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.     

Transport of flavonolignans to the culture medium of elicited cell suspensions of Silybum marianum

Cell suspension cultures of Silybum marianum are able to excrete silymarin compounds into the medium upon elicitation with methyl jasmonate or cyclodextrins. Knowledge of transport mechanism is important to understand Sm metabolism and to develop strategies aimed at increasing production by means of cell cultures. For these reasons, a pharmacological approach was undertaken in this work in order to elucidate the possible mechanism involved in the release of this class of secondary metabolites into the extracellular medium of suspensions.     

Silybum marianum: non‐medical exploitation of the species

Silybum marianum (milk thistle) is currently utilised as a medicinal plant which provides raw material for the production of silymarin. Silymarin is composed of a group of flavonolignans that are well known and studied for their medicinal properties. However, the biomass yield potential and the chemical composition of its biomass suggest opportunities for much broader utilisation of S. marianum. This paper reviews the available literature about S. marianum biomass productivity and composition and about properties of products and byproducts of silymarin extraction. Alternative uses of whole plant biomass include fodder, bioenergy production and phytoremediation. Byproducts of silymarin extraction such as oil and flour have possible applications in food, feed and cosmetics. Moreover, the review explores potential alternative applications of silymarin. We conclude that a pivotal issue for further utilisation of S. marianum is the development of improved cultivars suited for the different possible utilisations.     

Differential accumulation of monolignol-derived compounds in elicited flax (Linum usitatissimum) cell suspension cultures

Lignin and lignans share monolignols as common precursors and are both potentially involved in plant defence against pathogens. In this study, we investigated the effects of fungal elicitors on lignin and lignan metabolism in flax (Linum usitatissimum) cell suspensions. Cell suspension cultures of flax were treated with elicitor preparations made from mycelium extracts of Botrytis cinerea, Phoma exigua and Fusarium oxysporum F ssp lini. Elicitors induced a rapid stimulation of the monolignol pathway, as confirmed by the increase in PAL (phenylalanine ammonia-lyase, EC 4.1.3.5), CCR (cinnamoyl-CoA reductase EC 1.2.1.44) and CAD (cinnamyl alcohol dehydrogenase EC 1.1.1.195) gene expression and PAL activity. At the same time, CCR activity only increased significantly in F. oxysporum-treated cells 24 h post elicitation. On the other hand, CAD activity measured for coniferyl alcohol formation was transiently decreased but a substrate-specific activation of CAD activity was observed in F. oxysporum-treated cells when using sinapyl alcohol as substrate. The accumulation of monolignol-derived products varied according to the elicitor used. B. cinerea or P. exigua-elicited cell cultures were characterised by a reinforcement of the cell wall by a deposit of 8-O-4′-linked non-condensed lignin structures and phenolic monomers, while at the same time no stimulation of 8-8′-linked lignan or 8-5′-linked phenylcoumaran lignan accumulation was observed. Additionally, elicitation of cell cultures with F. oxysporum extracts even triggered a strong incorporation of monolignols in the non condensed labile ether-linked lignin fraction concomitantly with a decrease in lignan and phenylcoumaran lignan accumulation. Several hypotheses are proposed to explain the putative role of these compounds in the defence response of flax cells against pathogens. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users. C. Hano and M. Addi contributed equally to this work.     

Biosynthesis of plant-specific phenylpropanoids by construction of an artificial biosynthetic pathway in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Biological synthesis of plant secondary metabolites has attracted increasing attention due to their proven or assumed beneficial properties and health-promoting effects. Phenylpropanoids are the precursors to a range of important plant metabolites such as the secondary metabolites belonging to the flavonoid/stilbenoid class of compounds. In this study, engineered Escherichia coli containing artificial phenylpropanoid biosynthetic pathways utilizing tyrosine as the initial precursor were established for production of plant-specific metabolites such as ferulic acid, naringenin, and resveratrol. The construction of the artificial pathway utilized tyrosine ammonia lyase and 4-coumarate 3-hydroxylase from Saccharothrix espanaensis, cinnamate/4-coumarate:coenzyme A ligase from Streptomyces coelicolor, caffeic acid O-methyltransferase and chalcone synthase from Arabidopsis thaliana, and stilbene synthase from Arachis hypogaea.     

Characterization of basic <Emphasis Type="Italic">p</Emphasis>-coumaryl and coniferyl alcohol oxidizing peroxidases from a lignin-forming <Emphasis Type="Italic">Picea abies</Emphasis> suspension culture

A Norway spruce (Picea abies) tissue culture line that produces extracellular lignin into the culture medium has been used as a model system to study the enzymes involved in lignin polymerization. We report here the purification of two highly basic culture medium peroxidases, PAPX4 and PAPX5, and isolation of the corresponding cDNAs. Both isoforms had high affinity to monolignols with apparent K_m values in μM range. PAPX4 favoured coniferyl alcohol with a six-fold higher catalytic efficiency (V_max/K_m) and PAPX5 p-coumaryl alcohol with a two-fold higher catalytic efficiency as compared to the other monolignol. Thus coniferyl and p-coumaryl alcohol could be preferentially oxidized by different peroxidase isoforms in this suspension culture, which may reflect a control mechanism for the incorporation of different monolignols into the cell wall. Dehydrogenation polymers produced by the isoforms were structurally similar. All differed from the released suspension culture lignin and milled wood lignin, in accordance with previous observations on the major effects that e.g. cell wall context, rate of monolignol feeding and other proteins have on polymerisation. Amino acid residues shown to be involved in monolignol binding in the lignification-related Arabidopsis ATPA2 peroxidase were nearly identical in PAPX4 and PAPX5. This similarity extended to other peroxidases involved in lignification, suggesting that a preferential structural organization of the substrate access channel for monolignol oxidation might exist in both angiosperms and gymnosperms.     

Are polyamines directly involved in silymarin production in the milk thistle [<Emphasis Type="Italic">Silybum marianum</Emphasis> (L.) Gaernt]?

The possible role of polyamines (PAs) in the regulation of silymarin (Sm) production in milk thistle [Silybum marianum (L.) Gaernt] cell suspension cultures was studied in a young cell culture line (H2 line) and in a synchronized cell line (>3 years; H1 line). The effect of two exogenous PAs, putrescine (Put) and spermidine (Spd), and a number of metabolic inhibitors (L-canavanine, DL-α-difluoromethylornithine, methylglyoxal-bis-guanylhydrazone, cyclohexylamine) on the production of Sm during the growth cycle were analyzed. The results suggest that PAs are not directly involved in the Sm synthesis pathway. In our cell culture system, Sm production and PA contents were determined by the age of the suspension culture cells: with increasing age, the suspension culture cells showed a decreasing capacity to reach the stationary phase during prolonged subculture that was associated with a decreased production of Sm, a steady increase in PA content, and a constant Put/Spd ratio. The synchronization of dividing cells from the S. marianum H1 line did not modify this behaviour. In young cell suspensions, maximum Sm production occurred in the stationary phase, concurrent with the cellular PA contents reaching their minimum value. At the start of the stationary phase, the high percentage of cells in the growth phases (G₀/G₁) and a transient increase in the Put/Spd ratio were accompanied by maximum Sm production and a blockade of cell division.     

Methyl jasmonate increases silymarin production in Silybum marianum (L.) Gaernt cell cultures treated with β-cyclodextrins

Silymarin (Sm) from the fruit of Silybum marianum is an isomeric mixture of pharmacologically active flavonolignans which are formed by oxidative coupling of taxifolin (Tx) and coniferyl alcohol (CA). Suspension cultures of this plant constitutively secrete small amounts of Sm into the extracellular medium. Production can be increased by inclusion of cyclodextrins (CDs) in cultures. Both hydroxylated (RHCD) and dimethylated (RMCD) CDs strongly induced prompt accumulation of CA in the medium followed by a late production of flavonolignans. Simultaneous addition of methyl jasmonate (MJ) and RMCD to cells did not significantly modify CA release or flavonolignan accumulation. Delayed addition of MJ to cultures subcultivated in medium containing RMCD markedly influenced Sm production by promoting conversion of the previously formed CA precursor.     

Modulation of antioxidant enzymes,SIRT1 and NF‐κB by resveratrol and nicotinamide in alcohol‐aflatoxin B1‐induced hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the fifth most commonly diagnosed cancer worldwide and is associated with poor prognosis. The current study aimed to assess the therapeutic efficacy of resveratrol when administered alone and in combination with nicotinamide against alcohol‐aflatoxin B1‐induced HCC. Results reveal that during the development and progression of cancer, there was a decline in the level of antioxidant enzymes catalase, glutathione peroxidase, glutathione reductase (GR), antioxidant glutathione, and glutathione S‐transferase, which is an enzyme of detoxification pathways. Treatment of resveratrol restored the level of catalase and glutathione peroxidase toward normal in alcohol‐aflatoxin B1‐induced HCC; however, nicotinamide worked in concert with resveratrol only in upregulating the activity of glutathione reductase, glutathione level, and glutathione S‐transferase. SIRT1 agonist resveratrol was observed to modulate the activity of antioxidant enzymes by negatively regulating the expression of nuclear factor‐κB (NF‐κB) in alcohol‐aflatoxin B1‐induced HCC, thereby suggesting a cross‐talk between antioxidant enzymes SIRT1 and NF‐κB during the development and progression of HCC and its therapeutics by resveratrol and nicotinamide.     

Production of highly bioactive resveratrol analogues pterostilbene and piceatannol in metabolically engineered grapevine cell cultures

Grapevine stilbenes, particularly trans‐resveratrol, have a demonstrated pharmacological activity. Other natural stilbenes derived from resveratrol such as pterostilbene or piceatannol, display higher oral bioavailability and bioactivity than the parent compound, but are far less abundant in natural sources. Thus, to efficiently obtain these bioactive resveratrol derivatives, there is a need to develop new bioproduction systems. Grapevine cell cultures are able to produce large amounts of easily recoverable extracellular resveratrol when elicited with methylated cyclodextrins and methyl jasmonate. We devised this system as an interesting starting point of a metabolic engineering‐based strategy to produce resveratrol derivatives using resveratrol‐converting enzymes. Constitutive expression of either Vitis vinifera resveratrol O‐methyltransferase (VvROMT) or human cytochrome P450 hydroxylase 1B1 (HsCYP1B1) led to pterostilbene or piceatannol, respectively, after the engineered cell cultures were treated with the aforementioned elicitors. Functionality of both gene products was first assessed in planta by Nicotiana benthamiana agroinfiltration assays, in which tobacco cells transiently expressed stilbene synthase and VvROMT or HsCYP1B1. Grapevine cell cultures transformed with VvROMT produced pterostilbene, which was detected in both intra‐ and extracellular compartments, at a level of micrograms per litre. Grapevine cell cultures transformed with HsCYP1B1 produced about 20 mg/L culture of piceatannol, displaying a sevenfold increase in relation to wild‐type cultures, and reaching an extracellular distribution of up to 45% of total production. The results obtained demonstrate the feasibility of this novel system for the bioproduction of natural and more bioactive resveratrol derivatives and suggest new ways for the improvement of production yields.     

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.     

Unexpected behavior of coniferin in lignin biosynthesis of Ginkgo biloba L

To gain insight into the behavior of monolignol glucoside in Ginkgo biloba L., we examined glucosides potentially involved in lignin biosynthetic pathway. Coniferin (coniferyl alcohol 4O--D-glucoside) is a strong candidate for the storage form of monolignol. Coniferaldehyde glucoside may also have a role in lignin biosynthesis; this was examined with tracer experiments using labeled glucosides fed to stem segments. A series of tracer experiments showed that coniferin and coniferaldehyde glucoside were modified into coniferyl alcohol and then efficiently incorporated into lignin under the experimental conditions used. Interestingly, more than half of the administered coniferin underwent an oxidation to the aldehyde form before its aglycone; coniferyl alcohol was polymerized into lignin. This suggests that there is an alternative pathway for coniferin to enter the monolignol biosynthetic pathway, in addition to the direct pathway beginning with the deglucosylation of coniferin catalyzed by -glucosidase. Enzymatic assays revealed that coniferaldehyde glucoside was produced enzymatically from coniferin, and that coniferaldehyde glucoside can be deglucosylated to yield coniferaldehyde, which could be fated to become coniferyl alcohol . Albeit the findings cannot be taken as proof for the in-planta functioning, these results present a possibility for the existence of alternative pathway in which some of the stored coniferin is oxidized to coniferaldehyde glucoside, which is deglucosylated to generate coniferaldehyde that joins the monolignol biosynthesis pathway.     

Small Glycosylated Lignin Oligomers Are Stored in Arabidopsis Leaf Vacuoles

Lignin is an aromatic polymer derived from the combinatorial coupling of monolignol radicals in the cell wall. Recently, various glycosylated lignin oligomers have been revealed in Arabidopsis thaliana. Given that monolignol oxidation and monolignol radical coupling are known to occur in the apoplast, and glycosylation in the cytoplasm, it raises questions about the subcellular localization of glycosylated lignin oligomer biosynthesis and their storage. By metabolite profiling of Arabidopsis leaf vacuoles, we show that the leaf vacuole stores a large number of these small glycosylated lignin oligomers. Their structural variety and the incorporation of alternative monomers, as observed in Arabidopsis mutants with altered monolignol biosynthesis, indicate that they are all formed by combinatorial radical coupling. In contrast to the common believe that combinatorial coupling is restricted to the apoplast, we hypothesized that the aglycones of these compounds are made within the cell. To investigate this, leaf protoplast cultures were cofed with ¹³C₆-labeled coniferyl alcohol and a ¹³C₄-labeled dimer of coniferyl alcohol. Metabolite profiling of the cofed protoplasts provided strong support for the occurrence of intracellular monolignol coupling. We therefore propose a metabolic pathway involving intracellular combinatorial coupling of monolignol radicals, followed by oligomer glycosylation and vacuolar import, which shares characteristics with both lignin and lignan biosynthesis.     

Expression of a bacterial 3‐dehydroshikimate dehydratase reduces lignin content and improves biomass saccharification efficiency

Lignin confers recalcitrance to plant biomass used as feedstocks in agro‐processing industries or as source of renewable sugars for the production of bioproducts. The metabolic steps for the synthesis of lignin building blocks belong to the shikimate and phenylpropanoid pathways. Genetic engineering efforts to reduce lignin content typically employ gene knockout or gene silencing techniques to constitutively repress one of these metabolic pathways. Recently, new strategies have emerged offering better spatiotemporal control of lignin deposition, including the expression of enzymes that interfere with the normal process for cell wall lignification. In this study, we report that expression of a 3‐dehydroshikimate dehydratase (QsuB from Corynebacterium glutamicum) reduces lignin deposition in Arabidopsis cell walls. QsuB was targeted to the plastids to convert 3‐dehydroshikimate – an intermediate of the shikimate pathway – into protocatechuate. Compared to wild‐type plants, lines expressing QsuB contain higher amounts of protocatechuate, p‐coumarate, p‐coumaraldehyde and p‐coumaryl alcohol, and lower amounts of coniferaldehyde, coniferyl alcohol, sinapaldehyde and sinapyl alcohol. 2D‐NMR spectroscopy and pyrolysis‐gas chromatography/mass spectrometry (pyro‐GC/MS) reveal an increase of p‐hydroxyphenyl units and a reduction of guaiacyl units in the lignin of QsuB lines. Size‐exclusion chromatography indicates a lower degree of lignin polymerization in the transgenic lines. Therefore, our data show that the expression of QsuB primarily affects the lignin biosynthetic pathway. Finally, biomass from these lines exhibits more than a twofold improvement in saccharification efficiency. We conclude that the expression of QsuB in plants, in combination with specific promoters, is a promising gain‐of‐function strategy for spatiotemporal reduction of lignin in plant biomass.     

Caffeoyl coenzyme A O-methyltransferase and lignin biosynthesis.

Lignin, a complex phenylpropanoid compound, is polymerized from the monolignols p-coumaryl alcohol, coniferyl alcohol and sinapyl alcohol. These three monolignols differ only by the 3- and 5-methoxyl groups. Therefore, enzymatic reactions controlling the methylations of the 3- and 5-hydroxyls of monolignol precursors are critical to determine the lignin composition. Recent biochemical and transgenic studies have indicated that the methylation pathways in monolignol biosynthesis are much more complicated than we have previously envisioned. It has been demonstrated that caffeoyl CoA O-methyltransferase plays an essential role in the synthesis of guaiacyl lignin units as well as in the supply of substrates for the synthesis of syringyl lignin units. Caffeic acid O-methyltransferase has been found to essentially control the biosynthesis of syringyl lignin units. These new findings have greatly enriched our knowledge on the methylation pathways in monolignol biosynthesis.     

Evaluation of the yeast-extract signaling pathway leading to silymarin biosynthesis in milk thistle hairy root culture

The biosynthesis of silymarin, a potent antihepatotoxic compound, from the dried fruits of Silybum marianum L. Gaertn in hairy root cultures can be stimulated by a yeast extract elicitor. These results correlated with culture time, and the biosynthesis reached a maximum of 0.47 mg g^?1 DW by 72 h after culture (2-fold higher than the control). Lipoxygenase activity and linoleic acid content were stimulated by this treatment, suggesting that the jasmonate pathway may mediate the elicitor-induced accumulation of silymarin. The H₂O₂ content increased 24 h after elicitation and did not have marked changes between 48 and 72 h. In addition, the tocopherol content (especially α- and δ-tocopherols) increased 72 h after elicitation in comparison with non-treated cultures. Ascorbate had trace changes during feeding time and was lower than the control. The antioxidant activity was assayed by the 1-1-diphenyl-2-picrylhydrazyl stable free radical method and results were calculated base on an IC₅₀ that increased upon treatment, especially 24 h after treatment, with changes related to H₂O₂ content. These observations suggested that reactive oxygen species may mediate elicitor signals to the jasmonate pathway that lead to the production of silymarin.     

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.