Plant Cell, Tissue and Organ Culture (PCTOC) https://doi.org/10.1007/s11240-018-1390-0

ORIGINAL ARTICLE

Lignan accumulation in two-phase cultures of Taxus x media hairy roots

K. Sykłowska‑Baranek1 · K. Łysik1 · M. Jeziorek1 · A. Wencel1 · M. Gajcy1 · A. Pietrosiuk1

Received: 3 August 2017 / Accepted: 6 February 2018 © The Author(s) 2018. This article is an open access publication

Abstract The biosynthetic potential for six lignans accumulation in two lines of Taxus x media hairy roots was investigated. The cul- tures 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 sup- ported 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.

Keywords Taxus · Hairy root cultures · Lignans · Elicitation · Perfluorodecalin · In situ extraction

Introduction Onrubia et al. (2013). Using various biotechnological meth- ods to enhance paclitaxel production the exceptional pro- Taxus spp. cell, tissue and organ culture systems have been gress has been made and finally the process was commer- developed for the production of paclitaxel and other taxanes, cialized (Frense 2007; Expósito et al. 2009). an important class of anticancer agents (Yared and Tkac- Lignans represent an abundant and widespread group zuk 2012), which was reviewed in detail by Zhong (2002), of compounds demonstrating a wide range of biological Tabata (2004, 2006), Maheshwari et al. (2008), Expósito activities, such as anticancer (review by Landete 2012), et al. (2009), Sabater-Jara et al. (2010), Malik et al. (2011), antimicrobial, anti-inflammatory (Küpeli et al.2003 ; Dur- ing et al. 2012), hypoglycemic (Banskota et al. 2006; reviews by: Erdemoglu et al. 2004; Saleem et al. 2005; Topcu and Communicated by Sergio J. Ochatt. Demirkiran 2007; Dar and Arumugam 2013), antioxidant (Masuda et al. 2010), cardioprotective (Zanwar et al. 2011) Electronic supplementary material The online version of this article (https​://doi.org/10.1007/s1124​0-018-1390-0) contains and anti-osteoporotic (Yin et al. 2006). Lignan food ingre- supplementary material, which is available to authorized users. dients such as secoisolariciresinol, matairesinol, laricires- inol and pinoresinol are conversed by intestinal bacteria * M. Jeziorek to enterolignans: enterodiol and enterolactone and were [email protected] reported to reduce the risk of some cancers and cardiovas- 1 Department of Pharmaceutical Biology and Medicinal cular diseases (Milder et al. 2007; Ghisalberti 1997; Landete Plant Biotechnology, Faculty of Pharmacy with Division 2012) and exhibited estrogen activity (Cassidy et al. 2000; of Laboratory Medicine, Medical University of Warsaw, 1 Aehle et al. 2011). Banacha, 02‑097 Warsaw, Poland

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Apart taxanes about 50 lignans were isolated from eight creating in the culture vessel an auxiliary phase below the Taxus species, and in the roots of T. x media the presence aqueous phase of the medium (Lowe et al. 2003; Davey et al. of hydroxymatairesinol and epi-nortrachelogenin was con- 2005). firmed (Topcu and Demirkiran2007 ). To our best knowledge, the current research represents an Lignans are composed of two phenylpropanoid ­C6C3 units original approach for an examination of the effectiveness of linked by central carbon (C8) of their propyl side chains various biotechnological methods, applied to enhance lignan (Umezawa 2003; Suzuki and Umezawa 2007; Topcu and production in Taxus x media hairy root cultures. In the first Demirkiran 2007). Their biosynthesis involves the shikimate stage of our investigation, carried out in ATMA root cul- pathway providing l-phenylalanine and required multiple tures, we examined the influence of various concentrations enzymatic steps (Vogt 2010; Umezawa 2010; Thoge et al. of CA added as a unique agent or in combination with MeJa 2013; Wang et al. 2013). This pathway originated with a and PHEN, on biomass and lignan accumulation. In the sec- chiral coniferyl alcohol and two units of it are enantioselec- ond stage the two-phase cultures created by perfluorodecalin tively coupled by dirigent protein resulting with pinoresinol. application, aerated or not, together with the most effective Pinoresinol is reduced through lariciresinol to secoisola- elicitation regime were applied to both ATMA and KT hairy riciresinol and eventually oxidized to matairesinol (Suzuki root cultures to determine their influence on root growth and and Umezawa 2007). Coniferyl alcohol and p-coumaryl lignan production. alcohol are the major building units of conifer lignin, a het- erogeneous polymer deposited during cell differentiation and secondary cell wall formation (Marjamaa et al. 2007; Materials and methods Wagner et al. 2012). Plant sources of lignans are considered to be limited Hairy root cultures due to insufficient methods of cultivation, long growth and development phases, and low lignan content (Satake et al. Two lines of Taxus x media hairy roots were investigated for 2015). Plant biotechnology methods offers the possibility their potential for lignan accumulation. The first hairy root to produce valuable plant secondary metabolites in cell and line—KT was established by transformation with wild type organ cultures derived from plants which are rare or con- Agrobacterium rhizogenes LBA 9402 strain (Furmanowa sidered as endangered due to their overexploitation from and Syklowska-Baranek 2000). While the second hairy natural sources (Bunn et al. 2011). Lignans, such as podo- root line—ATMA was obtained as a result of direct inocu- phyllotoxin and 5-methoxypodophyllotoxin, which are of lation of 10-year-old Taxus plantlets cultivated in vitro with special interest, since their derivatives etoposide and teni- the C58C1 strain of Agrobacterium tumefaciens carrying poside are used in cancer therapy (Bohlin and Rosén 1996), the plasmid RiA4 of A. rhizogenes and the binary plasmid could be accumulated in considerable amounts in in vitro pCAMBIA-TXS-His, harbouring the taxadiene synthase cultures of various species from Podophyllum and Linum (txs) gene of T. baccata (GenBank accession: AY424738), genera (Petersen and Alfermann 2001; Fuss 2003; Majumder under the control of the 35S CaMV promoter, and the hygro- and Jha 2009). The enhancement of lignan production in in mycin phosphotransferase gene (hptII) as a resistance marker vitro culture systems of various species was achieved by (Sykłowska-Baranek et al. 2015a). The cultures of KT and optimization of culture conditions including elaboration of ATMA hairy root lines were established in 1996 and 2008, the most suitable for product improvement carbon source, respectively. These hairy root lines were subsequently sub- phytohormones, culture conditions like light or dark regime, cultured into fresh hormone-free DCR-M medium every four elicitor treatments (reviewed in Fuss 2003), developing of weeks (250 ml Erlenmeyer flasks with 35 ml of medium, hairy root cultures (Kumar et al. 2012; Bahabadi et al. 2014; inoculum 0.51 ± 0.016 g FW) (Syklowska-Baranek et al. Wawrosch et al. 2014; Gabr et al. 2016, 2017; Tashackori 2009). The cultures were maintained at 25 ± 1 °C in the dark et al. 2016; Chashimi et al. 2016) and metabolic engineering on an INFORS AG TR 250 shaker (Switzerland) at 105 rpm. approaches (reviewed in Satake et al. 2015). There has been also one paper on in vitro cultivated Taxus x media cell sus- Elicitor and precursor feeding experiments pension and shoots where pinoresinol has been determined and revealed solely in shoots (Mistrzak et al. 2015). The cultures of ATMA 28-day old hairy roots growing in Our current studies were undertaken to elucidate the Erlenmeyer flasks with 35 ml DCR-M were supplemented influence of precursors and elicitor feeding on six lignans with precursors in lignan biosynthesis pathway: l-phenyla- accumulation in two lines of Taxus x media hairy roots. lanine (PHEN, 100 µM, Sigma-Aldrich), coniferyl alcohol Moreover we have applied the two-phase culture system con- (CA, Sigma-Aldrich) and elicitor: methyl jasmonate (MeJa; taining perfluorodecalin (PFD), fully synthetic, biochemi- 100 µM, Sigma-Aldrich). The following experiments were cally inert and immiscible with aqueous phases compound, carried out: (i) medium supplementation with CA at doses

1 3 Plant Cell, Tissue and Organ Culture (PCTOC) of 1, 10 or 100 µM; (ii) medium supplementation with CA was aseptically saturated with atmospheric air for 15 min. at doses of 1, 10 or 100 µM and MeJa 100 µM; (iii) medium according to the procedure described previously (Pilarek supplementation with CA at doses of 1, 10 or 100 µM, each and Szewczyk 2008). Afterwards, 20 ml of PFD, degassed concentration of CA with PHEN 100 µM and MeJa 100 µM. or aerated, was added to 35 ml of sterile culture medium Each precursor and elicitor, according to the experiment on the 14th day of culture. type, was added to the 28-day old hairy root culture. The PHEN was dissolved in distilled water and filtered through Lignan determination Sartorius 0.20 µm PTFE filter prior to application while MeJa and CA were dissolved in EtOH (Avantor Performance The determination of lignan aglyca and their glycosides was Materials Poland S.A., Poland) aseptically and applied to performed: (−)-hydroxymatairesinol, secoisolariciresinol the cultures. The amount of PHEN, CA and MeJa has been (racemic), lariciresinol (racemic) manufactured by Chroma- given as final concentration in medium. Dex, (−)-matairesinol and (+)-pinoresinol manufactured by The samples were harvested after 1 and 2 weeks after Phyto-Lab. All standards were purchased in LCG Standards application of precursors and/or elicitor. At each time point, (Poland). The content of CA was also determined. the roots were harvested, gently pressed on filter paper, and For the extraction of lignans from hairy roots a modified their fresh weight (FW) was recorded. After lyophilization, method of Wichers et al. (1990) after Schmitt and Petersen the dry weight (DW) of roots was determined. The fresh and (2002) was applied. 0.09 g DW ± 0.024 of lyophilized dry biomass increase was expressed as the ratio of initial powdered tissue of roots was suspended in 2 ml of metha- weight to final weight. nol (MeOH, Avantor Performance Materials Poland S.A., Poland) and extracted twice in an ultrasonic bath on ice for Perfluorodecalin phase supported experiments 15 min. Afterwards 8 ml of ­H2O (pH = 4.5) was added to the samples and next 10 ml of dichloromethane (CH­ 2Cl2, The roots of both hairy root lines were cultivated in liquid Avantor Performance Materials Poland S.A., Poland) and hormone-free DCR-M medium for 14 days and on the mixed for 30 min. The organic phase was separated and 14th day PFD-degassed or PFD-aerated were added to evaporated to dryness and stored at − 20 °C till HPLC analy- the cultures. After the subsequent 14 days, on day 28th sis. The remaining water phase (10 ml), containing lignan of culture, the mixture of 100 µM of PHEN, 1 µM of CA glycosides, was incubated with 1 mg of β-glucosidase from and 100 µM of MeJa were added to the cultivated with almonds (Sigma-Aldrich, Poland) for 3.5 h at 35 °C and then PFD hairy roots. extracted with 10 ml of ­CH2Cl2 as described above. The experiments with the application of perfluorodecalin Samples of water and organic phases were analyzed (PFD; C­ 10F18; 98% equimolar mixture of cis-/trans- isomers; by the HPLC-UV-DAD method. Prior to HPLC analysis, ABCR GmbH & Co. KG, Karlsruhe, Germany) were per- samples were re-dissolved in 100% MeOH (1 ml) and 20 formed in ATMA and KT transformed root cultures. µl underwent HPLC analysis using the DIONEX (USA) The samples were harvested at three time points:(i) after system with a UVD 340S diode array detector and an auto- 14 days of cultivation with PFD-degassed or PFD-aerated mated sample injector (ASI-100). A NovaPak Phenyl col- before medium supplementation with precursors (PHEN umn (150 × 3.9 mm) was eluted employing the following − 1 and CA) and elicitor (MeJa); (ii) one week after applica- gradient: flow 1 mlmin ­ , solvent A: H­ 2O with 0.04 M tion of precursors and elicitor; (iii) after 2 weeks past the of H­ 3PO4; solvent B: acetonitrile (Sigma-Aldrich, Poland). application of precursors and elicitor. The roots from three Gradient program: 0–20 min 40% B, 20–25 min 50% B. flasks were harvested, gently pressed on paper, and their Eluting substances were monitored by UV absorption at fresh weight was recorded. After lyophilization at vacuum 230 nm (Fig. 1S). 0.340 mbar and initial product temperature − 20 °C (lyo- All experiments were performed in triplicate and all results philizer Christ ALPHA1-4 LSC, Germany) the dry weight represent the mean of three repetitions ± SD. The statistical (DW) of roots was determined. The fresh and dry biomass significance between means was assessed using analysis of increase was expressed as the ratio of initial weight to final variance (ANOVA) and Tukey’s multiple range test. For weight. pairwise analysis Student’s t test was used. A probability of As a control the cultures of both line of transformed roots P < 0.05 was considered significant. cultivated in the medium without any supplementations were used. The PFD phase were prepared as described earlier (Sykłowska-Baranek et al. 2015b). In brief, PFD was autoclaved at 121 °C for 20 min to ensure aseptic condi- tions and degassing of PFD. To obtain the PFD-aerated, it

1 3 Plant Cell, Tissue and Organ Culture (PCTOC)

Results and discussion experiments—day 42th. At this time point the growth increase amounted to 1.9-fold which was over 2-times The influence of different culture conditions higher than in cultures treated solely with CA or CA on hairy roots growth and MeJa, regardless of the CA concentration (Fig. 1a, b). However, the growth reduction was less pronounced Coniferyl alcohol, l‑phenylalanine and methyl jasmonate when culture media were supplemented with CA accom- feeding experiments in ATMA hairy root line cultures panied with MeJa and PHEN (Fig. 1a). Moreover, in the presence of 1 µM CA together with MeJa and PHEN, the The fresh and dry biomass growth was investigated root biomass increase achieved 1.45-fold and 1.52-fold in through the course of the 42-day lasting cultures. The fresh and dry mass, respectively which means more than highest growth index was achieved in control cultures doubled values obtained in cultures treated only with 1 where the root growth was observed till the end of the µM CA and MeJa, 0.58-fold and 0.71-fold on the basis of FW and DW respectively. The detrimental to biomass

Fig. 1 The fresh and dry a biomass increase of: a ATMA 5.0 hairy root line cultivated for ] ATMA hairy root line 42 days in (i) control untreated 4.5 cultures; cultures supplemented 4.0 with coniferyl alcohol (CA at doses 1, 10 or 100 µM); (ii) 3.5 cultures supplemented with CA (at doses 1, 10 or 100 µM) and 3.0 methyl jasmonate (MeJA 100 µM) and (iii) cultures supple- 2.5 mented with CA (at doses 1, ease [final weight/initial weight * * 10 or 100 µM) combined with 2.0 l-phenylalanine (100 µM) and 1.5 methyl jasmonate (100 µM) at each CA concentration; b the 1.0 * * * * * * * fresh and dry biomass increase The biomass incr * * * * * * * * * of ATMA hairy root line and 0.5 KT hairy root line cultivated for 42 days in: (i) control untreated 0.0 cultures; (ii) cultures elicited FW DW FW DW FW DW FW DW FW DW FW DW FW DW FW DW FW DW FW DW without any PFD phases; (iii) ControlCA 1 µM CA 10 µM CA 100 µM CA 1 µM + CA 10 µM CA 100 µM CA 1 µM + CA 10 µM CA 100 µM MeJa + MeJa +MeJa PHEN + + PHEN + + PHEN + cultures unelicited supple- MeJa MeJa MeJa mented with PFD-aerated or PFD-degassed PFD phases and 28 DAY 35 DAY 42 DAY (iv) cultures elicited (with CA b 100 µM, l-phenylalanine 100 5.0 ] * µM and methyl jasmonate 100 ATMA nad KT hairy root lines 4.5 µM) supplemented with PFD- aerated or PFD-degassed PFD 4.0 phases. Asterisks (*) indicate a * significant difference P < 0.05 3.5 3.0 *** * 2.5 ***** * * ** ** * ease [final weight/initila weight

2.0 **** * ****

1.5 **

** * 1.0

The biomass incr 0.5

0.0 FW DW FW DW FW DW FW DW FW DW FW DW FW DW FW DW FW DW FW DW FW DW FW DW ATMA KT ATMA KT ATMA KT ATMA KT ATMA KT ATMA KT CONTROLCULTURES PFD DEGASSED PFD AERATED PFD DEGASSED PFD AERATED ELICITED UELICITIED UNELICITED ELICITED ELICITED WITHOUT PFD PHASES 14 DAY 28 DAY 35 DAY 42 DAY

1 3 Plant Cell, Tissue and Organ Culture (PCTOC) accumulation effect of precursor and/or elicitor treatments a non sufficient manner from hairy roots. So the dry biomass was statistically significant (P < 0.05) in comparison with growth is considered to be more appropriate to describe the control cultures with the exception of 1 µM CA together influence of investigated culture conditions. The fresh root with MeJa and PHEN simultaneous treatment. In earlier growth significantly (P < 0.05) has not been lower than in studies of our group where we examined the influence of the control when elicitors were added to the medium supple- precursor or/and elicitor treatment on taxane content in mented with PFD-degassed or PFD-aerated (Fig. 1b) while KT root line cultures, we also observed the detrimental the final fresh biomass increase was at the same level in elic- effect of MeJA or PHEN with MeJa (both in a dose of 100 ited and PFD-phases supported elicited cultures. The com- µM) on root growth (Furmanowa and Syklowska-Baranek parison of growth rates obtained in PFD-supported unelic- 2000; Syklowska-Baranek et al. 2009). ited and PFD-supported elicited cultures revealed higher fresh mass growth at the end of the culture in the first ones. PFD‑phases supported cultures of ATMA and KT However, on day 35 of cultivation, one week after elicitors hairy root lines addition, both fresh and dry biomass increase was higher in PFD-supported elicited cultures. This phenomena could be The fresh weight of ATMA hairy roots growing in medium connected with the acting of MeJa activating primary and supplemented with PFD-degassed or aerated (Fig. 2a–c) but secondary metabolism which was discussed on the proteome without elicitors was augmented in comparison to cultures level (Bhattacharyya et al. 2012). elicited (treated with mixture of 1 µM CA, 100 µM PHEN The highest fresh and dry biomass increase of KT line and 100 µM MeJa) but lower than in the control, however the roots was achieved in control cultures and amounted to 4.50- differences were not significant atP < 0.05 (Fig. 1b). When fold and 3.32-fold, respectively over their initial weight. It the root dry mass was taken into consideration the biomass was 2.36-fold and 1.88-fold more than the fresh and dry decrease was noted in comparison to the control and cultures growth rates of ATMA roots, respectively in control cul- elicited but without PFD phases. Such discrepancy noted in tures (Fig. 1b). The addition of precursors and MeJa to the growth indexes could be attributed to the post-harvesting medium caused over twofold and 1.5-fold decrease in fresh manipulation procedure when the medium was removed in and dry root biomass growth, respectively, in relation to the

Fig. 2 Taxus x media hairy roots cultivated under various condi- control untreated culture on the 28th day of cultivation; e KT root line tions: a ATMA root line in control untreated culture on the 28th day on the 14th day of cultivation after PFD-degassed application; f KT of cultivation; b ATMA root line on the 14th day of cultivation after root line on the 42th day of cultivation in presence of PFD-degassed PFD-degassed application; c ATMA root line on the 42th day of cul- and elicitors. (Photo I. Rudnicki) tivation in presence of PFD-degassed and elicitors; d KT root line in

1 3 Plant Cell, Tissue and Organ Culture (PCTOC) control. The application of PFD phases with elicitors, did were supplemented with precursors and elicitor on the 28th not hamper significantly (P < 0.05) the root growth on the day of cultivation while PFD aerated or degassed phases basis of their fresh and dry mass in comparison to elicited were added to appropriate culture variants on day 14. cultures without and PFD-phases (Fig. 2d–f). In the previ- Lignans were detected neither in aqueous nor PFD phases ous investigation of our group performed in ATMA root line in any of the cultures variants (Fig. 2S d, e). cultures the diminished biomass growth was noted as a result of PFD-aerated or degassed applied alone or in combina- Lignan accumulation in control cultures of ATMA and KT tion with 100 µM MeJa with a marked tendency of more hairy root lines pronounced growth suppression in PFD-aerated supported cultures (Sykłowska-Baranek et al. 2015a). The chemical investigation of ATMA line roots growing in Up to now, published research on Taxus spp. cell, tissue control cultures revealed the presence of secoisolariciresinol and organ cultures reported diversified responses of cells as lignan aglyca. Its content amounted to 2.35 ± 0.36 µg/g to MeJa elicitation (review in Tabata 2004, Vongpaseuth DW, 2.03 ± 0.19 µg/g DW and 1.56 ± 0.44 µg/g DW on the and Roberts 2007, Ramirez-Estrada et al. 2016), although 28th, 35th and 42nd day of culture, respectively. While in the the decrease in cell growth is mainly described. Exposito form of glucoside only lariciresinol was present and detected et al. (2010) in suspension cultures of T. x media, compar- in a concentration of 1.43 ± 0.31 µg/g DW, 1.44 ± 0.21 µg/g ing the MeJa effect on the growth of two transformed cell DW and 1.28 ± 0.27 µg/g DW on the 28th, 35th and 42nd lines, one of them was carrying additional txs gene and rol day of culture, respectively. HMR and pinoresinol was deter- genes another only rol genes, observed a slight growth sup- mined as glucosides and aglyca (Table 1; Fig. 2S). The con- pression in comparison to the control, however the cell line tent of HMR glucoside determined on the 28th day was over with txs gene was more sensitive to MeJa treatment. The threefold higher than that of its aglyca. After 14 subsequent authors reported, in the first stage of cultures carried out in days of cultivation HMR was detected only as aglyca and a medium advantageous for cell growth, the significantly its yield increased over 13-fold, from 0.2 ± 0.01 µg/g DW higher biomass increase in rol genes carrying cell suspen- up to 2.61 ± 0.09 µg/g DW. Pinoresinol was detected only sion cultures than in the cultures of cells carrying txs and on the 14th day of culture and the ratio of glucoside to its rol genes. These results corroborate with our observation of aglyca was 1.8. In transformed roots of ATMA line, after current experiments (Fig. 1). The growth decline under MeJa β-glucosidase hydrolysis, 63.20 ± 2.80 µg/g DW of CA in treatment was further reported in cell suspension cultures root tissues growing for 28 days was also noted. of T. x. media carrying the additional txs gene by Onrubia In untreated control roots of KT line pinoresinol and et al. (2012). The results of studies on the exploration of the matairesinol were present predominantly in the form of protein profile of MeJa elicited cell suspension cultures of glucosides and determined only on the 14th day of cultiva- Podophyllum hexandrum, revealed the up-accumulation of tion (Table 1). The most abundant was matairesinol and its defense/stress—related protein, proteins engaged in second- content was 75.40 ± 3.15 µg/g DW while 9.70 ± 1.01 µg/g ary metabolites production and signal transduction (Bhat- DW of pinoresinol was noted. Starting from the 28th day tacharyya et al. 2012). However, the authors also noted the HMR was found as glucoside and aglyca (Fig. 3S a–c), with down-regulation of some proteins involved in primary cell its highest total yield on the 42nd day. Although the major metabolism, for example cyclin A protein connected with form of HMR was its aglyca (Table 1) which exceeded the the cell division cycle which could be correlated with cell level of HMR glucoside almost ninefold, 20.40 ± 2.32 µg/g growth suppression caused by MeJa. Recently, in Taxus cus- DW and 2.30 ± 0.23 µg/g DW, respectively. pidata suspension cultures the growth suppression caused by In the control cultures of KT root line CA was detected, MeJa was reported. The authors demonstrated that growth also after hydrolysis and in this case probably in the form of depletion was connected with the inhibition of cell cycle coniferin which is considered to be a storage or transported progression rather than cell death and impaired the G1/S form of CA (Savidge 1989; Whetten and Sederoff 1995) transition as observed by an increase in G0/G1 phase cells, hydrolyzed by specific β-glucosidase to CA (Steevens at al. and decreased the number of actively dividing cells (Patil 2001). The CA/coniferin was noted in roots from the 14th et al. 2013). till the 42nd day of cultivation, however simultaneously with its aglyca – CA only till the 35th day (Table 1). While on the The lignan accumulation under various culture 14th day 5.60 ± 0.82 µg/g DW of CA was solely determined. conditions On day 28 of the culture coniferin/CA and CA levels were comparable, 22.40 ± 2.07 µg/g DW and 20.50 ± 5.26 µg/g The content of six lignans was examined in hairy root tis- DW, respectively. On day 35 the ratio coniferin/CA to CA sues, aqueous and PFD phases during six weeks of cultiva- was changed in favour of coniferin/CA and finally on day 42 tion of two Taxus x media hairy root lines. The root cultures only 30.40 ± 3.77 µg/g DW of coniferin/CA was detected.

1 3 Plant Cell, Tissue and Organ Culture (PCTOC)

Table 1 Lignan content (µg/g DW) in control untreated cultures of ATMA and KT hairy root lines Day of culture CA/Coniferin HMR Secoisolariciresinol Aglyca Glucosides Aglyca Glucosides Aglyca Glucosides

ATMA root line 14 – – – – – – 28 – 63.20 ± 2.80 0.20 ± 0.01a,b 0.61 ± 0.05a 2.35 ± 0.36 – 35 – – – – 2.03 ± 0.20 – 42 – – 2.61 ± 0.09b – 1.56 ± 0.44 – KT root line 14 – 5.60 ± 0.82d,e – – – – 28 20.50 ± 5.26 22.40 ± 2.07d,e 1.01 ± 0.16f 0.20 ± 0.01h – – 35 13.80 ± 3.16 35.20 ± 2.48d 14.20 ± 2.65f,g 1.10 ± 0.11h,i – – 42 – 30.40 ± 3.77e 20.40 ± 2.32f,g 2.30 ± 0.23h,i – – Day of culture Pinoresinol Matairesinol Lariciresinol Aglyca Glucosides Aglyca Glucosides Aglyca Glucosides

ATMA root line 14 6.76 ± 2.63c 12.28 ± 1.27c – – – – 28 – – – – – 1.43 ± 0.31 35 – – – – – 1.44 ± 0.21 42 – – – – – 1.28 ± 0.27 KT root line 14 – 9.70 ± 1.01 – 75.40 ± 3.15 – – 28 – – – – – – 35 – – – – – – 42 – – – – – –

All values represents means of 3 replicates ± SD. The same letters indicates a significant difference P < 0.05; “–” not detected

The two investigated Taxus x media hairy root lines dif- of glucose from UDPG to coniferyl alcohol in cambium and fered in the lignan production profiles (Table 1). Roots of KT developing xylem, is closely coupled with cambium activity line did not accumulate secoisolariciresinol and laricires- (Savidge and Förster 1998). inol while ATMA roots did but were unable to accumulate Up to now there has been one report on lignan production matairesinol. Similar observations on the differences in bio- in shoot and cell suspension cultures of T. x media where synthetic potential of Taxus x media cell suspension cultures only pinoresinol was detected in cultivated in vitro shoots derived from callus tissues, one carried rol and txs genes and (Mistrzak et al. 2015). The pinoresinol reported yield ranged the second only rol genes, was reported by Exposito et al. from 0.51 to 0.69 mg/g DW of total content, depending on (2010), who noted significant variations in the percentage culture conditions and was detected mainly as its glucoside participation of five determined taxanes depending on the the amount of which exceeded its aglyca by about 3-times. cell line. In our study the divergence in coniferin/CA accu- mulation followed by the higher abundance of lignan con- Lignan accumulation in supplemented with precursors tent in KT root line cultures in comparison to ATMA roots and elicitor cultures of ATMA hairy root line could be also explained by KT roots more intensive biomass increase as a result of prolific cell divisions (Fig. 1b). It was The medium supplementation with CA at dose of 1 µM used previously demonstrated that in Pinus spp. cambium CA as a single biosynthesis stimulating factor, resulted in the content began to increase immediately before the initiation accumulation solely of lariciresinol after one week of treat- of cell-division activity in late winter (Savidge and Förster ment. Lariciresinol was present mainly as a free lignan at the 2001) while the coniferin was found in the cambial zone level of 4.41 ± 0.10 µg/g DW and after hydrolysis at the con- only during cambial growth and became undetectable at the centration of 2.74 ± 0.28 µg/g DW. However, after one week beginning of cambial dormancy (reviewed by Steevens at al. of 1 µM CA treatment 3.07 ± 1.07 µg/g DW of lariciresinol 2001). Moreover, the activity of UDPG: coniferyl alcohol glucoside was noted. After two weeks of precursor treat- glucosylotransferase, an enzyme that catalyses the transfer ment secoisolariciresinol was detected under the influence

1 3 Plant Cell, Tissue and Organ Culture (PCTOC) d,e,k,r f h 1.60 ± 0.61 – – – – – – – – – – – 42 42 – 42 2.09 ± 0.61 5.30 ± 1.93 1.49 ± 0.27 1.00 ± 0.39 1.53 ± 0.02 MeJa + PHEN + j,l f,q g q – – – 3.19 ± 0.28 1.93 ± 0.34 – – – – – – – – – 35 35 – 35 – CA 100 µM CA CA 100 µM + MeJa 2.75 ± 1.38 CA 100 µM CA 0.81 ± 0.30 b,c,e,n h,m,n,r 1.38 ± 0.11 3.79 ± 0.31 – – – – – – – – – – – – 42 42 – 42 – 24.29 ± 4.47 11.44 ± 2.87 MeJa + PHEN b g,l + – – – – 1.69 ± 1.34 – – – – – – – – – 35 35 – 35 – CA 10 µM CA CA 10 µM + MeJa 12.86 ± 2.71 CA 10 µM CA 10.11 ± 2.56 c,d,i,m h,i,k a 3.07 ± 1.07 4.72 ± 1.25 – – – – 37.88 ± 4.91 – – – – – – – – 42 42 1.28 ± 0.13 42 2.74 ± 0.28 14.43 ± 4.40 MeJa + PHEN a g,j + 4.41 ± 0.10 – – – – 1.73 ± 0.69 – – 9.20 ± 0.64 – – – – – – – – CA 1 µM CA CA 1 µM + MeJa 35 35 CA 1 µM CA – 35 SD. The same letters indicates a significant difference P < 0.05 indicates a significant difference SD. The same letters ± Aglyca Glucosides Aglyca Glucosides Glucosides Glucosides Aglyca Aglyca Aglyca Aglyca Aglyca Glucosides Glucosides Glucosides Culture conditions Culture conditions Culture Glucosides Culture conditions Culture Glucosides Aglyca Day of culture Day of culture Day Aglyca Day of culture Day methyl jasmonate, “–” not detected jasmonate, “–” not MeJa methyl - phenylalanine, l Lignan production (µg/g DW) in ATMA line hairy roots cultivated in cultures supplemented with in cultures and elicitor lignan precursors line hairy cultivated roots in ATMA (µg/g DW) Lignan production coniferyl alcohol, PHEN- coniferyl Lariciresinol Secoisolariciresinol Lariciresinol Lariciresinol HMR HMR HMR 2 Table means of 3 replicates represents All values CA Compound Compound Secoisolariciresinol Compound Secoisolariciresinol

1 3 Plant Cell, Tissue and Organ Culture (PCTOC) of 1 and 10 µM CA, 4.72 ± 1.25 and 3.79 ± 0.31 µg/g DW, Schmitt and Petersen (2002) in Forsythia x intermedia sus- respectively. While 100 µM of CA caused the produc- pension cultures. Lignan yield was enhanced 3- and sev- tion of 2.09 ± 0.61 µg/g DW and 1.60 ± 0.61 µg/g DW of enfold, respectively by MeJa while CA enhanced only the lariciresinol aglyca and glucoside, respectively. The yield of pinoresinol content by over ninefold. The authors reported lariciresinol under the influence of 1 or 10 µM CA increased also the presence of coniferin/CA in cell extracts obtained about twofold and over twofold compared to the control after from CA treated cultures whereas in our studies coniferin/ 1 and 2 weeks of treatment, respectively (Table 2). Moreover CA was not detected in root cultures subjected to CA or its aglycone was revealed that was undetectable in the con- CA, PHEN and MeJa treatments. Phenylpropanoids were trol. The content of secoisolariciresinol was enhanced over reported to play an important role in plant response to abiotic threefold and 2.4-fold under the same culture conditions, (Cabane et al. 2012) and biotic stresses (Dixon et al. 2002; respectively. Naoumkina et al. 2010) and the biosynthesis of CA was The application simultaneously of MeJa and CA at found to be induced as a result of wounding (Savidge and doses of 10 or 100 µM brought about the biosynthesis of Förster 2001). Anterola et al. (2002) reported that CA and hydroxymatairesinol (HMR) (Table 2). None of the other p-coumaryl alcohol biosynthesis were enhanced with pro- lignans were detected under the described conditions while viding cell cultures of Pinus taeda with saturating amounts after hydrolysis only 3.19 ± 0.28 and 1.49 ± 0.27 µg/g DW of PHEN and Cabane et al. (2012) concluded that effective of lariciresinol was revealed on 35th and 42nd day, respec- stimulation of phenylpropanoid pathway takes place when tively. Simultaneous treatments with CA and MeJa caused PHEN availability from the shikimate pathway is sufficient. exclusively the production of HMR aglyca (Table 2). The yield of HMR was considerably enhanced by precursor and Lignan accumulation in cultures of ATMA and KT hairy elicitor addition in a dose dependent manner in comparison root lines cultivated in two‑phase cultures supplemented to control. The most pronounced HMR content increase, with PFD‑phases over 14-times, was noted under 1 µM CA and MeJa two week treatment. While 10 or 100 µM CA and MeJa two The results obtained in previous research performed by week treatments resulted in over 9 and twofold HMR content our group indicated that direct application of PFD to the augmentation, respectively. Hydrolysis of extracts showed culture system constitutes simple and efficient conditions solely the presence of lariciresinol, 1.49 ± 0.27 µg/g DW and for the enhancement of secondary metabolites production its amount was not affected by culture elicitation. (Sykłowska-Baranek et al. 2014, 2015a). In cultures of KT The investigation of the effect of the combined two pre- root line supplemented with PFD-aerated or degassed but cursors: PHEN and CA together with MeJa resulted in the without elicitors no lignan content was detected (Table 4). accumulation of HMR and lariciresinol as free lignans and ATMA roots in cultures supported with PFD-degassed accu- lariciresinol in the form of glucoside (Table 2). The simul- mulated CA/coniferin 3.06 ± 0.87 µg/g DW (Table 3). Also taneous application of two lignan biosynthesis precursors aglycone of HMR and glycoside of pinoresinol were present, and elicitor resulted in higher HMR and lariciresinol accu- 0.24 ± 0.11 µg/g DW on the 35th day and 2.22 ± 0.43 µg/g mulation than in the control, however the influence of the- DW on the 28th day, respectively (Table 3). When PFD- ses culture conditions was less pronounced in comparison aerated was added to the cultures of ATMA hairy root line to CA and MeJa treatments. HMR yield augmentation was and after subsequent elicitor feeding a significant increase also dose dependent, although it was only 5.5-fold, 4.4-fold in lignan content was noted (Table 3). The highest CA/ higher than in the control when CA at a concentration of 1 or coniferin content was stated in KT hairy root line cul- 10 µM were applied. Moreover HMR content was even lower tures, 72.77 ± 0.34 µg/g DW and 134.60 ± 12.13 µg/g DW than in the control in the presence of 100 µM CA, PHEN and on day 35 and 42 respectively. At the same time points, in MeJa. The opposite effect was exerted by the mentioned pre- ATMA line cultures CA/coniferin concentration amounted cursors and elicitor on lariciresinol content which increased to 23.81 ± 1.49 and 15.50 ± 2.57 µg/g DW, respectively. up to 1.93 ± 0.34 µg/g DW and represents over a 1.3-fold Both ATMA and KT hairy root cultures accumulated HMR increase in relation to the control. We observed variability mainly as its aglyca (Tables 3, 4). KT roots produced also in respect to lignan content between flasks that was earlier lariciresinol in the form of its aglycone (0.59 ± 0.02 µg/g reported and investigating in details for paclitaxel production DW and 0.68 ± 0.10 µg/g DW) on day 35 and 42, respec- in suspension cultures of T. cuspidata by Kim et al. (2004). tively. In ATMA roots cultivated in the presence of PFD- The authors’ results indicated that there may be other fac- aerated and elicitors the most spectacular was the occur- tors affecting paclitaxel accumulation than MeJa like protein rence of matairesinol aglyca which at the same time proved signal compounds. to be the most abundant among all investigated lignans The pinoresinol and matairesinol content increase after (Table 3). The content of matairesinol aglyca amounted 100 µM MeJa or 3 mM CA treatment was described by to 159.79 ± 10.91 µg/g DW and 199.86 ± 22.55 µg/g DW,

1 3 Plant Cell, Tissue and Organ Culture (PCTOC)

g,h on day 35 and 42 respectively. In KT roots matairesinol

h was determined only in elicited cultures without any PFD phases (Table 4). ATMA roots cultivated in the medium supported with PFD-degassed and elicited accumulated 31.05 ± 7.71 42 – – – 31.83 ± 2.89 – 19.06 ± 5.14 160.25 ± 32.33 significantly lower levels of matairesinol aglyca (P < 0.05), g

e 27.86 ± 3.86 µg/g DW and 160.25 ± 32.33 µg/g DW, on day 35 and 42, respectively. Matairesinol glucosides were detected only on day 42, 4.47 ± 1.10 µg/g DW and – 35 – – – – 27.86 ± 3.86 2.30 ± 0.12 Cultures elicited supplemented withCultures PFD-degassed – 31.83 ± 2.89 µg/g DW during cultivation in elicited cultures

c supported with PFD-aerated or degassed, respectively. In suppoered with PFD-degassed elicited cultures, CA/conif- erin 25.71 ± 4.67 µg/g DW was found in KT roots on the – 42 – – – 3.06 ± 0.87 – – – 42nd day (Table 4) while it was not detected in ATMA roots

e (Table 3). Under these culture conditions HMR, solely in the form of its aglyca, was more intensively produced by ATMA roots than KT ones, 2.30 ± 0.12 and 19.06 ± 5.14 µg/g DW – 35 – – – – 0.24 ± 0.11 – – versus 48.04 ± 8.92 and 59.29 ± 0.95 µg/g DW, on the 35th and 42nd day, respectively. In ATMA roots also aglyca of pinoresinol (31.06 ± 7.71 µg/g DW) was found on the 42nd – 28 – 2.22 ± 0.43 – Cultures unelicited supplemented withCultures PFD- degassed – – – – day of cultivation (Table 3). Whereas in KT root line cul-

f tures pinoresinol was noted only in elicited cultures without j,k a,b,c i,j,k f PFD phases (Table 4). For the first time our group developed PFD-supported cultures systems for plant cell and organ cultures in the 4.47 ± 1.10 28.47 ± 8.46 42 – 199.86 ± 22.55 – 15.50 ± 2.57 0.85 ± 0.20 15.57 ± 0.76

0.05; “–” not detected 0.05; “–” not simultaneous function of gas carrier and in situ extrahent. < a,b,c i,j,k i,j,l PFD-aerated applied to the cell suspension cultures of Arnebia euchroma allowed to enhance naphthoquinone yield about 50% in comparison to control cultures (Sykłowska- – 23.78 ± 4.92 35 – 159.79 ± 10.91 – 0.18 ± 0.02 6.16 ± 0.31 23.81 ± 1.49 Cultures elicited supplemented Cultures with elicited PFD-aerated Baranek et al. 2014). The influence of PFD application at two time points: the day of inoculation (day 0) and the 14th day of culture together with elicitation (100 µM PHEN and MeJa) in ATMA transformed root cultures was studied – – 42 – – – 0.46 ± 0.01 0.35 ± 0.23 – (Sykłowska-Baranek et al. 2015a). It was demonstrated that b the most suitable time point for PFD addition is the 14th day of culture and the highest total (i.e. intracellular + extracellu- – – 35 – – – 1.64 ± 0.24 – 1.58 ± 1.25 lar: both in aqueous and PFD phases) yield of paclitaxel was obtained in PFD-degassed carrying cultures after two weeks b d d of elicitation. Under the conditions of the current study we also observed in most of the cases higher lignan contents – 1.34 ± 0.21 – 2.62 ± 0.46 28 – – – Cultures unelicited supplemented withCultures PFD- aerated 2.61 ± 0.72 after two week elicitation, however, the PFD-aerated sup- SD. The same letters indicates a significant difference P indicates a significant difference SD. The same letters ported elicited cultures seemed to be more favourable for ± b lignan accumulation (Tables 3, 4) likely due to generated to a higher extent than in PFD-degassed supported cultures – 2.72 ± 0.64 – 0.26 ± 0.15 0.29 ± 0.16 42 – – – oxidative stress which is believed to affect plant secondary metabolism (Shilpa et al. 2010; Kolewe et al. 2010). PFD application in the function of in situ extrahent could also contribute to the lower metabolite feedback inhibition and – – 0.60 ± 0.06 – 1.33 ± 0.86 35 – – Cultures elicited withoutCultures PFD phase – diminished cytotoxic metabolites accumulation in aqueous phases of the medium which seemed to be often the reason sides sides sides sides Gluco - Gluco - Gluco - Aglyca Aglyca Day of Day culture Aglyca Gluco - Culture Culture condi - tions Aglyca of limited secondary metabolites production (Malik et al. Lignan production (µg/g DW) in ATMA line hairy roots cultivated in two-phase PFD-supported cultures in two-phase line hairy cultivated roots in ATMA (µg/g DW) Lignan production 2013; Kolewe et al. 2010). Taxus hairy root cultures were used for lignan production inol erin inol Matares - - CA/Conif HMR - Pinores 3 Table means of 3 replicates represents All values Com - pound for the first time under conditions of current investigations.

1 3 Plant Cell, Tissue and Organ Culture (PCTOC)

b The applied various elicitation strategies combined with PFD-phases application caused mostly dose- and treat- ment dependent enhancement of lignan accumulation over – 42 9.29 ± 0.95 – – – – 25.71 ± 4.67 – the control in T. x media hairy root cultures, however their level were lower than those reported up to now in hairy root cultures, dealing mainly with species of Linum genus. In hairy root cultures of Linum album lignan accumulation was successfully enlarged by fungal elicitor usage, and laricires- 48.04 ± 8.92 – 35 PFD-degassed elicited PFD-degassed – – – – – – inol content rose threefold over control up to 260 µg/g DW (Bahabadi et al. 2014). Similary Tashackori et al. (2016), – 42 – – – – – – – examining various lignan production in L. album hairy roots elicited with mycelium extract of Piriformospora – 35 – – – – – – – indica, reported an 1.6-fold increase (up to 157.3 µg/g DW) over control in lariciresinol content. Chashimi et al. (2016) – 28 – – – – PFD-degassed PFD-degassed unelicited – – – demonstrated that coniferyl aldehyde (2 mM) significantly stimulated larici- and pinoresinol production up to 14.8- and

a,b 8.7-fold, respectively. Gabr et al. (2017) demonstrated that

c c,d the total lignan content (secoisolariciresinol diglucoside, secoisolariciresinol and matairesinol) was 55% higher in hairy root cultures of L. usitatissimum than in non-trans- 1.34 ± 0.10 – 42 – – – – 134.60 ± 12.13 3.26 ± 0.28 formed roots. Under conditions of reported herein experiments, the a d potential of two Taxus x media hairy root lines for lignan production have been demonstrated, however for the lignan

0.05; “–" not detected 0.05; “–" not production enhancement further biotechnological investiga- < – – 72.77 ± 23.34 35 – – – – PFD-aerated elicited PFD-aerated 14.95 ± 2.73 tions are needed. – – – 42 – – – – – Summary – – – 35 – – – – – Two yew hairy root lines were examined for their potential to lignan accumulation. It was demonstrated that the hairy – – – 28 PFD-aerated unelicited PFD-aerated – – – – – root lines subjected to the investigations differed in lig- nan production profiles. Among six lignans, in the control

e e untreated cultures, roots of KT line did not accumulate secoisolariciresinol and lariciresinol while ATMA roots were unable to accumulate matairesinol. The elicitation of – – – 42 4.60 ± 0.24 6.50 ± 3.93 – 0.26 ± 0.18 – ATMA root line cultures with CA at different doses also resulted solely in the production of lariciresinol and secoi- solariciresinol while under simultaneous CA and MeJA SD. The same letters indicates a significant difference P indicates a significant difference SD. The same letters treatment HMR was also accumulated. The addition of ± PHEN together with CA and MeJa neither changed the – – Cultures elicited withoutCultures PFD phase – 35 5.20 ± 0.82 9.50 ± 3.86 – – – accumulated lignan profile nor enhanced their production. The most beneficial conditions for lignan production under the conditions of the current experiments, were created by two-phase cultures supplemented with PFD-aerated or degassed. In ATMA hairy roots HMR, pinoresinol and Day of culture Day Glucosides Aglyca Glucosides Glucoides Glucosides Aglyca Aglyca Aglyca matairesinol were present predominantly as their aglyca while in KT hairy roots pinoresinol and matairesinol were mainly detected as glucosides. In both root lines CA was Lignan production (µg/g DW) in KT line hairy roots cultivated in two-phase PFD-supported cultures in two-phase line hairy in KT cultivated roots (µg/g DW) Lignan production found only in its glucosylated form. 4 Table Compound means of 3 replicates represents All values HMR CA/Coniferin Pinoresinol Mataresinol

1 3 Plant Cell, Tissue and Organ Culture (PCTOC)

Acknowledgements This study was supported by research Grant Cabane M, Afif D, Hawkins S (2012) Lignins and abiotic stress. Adv No. N N405 362537 from the Polish Ministry of Science and Higher Bot Res 61:219–262. https://doi.org/10.1016/B978-0-12-41602​ ​ Education and this work was supported by the Statutory Budget 3-1.00007​-0 [FW21/N/2017] of the Medical University of Warsaw. The authors are Cassidy A, Hanley B, Lamuela-Raventos RM (2000) Isoflavones, grateful to Alina Łukasiewicz and Bożenna Sztyber for their technical lignans and stilbenes–origins,metabolism and potential impor- assistance. We are also grateful to Paulina Mistrzak for the inspiring tance to human health. J Sci Food Agric 80:1044–1062. https://​ talk which led us to apply in our investigations methods developed by doi.org/10.1002/(SICI)1097-0010(20000​515)80:7 Prof. Petersen and Dr. Schmitt for examinations of lignan production Chashimi NA, Sharifi M, Behmanesh M (2016) Lignan enhancement in Forsythia x intermedia in vitro cultures. in hairy root cultures of Linum album using coniferaldehyde and methylenedioxycinnamic acid. Prep Biochem Biotechnol Author contributions KSB planning of the investigations, perfluoro- 46:454–460. https​://doi.org/10.1080/10826​068.2015.10688​02 decalin application, implementation of HPLC-UV-DAD analysis, Dar AA, Arumugam N (2013) Lignans of sesame: purification meth- elaboration of the results and preparation of the manuscript. KŁ car- ods, biological activities and biosynthesis - a review. Bioorg Chem rying out in vitro cultures of KT root line and sample preparations. 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