Lignans in plant cell and organ cultures: An overview

Lignans are found in a wide variety of plant species. The lignan podophyllotoxin is of special interest, since its derivatives like e.g. etopophos® are used in anticancer therapy. As chemical synthesis of podophyllotoxin is not yet economic, it still has to be isolated from wild growing Podophyllum species, some of which are considered to be endangered species. Therefore plant in vitro cultures may serve as alternative sources for podophyllotoxin and for other types of lignans as well. This review describes the establishment of plant cell and tissue cultures for lignan production and the experiments to improve product yields by changing the cultivation parameters, addition of elicitors and feeding of precursors. It also summarizes the use of plant cell and organ cultures to study the biosynthesis of lignans on enzymological level. Abbreviations: DOP – deoxypodophyllotoxin; LARI – lariciresinol; MATAI – matairesinol; 6MPTOX – 6-methoxypodophyllotoxin; PINO – pinoresinol; PTOX – podophyllotoxin; SECO – secoisolariciresinol     

Lignan accumulation in two-phase cultures of <Emphasis Type="Italic">Taxus</Emphasis> x <Emphasis Type="Italic">media</Emphasis> hairy roots

The biosynthetic potential for six lignans accumulation in two lines of Taxus x media hairy roots was investigated. The cultures of KT and ATMA hairy root lines were supplemented with precursors: coniferyl alcohol (CA 1, 10 or 100 µM) and/or l-phenylalanine (100 µM PHEN) and/or methyl jasmonate (100 µM MeJa). Moreover the two-phase in vitro cultures supported with perfluorodecalin (PFD) as a gas carrier and in situ extrahent were used. The hairy root lines differed in lignan production profiles. In the control untreated cultures KT roots did not accumulate secoisolariciresinol and lariciresinol while ATMA roots did not accumulate matairesinol. In ATMA roots the treatment with CA (1 or 10 µM) resulted in the production of lariciresinol and secoisolariciresinol whereas solely lariciresinol was present after 100 µM CA application. Elicitation with 1 µM CA and MeJa yielded with hydroxymatairesinol aglyca and lariciresinol glucosides with their highest content 37.88 and 3.19 µg/g DW, respectively. The stimulatory effect of simultaneous treatment with 1 µM CA, PHEN and MeJa on lignan production was observed when the cultures were supplemented with PFD-aerated or degassed. In ATMA root cultures these applied conditions were the most favourable for matairesinol content which amounted to 199.86 and 160.25 µg/g DW in PFD-aerated and PFD-degassed supported cultures, respectively. In KT root cultures solely, hydroxymatairesinol and coniferin/CA content was enhanced with their highest yield 59.29 and 134.60 µg/g DW in PFD-aerated and PFD-degassed cultures, respectively.     

Phenylpropanoid derivatives and biflavones at different stages of differentiation and development of Araucaria angustifolia

Chemical investigations carried out with tissues at different developmental stages of Araucaria angustifolia established the presence of E and Z isomers of octadecyl p-coumarate and octadecyl ferulate in undifferentiated callus; in the seedling stems, the source of explants, three biflavones of the amentoflavone-type were isolated, whereas the diterpene, trans-communic acid, was obtained from the seedling roots. Adult stems accumulated the benzaldehydes, vanillin, p-hydroxybenzaldehyde and coniferaldehyde; the lignans, pinoresinol, eudesmin and lariciresinol; and the isoflavones, cabreuvine and irisolidone.     

Significant enhancement of lignan accumulation in hairy root cultures of Linum album using biotic elicitors

Linum album has been shown to accumulate some lignans with antiviral and anticancer properties such as podophyllotoxin (PTOX) and 6-methoxy podophyllotoxin (MPTOX). In this research, we examined the effects of fungal elicitors on the production of lignans in L. album hairy root cultures. The biosynthesis of lignans was differentially affected by fungal elicitors. Fusarium graminearum extract induced the highest increase of PTOX, 190 μg g^?1 dry weight (DW), and lariciresinol, 260 μg g^?1 DW, which was two-fold and three-fold greater than the untreated control, respectively, while Trichoderma viride extract enhanced the accumulation of MPTOX, instead of PTOX, up to 160 µg g^?1 DW, which was 2.4-fold greater than the control. The enhancing effects of fungal elicitors on lignans production was correlated with the increased expression of some key genes involved in the biosynthesis of these compounds, phenylalanine ammonia-lyase, cinnamoyl-CoA reductase, cinnamyl-alcohol dehydrogenase and pinoresinol-lariciresinol reductase.     

Diversity in lignan biosynthesis

Lignans are phenylpropanoid dimers, where the phenylpropane units are linked by the central carbon (C8) of their side chains. Ligans vary substantially in oxidation level, substitution pattern, and the chemical structure of their basic carbon framework. In addition to structural diversity, lignans show considerable diversity in terms of enantiomeric composition, biosynthesis, and phylogenetic distribution. In this review, these diversities are outlined and the phylogenetic distribution of plants producing 66 typical lignans is listed. The distribution is correlated with the putative biosynthetic pathways of the lignans and discussed from evolutionary aspects. Abbreviations: SIRD – Secoisolariciresinol dehydrogenase; PLR – pinoresinol lariciresinol reductase; DP – dirigent protein     

Influence of methyl jasmonate and coniferyl alcohol on pinoresinol and matairesinol accumulation in a Forsythia × intermedia suspension culture

The accumulation of the lignans pinoresinol and matairesinol (both predominantly as glucosides) in a Forsythia 2 intermedia cell suspension culture was enhanced about three- and sevenfold, respectively, by the addition of methyl jasmonate to the cell culture medium. Cells accumulated 0.86ǂ.19 mg/g dry weight pinoresinol and 2.24ǃ.00 mg/g dry weight matairesinol. Feeding experiments with the precursor coniferyl alcohol resulted in a fast increase in the pinoresinol content, but matairesinol accumulation was not influenced. The racemic ratio of pinoresinol was 77dž% (+)-pinoresinol in methyl jasmonate-treated cells and 21Dž% (+)-pinoresinol in cells fed with coniferyl alcohol.     

Naphthohydroquinones and lignans from the roots of Plocama pendula, a Canary Island paleoendemism

Two naphthohydroquinones, mollugin 6-methyl ether and plocanaphthin, have been obtained from the roots of Plocama pendula (Rubiaceae), a paleoendemism of the Canary Islands. The former is a new compound, while the latter has now been isolated for the first time from nature. Other compounds obtained from this species were the lignans syringaresinol, pinoresinol and lariciresinol, and the coumarin scopoletin.     

RNAi-mediated pinoresinol lariciresinol reductase gene silencing in flax (Linum usitatissimum L.) seed coat: Consequences on lignans and neolignans accumulation

RNAi technology was applied to down regulate LuPLR1 gene expression in flax (Linum usitatissimum L.) seeds. This gene encodes a pinoresinol lariciresinol reductase responsible for the synthesis of (+)-secoisolariciresinol diglucoside (SDG), the major lignan accumulated in the seed coat. If flax lignans biological properties and health benefits are well documented their roles in planta remain unclear. This loss of function strategy was developed to better understand the implication of the PLR1 enzyme in the lignan biosynthetic pathway and to provide new insights on the functions of these compounds. RNAi plants generated exhibited LuPLR1 gene silencing as demonstrated by quantitative RT-PCR experiments and the failed to accumulate SDG. The accumulation of pinoresinol the substrate of the PLR1 enzyme under its diglucosylated form (PDG) was increased in transgenic seeds but did not compensate the overall loss of SDG. The monolignol flux was also deviated through the synthesis of 8-5′ linked neolignans dehydrodiconiferyl alcohol glucoside (DCG) and dihydro-dehydrodiconiferyl alcohol glucoside (DDCG) which were observed for the first time in flax seeds.     

Successful expression of a novel bacterial gene for pinoresinol reductase and its effect on lignan biosynthesis in transgenic Arabidopsis thaliana

Pinoresinol reductase and pinoresinol/lariciresinol reductase play important roles in an early step of lignan biosynthesis in plants. The activities of both enzymes have also been detected in bacteria. In this study, pinZ, which was first isolated as a gene for bacterial pinoresinol reductase, was constitutively expressed in Arabidopsis thaliana under the control of the cauliflower mosaic virus 35S promoter. Higher reductive activity toward pinoresinol was detected in the resultant transgenic plants but not in wild-type plant. Principal component analysis of data from untargeted metabolome analyses of stem, root, and leaf extracts of the wild-type and two independent transgenic lines indicate that pinZ expression caused dynamic metabolic changes in stems, but not in roots and leaves. The metabolome data also suggest that expression of pinZ influenced the metabolisms of lignan and glucosinolates but not so much of neolignans such as guaiacylglycerol-8-O-4′-feruloyl ethers. In-depth quantitative analysis by liquid chromatography–tandem mass spectrometry (LC-MS/MS) indicated that amounts of pinoresinol and its glucoside form were markedly reduced in the transgenic plant, whereas the amounts of glucoside form of secoisolariciresinol in transgenic roots, leaves, and stems increased. The detected levels of lariciresinol in the transgenic plant following β-glucosidase treatment also tended to be higher than those in the wild-type plant. Our findings indicate that overexpression of pinZ induces change in lignan compositions and has a major effect not only on lignan biosynthesis but also on biosynthesis of other primary and secondary metabolites.     

Generation of Triple-Transgenic Forsythia Cell Cultures as a Platform for the Efficient,Stable, and Sustainable Production of Lignans

Sesamin is a furofuran lignan biosynthesized from the precursor lignan pinoresinol specifically in sesame seeds. This lignan is shown to exhibit anti-hypertensive activity, protect the liver from damages by ethanol and lipid oxidation, and reduce lung tumor growth. Despite rapidly elevating demand, plant sources of lignans are frequently limited because of the high cost of locating and collecting plants. Indeed, the acquisition of sesamin exclusively depends on the conventional extraction of particular Sesamum seeds. In this study, we have created the efficient, stable and sustainable sesamin production system using triple-transgenic Forsythia koreana cell suspension cultures, U18i-CPi-Fk. These transgenic cell cultures were generated by stably introducing an RNAi sequence against the pinoresinol-glucosylating enzyme, UGT71A18, into existing CPi-Fk cells, which had been created by introducing Sesamum indicum sesamin synthase (CYP81Q1) and an RNA interference (RNAi) sequence against pinoresinol/lariciresinol reductase (PLR) into F. koreanna cells. Compared to its transgenic prototype, U18i-CPi-Fk displayed 5-fold higher production of pinoresinol aglycone and 1.4-fold higher production of sesamin, respectively, while the wildtype cannot produce sesamin due to a lack of any intrinsic sesamin synthase. Moreover, red LED irradiation of U18i-CPi-Fk specifically resulted in 3.0-fold greater production in both pinoresinol aglycone and sesamin than production of these lignans under the dark condition, whereas pinoresinol production was decreased in the wildtype under red LED. Moreover, we developed a procedure for sodium alginate-based long-term storage of U18i-CPi-Fk in liquid nitrogen. Production of sesamin in U18i-CPi-Fk re-thawed after six-month cryopreservation was equivalent to that of non-cryopreserved U18i-CPi-Fk. These data warrant on-demand production of sesamin anytime and anywhere. Collectively, the present study provides evidence that U18i-CP-Fk is an unprecedented platform for efficient, stable, and sustainable production of sesamin, and shows that a transgenic and specific light-regulated Forsythia cell-based metabolic engineering is a promising strategy for the acquisition of rare and beneficial lignans.     

Hinokinin biosynthesis in Linum corymbulosum Reichenb

Due to their peculiar stereochemistry and numerous biological activities, lignans are of widespread interest. As only a few biosynthetic steps have been clarified to date, we aimed to further resolve the molecular basis of lignan biosynthesis. To this end, we first established that the biologically active lignan (−)-hinokinin could be isolated from in vitro cultures of Linum corymbulosum. Two hypothetical pathways were outlined for the biosynthesis of (−)-hinokinin. In both pathways, (+)-pinoresinol serves as the primary substrate. In the first pathway, pinoresinol is reduced via lariciresinol to secoisolariciresinol by a pinoresinol–lariciresinol reductase, and methylenedioxy bridges are formed later. In the second pathway, pinoresinol itself is the substrate for formation of the methylenedioxy bridges, resulting in consecutive production of piperitol and sesamin. To determine which of the proposed hypothetical pathways acts in vivo , we first isolated several cDNAs encoding one pinoresinol-lariciresinol reductase ( PLR-Lc1 ), two phenylcoumaran benzylic ether reductases ( PCBER-Lc1 and PCBER-Lc2 ), and two PCBER-like proteins from a cDNA library of L. corymbulosum. PLR-Lc1 was found to be enantiospecific for the conversion of (+)-pinoresinol to (−)-secoisolariciresinol, which can be further converted to give (−)-hinokinin. Hairy root lines with significantly reduced expression levels of the plr-Lc1 gene were established using RNAi technology. Hinokinin accumulation was reduced to non-detectable levels in these lines. Our results strongly indicate that PLR-Lc1 participates in (−)-hinokinin biosynthesis in L. corymbulosum by the first of the two hypothetical pathways via (−)-secoisolariciresinol.     

Characterization of Arabidopsis thaliana pinoresinol reductase, a new type of enzyme involved in lignan biosynthesis

A lignan, lariciresinol, was isolated from Arabidopsis thaliana, the most widely used model plant in plant bioscience sectors, for the first time. In the A. thaliana genome database, there are two genes (At1g32100 and At4g13660) that are annotated as pinoresinol/lariciresinol reductase (PLR). The recombinant AtPLRs showed strict substrate preference toward pinoresinol but only weak or no activity toward lariciresinol, which is in sharp contrast to conventional PLRs of other plants that can reduce both pinoresinol and lariciresinol efficiently to lariciresinol and secoisolariciresinol, respectively. Therefore, we renamed AtPLRs as A. thaliana pinoresinol reductases (AtPrRs). The recombinant AtPrR2 encoded by At4g13660 reduced only (-)-pinoresinol to (-)-lariciresinol and not (+)-pinoresinol in the presence of NADPH. This enantiomeric selectivity accords with that of other PLRs of other plants so far reported, which can reduce one of the enantiomers selectively, whatever the preferential enantiomer. In sharp contrast, AtPrR1 encoded by At1g32100 reduced both (+)- and (-)-pinoresinols to (+)- and (-)-lariciresinols efficiently with comparative k(cat)/K(m) values. Analysis of lignans and spatiotemporal expression of AtPrR1 and AtPrR2 in their functionally deficient A. thaliana mutants and wild type indicated that both genes are involved in lariciresinol biosynthesis. In addition, the analysis of the enantiomeric compositions of lariciresinol isolated from the mutants and wild type showed that PrRs together with a dirigent protein(s) are involved in the enantiomeric control in lignan biosynthesis. Furthermore, it was demonstrated conclusively for the first time that differential expression of PrR isoforms that have distinct selectivities of substrate enantiomers can determine enantiomeric compositions of the product, lariciresinol.     

Establishment of Gymnema sylvestre hairy root cultures for the production of gymnemic acid

Gymnema sylvestre is an important medicinal plant that bears bioactive compound namely gymnemic acid. In the present study, G. sylvestre was transformed by Agrobacterium rhizogenes. Seedling explants namely roots, stems, hypocotyls, cotyledonary nodal segments, cotyledons and young leaves were inoculated with A. rhizogenes strain KCTC 2703. Transformed (hairy) roots were induced from cotyledons and leaf explants. Six transgenic clones of hairy roots were established and confirmed by polymerase chain reaction (PCR) and RT-PCR using rolC specific primers. Hairy roots cultured using MS liquid medium supplemented with 3 % sucrose showed highest accumulation of biomass (97.63 g l^?1 FM and 10.92 g l^?1 DM) at 25 days, whereas highest accumulation of gymnemic acid content (11.30 mg g^?1 DM) was observed at 20 days. Nearly 9.4-fold increment of biomass was evident in suspension cultures at 25 days of culture and hairy root biomass produced in suspension cultures possessed 4.7-fold higher gymnemic acid content when compared with the untransformed control roots. MS-based liquid medium was superior for the growth of hairy roots and production of gymnemic acid compared with other culture media evaluated (B5, NN and N6), with MS-based liquid medium supplemented with 3 % sucrose was optimal for secondary metabolite production. The current results showed great potentiality of hairy root cultures for the production of gymnemic acid.     

Enhanced production of phytoestrogenic isoflavones from hairy root cultures of Psoralea corylifolia L. Using elicitation and precursor feeding

The effect of biotic elicitors (yeast extract, chitosan), signaling molecule (salicylic acid), and polyamines (putrescine and spermidine) was studied with respect to isoflavones accumulation in hairy root cultures of Psoralea corylifolia L. Untreated hairy roots (control) accumulated 1.55% dry wt of daidzein and 0.19% dry wt of genistein. In precursor feeding experiment, phenylalanine at 2 mM concentration led to 1.3 fold higher production of daidzein (1.91% dry wt) and genistein (0.27% dry wt). In biotic elicitors, chitosan (2 mg/L) was found to be the most efficient elicitor to induce daidzein (2.78% dry wt) and genistein (0.279% dry wt) levels in hairy roots. Salicylic acid at 1 mM concentration stimulated the maximum accumulation of daidzein (2.2% dry wt) and genistein (0.228% dry wt) 2 days after elicitation. In case of polyamines, putrescine (50 mM) resulted in highest accumulation of daidzein (3.01% dry wt) and genistein (0.227% dry wt) after 5 days of addition. Present results indicated the effectiveness of elicitation and precursor feeding on isoflavones accumulation in hairy roots of P. corylifolia. This is the first report of elicitation on isoflavones production by hairy roots of P. corylifolia.     

Stereochemistry of lignans in Phaleria macrocarpa (Scheff.) Boerl

Phaleria macrocarpa (Scheff.) Boerl., a member of the Thymelaeaceae, is traditionally used in Indonesia as medicinal plant against cancer. In this context, we isolated the lignans pinoresinol, lariciresinol and matairesinol from different parts of this plant. The enantiomeric composition of these lignans was determined by chiral column analysis. Pinoresinol and lariciresinol were mixtures of both enantiomers with (79 +/- 4)% and (55 +/- 6)% enantiomeric excess for the (-)-enantiomers, respectively, whereas matairesinol was found as pure (+)-enantiomer.     

ENHANCEMENT OF RUTIN PRODUCTION IN Fagopyrum tataricum HAIRY ROOT CULTURES WITH ITS ENDOPHYTIC FUNGAL ELICITORS

Tartary buckwheat (Fagopyrum tataricum) is a potentially important source of rutin, a natural bioactive flavonoid with antihyperglycemic, antioxidative, antihypertensive, and anti-inflammatory properties. This study examines the effects of endophytic fungi on rutin production in the hairy root cultures of F. tataricum. Without obvious changes in the appearance of the hairy roots, the exogenous fungal mycelia elicitors efficiently stimulated the hairy root growth and rutin biosynthesis, and the stimulation effect was mainly dependent on the mycelia elicitor species, as well as its treatment dose. Two endophytic fungal isolates Fat9 (Fusarium oxysporum) and Fat15 (Alternaria sp.) were screened as promising candidates for promoting F. tataricum hairy root growth and rutin production. With application of polysaccharide (PS) of endophyte Fat9 (200 mg/L), and PS of endophyte Fat15 (100 mg/L) to the hairy root cultures on day 25, the rutin yield was increased to 45.9 mg/L and 47.2 mg/L, respectively. That was about 3.1- to 3.2-fold in comparison with the control level of 14.6 mg/L. Moreover, the present study revealed that the accumulation of rutin resulted from the stimulation of the phenylpropanoid pathway by mycelia PS treatments. This may be an efficient strategy for enhancing rutin production in F. tataricum hairy root culture provided with its endophytic mycelia elicitors.     

Hairy roots of Dracocephalum moldavica: rosmarinic acid content and antioxidant potential

Hairy roots of Dracocephalum moldavica L. were induced using Agrobacterium rhizogenes strain A4. Transformed roots were obtained from shoot explants with low transformation frequency of up to 3 %. The effects of different liquid media: Murashige and Skoog (MS), Gamborg et al. (B5) and Woody Plant (WP) with full- and half-strength (½MS, ½B5, ½WP), on biomass accumulation and rosmarinic acid (RA) content were investigated. The hairy roots were cultured in photoperiod (16 h light/8 h dark) and darkness. Biomass of D. moldavica hairy roots was the highest (7.23 g flask^?1 of fresh weight and 0.89 g flask^?1 of dry weight) in the cultures grown in WP medium under periodic light. Ultra performance liquid chromatography analysis revealed the highest RA content (78 mg g^?1 dry wt) in roots cultured in ½B5 medium under photoperiod conditions. It was about tenfold higher compared to roots of field-grown mother plants. Antioxidant activities and total phenolic contents of methanolic extracts of D. moldavica hairy roots cultured in ½B5 and WP media under photoperiod and darkness and roots of field grown plants were compared. All extracts were investigated using 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging and phosphomolybdenum reduction assays. Total phenolic contents were estimated by the Folin–Ciocalteu method. The methanolic extract of D. moldavica hairy roots grown in ½B5 medium under photoperiod possessed the strongest effects on reducing Mo and DPPH radical scavenging. The activities were significantly higher (p ≤ 0.05) than those of methanolic extract of roots of intact plants grown in the field. The most active methanolic extract of hairy roots was characterized by the highest level of rosmarinic acid and total content of phenolic compounds.