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Effects of salicylic acid on fungal elicitor-induced membrane-lipid peroxidation and taxol production in cell suspension cultures of Taxus chinensis
Process Biochemistry 37 (2001) 477– 482 www.elsevier.com/locate/procbio
Effects of salicylic acid on fungal elicitor-induced membrane-lipid peroxidation and taxol production in cell suspension cultures of Taxus chinensis Long-Jiang Yu *, Wen-Zhi Lan, Wen-Min Qin, Hui-Bi Xu School of Life Science and Technology, Huazhong Uni6ersity of Science and Technology, Wuhan 430074, China Received 14 February 2001; received in revised form 1 June 2001; accepted 9 June 2001
Abstract Effects of three treatments of 50 mg l − 1 fungal elicitors (F5), 50 mg l − 1 salicylic acid (SA) and 50 mg l − 1 F5 + 50 mg l − 1 SA were studied on membrane-lipid peroxidation. Peroxidase (POD), glucose-6-phosphate dehydrogenase (G6PDH) and taxol production in cell suspension cultures of Taxus chinensis were investigated. The results showed membrane-lipid peroxidation caused by fungal elicitor F5, prepared from fungus isolated the inner bark of T. chinensis, was decreased by the addition of salicylic acid, even if the latter also induced cell membrane-lipid peroxidation. F5 + SA resulted in improving the activity of POD and G6PDH compared to single F5 treatment, and achieved the greatest taxol production of 11.5 mg l − 1, being 1.5, 2.3 and 7.5 times higher than that of F5, SA and the control. By analysis of membrane-lipid peroxidation, POD and G6PDH activity, the reason combined treatment F5 and SA improved taxol production was reduction of membrane-lipid peroxidation induced by F5. The studies showed that treatment of F5 +SA on T. chinensis cultures not only gained more biomass than single F5 treatment, but also achieved the highest taxol production. © 2001 Elsevier Science Ltd. All rights reserved. Keywords: Taxus chinensis cell; Salicylic acid; Taxol; Membrane-lipid peroxidation; Fungal elicitor; Peroxidase (POD); Glucose-6-phosphate dehydrogenase (G6PDH)
1. Introduction Taxol, a novel diterpenoid secondary product from the bark of yew species, has been proved an efficient anti-cancer drug [1,2]. In vitro cell culture is a promising method to produce taxol and related taxane compounds [3]. It has been reported that elicitation induced by biotic and abiotic elicitors is one of the most effective methods to enhance production of taxol in Taxus cells [4]. Even the combination of elicitors can enhance more taxol production than that of a single elicitor [5]. To our knowledge, all previous research has focused on selecting the effective elicitors to improve taxol production, but few have provided detailed information concerning physiological and biochemical changes, especially the membrane-lipid peroxidation in suspen* Corresponding author: Fax: + 86-27-8754-0184. E-mail address: [email protected] (L.-J. Yu).
sion cultured Taxus cells under the condition of elicitation. In this study, we applied a fungal elicitor prepared from fungus isolated from the inner bark of Taxus chinensis to study its effects on membrane-lipid peroxidation in suspension culture cells of T. chinensis. Salicylic acid (SA) has been reported to be involved in regulating a number of processes in plants [6]. One of its well-known functions is that SA plays an important role in plants disease resistance against pathogens. SA is shown to regulate the expression of pathogen protein (PR) genes to mediate a hypersensitive response (HR) and a systemic acquired resistance response (SAR) [7,8]. At present, the protective role of exogenous SA on plant cells treated with elicitors to improve secondary metabolites is rarely reported. In this document, we also studied effects of SA on membrane-lipid peroxidation induced by fungal elicitor and production of taxol in suspension cell cultures of T. chinensis, in order to obtain higher biomass and production of taxol.
0032-9592/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 2 - 9 5 9 2 ( 0 1 ) 0 0 2 4 3 - 6
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L.-J. Yu et al. / Process Biochemistry 37 (2001) 477–482
2. Materials and methods
calculated from the extinction coefficient 155 mM − 1 cm − 1.
2.1. Plant materials and culture conditions 2.4. Peroxidase (POD) acti6ity determination T. chinensis cell line, initiated from zygote embryo, was maintained in modified Gamborg’s B5 liquid medium according to Zhang et al. [5] supplemented with 3% sucrose and 10 mmol l − 1 a-naphthalene acetic acid (NAA). Cultures were shaken in a gyratory shaker at 25 °C in the dark at an agitation speed of 120 rpm. In the treatment experiments, about 100 g fresh weight 14-day-old cells were inoculated to 1 l of fresh modified Gamborg’s B5 liquid medium supplemented with 3% sucrose and 10 mmol l − 1 NAA. The cells were grown in the dark on a rotary shaker at 120 rpm for 10 days before treatments. On the 10th day, 50 mg l − 1 fungal elicitor F5, 50 mg l − 1 salicylic acid (SA), 50 mg l − 1 F5 plus 50 mg l − 1 SA were added into the cultures. Samples were harvested at various time points after treatment to measure malondialdehyde (MDA) content, peroxidase (POD) and glucose-6-phosphate dehydrogenase (G6PDH) activity.
2.2. Preparation of fungal elicitor The fungus, isolated from the inner bark of T. chinensis, was grown in potato-dextrose broth for 7 days at 30 °C in a 130 rpm shaking incubator. After suspension cultures had been filtered, mycelial walls were resuspended in water, and filtered again. A total of 40 g (fresh weight) mycelial walls were homogenized in a blender for 10 min, and then were blended with ethyl acetate in a mixture at room temperature for 24 h. Mixture was filtered and the filtrate containing lipids was discarded. The remains were collected, and 100 ml deionized water was added, then the pH value was adjusted to 2 with 1 mol l − 1 HCl, and then it was autoclaved at 121 °C for 2 h. The suspension was filtered and filtrate was adjusted to pH 5.8 with 0.5 mol l − 1 NaOH and termed as elicitor F5. Its carbohydrate concentration was determined by the orcinol-sulfuric acid method [9].
2.3. Malondialdehyde (MDA) assay Lipid peroxidation was determined by estimating MDA content following the method described by Heath and Pack [10] with sight modification. One gram of fresh cells was macerated in 10 ml of 0.5% thiobarbituric acid (TBA) in 20% frichloracetic acid. The mixture was incubated at 95 °C water for 30 min, and quickly cooled in an ice-bath. The samples were centrifuged at 10 000×g for 5 min, and the absorbency of supernatant was read at 532 nm. The value for non-specific absorption at 600 nm was subtracted. The amount of MDA-TBA complex (red pigment) was
A total of 200 mg of sample was homogenized with 2 ml of 0.1 mol l − 1 phosphate buffer (pH 6.0) containing 0.1% (w/v) polyvinylpyrrolindone 4.0, and centrifuged at 15 000× g in a refrigerated centrifuge. The supernatant was taken as the enzyme extract. Peroxidase activity was determined following the method [11] with sight modification. The assay mixture for the POD activity contained 1 ml 0.1 mol l − 1 phosphate buffer (pH 6.0), 1 ml 15 mM pyrogallol, 1 ml 3 mmol l − 1 H2O2 and 5 ml enzyme extract. The mixture was incubated for 5 min at 25 °C. One POD unit (U) was defined as the change of 0.01 OD per minute under the assay conditions. The enzyme activity was expressed as U g fw − 1 (fresh weight cells).
2.5. Assay for glucose-6 -phosphate dehydrogenase (G6PDH) acti6ity Two gram (fresh weight) of each sample was homogenized with 4 ml of enzymatic extract reagent (0.005 mol l − 1 KCl, 0.005 mol l − 1 MgSO4, 0.02 mol l − 1 Tris–HCl, pH 7.5) and centrifuged at 6000 rpm in a refrigerated centrifuger. The supernatant was taken as the enzyme extract. G6PDH activity determined in the supernatant by the spectrophotometric reduction of NADP+ at 30 °C, accordingly to Deutsch [12]. The molar extinction coefficient of NADP+ at 340 nm is 6.22 mM − 1 cm − 1.
2.6. Biomass and taxol determination Biomass was determined by suction filtration of samples taken at the 50th h after treatment. The cells were washed with deionized water to remove residual medium and freeze-dried for 30 h for dry weight determinations. Samples harvested at the 50th h after treatment were extracted with methanol chloride (1:1 v/v), The CH2Cl2 phase was separated from the aqueous phase and evaporated in a rotary evaporator equipped with a condenser for solvent recovery. The residue was resuspended in and centrifuged at 8000× g for 5 min. The supernatant was analyzed for taxol production by HPLC as previously described [9]. Samples of 5 ml from the free media were extracted with 2 ml CH2Cl2 for three times. The combined CH2Cl2 fraction was dried and redissolved in 2 ml methanol, then centrifuged for HPLC analysis [5]. Taxol concentrations in the samples were the combination of taxol in cells and media.
L.-J. Yu et al. / Process Biochemistry 37 (2001) 477–482
3. Results
3.1. Effect of F5, F5 + SA, SA on malondialdehyde (MDA) concentration MDA is the terminal product and important landmark of cells membrane-lipid peroxidation. Fig. 1 shows that three treatments of F5, SA, and F5 +SA all enhanced the MDA content indicating the appearance of cells membrane-lipid peroxidation, but the time points of reaching their maximum were different. Treatment of F5 caused the MDA content to improve rapidly and reached a peak 15 h after treatment. Treatment of SA induced MDA content increased slowly and reached its maximum until 40 h after treatment. The concentration of MDA induced by the treatment of F5 +SA was not the summation of that of F5 treatment and SA treatment, being was less than that of single SA or F5. Therefore, the addition of 50 mg l − 1 SA was beneficial to reduce membrane lipid peroxidation when elicitor was applied to improve taxol production.
3.2. Effect of F5, F5 + SA, SA on peroxidase (POD) acti6ity The changes of POD activity in T. chinensis cell cultures induced with different treatments are demonstrated in Fig. 2. Treatment of F5 gained its peak at the 10th h after treatment. POD activity in cultures treated with SA kept increasing slowly during treatment. Treatment of F5 +SA attained a maximum value of POD activity 32 h, and the value was more than that of treatment of F5, SA and the control.
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3.3. Effect of F5, F5 + SA, SA on glucose-6 -phosphate dehydrogenase (G6PDH) acti6ity The changes of G6PDH activity in cells suspension cultures T. chinensis treated with 50 mg l − 1 F5, 50 mg l − 1 SA and 50 mg l − 1 F5 + 50 mg l − 1 SA are shown in Fig. 3. Three treatments all led to G6PDH activity improvement compared to the control, however, both the degree of improvement and the time when G6PDH reached the activity maximum were different. Single SA treatment induced the G6PDH activity slightly greater than that of the control during the process of treatment. G6PDH activity induced by F5 rapidly increased in short time, and gained its maximum value at the 10th h, then decreased quickly and maintained a low level for the rest time. Treatment of F5 + SA obtained its highest value at the 40th h, and G6PDH activity kept higher level during the time of the 20th and 40th h.
3.4. Effect of F5, F5 + SA, SA on biomass and production of taxol The effects of three treatments on biomass accumulations and production of taxol in T. chinensis suspension cultures were shown in Fig. 4. All treatments led to biomass to decrease. The biomass of F5, SA, F5 +SA was 82, 90, and 85% that of the control, respectively. The reduction of biomass might be due to membranelipid peroxidation induced by the treatments, as the degree of biomass reduction was actively parallel to membrane-lipid peroxidation (Figs. 1 and 4). Addition of SA relieved membrane-lipid peroxidation caused by F5, and the treatment of F5 + SA achieved more
Fig. 1. Time course of MDA content in cell suspension cultures of Taxus chinensis using F5, SA, and F5 +SA treatment. Values are means of triplicate results and error bars represent stand errors. Symbol: , control; , F5, 50 mg l − 1 fungal elicitor; , F5 +SA, 50 mg l − 1 fungal elicitor plus 50 mg l − 1 salicylic acid; × , SA, 50 mg l − 1 salicylic acid.
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Fig. 2. Time course of peroxidase (POD) activity in cell cultures suspension of Taxus chinensis using F5, SA, and F5 +SA treatment. Values are means of triplicate results and error bars represent stand errors. Symbol: , control; , F5, 50 mg l − 1 fungal elicitor; , F5 +SA, 50 mg l − 1 fungal elicitor plus 50 mg l − 1 salicylic acid; × , SA, 50 mg l − 1 salicylic acid.
biomass than that of single F5 treatment. In spite of the biomass decrease, three treatments resulted in the higher production of taxol compared to the control. Especially, combined treatment of F5 and SA caused significant improvement in taxol production of 11.5 mg l − 1. This was about 1.5 times higher than that of the culture elicited with F5, 2.3 times higher than that of the culture exposed to SA, 7.5 times higher than that of the control. Therefore, combined treatment of F5 +SA on T. chinensis cell suspension cultures not only gained more biomass but also achieved higher production of taxol compared to F5 treatment.
4. Discussion During the long-term process of plant-pathogen interaction, plants gradually formed a series of complex and effective protection mechanisms to defend against the incursion of pathogens. Cell death of infected tissues activates a plant hypersensitively reaction as one of the effective ways [13]. Membrane-lipid peroxides are generated from membrane fractions of dying or dead cells. The occurrence of lipid peroxidation indicates that cell death is not impaired even if the presence of lipid antioxidant [14]. Results showed that fungal elicitor F5, prepared from fungus isolated the inner bark of T. chinensis, caused cells membrane-lipid peroxidation to some degree (Fig. 1), and gained less biomass than that of the control (Fig. 4). It’s obvious that the treatment of F5 has a damaging effect on T. chinensis. Therefore, it is necessary to play attention to the elicitor’s negative effect when it is applied to improve taxol production. Rao et al. [15] have reported that exogenous SA treatment enhanced lipid peroxidation, and oxidative damage to proteins in Arabidopsis thaliana. Yu et al.[16] also observed membrane-lipid peroxidation when
tabacco cultures were treated with SA. Our results also showed that SA treatment enhanced membrane-lipid peroxidation in T. chinensis (Fig. 1). However, the addition of SA reduced the level of membrane-lipid peroxidation induced by F5. SA is regarded as a molecular signal playing the important roles in SAR. Kawano and Muto reported that SA could protect cells from highly reactive hydroxyl radicals, while producing the less reactive superoxide and H2O2 through a POD-catalyzed reaction [17]. We found that a combination of F5 and SA improved POD activity compared to that of F5, SA, or the control, which contributed to reduce membrane-lipid peroxidation induced by F5. Figs. 1 and 4 showed that the treatment of F5 + SA induced a lower level of membrane-lipid peroxidation, while attaining higher biomass and the highest production of taxol compared with F5. Therefore, the reduction of membrane lipid peroxidation would be in favour of cell growth and taxol synthesis. The oxidation of glucose-6-phosphate by the pentose phosphate pathway (PPP) is an important branch point in carbohydrate metabolism. This pathway provides ribose-5-phosphate for nucleic acid synthesis and generates NADPH. G6PDH is the key enzyme in the PPP. Recently G6PDH was reported to be involved in the elicitor-induced responses in plants [18]. Enhancement of G6PDH by stress responses could contribute by providing more NADPH [18]. NADPH is required for detoxification of free radicals and peroxides [19]. The improvement of PPP could contribute to reducing the level of active oxygen specials and nucleic synthesis to enhance cell vigour. Figs. 3 and 4 showed that the treatment of F5 + SA induced the higher G6PDH activity and got higher biomass and production of taxol compared to F5. SA was recently regarded as a signal molecule in inducing secondary metabolite biosynthesis [20]. Our
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Fig. 3. Time course of glucose-6-phosphate dehydrogenase (G6PDH) activity in cell suspension cultures of Taxus chinensis using F5, SA, and F5 +SA treatment. Values are means of triplicate results and error bars represent stand errors. Symbol: , control; , F5, 50 mg l − 1 fungal elicitor; , F5 + SA, 50 mg l − 1 fungal elicitor plus 50 mg l − 1 salicylic acid; ×, SA, 50 mg l − 1 salicylic acid.
References
Fig. 4. Effect of F5, F5 +SA, and SA on biomass and taxol production of cell suspension cultures of Taxus chinensis using F5, SA, and F5 +SA treatment. Samples were taken at 50 h after treatments. Values are means of triplicate results and error bars represent stand errors. Symbol: control, untreated; F5, 50 mg l − 1 fungal elicitor; F5 +SA, 50 mg l − 1 fungal elicitor plus 50 mg l − 1 salicylic acid; SA, 50 mg l − 1 salicylic acid.
results indicated that F5 or SA resulted in the increasing production of taxol, which implied F5 or SA could activate the taxol biosynthesis. It’s possible that different elicitor signals induce different parts of the defense response [21], and F5 or SA represented ‘cross-talk’ signal in inducing taxol biosynthesis in T. chinensis cell suspension. The synergistic accumulative effect of combination of F5 and SA on taxol biosynthesis was probably one of reasons for improving production of taxol.
Acknowledgements This work was financially supported by the Ministry of Education (2000 year excellent youth teacher fund). We thank NCI for presenting taxol standard sample.
[1] Woo DDL, Miao SYB, Pelayo JC, et al. Taxol inhibits progression of congenital policystic kidney disease. Nature 1994;368:750 – 3. [2] Crown JO, Leary M. The taxanes: an update. Lancet 2000;355:1176 – 8. [3] Gibson DM, Ketchum REB, Vance NC, et al. Initiation and growth of cell lines of Taxus bre6ifolia (pacific yew). Plant Cell Rep 1993;12:479 – 82. [4] Ciddi V, Srinivasan V, Shuler ML. Elicitation of Taxus sp cell cultures for production of taxol. Biotechnol Lett 1995;17:1343 – 6. [5] Zhang CH, Mei XG, Liu L, Yu LJ. Enhanced paclitaxel production induced by the combination of elicitors in cell suspension cultures of Taxus chinensis. Biotechnol Lett 2000;22:1561 –4. [6] Raskin I. Role of salicylic acid in plants. Ann Rev Plant Physiol Mol Biol 1992;43:439 – 63. [7] Yin SH, Mei L, Newman J, et al. Regulation of sesquiterpene cyclase gene expression. Characterization of sesquiterpene cyclase gene expression. Plant Physiol 1997;115:437 – 51. [8] Delaney TP, Uknes S, Vernooij B. A central role of salicylic acid in plant disease resistance. Science 1994;15188:1247 – 50. [9] Francois C, Marshall RD, Neuberger A. Carbohydrates in proteins 4. The determination of mannose in hen’s egg albumin by radioisotope dilution. Biochem J 1962;57:760 – 5. [10] Heath RL, Packer L. Photoperoxidation in isolated chloroplasts. I-kinetics and boichiometry of fatty acid peroxidation. Arch Biochem Biophys 1968;125:189 – 98. [11] Kim Y, Yoo YJ. Peroxidase production from carrot hairy root cell culture. Enzyme Microb Technol 1996;18:531. [12] Deytsh J. Glucose-6-phosphate dehydrogenase. In: Bergmeyer HU, Grabl M, editors. Methods in Enzymatic Analysis. Weinheim: Verlag Chemie, 1983:222 – 3. [13] Mittler R, Herr E, Orvar BL, et al. Transgenic tobacco plants with reduced capability to detoxity reactive oxygen intermediates are hyper responsitive to pathogen infection. Proc Natl Acad Sci USA 1999;96:14165 – 70. [14] Keppler LD, Novacky A. Involvement of membrane lipid peroxidation in the development of bacterially induced hypersensitive reaction. Phytopathology 1986;76:104 – 8. [15] Rao MV, Paliyath G, Ormrod DP, et al. Influence of salicylic acid on H2O2 production, oxidative stress, and H2O2-metabolizing enzymes. Plant Physiol 1997;115:137 – 49.
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[16] Yu DQ, Cen C, Yang ML, et al. Studies on the salicylic acid induced lipid peroxidation and defense gene expression in tabacco cell culture. Acta Bot Sin 1999;41:977 – 82. [17] Kawano T, Muto S. Mechanism of peroxidase actions for salicylic acid-induced generation of active oxygen species and increasing in cytosolic calcium in tabacco cell suspension culture. J Exp Bot 1999;345:685 –93. [18] Fahrendorf T, Ni W, Shorroosh BS, Dixon RA. Stress responses in alfalfa (Medicago sati6a L.) XIX. Transcriptional activation of oxidative pentose phosphate pathway genes at the onset of the isoflavanoid phytoalexin response. Plant Mol Biol 1995;28:885 – 900.
[19] Kuo WY, Tang TK. Effects of G6PDH overexpression in NIH3T3 cells treated with tert-butyl hydroperoxide of papaquat. Free Radic Biol Med 1998;24:1130 – 8. [20] Chen H, Chen F. Effect of methyl jasmonate and salicylic acid on cell growth and crytotanshinone formation in Ti transformed Sali6ia miltiorrhiza cell suspension cultures. Biothch Lett 1999;21:803 – 7. [21] Srinivasan V, Ciddi V, Bringi V, Shuler ML. Metabolic inhibitors and precursors as tools for probing yield limitation in taxane production by Taxus chinensis cell culture. Biotech Prog 1996;12:457 – 65.
Effects of salicylic acid on fungal elicitor-induced membrane-lipid peroxidation and taxol production in cell suspension cultures of Taxus chinensis Long-Jiang Yu *, Wen-Zhi Lan, Wen-Min Qin, Hui-Bi Xu School of Life Science and Technology, Huazhong Uni6ersity of Science and Technology, Wuhan 430074, China Received 14 February 2001; received in revised form 1 June 2001; accepted 9 June 2001
Abstract Effects of three treatments of 50 mg l − 1 fungal elicitors (F5), 50 mg l − 1 salicylic acid (SA) and 50 mg l − 1 F5 + 50 mg l − 1 SA were studied on membrane-lipid peroxidation. Peroxidase (POD), glucose-6-phosphate dehydrogenase (G6PDH) and taxol production in cell suspension cultures of Taxus chinensis were investigated. The results showed membrane-lipid peroxidation caused by fungal elicitor F5, prepared from fungus isolated the inner bark of T. chinensis, was decreased by the addition of salicylic acid, even if the latter also induced cell membrane-lipid peroxidation. F5 + SA resulted in improving the activity of POD and G6PDH compared to single F5 treatment, and achieved the greatest taxol production of 11.5 mg l − 1, being 1.5, 2.3 and 7.5 times higher than that of F5, SA and the control. By analysis of membrane-lipid peroxidation, POD and G6PDH activity, the reason combined treatment F5 and SA improved taxol production was reduction of membrane-lipid peroxidation induced by F5. The studies showed that treatment of F5 +SA on T. chinensis cultures not only gained more biomass than single F5 treatment, but also achieved the highest taxol production. © 2001 Elsevier Science Ltd. All rights reserved. Keywords: Taxus chinensis cell; Salicylic acid; Taxol; Membrane-lipid peroxidation; Fungal elicitor; Peroxidase (POD); Glucose-6-phosphate dehydrogenase (G6PDH)
1. Introduction Taxol, a novel diterpenoid secondary product from the bark of yew species, has been proved an efficient anti-cancer drug [1,2]. In vitro cell culture is a promising method to produce taxol and related taxane compounds [3]. It has been reported that elicitation induced by biotic and abiotic elicitors is one of the most effective methods to enhance production of taxol in Taxus cells [4]. Even the combination of elicitors can enhance more taxol production than that of a single elicitor [5]. To our knowledge, all previous research has focused on selecting the effective elicitors to improve taxol production, but few have provided detailed information concerning physiological and biochemical changes, especially the membrane-lipid peroxidation in suspen* Corresponding author: Fax: + 86-27-8754-0184. E-mail address: [email protected] (L.-J. Yu).
sion cultured Taxus cells under the condition of elicitation. In this study, we applied a fungal elicitor prepared from fungus isolated from the inner bark of Taxus chinensis to study its effects on membrane-lipid peroxidation in suspension culture cells of T. chinensis. Salicylic acid (SA) has been reported to be involved in regulating a number of processes in plants [6]. One of its well-known functions is that SA plays an important role in plants disease resistance against pathogens. SA is shown to regulate the expression of pathogen protein (PR) genes to mediate a hypersensitive response (HR) and a systemic acquired resistance response (SAR) [7,8]. At present, the protective role of exogenous SA on plant cells treated with elicitors to improve secondary metabolites is rarely reported. In this document, we also studied effects of SA on membrane-lipid peroxidation induced by fungal elicitor and production of taxol in suspension cell cultures of T. chinensis, in order to obtain higher biomass and production of taxol.
0032-9592/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 2 - 9 5 9 2 ( 0 1 ) 0 0 2 4 3 - 6
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L.-J. Yu et al. / Process Biochemistry 37 (2001) 477–482
2. Materials and methods
calculated from the extinction coefficient 155 mM − 1 cm − 1.
2.1. Plant materials and culture conditions 2.4. Peroxidase (POD) acti6ity determination T. chinensis cell line, initiated from zygote embryo, was maintained in modified Gamborg’s B5 liquid medium according to Zhang et al. [5] supplemented with 3% sucrose and 10 mmol l − 1 a-naphthalene acetic acid (NAA). Cultures were shaken in a gyratory shaker at 25 °C in the dark at an agitation speed of 120 rpm. In the treatment experiments, about 100 g fresh weight 14-day-old cells were inoculated to 1 l of fresh modified Gamborg’s B5 liquid medium supplemented with 3% sucrose and 10 mmol l − 1 NAA. The cells were grown in the dark on a rotary shaker at 120 rpm for 10 days before treatments. On the 10th day, 50 mg l − 1 fungal elicitor F5, 50 mg l − 1 salicylic acid (SA), 50 mg l − 1 F5 plus 50 mg l − 1 SA were added into the cultures. Samples were harvested at various time points after treatment to measure malondialdehyde (MDA) content, peroxidase (POD) and glucose-6-phosphate dehydrogenase (G6PDH) activity.
2.2. Preparation of fungal elicitor The fungus, isolated from the inner bark of T. chinensis, was grown in potato-dextrose broth for 7 days at 30 °C in a 130 rpm shaking incubator. After suspension cultures had been filtered, mycelial walls were resuspended in water, and filtered again. A total of 40 g (fresh weight) mycelial walls were homogenized in a blender for 10 min, and then were blended with ethyl acetate in a mixture at room temperature for 24 h. Mixture was filtered and the filtrate containing lipids was discarded. The remains were collected, and 100 ml deionized water was added, then the pH value was adjusted to 2 with 1 mol l − 1 HCl, and then it was autoclaved at 121 °C for 2 h. The suspension was filtered and filtrate was adjusted to pH 5.8 with 0.5 mol l − 1 NaOH and termed as elicitor F5. Its carbohydrate concentration was determined by the orcinol-sulfuric acid method [9].
2.3. Malondialdehyde (MDA) assay Lipid peroxidation was determined by estimating MDA content following the method described by Heath and Pack [10] with sight modification. One gram of fresh cells was macerated in 10 ml of 0.5% thiobarbituric acid (TBA) in 20% frichloracetic acid. The mixture was incubated at 95 °C water for 30 min, and quickly cooled in an ice-bath. The samples were centrifuged at 10 000×g for 5 min, and the absorbency of supernatant was read at 532 nm. The value for non-specific absorption at 600 nm was subtracted. The amount of MDA-TBA complex (red pigment) was
A total of 200 mg of sample was homogenized with 2 ml of 0.1 mol l − 1 phosphate buffer (pH 6.0) containing 0.1% (w/v) polyvinylpyrrolindone 4.0, and centrifuged at 15 000× g in a refrigerated centrifuge. The supernatant was taken as the enzyme extract. Peroxidase activity was determined following the method [11] with sight modification. The assay mixture for the POD activity contained 1 ml 0.1 mol l − 1 phosphate buffer (pH 6.0), 1 ml 15 mM pyrogallol, 1 ml 3 mmol l − 1 H2O2 and 5 ml enzyme extract. The mixture was incubated for 5 min at 25 °C. One POD unit (U) was defined as the change of 0.01 OD per minute under the assay conditions. The enzyme activity was expressed as U g fw − 1 (fresh weight cells).
2.5. Assay for glucose-6 -phosphate dehydrogenase (G6PDH) acti6ity Two gram (fresh weight) of each sample was homogenized with 4 ml of enzymatic extract reagent (0.005 mol l − 1 KCl, 0.005 mol l − 1 MgSO4, 0.02 mol l − 1 Tris–HCl, pH 7.5) and centrifuged at 6000 rpm in a refrigerated centrifuger. The supernatant was taken as the enzyme extract. G6PDH activity determined in the supernatant by the spectrophotometric reduction of NADP+ at 30 °C, accordingly to Deutsch [12]. The molar extinction coefficient of NADP+ at 340 nm is 6.22 mM − 1 cm − 1.
2.6. Biomass and taxol determination Biomass was determined by suction filtration of samples taken at the 50th h after treatment. The cells were washed with deionized water to remove residual medium and freeze-dried for 30 h for dry weight determinations. Samples harvested at the 50th h after treatment were extracted with methanol chloride (1:1 v/v), The CH2Cl2 phase was separated from the aqueous phase and evaporated in a rotary evaporator equipped with a condenser for solvent recovery. The residue was resuspended in and centrifuged at 8000× g for 5 min. The supernatant was analyzed for taxol production by HPLC as previously described [9]. Samples of 5 ml from the free media were extracted with 2 ml CH2Cl2 for three times. The combined CH2Cl2 fraction was dried and redissolved in 2 ml methanol, then centrifuged for HPLC analysis [5]. Taxol concentrations in the samples were the combination of taxol in cells and media.
L.-J. Yu et al. / Process Biochemistry 37 (2001) 477–482
3. Results
3.1. Effect of F5, F5 + SA, SA on malondialdehyde (MDA) concentration MDA is the terminal product and important landmark of cells membrane-lipid peroxidation. Fig. 1 shows that three treatments of F5, SA, and F5 +SA all enhanced the MDA content indicating the appearance of cells membrane-lipid peroxidation, but the time points of reaching their maximum were different. Treatment of F5 caused the MDA content to improve rapidly and reached a peak 15 h after treatment. Treatment of SA induced MDA content increased slowly and reached its maximum until 40 h after treatment. The concentration of MDA induced by the treatment of F5 +SA was not the summation of that of F5 treatment and SA treatment, being was less than that of single SA or F5. Therefore, the addition of 50 mg l − 1 SA was beneficial to reduce membrane lipid peroxidation when elicitor was applied to improve taxol production.
3.2. Effect of F5, F5 + SA, SA on peroxidase (POD) acti6ity The changes of POD activity in T. chinensis cell cultures induced with different treatments are demonstrated in Fig. 2. Treatment of F5 gained its peak at the 10th h after treatment. POD activity in cultures treated with SA kept increasing slowly during treatment. Treatment of F5 +SA attained a maximum value of POD activity 32 h, and the value was more than that of treatment of F5, SA and the control.
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3.3. Effect of F5, F5 + SA, SA on glucose-6 -phosphate dehydrogenase (G6PDH) acti6ity The changes of G6PDH activity in cells suspension cultures T. chinensis treated with 50 mg l − 1 F5, 50 mg l − 1 SA and 50 mg l − 1 F5 + 50 mg l − 1 SA are shown in Fig. 3. Three treatments all led to G6PDH activity improvement compared to the control, however, both the degree of improvement and the time when G6PDH reached the activity maximum were different. Single SA treatment induced the G6PDH activity slightly greater than that of the control during the process of treatment. G6PDH activity induced by F5 rapidly increased in short time, and gained its maximum value at the 10th h, then decreased quickly and maintained a low level for the rest time. Treatment of F5 + SA obtained its highest value at the 40th h, and G6PDH activity kept higher level during the time of the 20th and 40th h.
3.4. Effect of F5, F5 + SA, SA on biomass and production of taxol The effects of three treatments on biomass accumulations and production of taxol in T. chinensis suspension cultures were shown in Fig. 4. All treatments led to biomass to decrease. The biomass of F5, SA, F5 +SA was 82, 90, and 85% that of the control, respectively. The reduction of biomass might be due to membranelipid peroxidation induced by the treatments, as the degree of biomass reduction was actively parallel to membrane-lipid peroxidation (Figs. 1 and 4). Addition of SA relieved membrane-lipid peroxidation caused by F5, and the treatment of F5 + SA achieved more
Fig. 1. Time course of MDA content in cell suspension cultures of Taxus chinensis using F5, SA, and F5 +SA treatment. Values are means of triplicate results and error bars represent stand errors. Symbol: , control; , F5, 50 mg l − 1 fungal elicitor; , F5 +SA, 50 mg l − 1 fungal elicitor plus 50 mg l − 1 salicylic acid; × , SA, 50 mg l − 1 salicylic acid.
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Fig. 2. Time course of peroxidase (POD) activity in cell cultures suspension of Taxus chinensis using F5, SA, and F5 +SA treatment. Values are means of triplicate results and error bars represent stand errors. Symbol: , control; , F5, 50 mg l − 1 fungal elicitor; , F5 +SA, 50 mg l − 1 fungal elicitor plus 50 mg l − 1 salicylic acid; × , SA, 50 mg l − 1 salicylic acid.
biomass than that of single F5 treatment. In spite of the biomass decrease, three treatments resulted in the higher production of taxol compared to the control. Especially, combined treatment of F5 and SA caused significant improvement in taxol production of 11.5 mg l − 1. This was about 1.5 times higher than that of the culture elicited with F5, 2.3 times higher than that of the culture exposed to SA, 7.5 times higher than that of the control. Therefore, combined treatment of F5 +SA on T. chinensis cell suspension cultures not only gained more biomass but also achieved higher production of taxol compared to F5 treatment.
4. Discussion During the long-term process of plant-pathogen interaction, plants gradually formed a series of complex and effective protection mechanisms to defend against the incursion of pathogens. Cell death of infected tissues activates a plant hypersensitively reaction as one of the effective ways [13]. Membrane-lipid peroxides are generated from membrane fractions of dying or dead cells. The occurrence of lipid peroxidation indicates that cell death is not impaired even if the presence of lipid antioxidant [14]. Results showed that fungal elicitor F5, prepared from fungus isolated the inner bark of T. chinensis, caused cells membrane-lipid peroxidation to some degree (Fig. 1), and gained less biomass than that of the control (Fig. 4). It’s obvious that the treatment of F5 has a damaging effect on T. chinensis. Therefore, it is necessary to play attention to the elicitor’s negative effect when it is applied to improve taxol production. Rao et al. [15] have reported that exogenous SA treatment enhanced lipid peroxidation, and oxidative damage to proteins in Arabidopsis thaliana. Yu et al.[16] also observed membrane-lipid peroxidation when
tabacco cultures were treated with SA. Our results also showed that SA treatment enhanced membrane-lipid peroxidation in T. chinensis (Fig. 1). However, the addition of SA reduced the level of membrane-lipid peroxidation induced by F5. SA is regarded as a molecular signal playing the important roles in SAR. Kawano and Muto reported that SA could protect cells from highly reactive hydroxyl radicals, while producing the less reactive superoxide and H2O2 through a POD-catalyzed reaction [17]. We found that a combination of F5 and SA improved POD activity compared to that of F5, SA, or the control, which contributed to reduce membrane-lipid peroxidation induced by F5. Figs. 1 and 4 showed that the treatment of F5 + SA induced a lower level of membrane-lipid peroxidation, while attaining higher biomass and the highest production of taxol compared with F5. Therefore, the reduction of membrane lipid peroxidation would be in favour of cell growth and taxol synthesis. The oxidation of glucose-6-phosphate by the pentose phosphate pathway (PPP) is an important branch point in carbohydrate metabolism. This pathway provides ribose-5-phosphate for nucleic acid synthesis and generates NADPH. G6PDH is the key enzyme in the PPP. Recently G6PDH was reported to be involved in the elicitor-induced responses in plants [18]. Enhancement of G6PDH by stress responses could contribute by providing more NADPH [18]. NADPH is required for detoxification of free radicals and peroxides [19]. The improvement of PPP could contribute to reducing the level of active oxygen specials and nucleic synthesis to enhance cell vigour. Figs. 3 and 4 showed that the treatment of F5 + SA induced the higher G6PDH activity and got higher biomass and production of taxol compared to F5. SA was recently regarded as a signal molecule in inducing secondary metabolite biosynthesis [20]. Our
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Fig. 3. Time course of glucose-6-phosphate dehydrogenase (G6PDH) activity in cell suspension cultures of Taxus chinensis using F5, SA, and F5 +SA treatment. Values are means of triplicate results and error bars represent stand errors. Symbol: , control; , F5, 50 mg l − 1 fungal elicitor; , F5 + SA, 50 mg l − 1 fungal elicitor plus 50 mg l − 1 salicylic acid; ×, SA, 50 mg l − 1 salicylic acid.
References
Fig. 4. Effect of F5, F5 +SA, and SA on biomass and taxol production of cell suspension cultures of Taxus chinensis using F5, SA, and F5 +SA treatment. Samples were taken at 50 h after treatments. Values are means of triplicate results and error bars represent stand errors. Symbol: control, untreated; F5, 50 mg l − 1 fungal elicitor; F5 +SA, 50 mg l − 1 fungal elicitor plus 50 mg l − 1 salicylic acid; SA, 50 mg l − 1 salicylic acid.
results indicated that F5 or SA resulted in the increasing production of taxol, which implied F5 or SA could activate the taxol biosynthesis. It’s possible that different elicitor signals induce different parts of the defense response [21], and F5 or SA represented ‘cross-talk’ signal in inducing taxol biosynthesis in T. chinensis cell suspension. The synergistic accumulative effect of combination of F5 and SA on taxol biosynthesis was probably one of reasons for improving production of taxol.
Acknowledgements This work was financially supported by the Ministry of Education (2000 year excellent youth teacher fund). We thank NCI for presenting taxol standard sample.
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