Effects of Acetylsalicylic Acid and UV-B on Gene Expression and Tropane Alkaloid Biosynthesis in Hairy Root Cultures of Anisodus Luridus
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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 Á Elicitor Á Gene expression Á Alkaloid biosynthesis Á 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 Á L. Ma Á Y. Wang Á N. Wu (&) Á 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 sub- cultured 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.