Methyl Jasmonate Treatment Increases Podophyllotoxin Production in Podophyllum Hexandrum Roots Under Glasshouse Conditions
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Plant Soil (2017) 417:117–126 DOI 10.1007/s11104-017-3245-6 REGULAR ARTICLE Methyl jasmonate treatment increases podophyllotoxin production in Podophyllum hexandrum roots under glasshouse conditions Christel L. C. Seegers & Rita Setroikromo & Pieter G. Tepper & Peter Horvatovich & Ron Peters & Wim J. Quax Received: 29 November 2016 /Accepted: 30 March 2017 /Published online: 4 April 2017 # The Author(s) 2017. This article is published with open access at Springerlink.com Abstract methyl jasmonate on the podophyllotoxin production Background and aim The endangered Podophyllum was determined. hexandrum is an important industrial source of Results More root formation was observed in peat-perlite podophyllotoxin, which is a precursor for the anticancer soil than in sand soil. Furthermore, root formation was drugs etoposide and teniposide. Attempts to obtain higher at 15 °C than at 25 °C. This resulted in the highest podophyllotoxin through cell cultures or chemical syn- podophyllotoxin production per plant in peat-perlite at thesis have still a long way to go before being economical 15 °C (160 ± 22 mg/plant d.w.). Furthermore, methyl feasible. The objective of this study was to increase the jasmonate treatment of the leaves increased the root formation and podophyllotoxin production of podophyllotoxin production in the roots by 21%. P. hexandrum cultivated in a glasshouse. Conclusion We were able to cultivate P.hexandrum in a Methods Root formation and podophyllotoxin produc- glasshouse in the Netherlands and improve the root tion of P. hexandrum in sand or peat-perlite soil at 15 °C formation and podophyllotoxin production. This paves or 25 °C was determined. Furthermore, the influence of the way for large-scale cultivation of P. hexandrum in the temperate latitudes for the production of the phar- maceutical interesting podophyllotoxin. Responsible Editor: Hans Lambers. Keywords Podophyllum hexandrum . Electronic supplementary material The online version of this . article (doi:10.1007/s11104-017-3245-6) contains supplementary Podophyllotoxin Etoposide Soil Temperature material, which is available to authorized users. Methyl jasmonate C. L. C. Seegers : R. Setroikromo : P. G. Tepper : * W. J. Quax ( ) Introduction Department of Chemical and Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV, Groningen, The Netherlands The high demand for podophyllotoxin as precursor for e-mail: [email protected] the synthesis of important anticancer drugs (etoposide and teniposide) has led to the search for alternative P. Horvatovich Department of Analytical Biochemistry, Groningen Research sources (Imbert 1998). The commercially exploited nat- Institute of Pharmacy, University of Groningen, Groningen, ural sources of podophyllotoxin are Podophyllum The Netherlands hexandrum and Podophyllum peltatum (Guerram et al. 2012). Other podophyllotoxin producing plants in this R. Peters Proeftuin Ron Peters, Gantel 12, 7891 XAKlazienaveen, genus are Podophyllum sikkimensis and Podophyllum The Netherlands pleianthum (Jackson and Dewick 1985;Pauletal. 118 Plant Soil (2017) 417:117–126 2013). The highest concentration of podophyllotoxin was done on increasing root formation of mature plants was found in P. hexandrum roots, which varies between or increasing podophyllotoxin production in vivo. 0.025% and 9.53% (d.w.) depending on the geographic Several researchers reported that the plant morphology location (Purohit et al. 1999;Alametal.2009; Kitchlu and the geographic location, especially the altitude, are et al. 2011;Sharmaetal.2012; Liu et al. 2015;Pandey important factors for podophyllotoxin production et al. 2015). Podophyllotoxin production is not restricted (Purohit et al. 1999;Alametal.2009; Kitchlu et al. to the roots as low amounts of podophyllotoxin (0.003– 2011;Pauletal.2013;Pandeyetal.2015). Alam and 0.229% d.w.) are also found in P. hexandrum leaves Naik found that high podophyllotoxin production was (Pandey et al. 2013). Furthermore, several researchers correlated to low pH, high organic content and high reported podophyllotoxin production in the leaves of nitrogen levels in the soil (Alam and Naik 2009). P. peltatum (Bastos et al. 1996; Moraes et al. 2000, Another factor important for the production of 2002; Cushman et al. 2006; Zheljazkov et al. 2011). podophyllotoxin is temperature as some lignan biosyn- The most recent population study was by Zheljazkov thesis genes were found to exhibit a higher expression and coworkers who found up to 2.53% (d.w.) level at 15 °C rather than at 25 °C (Kumari et al. 2014). podophyllotoxin (Zheljazkov et al. 2011). The leaves Until now, the influence of soil composition and culti- of P. peltatum can be an attractive alternative source for vation temperature on podophyllotoxin production in P. podophyllotoxin due to its renewable properties. hexandrum roots has not been investigated in a P. hexandrum is an endangered species according glasshouse. to the Convention of International Trade in Besides cultivation conditions, hormone induction Endangered Species of Wild Fauna and Flora can be used to increase production of secondary metab- (https://www.cites.org/eng/app/appendices.php#hash2), olites. Elicitation of medicinal plant species by because of its excessive harvesting. Therefore, several jasmonates activates transcription factors followed by researchers focused on production of podophyllotoxin upregulation of the production of structurally divergent by chemical synthesis or in vitro cell cultures. The secondary metabolites, such as nicotine and artemisinin chemical synthesis is difficult due to the presence of (De Geyter et al. 2012). Most elicitation studies have four contiguous chiral centers, and the presence of a been performed on cell suspension and hairy root cul- base sensitive trans-lactone moiety (Canel et al. 2000). tures (van der Fits 2000;Häkkinenetal.2004; Baldi and Therefore, at least five chemical synthesis steps are Dixit 2008;Toddetal.2010;Shojietal.2010; necessary to convert the commercially available Suttipanta et al. 2011). Also the podophyllotoxin pro- bromopiperonal, a building block of the GPR30 recep- duction in P. hexandrum suspension cultures was in- tor antagonist (Dennis et al. 2009), into podophyllotoxin creased by seven to eight-fold after stimulation with or epipodophyllotoxin (Ting and Maimone 2014). As an methyl jasmonate (Bhattacharyya et al. 2012). alternative, podophyllotoxin production in cell suspen- Furthermore, methyl jasmonate treatment is also effec- sion cultures has been explored. This approach, howev- tive in vivo, as treatment of Nicotiana attenuata leaves er, provides a low yield with the highest production rate increased nicotine production in the roots (Baldwin of podophyllotoxin reaching 0.65% (d.w.) (Petersen and 1996). Whether methyl jasmonate can increase the Alfermann 2001; Ionkova et al. 2010). Untill now nei- amount of podophyllotoxin in vivo, and whether this ther the chemical synthesis nor the in vitro production of effect can be obtained by spraying the leaves, has not podophyllotoxin is economically competitive with the been reported up to now. extraction of podophyllotoxin from P. hexandrum. Our aim was to enhance the root formation and Therefore, we focused in this study on improving the podophyllotoxin production in P. hexandrum in a glass- cultivation conditions of P. hexandrum to ensure a sus- house in the Netherlands to meet the high demand of tainable supply of P. hexandrum roots for isolation of podophyllotoxin. The influence of soil, temperature and podophyllotoxin. Until now, most conservation studies methyl jasmonate was investigated in a systematic ap- focused on enhancing seed germination or the proach. In order to compare the podophyllotoxin pro- propagation/transplantation of the plants (Nadeem duction between conditions, a novel quick extraction et al. 2000; Guo et al. 2012). However, no research method was designed. Plant Soil (2017) 417:117–126 119 Materials and methods arrived in pots with peat soil enriched by addition of calcium and NPK (nitrogen, phosphorous, and potassi- Experimental design um) and were stored at 7–8 °C in the dark to prevent shoot formation. Plants were cultivated in the glass- P. hexandrum plants were cultivated under various house of Proeftuin Ron Peters (Klazienaveen, the growth conditions (soil, temperature and jasmonate treat- Netherlands) in sand soil or peat-perlite soil ment) to investigate root formation and podophyllotoxin (Table S1). The peat and perlite were mixed in a ratio production. The temperature study was done with 150 2:1 (w/w). For every condition and time point fifteen plants; 75 plants cultivated at 15 °C and 75 plants culti- plants were randomly harvested. vated at 25 °C. The soil study (sand versus peat-perlite) The soil and temperature study was done with the was performed at both temperatures with 30 plants for batch from Borculo (150 plants). The plants cultivated at each soil type. Sand and peat-perlite were previously 15 °C were grown in March and April of 2015 and the described to be successful for P. hexandrum, respectively plants cultivated at 25 °C in May and June 2015. The for the germination of seeds and in transplanting of root- average temperatures were 15 ± 2 °C and 22 ± 4 °C, lets into soil (Kharkwal et al. 2008; Guo et al. 2012). For respectively, and the average radiation sums were statistical analysis, a factorial design was used with three 1158 ± 450 joules/cm2 and 1554 ± 531 joules/cm2