Enhanced accumulation of flavonolignans in Silybum marianum cultured roots by methyl jasmonate

Root cultures of Silybum marianum (L.) Gaertn. (Asteraceae) were established from in vitro germinated sterile plantlets. The cultures grew in hormone-free Murashige and Skoog medium. The flavonolignan content in the cultured roots was determined by HPLC using 30% acetonitrile in acidified water (0.5% phosphoric acid). The major flavonolignans produced by the cultured roots were silychristin (74.2 μg g^?1 fresh weight (FW)) and silydianin (8.1 μg g^?1 FW). The flavonolignan precursor taxifolin was also detected in the cultured roots (40.8 μg g^?1 FW). Addition of methyl jasmonate to 7-days-old root cultures for 48 h increased the content of the produced flavonolignans and taxifolin to approximately 300% of the control cultures. Methyl jasmonate also enhanced about sixfold the accumulation of a compound identified as 3,3′,5,5′,7-pentahydroxyflavanone.     

Enhanced production of isoflavones by elicitation in hairy root cultures of Soybean

A combined treatment of sonication (2 min) and vacuum infiltration (2 min) stimulated isoflavones production of 75.26 mg g^?1 DW which was 15.11-fold higher than control hairy root line at optimal harvest time of 40 days. Addition of MeJ at 100 μM concentration with 72 h exposure time on 30 day-old hairy root culture further enhanced total isoflavones production of 53.16 mg g^?1 DW (10.67-fold) and SA at 200 μM concentration with 96 h exposure period enhanced the production of isoflavones (28.79 mg g^?1 DW; 5.78-fold). MeJ-treated hairy roots reduced biomass accumulation whereas sonication, vacuum infiltration and SA did not exhibit a negative effect on biomass growth.     

Molecular cloning and expression profiling of a chalcone synthase gene from hairy root cultures of Scutellaria viscidula Bunge

A cDNA encoding chalcone synthase (CHS), the key enzyme in flavonoid biosynthesis, was isolated from hairy root cultures of Scutellaria viscidula Bunge by rapid amplification of cDNA ends (RACE). The full-length cDNA of S. viscidula CHS, designated as Svchs (GenBank accession no. EU386767), was 1649 bp with a 1170 bp open reading frame (ORF) that corresponded to a deduced protein of 390 amino acid residues, a calculated molecular mass of 42.56 kDa and a theoretical isoelectric point (pI) of 5.79. Multiple sequence alignments showed that SvCHS shared high homology with CHS from other plants. Functional analysis in silico indicated that SvCHS was a hydrophilic protein most likely associated with intermediate metabolism. The active sites of the malonyl-CoA binding motif, coumaroyl pocket and cyclization pocket in CHS of Medicago sativa were also found in SvCHS. Molecular modeling indicated that the secondary structure of SvCHS contained mainly α-helixes and random coils. Phylogenetic analysis showed that SvCHS was most closely related to CHS from Scutellaria baicalensis. In agreement with its function as an elicitor-responsive gene, the expression of Svchs was induced and coordinated by methyl jasmonate. To our knowledge, this is the first report to describe the isolation and expression of a gene from S. viscidula.     

Alkaloid production by hairy root cultures in Atropa belladonna

Hairy roots were induced by inoculation of stems of sterile plants of Atropa belladonna with Agrobacterium rhizogenes. The axenic culture of the hairy roots isolated from the stems proliferated 60 fold as based on the initial fresh weight after one month of culture. The presence of atropine and scopolamine in hairy roots were examined by TLC and HPLC. Their amounts were analyzed by GLC. The results show that the amount of the two alkaloids in the axenic cultures was the same as or even higher than those of normal plants grown in the field.     

Identification of flavonolignans from Silybum marianum seeds as allosteric protein tyrosine phosphatase 1B inhibitors

Protein tyrosine phosphatase 1B (PTP1B) is an attractive molecular target for anti-diabetes, anti-obesity, and anti-cancer drug development. From the seeds of Silybum marianum, nine flavonolignans, namely, silybins A, B (1, 2), isosilybins A, B (3, 4), silychristins A, B (5, 6), isosilychristin A (7), dehydrosilychristin A (8), and silydianin (11) were identified as a novel class of natural PTP1B inhibitors (IC₅₀ 1.3 7–23.87?µM). Analysis of structure–activity relationship suggested that the absolute configurations at C-7" and C-8" greatly affected the PTP1B inhibitory activity. Compounds 1–5 were demonstrated to be non-competitive inhibitors of PTP1B based on kinetic analyses. Molecular docking simulations resulted that 1–5 docked into the allosteric site, including α3, α6, and α7 helix of PTP1B. At a concentration inhibiting PTP1B completely, compounds 1–5 moderately inhibited VHR and SHP-2, and weakly inhibited TCPTP and SHP-1. These results suggested the potentiality of these PTP1B inhibitors as lead compounds for further drug developments.     

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.     

In vitro cultures of Silybum marianum and silymarin accumulation

In this study, a protocol for initiation of callus and shoot cultures from leaves and shoot tips explants of different silybium genotypes collected from different locations in Egypt was established. Callus cultures were initiated from leaves explants and exposed to different concentrations of the precursor (coniferyl alcohol). Shoot cultures were initiated from shoot tips explants. Moreover, the produced plants of the different Silybium shoots as well as intact plants were subjected to protein screening using SDS–PAGE analysis.Results obtained revealed that the optimum medium for growth and maintenance of friable callus was MS medium supplemented with 0.25 mg L⁻¹ 2,4-Dichlorophenoxy acetic acid (2,4-D) + 0.25 mg L⁻¹ Kinetin (Kin). The best medium for proliferation of high number of shoots was MS-medium with 0.25 mg L⁻¹ each of Benzyl Adinine (BA) and Naphthalene Acetic Acid (NAA). Coniferyl alcohol in concentration of 30 μM caused an increase in accumulation of silymarin contents in most callus cultures. SDS–PAGE of different Silybium shoots revealed that the protein profiles of 100% of in vitro produced plantlets similar to their control.     

Alkaloid production in Duboisia hybrid hairy root cultures overexpressing the pmt gene

Putrescine:SAM N-methyltransferase (PMT) catalyses the N-methylation of the diamine putrescine to form N-methylputrescine, the first specific precursor of both tropane and pyridine-type alkaloids, which are present together in the roots of Duboisia plants. The pmt gene of Nicotiana tabacum was placed under the regulation of the CaMV 35S promoter and introduced into the genome of a scopolamine-rich Duboisia hybrid by a binary vector system using the disarmed Agrobacterium tumefaciens strain C58C1 carrying the rooting plasmid pRiA4. The presence of the foreign gene in kanamycin-resistant hairy roots and its overexpression were confirmed by polymerase chain reaction and Northern blot analysis respectively. The N-methylputrescine levels of the resulting engineered hairy roots increased (2-4-fold) compared to wild type roots, but there was no significant increase in either tropane or pyridine-type alkaloids.     

Indole alkaloid production by hairy root cultures of Catharanthus roseus

Hairy root cultures of Catharanthus roseus were established by infection with six different Agrobacterium rhizogenes strains. Two plant varieties were used and found to exhibit significantly different responses to infection. Forty-seven hairy root clones derived from normal plants and two derived from the flowerless variety were screened for their growth and indole alkaloid production. The growth rate and morphological appearance showed wide variations between the clones. The alkaloid spectra observed were qualitatively but not quantitatively very similar to that of the corresponding normal plant roots. No vindoline or deacetyltransferase activity could be detected in any of the cultures studied. O-acetylval-lesamine, an alkaloid which has not been previously observed in C. roseus was identified from extracts of hairy root clone No. 8. Two root clones were examined for their growth and alkaloid accumulation during a 26-day culture period. Alkaloid accumulation parallelled growth in both clones with ca. 2 mg ajmalicine and catharanthine per g dry weight being observed.Dedicated to Dr. Friedrich Constabel on the occasion of his 60th birthday     

Silymarin synthesis and degradation by peroxidases of cell suspension cultures of Silybum marianum

Treatment of Silybum marianum cell cultures with methyl jasmonate elicits the production of the antihepatotoxic drug silymarin and its release into the culture medium. In this work, we investigated the involvement of peroxidases (EC 1.11.1.7; donor hydrogen peroxidase oxido-reductase) in silymarin turnover in cell cultures as well as the influence of elicitation on the activity towards several substrates. Peroxidases from cell extracts and, to a higher degree from the spent medium, used the silymarin precursors taxifolin and coniferyl alcohol as substrates. Silymarin compounds were also degraded by suspension culture peroxidases; however, the oxidation efficiency was not modified by elicitation. S. marianum peroxidases were able to catalyse the oxidative coupling of taxifolin and coniferyl alcohol to silybinins. The synthetic activity was mainly associated with the extracellular compartment and as before, methyl jasmonate did not modify oxidative coupling activity. Changes in the isoenzyme profiles were not observed in elicited cultures.     

Enhanced glucotropaeolin production in hairy root cultures of Tropaeolum majus L. by combining elicitation and precursor feeding

We have studied the effects of precursor amino acids (phenylalanine and cystein), elicitors (salicylic and acetylsalicylic acids, methyl jasmonate, β-aminobutyric acid, yeast extract), PAL-inhibitor (1-amino-2-phenylethylphosphonic acid) applied alone or in combination on glucotropaeolin production and myrosinase activity in hairy root cultures of Tropaeolum majus. Short, 24-h treatment, and subsequent transfer of hairy roots to the fresh medium enabled avoiding detrimental effect of studied stimulators on biomass growth. In control cultures the highest glucotropaeolin content, 58.1 ± 6.7 mg g⁻¹ DW, was detected on the 3rd day after transfer of the roots to the fresh medium but glucotropaeolin yield (mg 100 ml^–1 culture volume) had been increasing until the 9th day after transfer as a result of continuous biomass growth. Glucotropaeolin content and yield were 2-fold enhanced after treatment with precursor amino acids or PAL-inhibitor alone, but their combination additively led to 4-fold increase in glucotropaeolin production. Among the studied elicitors acetylsalicylic acid induced the highest, 3-fold increase in glucotropaeolin production, it also enhanced myrosinase activity, but to a smaller extend (by about 50%). Acetylsalicylic acid also potentiated induced by precursors, PAL-inhibitor, methyl jasmonate and yeast extract production of glucotropaeolin. The highest, 4.8-fold increase in glucotropaeolin production was found after combined acetylsalicylic acid and precursors treatment. Additive effect of acetylsalicylic acid-combined treatment on myrosinase activity was not detected. The obtained results indicate that amino acid precursors, phenylalanine and cystein, availability may be a limiting factor in the process of stimulation of glucotropaeolin production in T. majus hairy root cultures.     

Bioreactor production of camptothecin by hairy root cultures of Ophiorrhiza pumila

A large-scale culture of hairy root of Ophiorrhiza pumila using a modified 3 l bioreactor was established. The hairy roots, incited by infection of Agrobacterium rhizogenes were grown in the bioreactor equipped with a stainless net. The final concentration of camptothecin was 0.0085% fresh wt of tissue, and the total production of camptothecin, an anti-neoplastic quinoline alkaloid, reached 22 mg over 8 weeks' culture in the reactor. Approx. 17% (3.6 mg) of the total camptothecin produced was excreted into the culture medium.     

Enhanced production of antimicrobial sesquiterpenes and lipoxygenase metabolites in elicitor-treated hairy root cultures of Solanum tuberosum

Potato (Solanum tuberosum) hairy root cultures, established by infecting potato tuber discs with Agrobacterium rhizogenes, were used as a model system for the production of antimicrobial sesquiterpenes and lipoxygenase (LOX) metabolites. Of the four sesquiterpene phytoalexins (rishitin, lubimin, phytuberin and phytuberol) detected in elicitor-treated hairy root cultures, rishitin (213 g g^–1 dry wt) was the most predominant followed by lubimin (171 g g^–1 dry wt). The elicitors also induced LOX activity (25-fold increase) and LOX metabolites, mainly 9-hydroxyoctadecadienoic acid and 9-hydroxyoctadecatrienoic acid, in potato hairy root cultures. The combination of fungal elicitor plus cyclodextrin was the most effective elicitor treatment, followed by methyl jasmonate plus cyclodextrin in inducing sesquiterpenes and LOX metabolites.     

Lipid composition of Silybum marianum cell cultures treated with methyl jasmonate

Elicitation of cell cultures of Silybum marianum with methyl jasmonate (MeJA) increases the production and release of the secondary metabolite silymarin into the culture medium and this process seems to be dependent on phospholipase D activity and its product phosphatidic acid (PA). However, MeJA did not alter total membrane lipid content or overall fatty acid composition. A progressive increase in some galactolipids was observed with elicitation time. Phospholipids were mainly represented by phosphatidylcholine (PC) followed by phosphatidylethanolamine (PE) and phosphatidylinositol (PI). MeJA caused losses of PC species that contain two unsaturated acyl species, 36:5 and 36:6 and an increase in 36:2 species. A drop in the ratio of compounds with 18:3 in PI and PE was also observed. The presence of the lysophospholipids (LP) LPC (16:0, 18:3, 18:2, 18:1) and LPE (16:0, 18:3, 18:2, 18:1) and the high contents of PA, represented by the molecular species 34:3, 34:2 and 36:5 and 36:4, indicates high basal level of phospholipase activity in cultures and a high phospholipid turnover. MeJA treatment did not quantitatively alter these lipid classes.     

Enhanced production of azadirachtin by hairy root cultures of Azadirachta indica A. Juss by elicitation and media optimization

Azadirachtin is one of the most potent biopesticides so far developed from a plant sources. Influence of different culture media and elicitation on growth and production of azadirachtin by hairy root cultures of Azadirachta indica was studied. Out of the three media tested, namely Ohyama and Nitsch, Gamborg's and Murashige and Skoog's basal media, hairy roots cultured on Ohyama and Nitsch's basal medium produced maximum yield of azadirachtin (0.0166% dry weight, DW). Addition of biotic elicitor enhanced the production of azadirachtin by approximately 5-fold (0.074% DW), while signal compounds such as jasmonic acid and salicylic acid showed a approximately 6 (0.095% DW) and approximately 9-fold (0.14% DW) enhancement, respectively, in the production of azadirachtin as compared to control cultures on Ohyama and Nitsch medium. Extracts from hairy roots were found to be superior to those from the leaves for antifeedant activity against the larvae of Spodoptera litura.     

Enhanced silymarin accumulation is related to calcium deprivation in cell suspension cultures of Silybum marianum (L.) Gaertn

Elimination of calcium ions from the medium of cell cultures of Silybum marianum (L.) Gaertn increased flavonolignan production. Silymarin accumulation was not altered by treatment of cultures with the calcium ionophore A23187. The specific Ca2+ chelator, EGTA, enhanced the silymarin content in cells by 200%, and its secretion by 3-4 times. The inorganic ion La3+, as well as the calcium channel inhibitor verapamil, also stimulated production. Several reagents known to block intracellular calcium movement, such as ruthenium red, thapsigargin and TMB-8 appreciably increased silymarin accumulation. These results suggest that inhibition of external and internal calcium fluxes plays a significant role in flavonolignan metabolism of S. marianum cell cultures.     

Yeast extract and methyl jasmonate-induced silymarin production in cell cultures of Silybum marianum (L.) Gaertn

The biosynthesis of the flavonolignan silymarin, a constitutive compound of the fruits of Silybum marianum with strong antihepatotoxic and hepatoprotective activities, is severely reduced in cell cultures of this species. It is well known that elicitation is one of the strategies employed to increase accumulation of secondary metabolites. Our study here reports on the effect of several compounds on the production of silymarin in S. marianum cultures. Yeast extract (YE), chitin and chitosan were compared with respect to their effects on silymarin accumulation in S. marianum suspensions and only yeast extract stimulated production. Jasmonic acid (JA) potentiated the yeast extract effect. One of the jasmonic acid derivatives, methyl jasmonate (MeJA), strongly promoted the accumulation of silymarin. Methyl jasmonate acted in a number of steps of the metabolic pathway of flavonolignans and its stimulating effect was totally dependent of de novo protein synthesis. Chalcone synthase (CHS) activity was enhanced by methyl jasmonate; however there did not appear to be a temporal relationship between silymarin accumulation and increase in enzyme activity. Also, this increase was not blocked by the protein synthesis inhibitor cycloheximide (CH). This study indicates that elicitor treatment promotes secondary metabolite production in S. marianum cultures and that jasmonic acid and its functional analogue plays a critical role in elicitation.     

Overexpression of the Saussurea medusa chalcone isomerase gene in S. involucrata hairy root cultures enhances their biosynthesis of apigenin

Saussurea involucrata is a medicinal plant well known for its flavonoids, including apigenin, which has been shown to significantly inhibit tumorigenesis. Since naturally occurring apigenin is in very low abundance, we took a transgenic approach to increase apigenin production by engineering the flavonoid pathway. A construct was made to contain the complete cDNA sequence of the Saussurea medusa chalcone isomerase (CHI) gene under the control of the cauliflower mosaic virus (CaMV) 35S promoter. Using an Agrobacterium rhizogenes-mediated transformation system, the chi overexpression cassette was incorporated into the genome of S. involucrata, and transgenic hairy root lines were established. CHI converts naringenin chalcone into naringenin that is the precursor of apigenin. We observed that transgenic hairy root lines grew faster and produced higher levels of apigenin and total flavonoids than wild-type hairy roots did. Over a culture period of 5 weeks, the best-performing line (C46) accumulated 32.1 mgL(-1) apigenin and 647.8 mgL(-1) total flavonoids, or 12 and 4 times, respectively, higher than wild-type hairy roots did. The enhanced productivity corresponded to elevated CHI activity, confirming the key role that CHI played for total flavonoids and apigenin synthesis and the efficiency of the current metabolic engineering strategy.