obtained from two‐week‐old cotyledon explants of H. reticulatus L. using the A7 strain of Agrobacterium rhizogenes. The effects of different concentrations of the signaling molecule nano‐zinc oxide (ZnO) (0, 50, 100 and 200 mg/L), with three exposure times (24, 48 and 72 h), on the growth rate, antioxidant enzyme activity, total phenol contents (TPC), tropane alkaloid contents and hyoscyamine‐6‐beta‐hydroxylase (h6h) gene expression levels were investigated. Growth curve analysis revealed a decrease in fresh and dry weight of ZnO‐treated hairy roots compared to the control. ANOVA results showed that the antioxidant activity of the enzymes catalase, guaiacol peroxidase and ascorbate peroxidase was significantly higher in the ZnO‐treated hairy roots than in the control, as was the TPC. The highest levels of hyoscyamine (37%) and scopolamine (37.63%) were obtained in hairy roots treated with 100 mg/L of ZnO after 48 and 72 h, respectively. Semi‐quantitative RT‐PCR analysis revealed the highest h6h gene expression was in hairy roots treated with 100 mg/L of ZnO after 24 h. It can be concluded that ZnO is as an effective elicitor of tropane alkaloids such as hyoscyamine and scopolamine due to its enhancing effect on expression levels of the biosynthetic h6h gene.     

A PIP-family protein is required for biosynthesis of tobacco alkaloids

Plants in the Nicotiana genus produce nicotine and related pyridine alkaloids as a part of their chemical defense against insect herbivores. These alkaloids are formed by condensation of a derivative of nicotinic acid, but the enzyme(s) involved in the final condensation step remains elusive. In Nicotiana tabacum, an orphan reductase A622 and its close homolog A622L are coordinately expressed in the root, upregulated by methyl jasmonate treatment, and controlled by the NIC regulatory loci specific to the biosynthesis of tobacco alkaloids. Conditional suppression of A622 and A622L by RNA interference inhibited cell growth, severely decreased the formation of all tobacco alkaloids, and concomitantly induced an accumulation of nicotinic acid β-N-glucoside, a probable detoxification metabolite of nicotinic acid, in both hairy roots and methyl jasmonate-elicited cultured cells of tobacco. N-methylpyrrolinium cation, a precursor of the pyrrolidine moiety of nicotine, also accumulated in the A622(L)-knockdown hairy roots. We propose that the tobacco A622-like reductases of the PIP family are involved in either the formation of a nicotinic acid-derived precursor or the final condensation reaction of tobacco alkaloids. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.