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Enhanced production of flavonoids by methyl jasmonate elicitation in cell suspension culture of Hypericum perforatum

Enhanced production of flavonoids by methyl jasmonate elicitation in cell suspension culture of... Background: Flavonoids of Hypericum perforatum are important secondary metabolites which have been widely utilized in medicine for a range of purposes. The use of methyl jasmonate (MeJA) elicitation for the enhancement of flavonoid production in cell suspension culture of H. perforatum would be an efficient alternative method for the flavonoid production. Results: MeJA influenced the cells growth and flavonoid production. The optimal elicitation strategy was treatment of the cell cultures with 100 μmol/L MeJA on day 15, which resulted in the highest flavonoid production (280 mg/L) and 2.7 times of control cultures. The activities of catalase (CAT) were inhibited after MeJA treatment in the cell cultures, while the activities of phenylalanine ammonia lyase (PAL) increased, which led to the enhancement of flavonoid production. Conclusion: MeJA elicitation is a useful method for the enhancement of flavonoid production in cell suspension culture of H. perforatum. Keywords: MeJA; Hypericum perforatum; Suspension culture; Flavonoids Background The consumption of H. perforatum-derived products Hypericum perforatum, commonly known as St. John's as pharmaceutical preparations or food additives has in- wort, is a perennial herb native to Europe and also a creased dramatically, and it is presently one of the most traditional medicinal plant which has been utilized in consumed medicinal plants in the world [4,7]. The extracts Chinese folk medicine for a range of purposes [1,2]. The of H. perforatum currently used in foods and pharmaceut- medicinal applications of H. perforatum, including skin ics, mostly composed of flavonoids, are mainly obtained wounds, eczema, burns, diseases of the alimentary tract, from the top aerial parts collected in the flowering stage and psychological disorders, have been related to the [6,8]. However, the quality of the flavonoid-rich extracts phenolic composition of the plant, particularly to hyperi- derived from field-grown plants may be affected by many cins, hyperforins, and flavonoids [3,4]. The efficacy of environmental factors as well as biological processes [5]. medical constituents of H. perforatum is based on the Furthermore, field cultivation of H. perforatum requires whole secondary metabolites, rather than the presence a long growth period and plant management, which is a of single chemical compound [5]. Flavonoids, specifically slow and laborious process [6]. Therefore, an alternative quercetin and its glycoside derivatives, comprise the major method for more efficient and controllable production group of biologically active metabolites in H. perforatum of flavonoids from H. perforatum is urgently required. and are important biochemical markers in authenticating Plant cell, tissue, and organ cultivation technology has the herbal plant materials [4,6]. been successfully applied to the production of pharma- ceutically valuable compounds and other fine chemicals of commercial interest in recent years [9]. For instance, * Correspondence: Lyyao1982@qq.com; luyanhua@ecust.edu.cn State Key Laboratory of Bioreactor Engineering, Department of cell suspension cultures of Panax ginseng have been widely Bioengineering, East China University of Science and Technology, 130 used for the production of saponin and other metabolites Meilong Rd., Shanghai 200237, China [10]. Plant-mediated green biomimetic synthesis of silver Full list of author information is available at the end of the article © 2015 Wang et al.; licensee Springer. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. Wang et al. Bioresources and Bioprocessing (2015) 2:5 Page 2 of 9 nanoparticle was considered a widely acceptable technol- Salicylic acid (SA), ammonium metavanadate (NH VO ), 4 3 ogy for rapid production of silver nanoparticles for suc- and nickel sulfate (NiSO ) were purchased from Shanghai cessfully meeting the excessive need and current market Zhong Lan Chemical Company (Shanghai, China). Aceto- demand and resulting in a reduction in the employment nitrile of HPLC grade was purchased from TEDIA Com- or generation of hazardous substances to human health pany, USA. All other chemicals (analytical grade) were and the environment [11]. Till now, a number of efforts purchased from Shanghai Chemical Co., Ltd. (Shanghai, have been made for the establishment of cell and/or ad- China). ventitious root cultures of H. perforatum for the produc- tion of hypericins, phenolics, and xanthones [12-15]. The Plant material and callus induction information on the factors affecting biomass and afore- H. perforatum plants were provided by Prof. Jie Qian mentioned metabolites of H. perforatum was also in- (Tongji University, China) and were cultivated in the vestigated through elicitation and culture optimization greenhouse. The fresh stem explants of H. perforatum [1,16-18]. However, there are few reports on the pro- were washed thoroughly with tap water, surface-sterilized duction of flavonoids from cell suspension culture of with 75% ethanol for 60 s, and then soaked in 10% sodium H. perforatum, and little is known about the strategies hypochlorite solution for 30 s and rinsed seven times in for hyperproduction of biomass and flavonoids. sterile deionized water. The clean and sterilized stem ex- Many cell cultures have been established from plants, plants were cut into 5-mm segments and inoculated on but they seldom produce sufficient amounts of the re- solid Murashige and Skoog (MS) [22] medium supple- quired secondary metabolites. Secondary metabolite bio- mented with 1.0 mg/L 2,4-D, 0.2 mg/L 6-benzyladenine synthesis in plants depends on environmental stresses; (BA), and 25 g/L sucrose to induce callus formation. After their accumulation can be stimulated by precursors and 1 month, the successfully induced callus was separated elicitors [19]. Precursors are intermediate compounds of from the explants and cultured separately until used for the secondary metabolite biosynthesis cycles, which would the establishment of cell suspensions. be toxic to the culture if not used at an appropriate stage or concentration [20]. Elicitors are biological (components Suspension cultures of H. perforatum cells of microbial cells and poly- and oligosaccharides), chemical Cell suspension cultures were initiated from the friable (heavy metals, pesticides, and the signaling compounds in callus and maintained in the liquid MS medium (pH plant defense responses), or physical (cold shock, UV, 5.8) supplemented with 1.0 mg/L 2,4-D, 0.2 mg/L 6- hyperosmotic stress, ultrasound, and pulsed electric field) benzyladenine (BA), and 25 g/L sucrose. A sterile standard factors that induce enzymatic activity against stress sieve with aperture of 300 μm was used to filter the initial [19-21]. Jasmonic acid (JA) and its methyl ester, methyl established suspension cells to obtain homogeneous cul- jasmonate (MeJA), have been proposed to be important tures. The pre-weighed cells were cultured in a 250-mL signaling compounds in the process of elicitation leading Erlenmeyer flask containing a 50-mL medium; cultures to the hyperproduction of various secondary metabolites were placed on a rotary shaker shaking at 120 rpm at [14]. They have also been reported to play a key role in 25 ± 2°C under continuous illumination. Every 20 days, signal transduction processes that regulate defense re- cells were subcultured to fresh media with 5.0-g fresh sponses in plants and shown effective to enhance the pro- weight (FW) in 250-mL flasks. duction of secondary metabolites in cell cultures [14,19]. In the present study, the effect of MeJA on cell growth Elicitation of cell suspension culture and flavonoid biosynthesis in the H. perforatum cell sus- Four elicitors including MeJA, SA, NH VO , and NiSO 4 3 4 pension culture was investigated in small batches. Some were used to study the elicitation effect on flavonoid parameters, such as elicitation time and MeJA concen- production by the cell suspension cultures of H. perfora- tration, on biomass and flavonoid production were stud- tum. MeJA and SA were dissolved in ethanol, NH VO 4 3 ied in detail. The activities of key enzymes (catalase and and NiSO were dissolved in water, and the four elicitors phenylalanine ammonia lyase) related to plant stress re- were filter-sterilized before adding into the suspension sponses and secondary metabolite biosynthesis were in- cultures. Based on the results of preliminary experi- vestigated as well. To the best of our knowledge, this is ments, the feeding concentration of MeJA, SA, NH VO , 4 3 the first report on the induction of flavonoid production and NiSO was 50, 100, 50, and 15 μM, respectively. As by MeJA in cell suspension culture of H. perforatum. a control, filter-sterilized ethanol was added to the cell suspension culture with a final concentration of 0.2%. Materials and methods According to the results of the above experiments, Chemicals MeJA was selected for further study. The MeJA induc- MeJA was supplied by Drug Institute of East China tion time and feeding concentration, known to be crucial University of Science and Technology (Shanghai, China). to plant cell culture, were investigated by single-factor Wang et al. Bioresources and Bioprocessing (2015) 2:5 Page 3 of 9 experimental design by varying a single factor at a time H O for 1 min at pH 7.0 and 25°C. Protein was estimated 2 2 and keeping other factors at a constant value. All experi- by the procedure of Lowry et al. [24], using crystalline bo- ments were performed in triplicate, and data are expressed vine albumin as standard. Phenylalanine ammonia lyase as the mean of three samples with standard deviation. (PAL) was extracted from fresh H. perforatum cells with borate buffer (pH 8.8). The cells were ground in the buffer Extraction and HPLC analysis of secondary metabolites (0.15 g/mL) for 2 min with a pestle and mortar on ice and H. perforatum extracts were prepared by soaking 0.5 g of then centrifuged at 10,000 rpm and 4°C for 20 min to ob- the dried cells in 10 mL of methanol and treated with 1 tain a solid-free extract. The PAL activity was determined h of ultrasonic extraction for two times. The extracts based on the conversion of L-phenylalanine to cinnamic were then centrifuged at 10,000 rpm at 4°C. The super- acid as described by Wu and Lin [21]. natant was combined and used for HPLC analysis and flavonoid determination. Results and discussion The HPLC system of Shimadzu LC-10Avp Plus with a Callus induction and cell suspension culture PDA detector (SPD-M20A) and a C column (4.6 mm × establishment 250 mm, 5 μm, Eclipse XDB C18) was used for the In the preliminary work, calyx, leaf, petal, and stem qualitative and quantitative analyses of flavonoids and segments of H. perforatum were used as explants for other metabolites in the methanol extracts. The mobile callus induction. It revealed that callus induction was phase was acetonitrile (A) and 0.1% trifluoroacetic acid significantly affected by the type of explants and the (B). A gradient method was used for the separation of stem was the best explant for the initiation of a friable the extracted samples: 0-25 min, 15%-40% A; 25-40 min, callus (Figure 1A,B,C). The stem-derived callus was in- 40%-100% A. The elution flow rate and detection wave- cubated at 25 ± 2°C under continuous illumination on length were set at 1.0 mL/min and 254 nm, respectively. a solid MS medium and subcultured every 4 weeks. The identity of peaks separated by HPLC was confirmed Cell suspension culture was initiated once the stable by the injection of standard, and UV spectral analysis cell lines were obtained on the solid MS medium. was carried out to confirm compound identification. Given the potential for deriving useful secondary me- tabolites from plant resource, cell suspension cultures Determination of biomass and flavonoid content of H. perforatum were established to produce hypericin, The plant cell biomass is expressed as the gram fresh naphtodianthrones, and phenylpropanoids [12,14,18]. In cell weight and/or dry cell weight per liter. Cell suspension addition, some efforts have been made for the establish- cultures were filtered and washed by deionized water, then ment of adventitious root cultures of H. perforatum for cells were collected and weighed to get the FW. The col- the production of flavonoids, phenolics, and xanthones lected cells were dried in the vacuum drying oven at 45°C [5,15-17]. In the present study, cell suspension culture was until constant weight (DW) was attained. successfully established (Figure 1D) from the stem-derived The flavonoid content was assayed using UV colori- calli of H. perforatum for further investigation of flavo- metric method as reported elsewhere [5,13]. Briefly, 0.5 noids' production performance. mL of the methanolic plant cell extract was mixed with To understand the growth and flavonoid accumulation 2 mL of distilled water and subsequently with 0.15 mL in H. perforatum cells for determining the optimal culti- of a 5% NaNO solution. After 6 min, 0.15 mL of a 10% vation time, cultures were analyzed throughout a 30-day AlCl solution was added and the mixture was allowed period (Figure 2A). The cell growth exhibited a lag phase to stand for a further 6 min before 2 mL of 4% NaOH or slow growth period in the first 10 days; subsequently, solution was added. Absorbance of the mixed solution the cells entered their exponential growth phase with was measured at 510 nm using a UV-Vis spectropho- the maximum DW of 8.2 g/L reached on day 20. As tometer (UV-1650PC, Shimadzu, Japan). Rutin was used shown in Figure 2B, the flavonoid production is related as standard compound for the quantification of total fla- to cell growth and its content reached maximum with a vonoid. Results were expressed as milligram of rutin value of 16 mg/g DW during stationary or declining equivalents per gram of dry cells. Data were expressed phase between day 20–25. The content of total flavonoids as means ± SD for three replications. in cell suspension culture of H. perforatum (16 mg/g DW) was lower than that of the adventitious root cultures (42.7 Enzyme activity analysis mg/g DW) as reported earlier [5]. However, the culture Suspension culture cells were harvested for evaluation of period for the suspension cells of H. perforatum (3 weeks) the catalase (CAT) activity level as described by Georgiew was much shorter than that of the adventitious root cul- et al [23]. The decomposition of H O was followed by tures (6 weeks). In addition, the flavonoid production in 2 2 measuring the decrease in absorbance at 240 nm. One cell suspension culture of H. perforatum can be further unit of CAT is the amount that decomposes 1 μmol of improved by the supplementation of effective elicitors Wang et al. Bioresources and Bioprocessing (2015) 2:5 Page 4 of 9 Figure 1 Callus induced from different explants of H. perforatum and established cell suspension cultures. (A) Callus induced from stem explants; (B) callus induced from leaf explants; (C) callus induced from calyx explants; (D) cell suspension culture. [18,19]. Therefore, it is crucial to screen potential elici- tors for the hyperproduction of flavonoids in a short- ened culture period by cell suspension culture of H. perforatum. HPLC was used to recognize the flavonoids biosynthe- sized in the cell cultures on the basis of the retention times and UV spectras with those of reference standards. As shown in Figure 3A,B,C,D, hyperin and quercetin were the main flavonoids obtained from the established cell suspension cultures, which was in accordance with a previous study [6]. In addition, the medium was exam- ined, and no flavonoids were detected. Selection of MeJA as the elicitor in cell suspension culture of H. perforatum Some known abiotic elicitors for plant secondary metab- olites such as signaling molecules (MeJA and SA) and inorganic salts (NH VO and NiSO ) were tested for 4 3 4 their effect on cell growth and flavonoid production by the H. perforatum cell suspension culture. Four treat- ments, MeJA (50 μM), salicylic acid (100 μM), NH VO 4 3 (50 μM), and NiSO (15 μM) were applied to the cell cultures on day 15 based on our previous lab results. As shown in Figure 4, the growth of cell cultures was inhib- ited by the addition of the four elicitors as compared to the control cultures. Similar findings were also observed by Dong and Zhong [25], in which the application of elicitation in suspension cell culture severely inhibited the growth of Taxus chinensis cells. However, elicitation treatments may have varied effects on different cell lines. Some researchers have reported that the growth of cell Figure 2 Determination of optimal culture time in H. perforatum cultures was not affected by elicitors [26]. The effects of cells. Kinetic profiles of cell growth (A) and content of flavonoids the elicitors on flavonoid production are also given in (B) in H. perforatum cell suspension cultures. Figure 4. Flavonoid content was promoted by MeJA and Wang et al. Bioresources and Bioprocessing (2015) 2:5 Page 5 of 9 Figure 3 Hyperin and quercetin obtained from the established cell suspension cultures. Chemical structures of hyperin and quercetin (A) and chromatographic profile of hyperin (B), quercetin (C) and the suspension cell extracts (D). The two insets are the UV spectrums of standard hyperin (B) and quercetin (C). Wang et al. Bioresources and Bioprocessing (2015) 2:5 Page 6 of 9 considering its further application. In the present investi- gation, the addition of MeJA (50 μM) resulted in max- imum flavonoid production, which indicated the potential of this abiotic elicitor for the enhancement of flavonoid biosynthesis in cell suspension culture of H. perforatum and was selected for further study. Effects of MeJA feeding time on cell growth and flavonoid production The elicitor induction time is one of the key factors that affect the cell growth and product yield for plant cell suspension culture [33]. The effects of MeJA addition time on cell growth and flavonoid accumulation was evaluated by the addition of 50 μM MeJA into the cell cultures on lag phase (day 5 and day 10) and exponential Figure 4 The effects of four abiotic elicitors on H. perforatum cell growth and flavonoid accumulation. 1 Control; 2 MeJA; 3 SA; phase (day 15 and day 20). Figure 5A,B,C showed the 4 NH4VO ; 5 NiSO (*P < 0.05, **P < 0.01, and ***P < 0.001 indicate 3 4 biomass and flavonoid production of H. perforatum cell statistical significance compared to the control). cultures after MeJA addition. As shown in Figure 5A,B, C, the cell growth and production of flavonoids were SA induction, which were 2.1 and 1.5 times higher in suppressed deleteriously when MeJA was added on the comparison to control cultures (15.36 mg/g DW), re- lag phase. MeJA treatment on the exponential phase spectively. Several studies on other plant cell cultures (day 15 and day 20) led to a slow decrease in DW have shown that MeJA and SA elicitation can enhance (19.0% and 9.7%, respectively); however, the flavonoid the production of secondary metabolites. For instance, content and production were significantly increased as MeJA was recognized as an effective elicitor that could compared to control cultures (Figure 5A,B,C). A similar increase the production of paclitaxel in Taxus candensis phenomenon was also observed by Huang and Zhong and T. cuspidate [27], anthocyanin in Tulipa gesneriana [30], in which elicitation treatment on the log phase of [28], and gymnemic acid in Gymnema sylvestre [29]. In P. ginseng reduced the DW and enhanced ginsenoside SA-elicited cell suspension culture of H. perforatum, the accumulation. Notably, MeJA treatment on day 15 re- production of both hypericin and pseudohypericin has sulted in the highest value of flavonoid content (38.26 doubled as compared to control cell suspension cultures mg/g DW) or production (229.79 mg/L). Therefore, [18]. As shown in Figure 4, MeJA and SA both induced MeJA treatment on day 15 was taken as the optimal flavonoid accumulation in H. perforatum cell suspension addition time for flavonoid production. cultures, but SA was less efficient than MeJA in pro- moting flavonoid accumulation under the tested feed- Effects of MeJA dosage on cell growth and flavonoid ing concentration. production Some literatures reported the enhancement of metab- A suitable elicitor concentration is important for the cell olite production by NH VO and NiSO elicitation [30]. growth and product yield in plant cell suspension cul- 4 3 4 However, the two elicitors revealed negative effects on ture process [34]. To study the effects of MeJA addition biomass and flavonoid accumulation in H. perforatum concentrations, cell suspension cultures of H. perfora- cell cultures (Figure 4). It was probably due to the sig- tum were treated with different levels (50-200 μM) of nificant biological toxicity caused by the inorganic salts MeJA after 15 days of cultivation. The DW and flavon- [30]. Some other elicitors such as fluoro- and hydroxyl- oid content on day 20 were shown in Figure 6A. It is containing derivatives of methyl jasmonate, which were clear that all the tested concentrations of MeJA had an proven more potent than methyl jasmonate in suspen- inhibitory impact on the cell growth; similar results were sion culture of Taxus [31,32], may also serve as possible also obtained in the SA-induced cell suspension culture options for the enhancement of flavonoid biosynthesis in of H. perforatum [18]. Induction with 50 or 100 μMof H. perforatum. In addition, some new elicitors have been MeJA decreased DW by 11.9%-23.1%, while higher con- reported to have significantly promoted secondary me- centration of MeJA (150 μM) severely decreased the DW tabolite biosynthesis by plant cell cultures [33]. However, from 7.0 ± 0.5 g/L (control cultures) to 5.0 ± 0.5 g/L. elicitation treatments may have varied effects among Addition of low dosage (<150 μM) of MeJA elicitor to the different cell lines as mentioned above. Therefore, efforts cultured cells of H. perforatum increased the flavonoid ac- devoted into the enhancement of flavonoid production by cumulation. As shown in Figure 6A, the flavonoid content H. perforatum cell suspension culture are still required was increased about 1.1-fold with a dosage of 50 μMand Wang et al. Bioresources and Bioprocessing (2015) 2:5 Page 7 of 9 Figure 6 Effects of MeJA dosage on cell growth and flavonoid content (A) and flavonoid production (B). *P < 0.05, **P < 0.01, and *** P < 0.001 indicate statistical significance compared to the control. about 2.3-fold with a dosage of 100 μM as compared to the control cultures. The flavonoid content reached max- imum (52.8 mg/g DW) at the elicitation dose of 100 μM, which was relatively higher than the previously reported values in the adventitious root culture (i.e., 42.7 and 48.6 mg/g DW) [5,17]. However, further dosage increase (150- 200 μM) decreased the flavonoid biosynthesis compared to moderate MeJA levels (50-100 μM). Sensitivity of suspension cell cultures to elicitor concentration differs with plant species [19,29]. The negative effect of MeJA at higher concentrations (200 μM) on cell growth and metabolite production was also reported in Gymnema sylvestre cell suspension cultures [29]. The best dose of the elicitor with maximum production (279.5 mg/L) of Figure 5 Effects of MeJA (50 μM) feeding time on cell growth flavonoids was 100 μMasshown in Figure 6B,which (A), flavonoid content (B), and flavonoid production (C). 5+, 10+, 15+, and 20+ indicate MeJA feeding on day 5, 10, 15, and 20. was about 2.7-fold the value of control cultures. The above-mentioned results indicate that the optimal MeJA elicitation strategy was treatment on day 15 with a dos- age of 100 μM. Wang et al. Bioresources and Bioprocessing (2015) 2:5 Page 8 of 9 Effects of MeJA addition on CAT and PAL activities accumulation of ROS and resulted in a promotion of The enzyme activity changes were often related to the secondary metabolites production. Similar results were secondary metabolite accumulation [35]. For a better observed on phytoalexin production in the suspension understanding of the elicitation effect of MeJA, the ac- cell culture of Glycine max, which indicated that the tivities of two important enzymes (CAT and PAL) under ROS mediated elicitor-induced accumulation of second- MeJA treatment were determined. Reactive oxygen spe- ary metabolites [38]. cies (ROS) are toxic intermediates resulting from succes- PAL is the first enzyme of phenylpropanoid biosynthesis sive steps in the reduction of molecular O in plant cells. in plants and plays an important role in the biosynthesis The ROS exerts various effects on plant defense re- of flavonoids, lignins, and many other compounds [36]. sponses, including cell wall reinforcement, hypersensitive An increase in PAL activity after elicitor treatment often cell death, defensive gene activation, as well as defensive results in enhanced secondary metabolism in plant cells compound induction [36]. CAT is a well-known intracel- [19]. Figure 7B shows the changes of intracellular PAL ac- lular enzyme which protects against the ROS generated tivity in the suspension cultures after the treatment by within cells [37]. As shown in Figure 7A, the enzyme ac- MeJA (100 μM). As shown in Figure 7B, MeJA induced tivity increased with the cultivation time and reached the increase of PAL activities and reached maximum (12.1 maximum on day 19, thereafter decreased. MeJA down- U/g FW) 4 days after treatment, thereafter decreased. regulated the CAT activity after its addition to the cell cul- However, cell cultures without MeJA treatment (control) tures on day 15 and kept at a relatively lower level as com- revealed little change of PAL activities. In the presence of pared to the control (Figure 7A), which led to a higher MeJA, PAL activity was much higher than that of control (Figure 7B), the elicitor-induced up-regulation of the PAL activity resulted in an improved flavonoid production in A the suspension cultures (Figure 6B). Zhao et al. [19] have observed similar results in Salvia miltiorrhiza cell cul- tures, which indicated that both biotic and abiotic elicit- ation treatment resulted in the up-regulation of PAL activity and improved tanshinone accumulation as com- pared to the control cultures. These results demonstrated that MeJA is an efficient elicitor for the induction of fla- vonoid production in the suspension cell culture of H. perforatum. Conclusions Plant cell suspension culture is an efficient alternative method for the production of useful biochemicals. In this work, MeJA-elicited cell suspension cultures of H. perfora- tum enhanced the production of flavonoids through the down-regulation of the CAT activity and up-regulation of the PAL activity. The flavonoid production reached 280 mg/L after the optimal elicitation conditions, which was 2.7-fold of the control cultures. These results would be useful for the hyperproduction of flavonoids from H. per- foratum and would be useful for the production of valu- able compounds from other plant cell cultures. Abbreviations CAT: Catalase; DW: Dry cell weight; FW: Fresh cell weight; MeJA: Methyl jasmonate; PAL: Phenylalanine ammonia lyase; SA: Salicylic acid. Competing interests The authors declare that they have no competing interests. Authors' contributions Figure 7 Dynamic changes of CAT (A) and PAL (B) activity with J W was in charge of the experiments and paper writing. LY Y participated in the experiments and paper writing. J Q kindly offered experimental plant 100 μM of MeJA added on day 15 in cell suspension culture of and guided the experiments. YH L directed the study as the tutor. All H. perforatum. authors read and approved the final manuscript. Wang et al. 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Enhanced production of flavonoids by methyl jasmonate elicitation in cell suspension culture of Hypericum perforatum

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Springer Journals
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2015 Wang et al.; licensee Springer.
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2197-4365
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10.1186/s40643-014-0033-5
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Abstract

Background: Flavonoids of Hypericum perforatum are important secondary metabolites which have been widely utilized in medicine for a range of purposes. The use of methyl jasmonate (MeJA) elicitation for the enhancement of flavonoid production in cell suspension culture of H. perforatum would be an efficient alternative method for the flavonoid production. Results: MeJA influenced the cells growth and flavonoid production. The optimal elicitation strategy was treatment of the cell cultures with 100 μmol/L MeJA on day 15, which resulted in the highest flavonoid production (280 mg/L) and 2.7 times of control cultures. The activities of catalase (CAT) were inhibited after MeJA treatment in the cell cultures, while the activities of phenylalanine ammonia lyase (PAL) increased, which led to the enhancement of flavonoid production. Conclusion: MeJA elicitation is a useful method for the enhancement of flavonoid production in cell suspension culture of H. perforatum. Keywords: MeJA; Hypericum perforatum; Suspension culture; Flavonoids Background The consumption of H. perforatum-derived products Hypericum perforatum, commonly known as St. John's as pharmaceutical preparations or food additives has in- wort, is a perennial herb native to Europe and also a creased dramatically, and it is presently one of the most traditional medicinal plant which has been utilized in consumed medicinal plants in the world [4,7]. The extracts Chinese folk medicine for a range of purposes [1,2]. The of H. perforatum currently used in foods and pharmaceut- medicinal applications of H. perforatum, including skin ics, mostly composed of flavonoids, are mainly obtained wounds, eczema, burns, diseases of the alimentary tract, from the top aerial parts collected in the flowering stage and psychological disorders, have been related to the [6,8]. However, the quality of the flavonoid-rich extracts phenolic composition of the plant, particularly to hyperi- derived from field-grown plants may be affected by many cins, hyperforins, and flavonoids [3,4]. The efficacy of environmental factors as well as biological processes [5]. medical constituents of H. perforatum is based on the Furthermore, field cultivation of H. perforatum requires whole secondary metabolites, rather than the presence a long growth period and plant management, which is a of single chemical compound [5]. Flavonoids, specifically slow and laborious process [6]. Therefore, an alternative quercetin and its glycoside derivatives, comprise the major method for more efficient and controllable production group of biologically active metabolites in H. perforatum of flavonoids from H. perforatum is urgently required. and are important biochemical markers in authenticating Plant cell, tissue, and organ cultivation technology has the herbal plant materials [4,6]. been successfully applied to the production of pharma- ceutically valuable compounds and other fine chemicals of commercial interest in recent years [9]. For instance, * Correspondence: Lyyao1982@qq.com; luyanhua@ecust.edu.cn State Key Laboratory of Bioreactor Engineering, Department of cell suspension cultures of Panax ginseng have been widely Bioengineering, East China University of Science and Technology, 130 used for the production of saponin and other metabolites Meilong Rd., Shanghai 200237, China [10]. Plant-mediated green biomimetic synthesis of silver Full list of author information is available at the end of the article © 2015 Wang et al.; licensee Springer. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. Wang et al. Bioresources and Bioprocessing (2015) 2:5 Page 2 of 9 nanoparticle was considered a widely acceptable technol- Salicylic acid (SA), ammonium metavanadate (NH VO ), 4 3 ogy for rapid production of silver nanoparticles for suc- and nickel sulfate (NiSO ) were purchased from Shanghai cessfully meeting the excessive need and current market Zhong Lan Chemical Company (Shanghai, China). Aceto- demand and resulting in a reduction in the employment nitrile of HPLC grade was purchased from TEDIA Com- or generation of hazardous substances to human health pany, USA. All other chemicals (analytical grade) were and the environment [11]. Till now, a number of efforts purchased from Shanghai Chemical Co., Ltd. (Shanghai, have been made for the establishment of cell and/or ad- China). ventitious root cultures of H. perforatum for the produc- tion of hypericins, phenolics, and xanthones [12-15]. The Plant material and callus induction information on the factors affecting biomass and afore- H. perforatum plants were provided by Prof. Jie Qian mentioned metabolites of H. perforatum was also in- (Tongji University, China) and were cultivated in the vestigated through elicitation and culture optimization greenhouse. The fresh stem explants of H. perforatum [1,16-18]. However, there are few reports on the pro- were washed thoroughly with tap water, surface-sterilized duction of flavonoids from cell suspension culture of with 75% ethanol for 60 s, and then soaked in 10% sodium H. perforatum, and little is known about the strategies hypochlorite solution for 30 s and rinsed seven times in for hyperproduction of biomass and flavonoids. sterile deionized water. The clean and sterilized stem ex- Many cell cultures have been established from plants, plants were cut into 5-mm segments and inoculated on but they seldom produce sufficient amounts of the re- solid Murashige and Skoog (MS) [22] medium supple- quired secondary metabolites. Secondary metabolite bio- mented with 1.0 mg/L 2,4-D, 0.2 mg/L 6-benzyladenine synthesis in plants depends on environmental stresses; (BA), and 25 g/L sucrose to induce callus formation. After their accumulation can be stimulated by precursors and 1 month, the successfully induced callus was separated elicitors [19]. Precursors are intermediate compounds of from the explants and cultured separately until used for the secondary metabolite biosynthesis cycles, which would the establishment of cell suspensions. be toxic to the culture if not used at an appropriate stage or concentration [20]. Elicitors are biological (components Suspension cultures of H. perforatum cells of microbial cells and poly- and oligosaccharides), chemical Cell suspension cultures were initiated from the friable (heavy metals, pesticides, and the signaling compounds in callus and maintained in the liquid MS medium (pH plant defense responses), or physical (cold shock, UV, 5.8) supplemented with 1.0 mg/L 2,4-D, 0.2 mg/L 6- hyperosmotic stress, ultrasound, and pulsed electric field) benzyladenine (BA), and 25 g/L sucrose. A sterile standard factors that induce enzymatic activity against stress sieve with aperture of 300 μm was used to filter the initial [19-21]. Jasmonic acid (JA) and its methyl ester, methyl established suspension cells to obtain homogeneous cul- jasmonate (MeJA), have been proposed to be important tures. The pre-weighed cells were cultured in a 250-mL signaling compounds in the process of elicitation leading Erlenmeyer flask containing a 50-mL medium; cultures to the hyperproduction of various secondary metabolites were placed on a rotary shaker shaking at 120 rpm at [14]. They have also been reported to play a key role in 25 ± 2°C under continuous illumination. Every 20 days, signal transduction processes that regulate defense re- cells were subcultured to fresh media with 5.0-g fresh sponses in plants and shown effective to enhance the pro- weight (FW) in 250-mL flasks. duction of secondary metabolites in cell cultures [14,19]. In the present study, the effect of MeJA on cell growth Elicitation of cell suspension culture and flavonoid biosynthesis in the H. perforatum cell sus- Four elicitors including MeJA, SA, NH VO , and NiSO 4 3 4 pension culture was investigated in small batches. Some were used to study the elicitation effect on flavonoid parameters, such as elicitation time and MeJA concen- production by the cell suspension cultures of H. perfora- tration, on biomass and flavonoid production were stud- tum. MeJA and SA were dissolved in ethanol, NH VO 4 3 ied in detail. The activities of key enzymes (catalase and and NiSO were dissolved in water, and the four elicitors phenylalanine ammonia lyase) related to plant stress re- were filter-sterilized before adding into the suspension sponses and secondary metabolite biosynthesis were in- cultures. Based on the results of preliminary experi- vestigated as well. To the best of our knowledge, this is ments, the feeding concentration of MeJA, SA, NH VO , 4 3 the first report on the induction of flavonoid production and NiSO was 50, 100, 50, and 15 μM, respectively. As by MeJA in cell suspension culture of H. perforatum. a control, filter-sterilized ethanol was added to the cell suspension culture with a final concentration of 0.2%. Materials and methods According to the results of the above experiments, Chemicals MeJA was selected for further study. The MeJA induc- MeJA was supplied by Drug Institute of East China tion time and feeding concentration, known to be crucial University of Science and Technology (Shanghai, China). to plant cell culture, were investigated by single-factor Wang et al. Bioresources and Bioprocessing (2015) 2:5 Page 3 of 9 experimental design by varying a single factor at a time H O for 1 min at pH 7.0 and 25°C. Protein was estimated 2 2 and keeping other factors at a constant value. All experi- by the procedure of Lowry et al. [24], using crystalline bo- ments were performed in triplicate, and data are expressed vine albumin as standard. Phenylalanine ammonia lyase as the mean of three samples with standard deviation. (PAL) was extracted from fresh H. perforatum cells with borate buffer (pH 8.8). The cells were ground in the buffer Extraction and HPLC analysis of secondary metabolites (0.15 g/mL) for 2 min with a pestle and mortar on ice and H. perforatum extracts were prepared by soaking 0.5 g of then centrifuged at 10,000 rpm and 4°C for 20 min to ob- the dried cells in 10 mL of methanol and treated with 1 tain a solid-free extract. The PAL activity was determined h of ultrasonic extraction for two times. The extracts based on the conversion of L-phenylalanine to cinnamic were then centrifuged at 10,000 rpm at 4°C. The super- acid as described by Wu and Lin [21]. natant was combined and used for HPLC analysis and flavonoid determination. Results and discussion The HPLC system of Shimadzu LC-10Avp Plus with a Callus induction and cell suspension culture PDA detector (SPD-M20A) and a C column (4.6 mm × establishment 250 mm, 5 μm, Eclipse XDB C18) was used for the In the preliminary work, calyx, leaf, petal, and stem qualitative and quantitative analyses of flavonoids and segments of H. perforatum were used as explants for other metabolites in the methanol extracts. The mobile callus induction. It revealed that callus induction was phase was acetonitrile (A) and 0.1% trifluoroacetic acid significantly affected by the type of explants and the (B). A gradient method was used for the separation of stem was the best explant for the initiation of a friable the extracted samples: 0-25 min, 15%-40% A; 25-40 min, callus (Figure 1A,B,C). The stem-derived callus was in- 40%-100% A. The elution flow rate and detection wave- cubated at 25 ± 2°C under continuous illumination on length were set at 1.0 mL/min and 254 nm, respectively. a solid MS medium and subcultured every 4 weeks. The identity of peaks separated by HPLC was confirmed Cell suspension culture was initiated once the stable by the injection of standard, and UV spectral analysis cell lines were obtained on the solid MS medium. was carried out to confirm compound identification. Given the potential for deriving useful secondary me- tabolites from plant resource, cell suspension cultures Determination of biomass and flavonoid content of H. perforatum were established to produce hypericin, The plant cell biomass is expressed as the gram fresh naphtodianthrones, and phenylpropanoids [12,14,18]. In cell weight and/or dry cell weight per liter. Cell suspension addition, some efforts have been made for the establish- cultures were filtered and washed by deionized water, then ment of adventitious root cultures of H. perforatum for cells were collected and weighed to get the FW. The col- the production of flavonoids, phenolics, and xanthones lected cells were dried in the vacuum drying oven at 45°C [5,15-17]. In the present study, cell suspension culture was until constant weight (DW) was attained. successfully established (Figure 1D) from the stem-derived The flavonoid content was assayed using UV colori- calli of H. perforatum for further investigation of flavo- metric method as reported elsewhere [5,13]. Briefly, 0.5 noids' production performance. mL of the methanolic plant cell extract was mixed with To understand the growth and flavonoid accumulation 2 mL of distilled water and subsequently with 0.15 mL in H. perforatum cells for determining the optimal culti- of a 5% NaNO solution. After 6 min, 0.15 mL of a 10% vation time, cultures were analyzed throughout a 30-day AlCl solution was added and the mixture was allowed period (Figure 2A). The cell growth exhibited a lag phase to stand for a further 6 min before 2 mL of 4% NaOH or slow growth period in the first 10 days; subsequently, solution was added. Absorbance of the mixed solution the cells entered their exponential growth phase with was measured at 510 nm using a UV-Vis spectropho- the maximum DW of 8.2 g/L reached on day 20. As tometer (UV-1650PC, Shimadzu, Japan). Rutin was used shown in Figure 2B, the flavonoid production is related as standard compound for the quantification of total fla- to cell growth and its content reached maximum with a vonoid. Results were expressed as milligram of rutin value of 16 mg/g DW during stationary or declining equivalents per gram of dry cells. Data were expressed phase between day 20–25. The content of total flavonoids as means ± SD for three replications. in cell suspension culture of H. perforatum (16 mg/g DW) was lower than that of the adventitious root cultures (42.7 Enzyme activity analysis mg/g DW) as reported earlier [5]. However, the culture Suspension culture cells were harvested for evaluation of period for the suspension cells of H. perforatum (3 weeks) the catalase (CAT) activity level as described by Georgiew was much shorter than that of the adventitious root cul- et al [23]. The decomposition of H O was followed by tures (6 weeks). In addition, the flavonoid production in 2 2 measuring the decrease in absorbance at 240 nm. One cell suspension culture of H. perforatum can be further unit of CAT is the amount that decomposes 1 μmol of improved by the supplementation of effective elicitors Wang et al. Bioresources and Bioprocessing (2015) 2:5 Page 4 of 9 Figure 1 Callus induced from different explants of H. perforatum and established cell suspension cultures. (A) Callus induced from stem explants; (B) callus induced from leaf explants; (C) callus induced from calyx explants; (D) cell suspension culture. [18,19]. Therefore, it is crucial to screen potential elici- tors for the hyperproduction of flavonoids in a short- ened culture period by cell suspension culture of H. perforatum. HPLC was used to recognize the flavonoids biosynthe- sized in the cell cultures on the basis of the retention times and UV spectras with those of reference standards. As shown in Figure 3A,B,C,D, hyperin and quercetin were the main flavonoids obtained from the established cell suspension cultures, which was in accordance with a previous study [6]. In addition, the medium was exam- ined, and no flavonoids were detected. Selection of MeJA as the elicitor in cell suspension culture of H. perforatum Some known abiotic elicitors for plant secondary metab- olites such as signaling molecules (MeJA and SA) and inorganic salts (NH VO and NiSO ) were tested for 4 3 4 their effect on cell growth and flavonoid production by the H. perforatum cell suspension culture. Four treat- ments, MeJA (50 μM), salicylic acid (100 μM), NH VO 4 3 (50 μM), and NiSO (15 μM) were applied to the cell cultures on day 15 based on our previous lab results. As shown in Figure 4, the growth of cell cultures was inhib- ited by the addition of the four elicitors as compared to the control cultures. Similar findings were also observed by Dong and Zhong [25], in which the application of elicitation in suspension cell culture severely inhibited the growth of Taxus chinensis cells. However, elicitation treatments may have varied effects on different cell lines. Some researchers have reported that the growth of cell Figure 2 Determination of optimal culture time in H. perforatum cultures was not affected by elicitors [26]. The effects of cells. Kinetic profiles of cell growth (A) and content of flavonoids the elicitors on flavonoid production are also given in (B) in H. perforatum cell suspension cultures. Figure 4. Flavonoid content was promoted by MeJA and Wang et al. Bioresources and Bioprocessing (2015) 2:5 Page 5 of 9 Figure 3 Hyperin and quercetin obtained from the established cell suspension cultures. Chemical structures of hyperin and quercetin (A) and chromatographic profile of hyperin (B), quercetin (C) and the suspension cell extracts (D). The two insets are the UV spectrums of standard hyperin (B) and quercetin (C). Wang et al. Bioresources and Bioprocessing (2015) 2:5 Page 6 of 9 considering its further application. In the present investi- gation, the addition of MeJA (50 μM) resulted in max- imum flavonoid production, which indicated the potential of this abiotic elicitor for the enhancement of flavonoid biosynthesis in cell suspension culture of H. perforatum and was selected for further study. Effects of MeJA feeding time on cell growth and flavonoid production The elicitor induction time is one of the key factors that affect the cell growth and product yield for plant cell suspension culture [33]. The effects of MeJA addition time on cell growth and flavonoid accumulation was evaluated by the addition of 50 μM MeJA into the cell cultures on lag phase (day 5 and day 10) and exponential Figure 4 The effects of four abiotic elicitors on H. perforatum cell growth and flavonoid accumulation. 1 Control; 2 MeJA; 3 SA; phase (day 15 and day 20). Figure 5A,B,C showed the 4 NH4VO ; 5 NiSO (*P < 0.05, **P < 0.01, and ***P < 0.001 indicate 3 4 biomass and flavonoid production of H. perforatum cell statistical significance compared to the control). cultures after MeJA addition. As shown in Figure 5A,B, C, the cell growth and production of flavonoids were SA induction, which were 2.1 and 1.5 times higher in suppressed deleteriously when MeJA was added on the comparison to control cultures (15.36 mg/g DW), re- lag phase. MeJA treatment on the exponential phase spectively. Several studies on other plant cell cultures (day 15 and day 20) led to a slow decrease in DW have shown that MeJA and SA elicitation can enhance (19.0% and 9.7%, respectively); however, the flavonoid the production of secondary metabolites. For instance, content and production were significantly increased as MeJA was recognized as an effective elicitor that could compared to control cultures (Figure 5A,B,C). A similar increase the production of paclitaxel in Taxus candensis phenomenon was also observed by Huang and Zhong and T. cuspidate [27], anthocyanin in Tulipa gesneriana [30], in which elicitation treatment on the log phase of [28], and gymnemic acid in Gymnema sylvestre [29]. In P. ginseng reduced the DW and enhanced ginsenoside SA-elicited cell suspension culture of H. perforatum, the accumulation. Notably, MeJA treatment on day 15 re- production of both hypericin and pseudohypericin has sulted in the highest value of flavonoid content (38.26 doubled as compared to control cell suspension cultures mg/g DW) or production (229.79 mg/L). Therefore, [18]. As shown in Figure 4, MeJA and SA both induced MeJA treatment on day 15 was taken as the optimal flavonoid accumulation in H. perforatum cell suspension addition time for flavonoid production. cultures, but SA was less efficient than MeJA in pro- moting flavonoid accumulation under the tested feed- Effects of MeJA dosage on cell growth and flavonoid ing concentration. production Some literatures reported the enhancement of metab- A suitable elicitor concentration is important for the cell olite production by NH VO and NiSO elicitation [30]. growth and product yield in plant cell suspension cul- 4 3 4 However, the two elicitors revealed negative effects on ture process [34]. To study the effects of MeJA addition biomass and flavonoid accumulation in H. perforatum concentrations, cell suspension cultures of H. perfora- cell cultures (Figure 4). It was probably due to the sig- tum were treated with different levels (50-200 μM) of nificant biological toxicity caused by the inorganic salts MeJA after 15 days of cultivation. The DW and flavon- [30]. Some other elicitors such as fluoro- and hydroxyl- oid content on day 20 were shown in Figure 6A. It is containing derivatives of methyl jasmonate, which were clear that all the tested concentrations of MeJA had an proven more potent than methyl jasmonate in suspen- inhibitory impact on the cell growth; similar results were sion culture of Taxus [31,32], may also serve as possible also obtained in the SA-induced cell suspension culture options for the enhancement of flavonoid biosynthesis in of H. perforatum [18]. Induction with 50 or 100 μMof H. perforatum. In addition, some new elicitors have been MeJA decreased DW by 11.9%-23.1%, while higher con- reported to have significantly promoted secondary me- centration of MeJA (150 μM) severely decreased the DW tabolite biosynthesis by plant cell cultures [33]. However, from 7.0 ± 0.5 g/L (control cultures) to 5.0 ± 0.5 g/L. elicitation treatments may have varied effects among Addition of low dosage (<150 μM) of MeJA elicitor to the different cell lines as mentioned above. Therefore, efforts cultured cells of H. perforatum increased the flavonoid ac- devoted into the enhancement of flavonoid production by cumulation. As shown in Figure 6A, the flavonoid content H. perforatum cell suspension culture are still required was increased about 1.1-fold with a dosage of 50 μMand Wang et al. Bioresources and Bioprocessing (2015) 2:5 Page 7 of 9 Figure 6 Effects of MeJA dosage on cell growth and flavonoid content (A) and flavonoid production (B). *P < 0.05, **P < 0.01, and *** P < 0.001 indicate statistical significance compared to the control. about 2.3-fold with a dosage of 100 μM as compared to the control cultures. The flavonoid content reached max- imum (52.8 mg/g DW) at the elicitation dose of 100 μM, which was relatively higher than the previously reported values in the adventitious root culture (i.e., 42.7 and 48.6 mg/g DW) [5,17]. However, further dosage increase (150- 200 μM) decreased the flavonoid biosynthesis compared to moderate MeJA levels (50-100 μM). Sensitivity of suspension cell cultures to elicitor concentration differs with plant species [19,29]. The negative effect of MeJA at higher concentrations (200 μM) on cell growth and metabolite production was also reported in Gymnema sylvestre cell suspension cultures [29]. The best dose of the elicitor with maximum production (279.5 mg/L) of Figure 5 Effects of MeJA (50 μM) feeding time on cell growth flavonoids was 100 μMasshown in Figure 6B,which (A), flavonoid content (B), and flavonoid production (C). 5+, 10+, 15+, and 20+ indicate MeJA feeding on day 5, 10, 15, and 20. was about 2.7-fold the value of control cultures. The above-mentioned results indicate that the optimal MeJA elicitation strategy was treatment on day 15 with a dos- age of 100 μM. Wang et al. Bioresources and Bioprocessing (2015) 2:5 Page 8 of 9 Effects of MeJA addition on CAT and PAL activities accumulation of ROS and resulted in a promotion of The enzyme activity changes were often related to the secondary metabolites production. Similar results were secondary metabolite accumulation [35]. For a better observed on phytoalexin production in the suspension understanding of the elicitation effect of MeJA, the ac- cell culture of Glycine max, which indicated that the tivities of two important enzymes (CAT and PAL) under ROS mediated elicitor-induced accumulation of second- MeJA treatment were determined. Reactive oxygen spe- ary metabolites [38]. cies (ROS) are toxic intermediates resulting from succes- PAL is the first enzyme of phenylpropanoid biosynthesis sive steps in the reduction of molecular O in plant cells. in plants and plays an important role in the biosynthesis The ROS exerts various effects on plant defense re- of flavonoids, lignins, and many other compounds [36]. sponses, including cell wall reinforcement, hypersensitive An increase in PAL activity after elicitor treatment often cell death, defensive gene activation, as well as defensive results in enhanced secondary metabolism in plant cells compound induction [36]. CAT is a well-known intracel- [19]. Figure 7B shows the changes of intracellular PAL ac- lular enzyme which protects against the ROS generated tivity in the suspension cultures after the treatment by within cells [37]. As shown in Figure 7A, the enzyme ac- MeJA (100 μM). As shown in Figure 7B, MeJA induced tivity increased with the cultivation time and reached the increase of PAL activities and reached maximum (12.1 maximum on day 19, thereafter decreased. MeJA down- U/g FW) 4 days after treatment, thereafter decreased. regulated the CAT activity after its addition to the cell cul- However, cell cultures without MeJA treatment (control) tures on day 15 and kept at a relatively lower level as com- revealed little change of PAL activities. In the presence of pared to the control (Figure 7A), which led to a higher MeJA, PAL activity was much higher than that of control (Figure 7B), the elicitor-induced up-regulation of the PAL activity resulted in an improved flavonoid production in A the suspension cultures (Figure 6B). Zhao et al. [19] have observed similar results in Salvia miltiorrhiza cell cul- tures, which indicated that both biotic and abiotic elicit- ation treatment resulted in the up-regulation of PAL activity and improved tanshinone accumulation as com- pared to the control cultures. These results demonstrated that MeJA is an efficient elicitor for the induction of fla- vonoid production in the suspension cell culture of H. perforatum. Conclusions Plant cell suspension culture is an efficient alternative method for the production of useful biochemicals. In this work, MeJA-elicited cell suspension cultures of H. perfora- tum enhanced the production of flavonoids through the down-regulation of the CAT activity and up-regulation of the PAL activity. The flavonoid production reached 280 mg/L after the optimal elicitation conditions, which was 2.7-fold of the control cultures. These results would be useful for the hyperproduction of flavonoids from H. per- foratum and would be useful for the production of valu- able compounds from other plant cell cultures. Abbreviations CAT: Catalase; DW: Dry cell weight; FW: Fresh cell weight; MeJA: Methyl jasmonate; PAL: Phenylalanine ammonia lyase; SA: Salicylic acid. Competing interests The authors declare that they have no competing interests. Authors' contributions Figure 7 Dynamic changes of CAT (A) and PAL (B) activity with J W was in charge of the experiments and paper writing. LY Y participated in the experiments and paper writing. J Q kindly offered experimental plant 100 μM of MeJA added on day 15 in cell suspension culture of and guided the experiments. YH L directed the study as the tutor. All H. perforatum. authors read and approved the final manuscript. Wang et al. Bioresources and Bioprocessing (2015) 2:5 Page 9 of 9 Acknowledgements and cell suspension cultures on production of naphtodianthrones and This work was supported by the National Special Fund for State Key phenylpropanoids in Hypericum perforatum L. Plant Cell Tiss Org 113:25–39 Laboratory of Bioreactor Engineering (2060204) and the Fundamental 19. Zhao JL, Zhou LG, Wu JY (2010) Effects of biotic and abiotic elicitors on cell Research Funds for the Central Universities (WF1113010). growth and tanshinone accumulation in Salvia miltiorrhiza cell cultures. Appl Microbiol Biotechnol 87:137–144 Author details 20. Gueven A, Knorr D (2011) Isoflavonoid production by soy plant callus State Key Laboratory of Bioreactor Engineering, Department of suspension culture. J Food Eng 103:237–243 Bioengineering, East China University of Science and Technology, 130 21. Lin L, Wu J (2002) Enhancement of shikonin production in single- and Meilong Rd., Shanghai 200237, China. 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Journal

"Bioresources and Bioprocessing"Springer Journals

Published: Dec 1, 2015

Keywords: Biochemical Engineering; Environmental Engineering/Biotechnology; Industrial and Production Engineering

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