Hydrogen Peroxide from the Oxidative Burst as fungal elicitors (Zhang et al., 2000; Yuan et al., is Not Involved in the Induction of Taxol 2001; 2002; Yu et al., 2002), methyl jasmonate Biosynthesis in Taxus chinensis Cells (Zhang et al., 2000; Spela et al., 2002) and heavy metal ion (Zhang et al., 2000) have been employed Wen Zhi Lana*, Wen Min Qinb, Long Jiang Yua, to induce the biosynthesis of taxol in Taxus cell cul- and Xi Yanga tures. Before the activation of de novo synthesis of taxol, the oxidative burst is also observed in elici- a School of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, tor-induced Taxus chinensis cultures (Yuan et al., China. Fax: +86-27-875436833. 2001, 2002; Yu et al., 2002). Yuan et al., (2001, 2002) E-mail: [email protected] concluded that the dependence of taxol production b Department of Biology, University of Waterloo, on the intensity of H O from oxidative burst fol- Ontario N2L 3G1,Canada 2 2 lowed a modified logistic curve, and inferred that * Author for correspondence and reprint requests the syntheses of the side chain and nucleus of taxol Z. Naturforsch. 58c, 605Ð608 (2003); were enhanced by low and high intensities of received January 13/March 17, 2003 H2O2, respectively. However, it is not clear In cell suspension cultures of Taxus chinensis, 40 mg/l whether inhibition or enhancement of H2O2 from fungal elicitor from Aspergillus niger and 20 µm HgCl2 elicited 5.7 and 3.6 mg/l taxol, which was a 9-fold and 5- the oxidative burst would affect taxol biosynthesis, fold increase vs. compared with the control, respectively. which is the main subject of the present paper. The fungal elicitor induced hydrogen peroxide (H2O2) accumulation but HgCl2 did not, indicating that H2O2 was not necessary for enhancement of taxol induced by Materials and Methods elicitor. Compared with the treatment with fungal elici- Plant materials and culture conditions tor alone, exogenous catalase, ascorbic acid, diphenylene iodonium and superoxide dismutase induced a 0.45, 0.4, Taxus chinensis cell lines, isolated from Taxus 0.7 and 1.4-fold H2O2, but elicited taxol production, chinensis zygote embryos, were maintained in which was 0.98, 1.2, 1.1 and 0.9-fold, respectively, vs. non-treated cells. Elicitor-induced taxol production was modified MS medium as previously described not accorded with the amount of H2O2 production. (Zhang et al., 2000). With 10 g (fresh weight) of Key words: Elicitor, Taxol, Taxus chinensis cells inoculated into a 250 ml Erlenmeyer flask containing 100 ml liquid modified MS media, 40 mg/l fungal elicitor, and 20 µm HgCl2 were added to the 10-day-old cultures. The flasks were Introduction shaken at 120 ð 5 rpm at 25 ð 1 ∞C. Oxidative burst, a transient increase in the pro- duction of reactive oxygen species (ROS), gen- Fungal elicitor preparation erally occurs in a number of plant pathogen in- The fungal strain Aspergillus niger was isolated teractions at the early stage. Hydrogen peroxide from the inner bark of Taxus chinensis. Preparation (H O ), the most stable compound among ROS, 2 2 of fungal elicitor was according to the method de- has been implicated in plant disease resistance scribed by Zhang et al. (2000). The elicitor dose was (Lamb and Dixon, 1997). However, reports linking measured by the total carbohydrate content of the H O from the oxidative burst to biosynthesis of 2 2 fungal homogenate, which was determined by the second metabolites, such as phytoalexin, have phenol-sulfuric acid method using glucose as stan- been contradictory, even with respect to experi- dard. ments performed on the same plant species, such as soybean (Levine et al., 1994; Mithöfer et al., 1997; Guo et al., 1998) and tobacco (Lamb and H2O2 measurement Dixon, 1997; Doreyl et al., 1999). Contents of H2O2 were measured by monitoring Presently there has been increasing interest in ex- the A415 of the titanium-peroxide complex accord- ploiting Taxus spp. cell cultures to produce the anti- ing to Brennan and Frenkel (1977). Absorbance cancer drug, taxol. A wide variety of elicitors such values were calibrated to the standard curve. 0939Ð5075/2003/0700Ð0605 $ 06.00 ” 2003 Verlag der Zeitschrift für Naturforschung, Tübingen · www.znaturforsch.com · D 606 Notes Taxol determination 300 Fungal elicitor HgCl 2 The cells after elicitation were washed with de- 270 Control ionized water to remove residual medium and filtrated under vacuum. The cells were then 240 freeze-dried for 30 h for dry wt determination. 210 Dried samples (100 mg) were mashed and ex- tracted with 4 ml methanol/dichloromethane (1:1, 180 v/v) with sonication for 1 h at room temperature production (nmol/g FW) 150 2 O for 3 times. The extract was evaporated to dryness 2 with a rotary evaporator equipped with a con- H 120 denser for solvent recovery, then redissolved in 90 2 ml methanol. The methanol extracts were centri- 0123456 fuged at 3400 ¥ g for 5 min prior to HPLC analysis. Time after treatment (h) Samples of 5 ml from the cell free medium were Fig. 1. H2O2 accumulation induced by fungal elicitor and extracted with 2 ml of dichloromethane for HgCl2 in Taxus chinensis suspension cells. The rate of 3 times. The combined dichloromethane fraction H2O2 accumulation was determined in cultures at vari- was vacuum dried and redissolved in 2 ml metha- ous times after addition of 40 mg/l fungal elicitor and 20 µM HgCl2, and in the untreated control. Elicitor was nol for HPLC analysis after centrifugation. taxol added into 10-d old cultures. FW represents fresh was analyzed by HPLC on a reverse-phase C18 col- weight. Each value is the mean ðSE from seven inde- umn at 227 nm at 25 ∞C using a mobile phase of pendent experiments. methanol/acetonitrile/water (25:30:30, v/v/v). The elution rate was kept at 1 ml minÐ1. Throughout little effect on H2O2 accumulation in white clover the experiment, all injection volumes were 10 µl. (Trifolium repens L.) suspension cultures (Devlin Taxol concentration (mg/l) in the samples was the et al., 1992). combination of taxol in cells and medium. Effect of elicitor on taxol production The time courses of taxol accumulation in Taxus Results and Discussion chinensis cultures treated with fungal elicitor and Effect of elicitor on H2O2 production HgCl2 were demonstrated in Fig. 2. Significant dif- Several suspension cultures plant cell have been 7 reported produce H2O2 during stimulation by eli- Fungal elicitor 6 HgCl citors from the fungus (Levine et al., 1994; Lamb 2 Control and Dixon, 1997; Mithöfer et al., 1997; Guo et al., 5 1998; Doreyl et al., 1999). Fig. 1 shows the effect of 4 the fungal elicitor from Aspergillus niger on H2O2 production in Taxus chinensis cell cultures. The 3 elicitor-induced H2O2 production started to in- crease 1 to 2 h after treatment, and reached a max- 2 imum of 240 nmol/gFW at about 4 h, and de- Taxol production (mg/l) 1 creased thereafter (Fig. 1). Treatment of Taxus chinensis cultures with fungal elicitor stimulated 0 the H2O2 accumulation, which agrees with the ob- 024681012141618 servations by Yuan et al. (2001; 2002) and Yu et Time after treatment (d) al. (2002). Taxus chinensis cultures without elicitor Fig. 2. Taxol accumulation induced by fungal elicitor and µm treatment accumulated little H2O2.20 HgCl2 HgCl2 in Taxus chinensis suspension cells. The rate of only induced a slight increase in H O accumula- H2O2 accumulation was determined in cultures at vari- 2 2 µm tion; however, the increase vs. free elicitor treat- ous times after addition of 40 mg/l elicitor and 20 HgCl2, and in the untreated control. Elicitor was added ment was not statistically significant (P < 0.05) to 10-d old cultures. Each value is the mean ð SE from (Fig. 1). Indeed, a low concentration of HgCl2 had five independent experiments. Notes 607 ferences in taxol concentrations between elicited 130 B cultures and the control appeared within 2 d. The 120 highest taxol concentrations under the influence 110 of fungal elicitor (5.7 mg/l) and HgCl2 (3.6 mg/l) 100 appeared on day 10 after treatment, which was 10- 90 fold and 6-fold of the control, respectively. 80 Although H2O2 acts as a second messenger for 70 the induction of some defense genes (Lamb and 60 Dixon, 1997), Levine et al. (1994) and Doreyl et al. Relative concentration of Taxol(%) 50 (1999) observed that manipulation of H2O2 of the 160 A (%) oxidative burst did not modify transcripts of phe- 2 140 O nylalanine ammonia-lyase and chalcone synthase, 2 120 respectively. Moreover, phytoalexin and terpenes, 100 secondary metabolites generally present in plant- 80 pathogen interactions, were independent on hy- 60 drogen peroxide in elicitor-treated tobacco and 40 soybean cell cultures (Devlin et al., 1992; Levine 20 et al., 1994; Mithöfer et al., 1997; Guo et al., 1998; Relative concentration of H 0 Doreyl et al., 1999). The present results showed Elicitor DPI CAT ASA SOD that HgCl2 did not induce H2O2 accumulation, but Fig. 3. Effect of inhibitors, exogenous SOD, and catalase enhanced taxol production similarly to what the on the production of H2O2 (A) taxol (B) in Taxus chi- nensis cell suspension cultures treated with fungal elici- fungal elicitor did. Therefore, we postulate that tor. H2O2 was not required for enhancement of taxol Catalase (CAT, 200 U/ml), ascorbic acid (ASA, 60 mg/ induced by elicitor. l), diphenylene iodonium (DPI, 500 µm) and superoxide dismutase (SOD, 300 U/ml) were added to cultures 10 min before the addition of elicitor.