Effects of Acetylsalicylic Acid and UV-B on Gene Expression and Tropane Alkaloid Biosynthesis in Hairy Root Cultures of Anisodus Luridus

Plant Cell Tiss Organ Cult (2014) 117:483–490 DOI 10.1007/s11240-014-0454-z

RESEARCH NOTE

Effects of acetylsalicylic acid and UV-B on gene expression and tropane alkaloid biosynthesis in hairy root cultures of Anisodus luridus

Baifu Qin • Lili Ma • Yaxiong Wang • Min Chen • Xiaozhong Lan • Nengbiao Wu • Zhihua Liao

Received: 4 October 2013 / Accepted: 20 February 2014 / Published online: 9 March 2014 Ó Springer Science+Business Media Dordrecht 2014

Abstract Anisodus luridus hairy root cultures were hairy root cultures treated with 1 mM ASA had the highest established to test biological effects of acetylsalicylic acid capacity of TAs biosynthesis, in which the content of (ASA) and ultraviolet ray-B (UV-B) on gene expression, scopolamine and hyoscyamine reached respectively 57.2 tropane alkaloid (TA) biosynthesis and efflux. The TAs- and 14.7 lgg-1 DW. Surprisingly, it was found that 1 mM pathway gene expression was ASA dosage dependant. The ASA dramatically induced the efflux of scopolamine. In the expression of PMT, TRI and CYP80F1 showed no signifi- liquid medium with 1 mM ASA, the content of scopol- cant difference in hairy root cultures in treatment of 0.01 amine was 153.4 lg flask-1, about 6.2 folds compared and 0.1 mM ASA, compared with those without ASA with that of control. At the same time, hyoscyamine was treatment; while 0.01 or 0.1 mM ASA slightly upregulated detected at trace levels in liquid medium. In the UV-B H6H expression. All the four genes including PMT, TRI, stressed hairy root cultures, all the four genes had a very CYP80F1 and H6H had a dramatic increase in 1 mM ASA- strong increase of gene expression that led to more accu- treated hairy root cultures compared with control. The mulation of scopolamine and lower accumulation of hyo- expressing levels of all the four genes were much signifi- scyamine. Only trace amounts of hyoscyamine and cantly higher in 1 mM ASA-treated hairy root cultures than scopolamine were detected in the liquid medium when those in 0.01 and 0.1 mM ASA-treated ones. As expected, hairy root cultures were stressed under UV-B, and this suggested that UV-B did not affect TAs efflux.

Baifu Qin and Lili Ma have contributed equally to this work. Keywords Anisodus luridus Elicitor Gene expression Alkaloid biosynthesis Efﬂux Electronic supplementary material The online version of this article (doi:10.1007/s11240-014-0454-z) contains supplementary material, which is available to authorized users. Introduction B. Qin L. Ma Y. Wang N. Wu (&) Z. Liao (&) Key Laboratory of Eco-environments in Three Gorges Reservoir Region (Ministry of Education), Chongqing Engineering Anisodus luridus, namely Himalayan Scopolia, is a peren- Research Center for Sweetpotato, School of Life Sciences, nial herbal plant native to Tibetan Plateau (Wang et al. Southwest University, Beibei, 400715 Chongqing, China 2010), which produces pharmaceutical tropane alkaloids e-mail: [email protected] (TAs) including hyoscyamine and scopolamine especially Z. Liao in root (Jovankovics 1966). TAs have been widely used as e-mail: [email protected] anticholinergic agents affecting systemic nervous para- M. Chen sympatheticum (De Luca and St Pierre 2000) and exclu- School of Pharmaceutical Sciences, Southwest University, sively exist in Solanaceous plants (Zhang et al. 2004). Beibei, 400715 Chongqing, China Scopolamine has higher pharmacological activities and fewer side effects than hyoscyamine, so the world demand X. Lan Agricultural and Animal Husbandry College, Tibet University, for scopolamine is much larger than hyoscyamine (Oks- Nyingchi, 860000 Tibet, China man-Caldentey 2000; Wang et al. 2011). Unfortunately, the 123 484 Plant Cell Tiss Organ Cult (2014) 117:483–490

Fig. 1 The biosynthetic pathway of tropane alkaloids and the plant of A. luridus. PMT putrescine N-methyltransferase, TRI tropinone reductase I, CYP80F1 cytochrome P450, H6H hyoscyamine 6b-hydroxylase. Source Li et al. (2006), Zhang et al. (2004) content of scopolamine in plants is very low and unable to hydroxylation of hyoscyamine and the epoxidation to meet the increasing needs of market. Because of the dif- scopolamine, which is regarded as the last committed step ficulty in industrial synthesis, TAs are mainly extracted (Hashimoto et al. 1993; Zarate et al. 2006). There is no from plants. So, it is eager to find new species to produce difference of TAs biosynthesis among TAs-producing TAs or find other alternative ways to improve the pro- medicinal plants at the biochemical level, but the regula- duction of TAs especially scopolamine. Anisodus luridus tion of TAs biosynthesis is quite different from species to belongs to Solanaceae family and currently has not been species. For example, methyl jasmonate (MeJA) dramati- researched at the levels of TAs biosynthesis and plant cally increased gene expression of both PMT and H6H in biotechnology. Hyoscyamus niger, which led to more production of sco- The TAs biosynthetic pathway is not fully veiled at the polamine (Zhang et al. 2007). However, the accumulation molecular level; however the key enzymatic steps are of scopolamine in Anisodus acutangulus did not obviously known (Fig. 1). Putrescine N-methyltransferase (PMT; EC response to MeJA treatment (Kai et al. 2012). 2.1.1.53) is the first committed enzyme and catalyzes the The hairy root technology offered many advantages, as N-methylation of putrescine (Hibi et al. 1992); the second hair roots have high genetic stability and rapidly grow in committed enzyme tropinone reductase I (TRI; EC hormone-free media (Guillon et al. 2006; Wu and Shi 1.1.1.206) converts tropinone to tropine heading to TAs 2008). Simultaneously, TAs are mainly synthesized in root biosynthesis (Kai et al. 2009; Portsteffen et al. 1994). and hairy roots could have much higher accumulation of Despite the detail biosynthesis during the conversion of TAs than in plants (Favali et al. 2004). All these advan- littorine to hyoscyamine is not completely understood, tages make hairy roots be an effective approach to produce Cytochrome P450 (CYP80F1; EC 1.6.2.4) is a key gene TAs (Kai et al. 2011; Pavlov et al. 2009; Teli and Timko involved in it (Li et al. 2006). Hyoscyamine 6b-hydroxy- 2004). Elicitors including biotic and abiotic ones, have lase (H6H; EC 1.14.11.11) is a bifunctional enzyme and outstanding effects on enhancing plant secondary metabo- catalyzes two consecutive oxidation reactions: the lite production, and the induction of hairy roots to increase

123 Plant Cell Tiss Organ Cult (2014) 117:483–490 485 the content of TAs has been established (el Jaber-Vazdekis RNA isolation and gene expression studies using real- et al. 2008; Kai et al. 2011). The physical stress ultraviolet time quantitative PCR ray-B (UV-B) has significant effects on increasing the plant secondary metabolites (Binder et al. 2009; Liu et al. 2012) Total RNAs of hairy roots were extracted by PlantRNA kit and the plant hormones acetylsalicylic acid (ASA) is also a (Tiangen, China). The quality and concentration of the well-known elicitor to boost the TAs yields in Atropa RNAs were checked by agarose gel electrophoresis and baetica (el Jaber-Vazdekis et al. 2008). So the hairy root spectrophotometer analysis (WFZUV-2100, Unico, Shang- cultures of A. luridus were established and used to test the hai, China). RNAs (about 400 ng) were used as templates to biological effects of ASA and UV-B on gene expression, generate cDNAs using PrimeScriptTM RT Kit (Takara, alkaloid biosynthesis and efflux in the present research. Japan). The relative gene expression was analyzed by Q-PCR. The full-length cDNAs of PMT, TRI, CYP80F1 and H6H were cloned from A. luridus for the first time by us (data Materials and methods not shown). The sequences of PMT (GenBank accession no. KC713799), TRI (GenBank accession no. KC713800), Establishment of hairy root cultures CYP80F1 (GenBank accession no. KC894914) and H6H (GenBank accession no. KC713802) of A. luridus were The seeds of A. luridus were collected from the northern area released in GenBank. The real-time qPCR was performed on of Tibet Himalaya Mountain and germinated into seedlings Bio-Rad real-time thermal cycler (Bio-Rad, USA) using the on MS medium. Sterile leaf discs of A. luridus were inocu- SYBRÒ Premix ExTaqII (Takara, Japan). The phospho- lated with A. tumefaciens strain C58C1 (pRiA4) and then glycerate kinase gene (PGK) was used as the reference gene plated on MS medium for co-cultivation in the dark for (Li et al. 2014) and the primers used for qPCR were listed in 2 days. After 2 days of co-culture, the infected leaf discs Supplemental Table 1. The experiments were repeated for were transferred onto MS medium containing 300 mg l-1 three times on independently isolated mRNA preparation. cefotaxime to eliminate bacteria. Roots generated at cutting edges were excised and cultured on MS medium with Extraction and HPLC analysis of tropane alkaloids 300 mg l-1 cefotaxime at 25 °C in dark and routinely subcultured every 28 days (Yang et al. 2011). To confirm the Tropane alkaloids including hyoscyamine and scopolamine integration of rol genes into the genome of hair roots, both in hairy root cultures (Zhang et al. 2004) and culture rolB (forward primer: 50-GCTCTTGCAGTGCTAGATTT- medium (Kang et al. 2004) were extracted, and analyzed by 30; reverse primer: 50-GAAGGTGCAAGCTACCTCTC-30) HPLC (Yang et al. 2011): the mobile phase consisted of and rolC (forward primer: 50-TAACATGGCTGAAGACG methanol and acetate (0.05 mol l-1 ammonium acetate ACC-30; reverse primer: 50-AAACTTGCACTCGCCATG solution added with 0.0025 M SDS and adjusted to pH 4.6) CC-30) genes were detected through genomic PCR analysis at a ratio 58:42. The column (Phenomenx GEMINI 5 lm (Zhang et al. 2007). C18 150 mm 9 4.6 mm, USA) was operated at a flow rate of 1 ml min-1 and temperature of 40 °C. The detecting Elicitation wavelength was 226 nm. The sample solution of injection was 20 ll each time. The authentic samples of hyoscya- About 50 mg fresh roots with 3–4 cm in length were inoc- mine and scopolamine (Sigma, USA) were prepared in ulated into 250 ml conical flasks containing 150 ml liquid methanol at a final concentration of 1,000 lgml-1 and MS medium and grew at 110 rpm, 25 °C in the dark. When diluted into 500, 250, 100, 50, 25, 10 and 5 lgml-1. The the hairy root cultures were cultured for 30 days, the liquid retention time of hyoscyamine and scopolamine was MS medium were replaced by fresh liquid MS medium respectively 8.896 and 7.245 min. added with ASA to the final concentrations of ASA1 (0.01 mM), ASA2 (0.1 mM) and ASA3 (1 mM), and each Statistical analysis culture contained 1 ml EtOH (as control because ASA was solved in EtOH solutions). All cultures grew at 110 rpm, All the experiments including culture of hairy root lines, 25 °C in the dark for 24 h and were harvested for the future PCR identification, qPCR analysis of gene expression, research. For UV-B treatment, the 30-day-old liquid MS HPLC analysis of metabolites were repeated three times. medium was renewed, and the cultures were exposed to UV- The relative expression levels of the targeted genes and B (a wavelength in the range of 280–315 nm and a high- product contents were presented as mean values ± SD. intensity setting of 9,000 UW cm-2 at the surface) for 24 h The statistical significance of gene expression in different and then harvested. All the experiments were conducted for treatments and contents of alkaloids were analyzed by one three times. way ANOVA. 123 486 Plant Cell Tiss Organ Cult (2014) 117:483–490

Fig. 2 Establishment of hairy root cultures of A. luridus. A Hairy roots induced from leaf; B cultivation of hairy root on solid MS medium; C 30-day-old hairy root cultures in liquid MS medium

Results and discussion Gene expressing profile, alkaloid accumulation and efflux in ASA-treated A. luridus hairy roots Establishment of Anisodus luridus hairy root cultures ASA acts as an abiotic elicitor of secondary metabolites in The transformed roots emerged at the wounded sites of medicinal plants. It was found for the first time that ASA leaves after 2 weeks (Fig. 2A) when the leaves were could remarkably elicit alkaloids (an increase of 505 %) infected by A. tumefaciens strain C58C1 (pRiA4) because and phenolics (an increase of 1,587 %) biosynthesis in the wounded plant cells secreted some low-molecular Catharanthus roseus cell suspension cultures (GodoyHer- phenolic signals that facilitated the plasmid to enter cells nandez and LoyolaVargas 1997). The following researches (Stachel et al. 1985). The independently transformed hairy also supported that ASA was an abiotic elicitor of sec- root lines were respectively excised and cultured on MS ondary metabolites in planta. ASA was the most potent medium with 300 mg l-1 cefotaxime at 25 °C in dark and elicitor among MeJA, SA and ASA in glucotropaeolin routinely subcultured every 28 days to eliminate bacteria production in hairy root cultures of Tropaeolum majus (Fig. 2B). The transformed roots showed several typical (Wielanek and Urbanek 2006). In hairy root cultures, ASA morphological features of hairy roots characterized by also enhanced production of withanolide in Withania lateral branching, plagiotropic growth and rapid growth somnifera (Doma et al. 2012) and ginseng saponin in on hormone-free MS medium (Giri and Narasu 2000; Lan Panax ginseng (Jeong et al. 2005). It was also found that and Quan 2010; Mano et al. 1986). Based on the typical ASA boosted tropane alkaloid yields in hairy root cultures morphologies, it could be concluded that the subcultured of Atropa baetica (el Jaber-Vazdekis et al. 2008). In the roots were hairy roots genetically transformed by the Ri present study, ASA at three different concentrations was plasmid, but that should have to be confirmed at molec- used to treat hairy root cultures of A. luridus to investigate ular level. The integration of Ri T-DNA into the genome gene expression of tropane alkaloid pathway (PMT, TRI, of plant cells caused the formation of hairy roots, in CYP80F1 and H6H), alkaloid production and efflux. which the rol genes were harbored (Dehghan et al. 2012). The qPCR efficiency (E) of the genes of interest ranged The members of rol gene families including rolB and from 96.3 to 109.1 %, which was reliable (Supplemental rolC were simultaneously amplified from the positive Table 1). The expressing levels of the three structural control C58C1 (pRiA4) and the transformed hairy root genes including PMT, TRI and CYP80F1 showed no sig- lines. The fragment of rolB and rolC was respectively 423 nificant difference in hairy root cultures in treatment of and 626 bp in length (Supplemental Fig. 1), and the 0.01 and 0.1 mM ASA, compared with those without ASA results were consistent with the previously reported pub- treatment (Fig. 3A–C); while 0.01 and 0.1 mM ASA lication (Yang et al. 2011). The fragments of rolB and slightly upregulated H6H expression (Fig. 3D). The higher rolC were not amplified from the genomic DNA of wild level of ASA (1 mM) dramatically upregulated gene type roots of A. luridus that was used as negative control. expression levels of all the four genes including PMT, TRI, Then, a growing-fast hairy root line confirmed by geno- CYP80F1 and H6H, which was respectively 14.8, 6.6, 3.1 mic PCR was selected for the following experiments. and 11.9 times compared with that in the EtOH control After 30-day growth in liquid MS medium, the biomass (Fig. 3A–D). The expressing levels of all the four genes of hairy roots almost reached to the maximum and used were much significantly higher in 1 mM ASA-treated hairy for elicitation with ASA and UV-B (Fig. 2C). root cultures than those in 0.01 and 0.1 mM ASA-treated 123 Plant Cell Tiss Organ Cult (2014) 117:483–490 487

Fig. 3 Expression proﬁle of tropane alkaloid biosynthetic genes in measured by Q-PCR: A PMT, B TRI, C CYP80F1 and D H6H. Data 30-day-old hairy roots after 24 h treatments with ASA1 (0.01 mM), presented as mean ± SD from triplicate experiments, statistically ASA2 (0.1 mM) and ASA3 (1 mM). Gene expression levels were signiﬁcant differences are indicated by asterisks

Fig. 4 The hyoscyamine and scopolamine contents in A. luridus hairy roots were treated for 24 h. A, B respectively, hyoscyamine and hairy roots and culture mediums supplied with chemical elicitors scopolamine content in hair roots; C scopolamine content in culture ASA1 (0.01 mM), ASA2 (0.1 mM) and ASA3 (1 mM), and each medium. The data were presented as the mean ± SD from three culture contain 1 ml ETOH, include the ethanol control. 30-day-old replicates, bars with asterisks represent signiﬁcant differences at 0.05 ones (Fig. 3A–D). This suggested that the upregulation of with that in the control (12.8 ± 4.2 lgg-1 DW) and 1 mM TAs-pathway gene expression by ASA might be dosage- ASA treated hairy root cultures (14.7 ± 3.4 lgg-1 DW); dependant. the content of hyoscyamine in 0.1 mM ASA treated hairy The hairy root cultures treated with 0.01 and 0.1 mM root cultures was 37.0 ± 7.4 lgg-1 DW (Fig. 4A) that was ASA showed much higher accumulation levels of hyoscya- 2.9 and 2.5 times respectively compared with that in the mine than the control and 1 mM ASA treated hairy root control and 1 mM ASA treated root cultures. 0.01 and cultures (Fig. 4A). The content of hyoscyamine in 0.01 mM 0.1 mM ASA also enhanced scopolamine production in ASA treated hairy root cultures was 32.0 ± 5.8 lgg-1 DW hairy root cultures of A. luridus. In the 0.01 mM ASA treated (Fig. 4A) that was 2.5 and 2.2 times respectively compared hairy root cultures, the content of scopolamine reached

123 488 Plant Cell Tiss Organ Cult (2014) 117:483–490

Fig. 5 Expression proﬁle of tropane alkaloid biosynthetic genes supplied with physical elicitor UV-B. Gene expression levels were measured by Q-PCR: A PMT, B TRI, C CYP80F1 and D AlH6H. Data presented as mean ± SD from triplicate experiments, statistically signiﬁcant differences are indicated by asterisks

Fig. 6 The hyoscyamine and scopolamine contents in A. luridus hairy roots and culture media treated with UV-B. 30-day-old cultures were exposed to UV-B for 24 h. A and B respectively, hyoscyamine and scopolamine content in hair roots; the data were presented as the mean ± SD from three replicates, bars with asterisks represent signiﬁcant differences at 0.05

51.6 ± 4.8 lgg-1 DW (Fig. 4B), about 1.5 times compared 1 mM ASA than those in hair root cultures treated with with that in control; in the 0.1 mM ASA treated hairy root 0.1 mM ASA, the scopolamine content in hairy root cultures cultures, the content of scopolamine reached 68.1 ± treated with 1 mM ASA was almost the same as that in hairy 10.4 lgg-1 DW (Fig. 4B), about 2 times compared with root cultures treated 0.1 mM ASA. This strongly suggested that in control. In the hairy root cultures treated with 1 mM that the scopolamine efflux might exist. Then, the HPLC ASA, the content of scopolamine was 57.2 ± 9.0 lgg-1 analysis was used to detect the tropane alkaloids in the liquid DW (Fig. 4B), without significant difference compared with medium. that in hairy root cultures treated with 0.1 mM ASA. Inter- As expected, a high level of scopolamine estingly, although all the four structural genes showed much (153.4 ± 16.7 lg flask-1) was found in the medium con- higher expression levels in hairy root cultures treated with taining 1 mM ASA, which was much higher than that in

123 Plant Cell Tiss Organ Cult (2014) 117:483–490 489 the medium of control as well as the 0.1 and 0.01 mM ASA In summary, we established the hairy root culture of A. contained media (Fig. 4C). When the scopolamine in hairy luridus, a Tibetan herbal plant that produced tropane root cultures and in the medium were considered together, alkaloids including hyoscyamine and scopolamine. It was it could be concluded that 1 mM ASA dramatically found that ASA (1 mM) as an abiotic elicitor had a strong enhanced biosynthesis of scopolamine by strongly upreg- effect on boosting TAs accumulation and promoting the ulating the four structural genes involved in tropane bio- scopolamine efflux. ASA might be a potent tool on the synthetic pathway and also strongly promoted the release industrialization of scopolamine production, and especially of scopolamine into the liquid medium. Due to much more promoting scopolamine efflux by ASA made it possible the scopolamine biosynthesis in 1 mM ASA treated hairy root scopolamine extraction from liquid medium; and ASA- cultures, hyoscyamine was consumed a lot leading to lower induced scopolamine efflux might open a window for hyoscyamine content in 1 mM ASA treated hairy root understanding transportation of scopolamine. In other cultures. At the same time, only trace amounts of hyo- hand, we also found that UV-B as a physical stress was also scyamine were detected in all the media and this meant that able to facilitate the accumulation of scopolamine. It can be ASA did not significantly affect hyoscyamine efflux. By deduced that ASA and UV-B might enhance the accumu- the way, the inducible release of scopolamine to medium lation of scopolamine or even hyoscyamine in the intact by 1 mM ASA made it a useful tool to identify the related plants of A. luridus, and this will be the next story in the transporter genes in A. luridus and it was possible to extract further study. scopolamine in liquid medium. Acknowledgments This work was financially supported by the Gene expressing profile, alkaloid accumulation in UV- NSFC Project (31370333), the National 863 Hi-Tech Plans (2011AA100605; 2011AA100607), Chongqing Sciences and Tech- B-treated Anisodus luridus hairy roots nology Project (CSTC2012GGYYJS80013), the Program for New Century Excellent Talents in University (NECT-12-0930) and the The UV-B radiation can enhance biosynthesis of different Fundamental Research Funds for the Central Universities plant secondary metabolites and it was intensively reviewed (XDJK2013A024). by Zhang and Bojo¨rn (2009). 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