Biologia 67/2: 352—359, 2012 Section Cellular and Molecular Biology DOI: 10.2478/s11756-012-0007-z

The effects of elicitation on the expression of key enzyme genes and on production of tropane alkaloids in Anisodus acutangulus plant

Xiuqin Luo1, Shiqiu Weng2, Xiaoling Ni3,XiaoyunWang2, Xueqing Fu1,JianboXiao1 &GuoyinKai1*

1Laboratory of Plant Biotechnology, College of Life and Environment Sciences, Shanghai Normal University, Shanghai 200234, People’s Republic of China; e-mail: [email protected]; [email protected] 2Department of Gynaecology and Obstetrics, Kunshan Hospital of Traditional Chinese Medicine, Kunshan, Jiangsu Province 215300, People’s Republic of China 3Department of General Surgery, Zhongshan Hospital, Fudan University, Shanghai 200032, People’s Republic of China

Abstract: Tropane alkaloids (TAs) including hyoscyamine, anisodamine, anisodine and scopolamine are widely used as anticholinergic agents, which act on the parasympathetic nervous system, and their clinical demands are increasing rapidly. Anisodus acutangulus is a solanaceous perennial plant that is endemic to China with good potential for TA production and classified as an endangered species in China. Therefore there is a need to improve the TA production in this resource plant by biology methods. In our research, three different important elicitors including methyl jasmonate (MJ), yeast elicitor (YE) and abscisic acid (ABA) were used to study their effects on TA production, and genes expressions in A. acutangulus plant under each treatment were examined. The results revealed that TA production was induced by all the three treatments. However, its quantity varies with inducers used. The effect induced by MJ and YE were significant, while the effect induced by ABA was not obviously. To our knowledge, this is the first report on the relationship between TA production and genes expression profile induced by different elicitors in cultivatedA. acutangulus plant, which should provide some useful information on how to improve TA production with the cultivated A. acutangulus plant in the near future. Key words: Anisodus acutangulus; elicitors; tropane alkaloids; hyoscyamine; scopolamine. Abbreviations: TA, tropane alkaloid; ABA, abscisic acid; DW, dry weight; H6H, hyoscyamine-6β-hydroxylase; MJ, methyl jasmonate; PCR, polymerase chain reaction; PMT, putrescine N-methyltransferase; TR I, tropinone reductase I; TR II, tropinone reductase II; YE, yeast extract.

Introduction methyltransferase 1 and 2), AaTR I, AaTR II (for tropinone reductase I and II), and AaH6H (for hyoscy- Tropane alkaloids (TAs) including hyoscyamine, aniso- amine-6β-hydroxylase) in biosynthesis pathway of TA damine, anisodine and scopolamine play a vital role in in A. acutangulus have been cloned in our laboratory stomach and intestinal colic, rheumatism joint pain, (Kai et al. 2007; Kai et al. 2009a; Kai et al. 2009b). kid crazes backleg pain. These TAs are mainly pro- It is a feasible way to enhance the production of TA duced in Solanaceae plants such as Anisodus, Atropa, by over-expressing the key gene/genes of TA biosyn- Datura, Duboisia, Hyoscyamus and Scopolia (Zhang et thesis in A. acutangulus hairy roots. For example, over- al. 2004; Zarate et al. 2007). However, TA produced expression of AaTR I in A. acutangulus hairy roots re- from these Solanaceae plants is too low to meet the in- sulted in 1.87-fold higher yield of hyoscyamine and 8- creasing needs of the pharmaceutical market. Anisodus fold higher yield of scopolamine compared with control acutangulus, which is endemic to China and is classi- hairy roots, and over-expression of both AaPMT and fied as an endangered species, could produce more TAs AaTR I resulted in the TA amount reaching 8.66-fold than other Solanaceae plants. Hence, it is an attractive higher than those of under controlled condition (Kai et resource for TA production (Wu et al. 1962; Kai et al. al. 2009a,2011). 2007; Li et al. 2008). The elicitor strategy has been developed for many The biosynthesis of TA in Solanaceae plants years and has been used not only to stimulate defense involves a complex process which includes several response, but also to induce accumulation of phytoalex- catalytic steps (Fig. 1). Fortunately, the key genes ins in plants, which is feasible and well documented for such as AaPMT1 and AaPMT2 (for putrescine N- enhanced production of metabolite from plant cell cul-

* Corresponding author

c 2012 Institute of Molecular Biology, Slovak Academy of Sciences Tropane alkaloids in Anisodus acutangulus plant 353

Fig. 1. The metabolic pathway of TA (including hyoscyamine, anisodine and scopolamine) synthesis in A. acutangulus (adapted according to Kai et al. 2012). ture recently (Boitel-Conti et al. 2000; Pitta-Alvarez (Kai et al. 2012). However, there is no report on the et al. 2000; Kang & Jung et al. 2004). Elicitors can be effects of elicitors on TA production in A. acutangulus subdivided into two categories: abiotic elicitors, such as plant and its possible molecular mechanism. As A. acu- metal ions and inorganic compounds, and biotic elici- tangulus plant has its advantages such as the ability to tors from fungi, bacteria, viruses, and chemicals (Zhao grow under traditional method (growing in the nature) et al. 2005). There are many studies about the rela- and cost saving (does not need equipments necessary tionship mechanism between secondary metabolic path- for hairy roots), it is also worth of investigating the ef- way and the elicitors. Methyl jasmonate (MJ) was re- fects of different elicitors on the production of TA and ported to be a signaling molecule in biotic and abiotic expression of related key genes of TA biosynthesis in A. stresses (Zhao et al. 2005; Arias Zabala et al. 2010). acutangulus plant. Lots of experiments have demonstrated that MJ was In this study, MJ, YE, ABA were introduced indi- able to stimulate the production of several compounds vidually to treat the wild A. acutangulus plant to study in many species (Ignatov et al. 1996; Rijhwani & Shaks the effects on TA production and the expression pro- 1998; Dong & Zhong 2002; Wang & Zhong 2002; Vas- file of the key enzyme genes in TA biosynthetic path- consuelo & Boland 2007). Yeast extract (YE), a kind of way. This is the first time when A. acutangulus plant polysaccharide, was also been reported to have the abil- was treated with different elicitors, which should pro- ity to regulate increase or decrease different secondary vide some valuable information on how to improve TA metabolites in many species, such as syringin, vale- production with wild A. acutangulus plant in the near portriates, podophyllotoxin and silymarin (Kittipong- future. patana et al. 2002; Shams-Ardakani et al. 2005; Xu et al. 2007; Hasanloo et al. 2009). Abscisic acid (ABA), Material and methods also called resistant inducer element, is a key element Plant cultivation which can balance the plant endogenous hormones and The plants were cultivated in Murashige and Skoog solid is relevant to growth-active substances. media under aseptic conditions for three weeks. Then they In order to improve the content of bioactive compo- were transferred to the soil for 200 days. At first, we take the nents, we apply genetic engineering method or elicita- plants out of aseptic conditions, washed away the Murashige tion or another way of “inducement”, such as the expo- and Skoog media attached to the roots and domesticated sure of the plant in conditions like limiting light, water, the young plants covered them with baggies for 10 days after taken from the aseptic conditions, and then uncovered ultraviolet, wound-induced, virus-induced (Saedler & ◦ Baldwin 2003). In order to identify the optimal method baggies for them to grow. The temperature was 25 Cand to improve the contents of TA in A. acutangulus,it illumination was 16 hours with them being watered every two days. is necessary to try out different strategies and analyze their effects on the production of TA, respectively. TA Elicitors preparation and elicitation production in A. acutangulus hairy roots is induced by Elicitation was carried out with three different elicitors, ethanol, MJ, Ag+ and salicylic acid in our laboratory MJ, YE and ABA, respectively. MJ (Sigma, St. Louis, MO, 354 X. Luo et al.

USA) was dissolved in dimethyl sulfoxide with the concen- quantities of the sample including the control group. Sam- tration of 4.4583 M, and was finally adjusted the concentra- ples were quantified using standard curves fit with linear tion of 100 µM with distilled water. ABA (Sigma, St. Louis, regression. MO, USA) was dissolved in distilled water giving a concen- tration of 0.7567 M, and finally diluted to 100 µM. YE (Bio Statistical analysis Basic, Inc.) was dissolved in distilled water with the con- All the data were repeated in three independent times and centration of 200 g/L, and then diluted the concentration analyzed by SPSS software. Standard deviation of the mean to8mg/L. was estimated to measure the precision of the estimate of After the three elicitors were prepared, the each basin the mean. The statistical significance of the treatments and plant was administrated at 0, 12, 24, 48 and 96 h, respec- control differences were analyzed using variance, and the tively. The control group did not add any elicitor. difference was significant (P<0.05).

RNA extraction and genes expression studies using RT-PCR Results Each basin of plant was treated with 50 mL of three dif- ferent elicitors with specific concentration, and harvested Effects of TA production and genes expression after MJ at specific time by dividing roots, stems and leaves. About treatment 0.12 g fresh weigh (roots, stems and leaves) were taken out The contents of hyoscyamine and scopolamine of A. for RNA extraction using the RNA pure Plant Kit (Tian- acutangulus root after MJ treatment were significantly gen Biotech Co., Ltd., Beijing, China) following the man- boosted; especially the content of hyoscyamine after ufacturer’s instructions. The quality and concentration of 24 h treatment was over 3-fold higher (1.572 mg/g − ◦ the extracted RNA were checked and stored at 80 Cfor of dry weight, DW) compared to that of control reverse transcription PCR as described before (Kai et al. (0.406 mg/g DW), and then decreased gradually. 2006). Scopolamine and anisodine also presented the same The RT-PCR was performed in 20 µLvolumeswith 4 µL5×M-MLV buffer containing 2 µL50µM primer, trend as hyoscyamine, with early increase followed by 1 µL10×dNTP mixture, 0.5 µL 200 U/µL RNase M-MLV a decreased (Fig. 2A). The expression profile of related (RNaseH−, Promega) and 0.5 µL40U/µL RNase inhibitor genes was shown (Fig. 3A) that several genes including (Takara, Japan) at 42 ◦C 1.5 h, followed by inactivation of AaPMT1, AaPMT2, AaTR I and AaTR II were induced RNase at 70 ◦C for 15 min. An aliquot of the cDNA obtained by MJ, expressed at the highest level at 24 h point and was amplified – AaPMT1, AaPMT2, AaTR I, AaTR II,and then decreased, except AaH6H. ◦ AaH6H by incubation at 94 C (5 min), followed by 30 cycles The TA contents after MJ treatment in the stem of 94 ◦C degeneration (45 s), 58 ◦C annealing (1 min), and ◦ ◦ of A. acutangulus were the same as those in the root, 72 C outspread (1 min) and a final elongation step of 72 C which increased first then decreased (Fig. 2B). Be- (10 min). All the reactions were performed in a total volume of 25 µL containing 2 µLof10µMdNTPs,0.5µLofeach cause of the less contents of TA in the stem, the ef- primer (10 µM) (Kai et al. 2012), 0.05 µg of cDNA, and 1.25 fect of induction was more obvious than in the root. units of Taq DNA polymerase (Shanghai Genebase Gene- Hyoscyamine reached 1.453 mg/g DW after 24 h treat- Tech Co., Ltd.) plus 2.5 µLof10×PCR buffer (no Mg2+), ment, which was much higher than that of control 1.5 µL25mMMgCl2. The plant 18SrRNAgene with the (0.005 mg/g DW). Scopolamine reached 0.373 mg/g specific primers 18SF: 5’-CCAGGTCCAGACATAGTAAG- DW, which was over 300–fold higher than that of con- 3’ and 18SR: 5’-GTACAAAGGGCAGGGACGTA-3’ was trol (0.001 mg/g DW). While the content of anisodine used as the reference gene. was very low and the trend was a little complex. The expression profile trend of genes (Fig. 3B) also showed Extraction and analysis of TAs the same performance as the content. The genes ex- The fresh plants were harvested at specific time by divid- pressed weaker in the stem than in the root, such as ◦ ing roots, stems and leaves, then dried in the oven at 50 C AaTR I,andAaH6H could even not be detected un- for two days. The TA was extracted according to our previ- der the ultraviolet light by the condition of 30 cycles of ous paper (Li et al. 2008). HPLC analyses were performed PCR. to determine the contents of hyoscyamine, anisodine and The contents of TA after MJ treatment in the leaf scopolamine using a Sepax reversed-phase symmetry col- umn (250 mm × 4.6 mm, 5 µM). The mobile phase con- of A. acutangulus were observed the same as in the sisted of 22% acetonitrile (HPLC grade) and 78% diethy- root (Fig. 2C). While the expression of some key en- lamine buffer (containing 0.7% diethylamine and adjusted zyme genes in the leaf (Fig. 3C) was very weak and pH to 7.2 with orthophosphoric acid). The flow rate was even cannot be detected under the ultraviolet by the 1.0 mL/min and the injection volume was 40 µL. The chro- condition of 30 cycles of PCR, such as AaPMT2, AaTR matogram was monitored at 220 nm on a Hitachi Diode I and AaH6H, the genes AaPMT1 and AaTR II nearly Array Detector L2455. Hyoscyamine and anisodine (Sigma) could not be regulated after MJ inducement. were dissolved in methanol with a concentration of 1 mg/mL (scopolamine, 0.5 mg/mL). The peak purity of the test sam- Effects of TA production and genes expression after YE ples was determined by comparing their UV spectra to that of the authentic standard. Quantification was based on the treatment peak area of the original sample injected. The recovery rate The contents of hyoscyamine (3.135 mg/g DW) and of extraction was evaluated by adding known quantities of scopolamine (0.351 mg/g DW) in the root of A. acutan- standards to the samples. The accuracy and reproducibil- gulus after YE treatment were improved obviously, and ity of HPLC analysis were confirmed by analyzing different especially at 48 h; they reached the highest point and Tropane alkaloids in Anisodus acutangulus plant 355

and AaTR II were increased gradually in 0, 12, 24 h, then decreased, and AaH6H expressed stronger gradu- ally in the range of 96 h (Fig. 5A). The TA contents in the stem of A. acutangulus after YE treatment were not improved in the range of 48 h, while they were improved dramatically at 96 h, especially the content of hyoscyamine (0.129 mg/g DW) was much higher than that of control (0.005 mg/g DW) (Fig. 4B). Most of the genes expressed weakly, expect for AaPMT1, which was expressed almost at similar level and could not be regulated by YE (Fig. 5B). The results in the leaf of A. acutangulus after YE treatment were different from the root and the stem, which showed that the contents of hyoscyamine and scopolamine were inhibited by YE, while the produc- tion of anisodine was improved (Fig. 4C). The expres- sion profile of the all genes was weaker than in root and stem, even it cannot be detected under the condi- tion of 30 cycles of PCR, except for AaTR II,which was stronger than in root and stem, especially after 48 htreatment(Fig.5C).

Effects of TA production and genes expression after ABA treatment The results after ABA treatment were a little different from the above two elicitors. The TA contents after ABA treatment in the root (Fig. 6A) indicated that the contents of hyoscyamine and anisodine were both improved, particularly the content of anisodine at 24 h and 96 h treatment, which reached hundreds of fold higher level (0.177 and 0.246 mg/g DW, respectively) than the control (0.004 mg/g DW). The contents of hyoscyamine and scopolamine were showed completely invert, one was increased and the other was decreased. The expression profile of related genes was inhibited, except for AaPMT2 (Fig. 7A). Fig. 2. The contents of TA (hyoscyamine, anisodine and scopo- lamine) at 0 h, 12 h, 24 h, 48 h, 96 h about the root (A), stem The contents of TA in the stem of A. acutan- (B) and leaf (C) in A. acutangulus plant after MJ treatment. gulus after ABA treatment are shown in Figure 6B. TA including hyoscyamine, anisodine and scopolamine were inhibited, and the expression profiles of the genes both were almost 10-fold higher than those of control (Fig. 7B) showed that the gene AaPMT1 expressed (0.406 and 0.077 mg/g DW, respectively), while aniso- weaker than control; the other genes expressed weakly, dine almost cannot be detected under the condition of even they cannot be detected under the ultraviolet by HPLC (Fig. 4A). The genesAaPMT1 and AaPMT2 ex- the condition of 30 cycles of PCR. pressed strongly especially after 48 h treatment, while The contents profiles in the leaf of A. acutangulus the other genes expressed weakly. The genes AaTR I after ABA treatment (Fig. 6C) also showed difference

Fig. 3. The expression profile of the key enzyme genes and the reference gene 18S rRNA at0h,12h,24h,48h,96habouttheroot (A), stem (B) and leaf (C) in A. acutangulus plant after MJ treatment. 356 X. Luo et al.

while the hyoscyamine content decreased gradually. The gene expression profile (Fig. 7C) illustrated that the gene AaTR II expressed stronger in the range of 48 h, and other genes except for AaPMT1 expressed weakly, even they could not be detected under the ultraviolet by the condition of 30 cycles of PCR.

Discussion

As common elicitors, MJ, YE and ABA are well stud- ied in a lot of species and organs including plant, hairy root or cell culture and so on. The relationship between the key genes expression profile in the pathways of TA biosynthesis and the TA production in wild A. acutan- gulus plant was carried out in our work. The results showed that the key genes could be regulated by MJ, and the production of TA variation was observed in the range of 96 h in wild A. acutangulus plant. Accord- ing to previous reports, MJ has been reported to stim- ulate the secondary metabolites production in many species, such as TA accumulation in Scopolia parviflora (Kang & Jung 2004), phenylpropanoid in Atropa bel- ladonna (Lee et al. 1998) and camptothecin accumula- tion inCamptotheca acuminate (Seung & Sang 1998). MJ has also been proven to be an effective signaling molecule, which can strongly boost taxane biosynthesis in cultured Taxus cells (Wang et al. 2001). Our results displayed that the exposure time was a very impor- tant factor for promoting the production of TAs and the contents of TA yield varied quickly as the change of point in time. It reached the highest point at 24 h treatment and then it decreased. The same happened with genesAaPMT1 and AaH6H expression. We specu- late that MJ is one of the key signaling molecules, which led to the changed activities of the key enzymes involved in the process of their induction inA. acutangulus,the 24 h being the highest point for MJ inducement. YE is composed of a variety of compounds, such as amino acids, vitamins, and minerals. It is also pos- Fig. 4. The contents of TA (hyoscyamine, anisodine and scopo- sible that the elicitation effects might be due to other lamine) at 0 h, 12 h, 24 h, 48 h, 96 h about the root (A), stem components, still not being identified (Ertola & Hours (B) and leaf (C) in A. acutangulus plant after YE treatment. 1998). In many species it has been proven to stimu- late or decrease different secondary metabolites, such as syringin, valeportriates, podophyllotoxin and sily- from the root and the stem. The contents of anisodine marin (Kittipongpatana et al. 2002; Shams-Ardakani and scopolamine were improved in the range of 24 h, et al. 2005; Xu et al. 2007; Hasanloo et al. 2009). Our

Fig. 5. The expression profile of the key enzyme genes and the reference gene 18S rRNA at0h,12h,24h,48h,96habouttheroot (A), stem (B) and leaf (C) in A. acutangulus plant after YE treatment. Tropane alkaloids in Anisodus acutangulus plant 357

ually boosted the contents of hyoscyamine and scopo- lamine in the root and stem in the range of 96 h treat- ment, the same as the key genes of TA biosynthesis. Although we do not have enough evidences to demon- strate it, we speculate that YE should contain some composition that is beneficial for the TA biosynthesis. There are many genes identified as ABA-respon- sive, including the genes encoding seed-storage pro- teins, late embryogenesis abundant (LEA) proteins, and various other proteins and protein families. Examples of these genes are EM in wheat, EM and RAB16 in rice, HVA1, HVA22 and dehydrins in barley, and EM and RD29 in Arabidopsis thaliana (Hattori et al. 1995; Mundy et al. 1990; Shen et al. 1996; Xu et al. 1996; Shen & Ho 1997; Narusaka et al. 2003). In plants, a combination with ABA specificity may result in emerg- ing a series of physiological reactions for adapting to or resisting to adversity. As a key factor for signal trans- mission between plant organs, the effect of ABA treat- ment was not as obvious as was the treatment of the two above-mentioned elicitors, neither MJ, nor YE. It seemed to be an inhibition factor in our work. The content of scopolamine decreased in the range of 96 h, while hyoscyamine was improved in the root group (Fig. 6A–C). The expression profiles of genes were not significantly induced, regardless of increase or decrease (Fig. 7A–C). The reason may be attributed to the fact that the genes do not play close roles in the develop- mental stages, but in the TA biosynthesis pathway of A. acutangulus plant. As indicated in the metabolic pathways in A. acu- tangulus (Fig. 1), hyoscyamine may be possibly trans- formed to anisodamine and anisodine, and scopolamine may be changed from anisodamine by AaH6H, so it is hard to say that with the improvement of hyoscyamine, the contents of anisodine and scopolamine will be im- proved, too. The AaTR II catalyzes a branch product convolvine (Kai et al. 2009), which exists inevitably and may consume some of hyoscyamine, therefore the AaTR Fig. 6. The contents of TA (hyoscyamine, anisodine and scopo- II was expressed during all the investigated period in lamine) at 0 h, 12 h, 24 h, 48 h, 96 h about the root (A), stem the whole plant in our experiment. It is not easy to (B) and leaf (C) in A. acutangulus plant after ABA treatment. distinguish the importance between two branch ways which one is dominant at the specific time and specific tissue, and there is nearly conformal trend in the con- current work revealed that YE is a stimulating factor tent of anisodine after elicitors’ treatment. Moreover, for TA production in A. acutangulus plant, which grad- there exists lagging regulated from genes to enzyme to

Fig. 7. The expression profile of the key enzyme genes and the reference gene 18S rRNA at0h,12h,24h,48h,96habouttheroot (A), stem (B) and leaf (C) in A. acutangulus plant after ABA treatment. 358 X. Luo et al. content in the transcriptome. The mechanism of plant Boitel-Conti M., Laberche J-C., Lanoue A., Ducrocq C. & secondary metabolism is very complex, which also leads Sangwan-Norreel B.S. 2000. Influence of feeding precursors to difficult analysis of the variation of TAs in different on tropane alkaloid production during an abiotic stress in Datura innoxia transformed roots. Plant Cell Tiss. Org. 60: tissues after inducement by various elicitors. 131–137. The above results showed that the mRNA expres- Dong H.D. & Zhong J.J. 2002. Enhanced taxane productivity sions levels of different genes in the pathway of TA in bioreactor cultivation of Taxus chinensis cells by combin- biosynthesis were various. Due to the regulatory effect ing elicitation, sucrose and ethylene incorporation. Enzyme Microb. Technol. 31: 116–121. lags behind, the contents of TAs were synthesized by all Ertola R.J. & Hours R.A. 1998. Role of yeast extract compo- genes. So the content was different in root, stem and leaf nents in microbial cultures not associated with amino acids, after different elicitor treatment. Furthermore, the TA vitamins and minerals: a review. Appl. Biol. Sci. 4: 1–15. contents and the profiles of genes expression presented Hasanloo T., Sepehrifar R., Rahnama H. & Shams M.R. 2009. Evaluation of the yeast-extract signaling pathway leading a correlative relationship. It may be attributed to each to silymarin biosynthesis in milk thistle hairy root culture. independent plant and also to existing diversity among World J. Microbiol. Biotechnol. 25: 1901–1909. different plants. Meanwhile, we speculate that there are Hattori T., Terada T. & Hamasuna S. 1995. 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