Applied Microbiology and Biotechnology

, Volume 68, Issue 1, pp 98–103 | Cite as

A novel synthetic fluoro-containing jasmonate derivative acts as a chemical inducing signal for plant secondary metabolism

  • Zhi-Gang Qian
  • Zhen-Jiang Zhao
  • Yufang Xu
  • Xuhong Qian
  • Jian-Jiang Zhong
Applied Microbial and Cell Physiology

Abstract

A novel fluoro-containing jasmonate derivative was chemically synthesized and evaluated as a potential elicitor with respect to the induction of plant defense responses and the biosynthesis of plant secondary metabolites. A bioactive taxuyunnanine C (Tc)-producing cell line of Taxus chinensis was taken as a model plant cell system. The presence of novel synthesized pentafluoropropyl jasmonate (PFPJA) induced two early and important events in plant defense responses, including an oxidative burst and activation of l-phenylalanine ammonia lyase. In addition, PFPJA was found to significantly increase Tc accumulation, without any inhibition of cell growth. Moreover, Tc accumulation was increased more in the presence of PFPJA compared with methyl jasmonate (MJA) and previously reported trifluoroethyl jasmonate (TFEJA). For example, addition of 100 μM PFPJA on day 7 led to a high Tc content (38.2±0.3 mg/g) at day 21, while the Tc content was 29.3±0.3 mg/g and 34.9±0.9 mg/g with the addition of 100 μM MJA and TFEJA, respectively. Quantitative structure–activity analysis of fluoro-containing jasmonates suggests that the increase in the fluoro-groups introduced into the carboxyl side-chain of MJA resulted in a higher stimulatory activity for Tc biosynthesis, which corresponds well with the markedly increased lipophilicity after fluorine introduction. These results indicate that newly synthesized fluoro-containing PFPJA can act as a powerful chemical inducing signal for secondary metabolism in plant cell cultures.

Introduction

Since the accumulation of most plant secondary metabolites is thought to be a biological response to specific external stimuli, various treatments or molecules that induce plant defense responses, generally referred to as elicitors, may stimulate the biosynthesis of secondary metabolites. In reality, the treatment of plant cells with various elicitors has been the best approach for dramatically enhancing secondary metabolite production in plant cell cultures (Wu and Lin 2003; Zhang and Wu 2003; Linden et al. 2001). The oxidative burst, corresponding to a rapid and transient production of active oxygen species (AOS), is an early and important event in plant defense responses. Involved in scavenging over-produced AOS in plant cells, l-phenylalanine ammonia lyase (PAL) is a key enzyme required for the biosynthesis of a defense phenolic compound. Therefore, PAL is recognized as a plant cell defense marker (Dorey et al. 1999; Jabs et al. 1997).

Previously used elicitors are mainly fungal cell materials (Yuan et al. 2002; Wang et al. 2001; Zhao et al. 2001b), heavy metals (Bonfill et al. 2003; Zhang et al. 2002), ultrasound (Wu and Ge 2004; Lin and Wu 2002), and various chemicals (Tabata 2004; Staniszewska et al. 2003; Zhang and Wu 2003; Aoyagi et al. 2001). There have been few studies on the use of synthetic chemicals to enhance plant secondary metabolism (Staniszewska et al. 2003). Recently, we have been searching for novel synthetic jasmonate elicitors that can significantly promote secondary metabolite biosynthesis by plant cells. Jasmonates, including jasmonic acid (JA) and methyl jasmonate (MJA), are a family of important signal transducers in plant defense responses. Exogenously applied MJA can stimulate secondary metabolism in a variety of plant species; and it is the most effective elicitor of taxane-ring formation (taxoid production) by various Taxus cell lines (Tabata 2004; Ketchum et al. 2003; Yukimune et al. 1996). We have found that transesterification of MJA carboxyl group with trifluoroethanol leads to a higher stimulatory effect on secondary metabolite production, compared with commercially available MJA (Qian et al. 2004a). However, it is unclear whether this fluoro-containing jasmonate elicits plant cells via a defense response mechanism like MJA. In addition, the structure activity relationship needs investigation to provide some useful information for the rational design of chemically synthesized elicitors.

As part of our continuing studies on the chemistry and biology of synthetic jasmonate derivatives, a novel fluoro-containing jasmonate was synthesized and evaluated by bioassay as a potential elicitor for the induction of plant defense responses and the biosynthesis of plant secondary metabolites. A high taxuyunnanine C (Tc)-producing cell line of Taxus chinensis was used as a model plant cell system. Tc is a physiologically active substance with a neuron growth factor-like activity. In addition, the structure–activity relationship of the fuoro-containing jasmonate elicitors was quantitatively analyzed.

Materials and methods

Synthesis of jasmonates

Pentafluoropropyl jasmonate (PFPJA) (Fig. 1) was synthesized by acylation of pentafluoropropanol (Sigma) with jasmonic chloride in anhydrous dichloromethane in the presence of triethylamine under argon (Fig. 2), as described for the synthesis of trifluoroethyl jasmonate (TFEJA; Qian et al. 2004a). Jasmonic chloride was prepared from JA and bis-(trichloromethyl) carbonate as described by Eckert and Forster (1987). JA was obtained by alkaline hydrolysis of MJA (Tokyo Kasei Co., Tokyo, Japan). Synthesized jasmonates were purified by silica gel (200–400 mesh, 60 Å; Aldrich) chromatography and checked for purity by GC-MS with a Hewlett–Packard model 6890 gas chromatograph and quadrupole mass selective detector (Agilent, Palo Alto, Calif.). All the jasmonates contained two asymmetric centers at C-3 and C-7 (Fig. 1) and the ratio of stereoisomers (i.e., cis-membered side-chains vs trans-membered side-chains) was determined to be about 6:44 in our case.
Fig. 1

Chemical structures of MJA, TFEJA, and the newly synthesized PFPJA

Fig. 2

Synthetic scheme to PFPJA from commercially available MJA. A previously reported TFEJA (Qian et al. 2004a) is also included

Cell subcultures

The T. chinensis cell line was maintained on MS medium (Murashige and Skoog 1962) supplemented (per liter) with 0.5 mg of 6-benzyladenine, 0.2 mg of 2,4-dichlorophenoxy-acetic acid, 0.5 mg of naphthaleneacetic acid, 100 mg of ascorbic acid, and 30 g of sucrose. The medium pH was adjusted to 5.8 before autoclaving. The cells were subcultured biweekly, by inoculating 18 g of fresh cells into 500-ml Erlenmeyer flasks containing 200 ml of medium. The flasks were capped with cotton-plug closures and were incubated on a rotary shaker (110 rpm) at 25°C in the dark.

Elicitation

PFPJA, MJA, and fluoro-containing TFEJA (Qian et al. 2004a) were employed in this work. About 5 g of fresh cells were incubated into a 250-ml Erlenmeyer flask containing 50 ml of medium with the same culture conditions as for subcultures. The type of closure used on the flasks was a cotton plug, the same as for subcultures. Each jasmonate derivative was added to the cultures in 1 μl of ethanol per 1 ml of culture medium and sterilized by filtering through 0.22-μm polyvinylidenedifluoride syringe filters (Millipore). Equal volumes of ethanol (50 μl) were added to all flask cultures. Here, ethanol was used to solubilize the jasmonate elicitors; and the effect of ethanol alone (at 1 μl/ml) on the T. chinensis cultures was tested by comparison with cultures without ethanol addition. It was confirmed that there were no side-effects of ethanol on cell growth, taxane accumulation, H2O2 production, and PAL activity (Qian et al. 2004b).

Measurement of biomass and taxane content

The samples from flasks were filtered under vacuum and washed with several volumes of distilled water to remove residual medium. The cells were weighed to obtain their fresh weight and 5 g of fresh cells were dried at 50°C to a constant weight for the measurement of cell dry weight (DW).

Extraction and determination of intracellular taxane was the same as described by Qian et al. (2004a). In our culture system, the Tc levels detected in liquid medium were less than 1% of the total production in all cultures and are not shown here. No paclitaxel and other known taxoids were detected obviously, therefore the titer of Tc production promoted by jasmonate derivatives also represents their total taxane-ring formation activity.

Assay of oxidative burst

Associated with the so-called oxidative burst, H2O2 originates from superoxide generated by a plasma membrane-associated NADH oxidase in challenged plant cells. H2O2 produced by the cells and released into the medium was determined according to Cazalé et al. (1998) by the scopoletin fluorescence oxidative quenching method (excitation wavelength 350 nm, emission 460 nm). To measure H2O2 accumulation, samples were taken at various intervals over the 180-min period following elicitation. Aliquots (4 ml) of extracellular medium were mixed with 40 μl of 5 mM scopoletin (Fluka) stock solution in DMSO (Sigma) or 40 μl of peroxidase stock solution (1 mg/ml; Sino-American Biotechnology Co., Shanghai), respectively. The concentration of H2O2 in the medium was calculated from the fluorescence decrease, using a calibration curve established in the presence of H2O2. A standard curve for adding scopoletin to solutions at different H2O2 concentrations was prepared using cell-free medium. It was confirmed that the various jasmonate elicitors did not affect the above peroxidase-dependent assay for H2O2 determination (data not shown).

Enzyme extraction and PAL assay

Cells were harvested as described above, and then samples (1 g) of fresh cells were frozen and then reduced to powder with a mortar and pestle in the presence of liquid nitrogen. Crude enzymes in the frozen powder were extracted by adding 50 mg of polyvinylpyrrolidone and 2 ml of prechilled buffer at pH 7.2 (0.1 M phosphate buffer, 2 mM ethylenediaminetetraacetic acid, 4 mM dithiothreitol) and were then homogenized at 4°C. The homogenate was centrifuged at 10,000 g for 30 min at 4°C to obtain a cell-free enzyme extract. The protein concentration of the enzyme extract was determined by the method of Bradford (1976).

The PAL assay was a modified method of Heide et al. (1989), based on the PAL conversion of l-phenylalanine to trans-cinnamic acid. For the assay, 200 μl of the enzyme extract was incubated with 120 μl of 0.1 M l-phenylalanine (in 0.1 M borate buffer at pH 8.8) and 280 μl of 0.1 M borate buffer at pH 8.8 and 30 °C for 60 min. The reaction was stopped by adding 50 μl of 5 N trichloroacetic acid. After centrifugation at 10,000 g for 30 min, the supernatant was analyzed on a Hewlett–Packard series 1100 HPLC system (Agilent) under the following conditions: water:methanol:acetic acid solvent (40:60:1, by vol.), flow 1 ml/min, detection at 280 nm, Zorbax ODS column (250 mm long, 4.6 mm diam., 5 μm film; Agilent), injection volume 20 μl. Genuine trans-cinnamic acid (Sigma) was used as an external standard. One unit of enzyme activity was defined as the amount of enzyme forming 1 pmol/min of trans-cinnamic acid from l-phenylalanine.

Statistical analysis

Experiments were done in a completely randomized layout. Each experiment was carried out in triplicate and three different sets of experiments reproduced the same result. Data were analyzed by one-way ANOVA. The means of each experiment were analyzed using Tukey’s honestly significant difference multiple-comparison test with a family error rate of 0.05. All differences are significant unless otherwise indicated.

Results

Novel jasmonate-induced secondary metabolite production

Preliminary experiments were conducted to study the elicitation timing and dose response for PFPJA, as reported for 2,3-dihydroxypropyl jasmonate in our recent work (Qian et al. 2004b). The results indicated that day 7 and 100 μM were the optimal elicitation time and concentration, respectively, for both cell growth and Tc accumulation. Figure 3 shows the typical trends of cell growth and Tc production in the control cultures and in cultures elicited with100 μM PFPJA on day 7.
Fig. 3

Kinetic profiles of cell mass (by dry weight) and Tc content in control and PFPJA-elicited T. chinensis suspension cultures. PFPJA (100 μM) was added to the cultures in 1 μl of ethanol per 1 ml of culture medium on day 7 of cultivation. Data-points are the means of three flasks and vertical bars show standard deviations. d Days, L liters

The data shown in Fig. 3 indicate that the cell mass in control and elicited cultures reached a peak at day 12 and declined thereafter. There was no significant difference in biomass yield between the control cultures and the cultures subjected to elicitor treatment. It can be seen that the Tc content of the elicited cultures was significantly higher than that of the control. The maximum Tc content obtained in the elicited cultures was 38.2±1.7 mg/g on day 21, while it was 13.0±0.1 mg/g for the control on the same day. In addition, a rapid and consistent stimulatory effect of elicitor addition on Tc biosynthesis was observed. These results indicated the significant contribution of elicitation by PFPJA to the efficient enhancement of secondary metabolite (Tc) yield.

Structure–activity relationship analysis

Data on the elicitation of Tc biosynthesis were collected to investigate the structure–activity relationship of the fluoro-containing jasmonate elicitors. PFPJA, MJA, and TFEJA (Fig. 1) were added to 7-day-old cell cultures at 100 μM, which was the optimal concentration for all elicitors. As shown in Fig. 4, a significantly higher Tc content was obtained after treatment with various elicitors than in the control. More importantly, the stimulatory activity of the newly synthesized elicitor was greater than that of TFEJA and MJA. For example, the addition of 100 μM PFPJA on day 7 led to a Tc content of 38.2±0.3 mg/g (at day 21), while the Tc content was 34.9±0.9 mg/g and 29.3±0.3 mg/g for the TFEJA- and MJA-treated cultures. The superior stimulatory activity of TFEJA over MJA coincides with that reported in our recent study (Qian et al. 2004a).
Fig. 4

Time-profiles of Tc content in T. chinensis suspension cultures elicited with MJA, TFEJA, and PFPJA. Each elicitor was added to the cultures as 100 μM in 1 μl of ethanol per 1 ml of culture medium on day 7 of cultivation. Data-points are the means of three flasks and vertical bars show standard deviations

A quantitative structure–activity analysis was conducted to acquire the relationship between the chemical structure of the fluoro-containing jasmonates and their stimulatory activity (Table 1). An AlogP98 value was calculated for the lipophilic properties of each molecule, performed on a Silicon Graphics workstation running on the IRIX 6.5 operating system. Relevant computational modules were accessed from the Drug discovery workbench of Cerius2 ver. 4.8 (Molecular Simulations, San Diego, Calif., USA). Molecular geometries were optimized under the universal forcefield provided by the Open force field within Cerius2. It appeared that an increase in the fluoro-groups introduced in the side-chain at the C-1 position resulted in a higher stimulatory activity for Tc biosynthesis. Fluorine, the H-bond acceptor, may facilitate interaction or coupling with the putative jasmonate acceptor localized on the plasma membrane of plant cells. This effect of fluorine introduction might also be due to the markedly increased lipophilicity represented as AlogP98. For example, the AlogP98 values of MJA, TFEJA, and PFPJA are estimated to be 2.22, 3.22, and 3.72, respectively. A relatively high level of lipophilicity is beneficial for the stimulatory activity because it is easier for an elicitor to transform from the water phase to the interaction site. This structure–activity relationship is in good agreement with that observed for hydroxyl-containing jasmonates in our recent study (Qian et al. 2004b).
Table 1

Quantitative analysis of the structure–activity relationship between the chemical structure of jasmonate elicitors and their stimulatory activity on Tc biosynthesis by suspension cultures of T. chinensis. Each elicitor was added to the cultures as 100 μM in 1 μl of ethanol per 1 ml of culture medium on day 7 of cultivation. The stimulatory activity is defined as the ratio of average Tc content under elicitation vs that of the control. The stimulating activity in this table was present on day 21, when the maximal Tc content was obtained for both elicited and control cultures

Elicitor

Number of fluorine atoms

H-bond acceptor

AlogP98

Stimulatory activity

MJA

0

3

2.22

2.50a

TFEJA

3

6

3.22

2.99

PFPJA

5

8

3.72

3.27

aIn this column, means are significantly different according to Tukey’s honestly significant difference multiple-comparison test with a family error rate of 0.05

Synthetic jasmonate-induced plant defense responses

Preliminary experiments were conducted to determine suitable time-intervals for the investigation of the elicitor-induced plant defense responses. The results indicated that periods of 180 min and 480 min after elicitor addition were suitable for the analysis of H2O2 production and PAL activity, respectively. PFPJA (100 μM) was added to the cultures on day 7 of the culture cycle, while a culture without elicitor treatment was used as the control.

After elicitor treatment, the H2O2 levels in T. chinensis cell cultures markedly changed (Fig. 5a). A significantly increased H2O2 production was observed 5 min after the addition of 100 μM PFPJA. The H2O2 level reached its maximum at 30 min after elicitor treatment and was nearly five-fold that of the control. Then, it dropped rapidly to a very low level at 80 min after treatment. In contrast, the H2O2 level of the control did not change over the period studied. The results suggest that an oxidative burst occurred in elicited T. chinensis cell cultures. Figure 5b indicates that PAL activity in PFPJA-elicited cultures significantly increased at 60 min after elicitation, peaking at approximately two-fold (compared with controls) at 120 min after elicitation. The increased PAL activity declined steadily to the basic level of the controls, which showed little change over the period studied.
Fig. 5

PFPJA-induced H2O2 production (A) and PAL activity (B) in T. chinensis suspension cultures. PFPJA was added to the cultures as 100 μM in 1 μl of ethanol per 1 ml of culture medium on day 7 of cultivation and elicited cell cultures were sampled at certain intervals for H2O2 and PAL analysis. Data-points are the means of three flasks and vertical bars show standard deviations. U Units of activity

Discussion

Our experimental results show that the treatment of plant cells with PFPJA induces the characteristic events of plant defense responses, the transient production of AOS and increased PAL activity, and the biosynthesis of secondary metabolites. AOS production was a universal response of plants to various exogenous elicitors, e.g., fungal elicitation (Yuan et al. 2002; Zhao et al. 2001a), physical elicitors (Wu and Ge 2004; Ye et al. 2004), and chemical elicitors (Qian et al. 2004b; Sharan et al. 1998). There exists increasing evidence that H2O2 can act as a diffusible signal to activate plant defense genes and the biosynthesis of secondary metabolites (Jabs et al. 1997; Mithofer et al. 1997; Mehdy 1994). Increased PAL activity provides additional evidence for the occurrence of an oxidative burst in elicited plant cell cultures. These findings suggest that the newly synthetic PFPJA may act as a chemical signal for plant secondary metabolism.

In the past decade, there have been numerous attempts to find more potent jasmonate elicitors for plant secondary metabolism. However, most modifications of MJA did not result in an enhanced stimulatory activity for the biosynthesis of secondary metabolites. Aoyagi et al. (2001) found that deletion of the methyl group or substitution by a n-propyl group in the MJA carboxyl group at the C-1 position led to a weaker stimulatory activity or almost no activity for saikosaponin biosynthesis. According to Yukimune et al. (1996), both the carboxyl group at the C-1 position and the keto group at the C-6 position are two important parts of the MJA structure for stimulating taxoid production by Taxus cell cultures. Our previous work suggested that the double bond at positions 9 and 10 played an important role in the promotion of ginsenoside production (Wang and Zhong 2002) and taxoid formation (Dong and Zhong 2001). In another work by Yukimune et al. (2000), the effects of the MJA configuration on both cell growth and taxoid production were recognized in Taxus suspension cultures. The commercially available MJA used in this work is a mixture of four stereoisomers, with the cis-membered side-chains and trans-membered side-chains at a ratio of about 6:44. It was confirmed by GC analysis that PFPJA and TFEJA had the same ratio of stereoisomers as MJA. The acylation reaction described in this work did not change the distribution of jasmonate stereoisomers, compared with the starting material (MJA). Therefore, it might be reasonable to neglect the effect of stereo-configuration. Under the same experimental conditions, the difference in stimulatory activity for Tc biosynthesis was attributed to differences in the chemical structure of jasmonate derivatives. Structure–activity analysis of the chemically synthesized fluoro-containing jasmonates implied that an increase in the fluoro-groups introduced into the side-chain at the C-1 position resulted in a higher stimulatory activity for Tc biosynthesis, which corresponds well with the markedly increased lipophilicity after fluorine introduction.

In summary, this work presents a novel fluoro-containing jasmonate derivative for eliciting plant secondary metabolism. An oxidative burst followed by increased PAL activity and taxoid overproduction was observed after synthetic PFPJA treatment of the model plant cell system, i.e., T. chinensis suspension cultures. These findings suggest that this synthetic fluoro-containing jasmonate can act as a new inducing signal for secondary metabolite production by plant cell cultures. Quantitative analysis of the structure–activity relationship is beneficial for the rational design of more potent chemically synthesized elicitors. Since the synthetic steps are simple and other chemical reagents are not expensive, compared with the starting material (MJA), it is considered that the new synthetic jasmonate analogue may be potentially applied to other cell culture systems and for the large-scale production of valuable plant secondary metabolites.

Notes

Acknowledgements

Financial support from the National Natural Science Foundation of China (NSFC projects 20225619, 20236040, 20376023), the National Key Project for Basic Research, the Ministry of Science and Technology of China (2003CB114400), and the Shanghai Leading Academic Discipline Program is gratefully acknowledged. Both J.J.Z. and X.H.Q. are recipients of the Cheung Kong Scholars Program Professorship, from the Ministry of Education of China

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Copyright information

© Springer-Verlag 2005

Authors and Affiliations

  • Zhi-Gang Qian
    • 1
  • Zhen-Jiang Zhao
    • 2
  • Yufang Xu
    • 2
  • Xuhong Qian
    • 2
  • Jian-Jiang Zhong
    • 1
  1. 1.State Key Laboratory of Bioreactor EngineeringEast China University of Science and TechnologyShanghaiChina
  2. 2.Shanghai Key Laboratory of Chemical Biology, Institute of Pesticides and PharmaceuticalsEast China University of Science and TechnologyShanghaiChina

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