Elicitor-enhanced syringin production in suspension cultures of Saussurea medusa

  • Chunming Xu
  • Bing Zhao
  • Yuan Ou
  • Xiaodong Wang
  • Xiaofan Yuan
  • Yuchun Wang
Original Paper

Abstract

Syringin production and related secondary metabolism enzyme activities in suspension cultures of Saussurea medusa treated with different elicitors (yeast extract, chitosan and Ag+) were investigated. All elicitors enhanced syringin production, and the optimal feeding protocol was the combined addition of 1.5% (v/v) yeast extract, 0.2 g l−1 chitosan and 75 μM Ag+ at the 15th day of the cell culture. The highest syringin production reached 741.9 mg l−1, which was 3.6−fold that of the control. The glucose−6-phosphate dehydrogenase (EC 1.1.1.49), phenylalanine ammonia lyase (EC 4.3.1.5) and peroxidase (EC 1.11.1.7) activities increased significantly after elicitor treatment. The maximum enzyme activities were obtained when the treatment time was 6 h.

Keywords

Elicitor G6PDH PAL POD Saussurea medusa Syringin Suspension culture 

Abbreviations

6BA

6-Benzylaminopurine

DTT

Dithiotreitol

G6PDH

Glucose-6-phosphate dehydrogenase

NAA

α-Naphthaleneacetic acid

PAL

Phenylalanine ammonia lyase

POD

Peroxidase

PPP

Pentose phosphate pathway

PVPP

Polyvinyl polypyrrolidone

ROS

Reactive oxygen species

Introduction

Syringin, extracted from the aerial part of Saussurea medusa, is effective for the treatment of psychogenic behavior disorder and has a hypnosis-inducing action, hepato-protective activities, anti-hypersensitivity effects as well as anti-inflammatory effects on auto-immune diseases (Yang et al. 2004). Because S. medusa has been listed as a protected plant by Chinese government and is difficult to obtain by cultivation field in China, the supply shortage of this medical plant has promoted enormous efforts to find alternative sources of supply (Chen et al. 1999). The use of plant cell culture is considered as a promising alternative for the efficient production of syringin. In plant cell cultures, the use of elicitors has been an important strategy for improving the production of plant secondary metabolites. Elicitors can trigger an array of defense or stress responses and activate specific genes for the enzymes involved in secondary metabolite biosynthesis, and improved the production of plant secondary metabolites (Rajendran et al. 1994). A number of elicitors such as silver nitrate, salicylic acid, glutathione and rare earth elements have been investigated for the enhancement of flavone production in the cell cultures of S. medusa (Yu et al. 2006; Zhao et al. 2005a; Yuan et al. 2002). Yu et al. (2006) reported that addition of 20 μM salicylic acid to S. medusa cell cultures, a 2.7-fold increase in syringin production was observed on the tenth day. There is no report about other elicitors affecting syringin production in S. medusa cell cultures.

The present work was to study the effect of different elicitors such as yeast extract, chitosan and Ag+ on syringin production. Addition time of elicitors and the change of glucose-6-phosphate dehydrogenase (G6PDH), phenylalanine ammonia lyase (PAL) and peroxidase (POD) activities were also studied.

Materials and methods

Cell line and cultivation conditions

The cell line was established from the leaf explants of S. medusa and maintained in our laboratory (Yuan et al. 2002). The callus was subcultured every 15 days. All cell cultures were maintained in MS medium supplemented with 2 mg α-naphthaleneacetic acid l−1, 0.5 mg 6-benzylaminopurine 1−1 (Murashige and Skoog 1962). The medium pH was adjusted to 5.85∼5.90. S. medusa callus, 1 g fresh weight (FW), was inoculated into 40 ml medium held in 100 ml shake flask on a rotary shaker (130 rev min−1). Varying concentrations of sterilized elicitors were added to the medium in a suitable growth phase. All cultures were cultured for 21 days and incubated under 27 μmol m−2 s−1 light provided by white cool fluorescent tube lamps in a photoperiod of 16 h at 25 ± 1°C.

Elicitor preparation

Yeast extract was dissolved in distilled water and ethanol (95%) was added. The solution was kept at 4°C for 4 days for a precipitate to form. The supernatant was decanted, and the remaining precipitate was redissolved in distilled water, sterilized by autoclaving at 121°C and used for elicitor. Chitosan was dissolved in glacial acetic acid. The pH of the solution was adjusted to 5.85 before autoclaving. Ag2SO4 was dissolved in distilled water and kept at 4°C.

Biochemical estimation

Two grams of fresh sampling tissue were homegenized in 0.1 M K-phosphate buffer (pH 7.4), containing 0.5 mM dithiothreitol, 2 mM l-cysteine, 2 mM ethylene diamine tetraacetic acid (EDTA), 8 mM β-mercaptoethanol and 0.5 g of polyvinyl polypyrrolidone (PVPP). G6PDH activity was measured in 50 mM Tris–HCl, pH 7.4, containing 5 mM MgCl2, 0.5 mM glucose−6-phosphate and 0.5 mM NADP, according to the method of Debnam and Emes (1999). The reaction was initiated by the addition of the enzyme and an increase in absorbance was recorded at 340 nm. The G6PDH activity was expressed as U340, where U340 = 1 μmol mg−1 protein min−1. To analyse PAL and POD activities, 2 g sampling tissue were well homogenized with 1 ml extracting buffer, pH 8.8, 0.2 M boric acid buffer containing 10% (w/v) PVPP, 1 mM EDTA, 50 mM β-mercaptoethanol for PAL, pH 7.5, 50 mM phosphate buffer containing 8% PVPP, 1 mM polyethyleneglycol, 1 mM phenylmethanesulfonyl fluoride and 0.01% (v/v) Triton X-100 for POD, and centrifuged at 13,000×g at 4°C for 20 min, and the supernatant collected. For the PAL assay (Koukol and Conn 1961), 300 μl of the extract was incubated with 1 ml 0.02 M l-phenylalanine and 2 ml of the PAL extracting buffer at 24°C for 2 min, and absorbance at 290 nm measured in an ultraviolet spectrophotometer. The PAL activity was expressed as U290, where U290 = 0.01ΔA290 mg−1 protein min−1. For the POD assay (Hammerschmidt and Kuc 1982), 100 μl of the extract was incubated with 2.5 ml 25 mM guaiacol and 200 μl 250 mM H2O2 at 24°C for 3 min, and absorbance at 470 nm measured with an ultraviolet spectrophotometer. The POD activity was expressed as U470, where U470 = 0.01ΔA470 g−1 protein min−1.

Analytical methods

For FW determinations, the cell suspensions were filtered through a filter paper and washed with distilled water. Subsequently, the cells were dried at 60°C to constant weight to determine the dry weight (DW). For syringin content determinations, dry cells (0.1 g) were extracted with 10 ml 70% (v/v) methanol at room temperature for 24 h and centrifuged at 4,000 rev min−1 for 10 min. The sample was filtered through a 0.2-μm Waters membrane filter for analysis. Twenty microliters of sample was injected into a HPLC system (Waters 2695) equipped with a C18 column (Diamonsil 250 mm × 4.6 mm, 5 μm particle size), fitted with a C18 guard cartridge. The mobile phase used for separation consisted of solvent A (water/acetic acid/triethylamine 99.1:0.8:0.1, v/v/v) and solvent B (methanol). The elution profile was: 0 min A/B (8:2, v/v), 10 min A/B (7:3, v/v), 15 min A/B (4:6, v/v). The flow rate was 1.0 ml min−1, column temperature was 30°C and pH was 3.8. UV detection was at a wavelength of 270 nm. A linear response (r = 0.9997) was observed in the range of 100–800 mg l−1. The precision and accuracy experiments showed good results, the relative standard deviation was <1%. The limit of quantification was 16 μg ml−1, and the limit of detection was 5 μg ml−1. The standard syringin was obtained from Delta Information Centre for Natural Organic Compounds, Anhui China. The purity of the standard was 99%.

Statistical analyses

All experiments were repeated three times. The data obtained were statistically analysed by PROC ANOVA of SAS version 6.12. Data were submitted to analysis of variance and mean were then compared with Duncan’s Multiple Range Test, and the term significant has been used to denote the differences for which p ≤ 0.05.

Results

Time course of Saussurea medusa cell in suspension culture

The time course of cell growth and syringin production in suspension culture are shown in Fig. 1. The lag phase of the cell growth ranged from the beginning to the ninth day. In this phase, cell growth and syringin production was very slowly. From the day 9 to the day 21, cell growth phase was the exponential phase. In this phase, cell grew fast and syringin accumulation increased rapidly. The cell DW and syringin production reached maximum at the end of exponential phase. The maximum was 13.97 g DW l−1 and 206.6 mg l−1, respectively.
Fig. 1

Time courses of S. medusa cell growth and syringin accumulation in suspension culture. S. medusa cell, 1 g FW, was inoculated into 40 ml medium held in 100 ml shake flask on a rotary shaker (130 rev min−1). Vertical bar represents standard error of three replications

Effects of elicitors on syringin production

Addition of yeast extract to the culture medium of S. medusa cells enhanced the syringin production with the increase of yeast extract concentration up to 1.5% (v/v). Above this concentration, syringin production decreased with the increase of yeast extract concentration. The highest value of syringin production (601.3 mg l−1) was obtained when the concentration was 1.5% (v/v), almost 2.9-fold increase over control cell (206.6 mg l−1) (Table 1). Chitosan significantly stimulated syringin biosynthesis. Relatively lower concentrations (<0.20 g l−1) were more effective stimulants than higher concentrations. All tested concentrations showed an increase of syringin production over that of control. The maximum of syringin production reached 628.2 mg l−1, which was 3.0-fold higher than that of control (Table 1). Ag+ at different concentrations provoked the syringin synthesis. Its lower concentrations of 25, 50 and 75 μM markedly elevated the production. Among the concentrations tested, the optimal concentration was 75 μM, and syringin production reached 602.7 mg l−1. The higher concentration did not elevate but rather decreased syringin production (Table 1). Combining elicitors had better effects on syringin production than a single elicitor. Among the various combinations of elicitors, addition of 1.5% (v/v) yeast extract, 0.2 g l−1 chitosan and 75 μM Ag+ showed the best stimulation of syringin biosynthesis. The highest syringin production reached 687.3 mg l−1, almost 3.4-fold of that in the control (Table 1).
Table 1

Effects of yeast extract (Y) (v/v), chitosan (C) and Ag+ on the cell growth and syringin accumulation of S. medusa in suspension culture

Elicitor

Syringin production (mg l−1)

Elicitor

Syringin production (mg l−1)

Control

206.6 ± 10.2

Ag+ 25 μM

323.2 ± 15.5

Y 0.5%

365.5 ± 17.2

Ag+ 50 μM

440.4 ± 17.1

Y 1.0%

480.7 ± 15.2

Ag+ 75 μM

602.7 ± 18.2

Y 1.5%

601.3 ± 14.8

Ag+ 100 μM

537.9 ± 15.8

Y 2.0%

519.2 ± 15.3

Ag+ 125 μM

495.1 ± 15.2

C 0.05 mg l−1

386.2 ± 16.4

Y + C 1.5% + 0.2 mg l−1

613.5 ± 16.9

C 0.10 mg l−1

397.3 ± 15.0

Y + Ag+ 1.5% + 75 μM

608.6 ± 17.4

C 0.15 mg l−1

509.6 ± 17.1

C + Ag+ 0.2 mg l−1 + 75 μM

625.8 ± 12.2

C 0.20 mg l−1

628.2 ± 17.4

Y + C + Ag+ 1.5% + 0.2 mg l−1 + 75 μM

687.3 ± 17.2

C 0.25 mg l−1

545.4 ± 16.8

  

Saussurea medusa callus, 1 g FW, was inoculated into 40 ml medium held in 100 ml shake flask on a rotary shaker (130 rev min−1) for 21 days. Feeding time of elicitors was the day 12 of the cell culture. Values represent means ± SE of three replications

Effects of feeding time on Saussurea medusa cell growth and syringin production

The next experiment was based on the above conditions. Different feeding times had significant effects on syringin production (Fig. 2). The optimal feeding time of the combined elicitors was the 15th day of suspension culture, which was the last exponential phase. When the elicitor was fed at the optimal time, addition of 1.5% (v/v) yeast extract, 0.2 g l−1 chitosan and 75 μM Ag+ gave rise to the highest syringin production, which reached 741.9 mg l−1, and was 3.6-fold of that of the control.
Fig. 2

Effects of feeding time on S. medusa cell growth and syringin production. S. medusa callus, 1 g FW, was inoculated into 40 ml medium held in 100 ml shake flask on a rotary shaker (130 rev min−1) for 21 days. Combining elicitors [1.5% (v/v) yeast extract, 0.2 g l−1 chitosan and 75 μM Ag+] were added to liquid medium. Vertical bar represents standard error of three replications

Effects of elicitors on G6PDH, PAL and POD activities of Saussurea medusa cell

Elicitors induced the increase of G6PDH activities, PAL activities and POD activities. The enzyme activity changes were related to syringin accumulation. When the elicitor concentration was suitable, the syringin content and the activities of G6PDH, PAL and POD reached a maximum. Different elicitors showed similar results in inducing enzyme activities (Fig. 3). The combined elicitors induced the increase of G6PDH activities, and reached a maximum (64.5 U g−1 FW) at about 6 h after treatment, thereafter decreased. Cell cultures without elicitor treatment did not have significant change of G6PDH activities (Table 2). As shown in Table 2, the PAL activities in S. medusa cell cultures supplied with combined elicitors changed significantly.
Fig. 3

Effects of different elicitors [yeast (a), chitosan (b), Ag+ (c), combining elicitors (d)] on related enzyme activities. S. medusa callus, 1 g FW, was inoculated into 40 ml medium held in 100 ml shake flask on a rotary shaker (130 rev min−1) for 21 days. Feeding time of elicitors was the day 15 of the cell culture. Enzymes assays were performed after 6 h of adding elicitors. Vertical bar represents standard error of three replications

Table 2

Effects of combining elicitors [1.5% (v/v) yeast extract, 0.2 g l1 chitosan and 75 μM Ag+] on related enzyme activities

Treatment time (h)

G6PDH (U g−1 FW)

PAL (U g−1 FW)

POD (U g−1 FW)

(−)

(+)

(−)

(+)

(−)

(+)

0

21.3 ± 0.67

21.3 ± 0.67

128.6 ± 4.11

128.6 ± 4.11

11.5 ± 0.57

11.5 ± 0.57

3

21.9 ± 0.80

46.7 ± 1.72

153.4 ± 4.68

285.7 ± 5.14

14.3 ± 0.61

31.7 ± 1.65

6

22.4 ± 0.98

64.5 ± 1.83

167.8 ± 4.02

412.9 ± 6.47

16.9 ± 0.64

44.9 ± 1.80

9

24.6 ± 0.84

51.9 ± 1.63

169.6 ± 4.52

369.7 ± 5.70

15.5 ± 0.59

39.8 ± 1.77

12

24.8 ± 0.88

39.8 ± 1.42

181.3 ± 4.74

301.2 ± 4.82

17.6 ± 0.56

27.6 ± 1.55

(−) represents without elicitor, (+) represents with elicitor. S. medusa callus, 1 g FW, was inoculated into 40 ml medium held in 100 ml shake flask on a rotary shaker (130 rev min−1) for 21 days. Feeding time of elicitors was the day 15 of the cell culture. Values represent means ± SE of three replications

However, no marked change was observed in the control experiments. PAL activities of S. medusa cells markedly increased when the elicitor treatment time was <6 h. The maximum of PAL activities was 2.18 times as high as that of control. After 6 h treatment, the PAL activities significantly decreased, but were still higher than that of the control. The change trend of POD activities was similar to that of PAL activities (Table 2). No marked change was observed in the control experiments. However, addition of elicitor induced the increase of POD significantly in short time. When the treatment time was 6 h, the POD activity reached 44.9 U g−1 FW, almost 2.66-fold of that in the control.

Discussion

In the suspension culture of S. medusa cells, addition of elicitors promoted syringin biosynthesis. The highest syringin production was obtained when added 1.5% (v/v) yeast extract, 0.2 g l−1 chitosan and 75 μM Ag+ to the liquid culture medium at the 15th day. The highest syringin production reached 741.9 mg l−1, which was 3.6-fold higher than that of control. Combining elicitors showed better effects on syringin production than a single elicitor. Considering the complexity of syringin production, different elicitors may induce different parts of the defense response system (Luo and He 2004). Yeast extract had significant effect on syringin production in present study. Similar results were obtained in betalain production by hairy root culture of Beta vulgaris (Savitha et al. 2006). The involvement of chitosan is intricate in the elicitation process. The mechanisms by which plant cells perceive it are not fully understood. However, many studies suggest that oligosaccharides and polysaccharide elicitor signals may be initiated by receptors on the plant plasma membrane (Savitha et al. 2006). In plant secondary metabolite biosynthesis process, an elicitor signal is perceived by a receptor on the surface of the plasma membrane and initiates a signal transduction network and it regulates the expression of biosynthesis genes involved in plant secondary metabolism and further produces key enzymes which catalyse the biosynthesis of the target secondary metabolites (Zhao et al. 2005b). Ag+ is a potent inhibitor of the ethylene signal transduction pathway. Ethylene at high concentrations may inhibit the biosynthesis of secondary metabolites, whereas at low concentration, it promotes the production of secondary metabolites (Pan et al. 2000). So, a positive effect of Ag+ on secondary metabolite production may be attributed to its inhibitory effect on ethylene synthesis or action (Zhang and Wu 2003). In the present study, it was observed that the optimal feeding time of elicitors was the 15th day of suspension culture, which was the last exponential phase. A similar observation was obtained in suspension culture of Plumbago rosea L. (Komaraiah et al. 2002). Oxidative burst is a significant event in plant defense responses when plant cells are exposed to abiotic or biotic stress such as elicitor treatment. G6PDH is the first enzyme of the pentose phosphate pathway (PPP), which provides precursors for phenolic secondary metabolite synthesis. Recently, G6PDH was reported to be involved in the elicitor-induced responses and contributes by providing more NADPH. NADPH is required for the detoxification of reactive oxygen species (ROS) and peroxides, which are harmful to plant cells. Acceleration of the PPP could result in a reduction of ROS (Fahrendorf et al. 1995). The phenylpropanoid pathway leads to biosynthesis of flavonoids and pigments, as well as lignin and phenolic compounds. PAL is a rate-limiting enzyme in this pathway to various products. The results obtained in this study showed that addition of combining elicitors significantly increased PAL activity, especially at 6 h after treatment. POD is protective enzyme of plant cells against a variety of physical, chemical and biological stresses by regulating the concentrations of O2- and H2O2. The H2O2 is an important toxic intermediate and mediates the elicitor-induced accumulation of secondary metabolites. H2O2 also induces expression of many defense genes and secondary metabolite biosynthetic genes, such as sesquiterpene cyclases and PAL (Mehdy 1994). H2O2-mediated non-enzymatic or enzymatic lipid peroxidation can initiate the octadecanoid pathway leading to biosynthesis of jasmonic acid and related compounds, and other oxylipins, which have an effective function in the induction of plant secondary metabolites (Thoma et al. 2003). In the present work, the G6PDH, PAL and POD activities increased significantly when added combining elicitors to liquid culture medium and it showed that elicitors resulted in oxidative burst, and mediated syringin biosynthesis.

Notes

Acknowledgment

This research is supported by Directional Project of Knowledge Innovation Engineering, CAS (KGCX2-SW−506).

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Copyright information

© Springer Science+Business Media B.V. 2006

Authors and Affiliations

  • Chunming Xu
    • 1
    • 2
  • Bing Zhao
    • 1
  • Yuan Ou
    • 1
    • 2
  • Xiaodong Wang
    • 1
  • Xiaofan Yuan
    • 1
  • Yuchun Wang
    • 1
  1. 1.State Key Laboratory of Biochemical Engineering, Institute of Process EngineeringChinese Academy of SciencesBeijingPeople’s Republic of China
  2. 2.Graduate University of Chinese Academy of SciencesBeijingPeople’s Republic of China

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