Directory UMM :Data Elmu:jurnal:P:PlantScience:PlantScience_Elsevier:Vol158.Issue1-2.Sept2000:

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Oligosaccharides potentiate methyl jasmonate-induced production

of paclitaxel in

Taxus canadensis

James C. Linden*, Muenduen Phisalaphong

Department of Chemical and Bioresource Engineering,Colorado State Uni6ersity,Fort Collins,CO80523,USA

Received 8 November 1999; received in revised form 23 May 2000; accepted 30 May 2000

Abstract

The interdependence of methyl jasmonate (MJ) with chitin and chitosan derived elicitors in formation of paclitaxel was studied using plant cell suspension cultures ofTaxus canadensis. Induction of paclitaxel biosynthesis was enhanced when MJ and elicitors were added 8 days after culture transfer compared to treatments in which only MJ or only elicitors were added. The enhancement of the paclitaxel biosynthesis response to MJ concentration was roughly linear between 0 and 200mM using colloidal chitin or oligosaccharides of chitin and chitosan as elicitors. MJ concentrations greater than 200 mM were inhibitory. In kinetic studies, culture growth and substrate utilization were inhibited when the cultures were elicited with 100mM MJ and with 0.63 mg l−1

N-acetylchitohexaose and with 100mM MJ alone; paclitaxel yields were 10-fold greater under the latter condition than the former. Ethylene biosynthesis by the cell cultures in response to elicitation is implicated in regulation of the response. © 2000 Published by Elsevier Science Ireland Ltd. All rights reserved.

Keywords:Taxus canadensis; Cell culture; Oligosaccharide; Elicitation; Methyl jasmonate; Paclitaxel; Ethylene

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1. Introduction

Various simple carbohydrates and lipids are proving important as signal induction mediators for regulation of plant growth and development. Fungal cell wall-derived oligosaccharides are one group of the former, while methyl jasmonate (MJ) is a lipid-derived elicitor. Both classes of com-pounds mediate signal transduction and regulate expression of genes for the production of phy-toalexins and other secondary metabolites. Oligosaccharides and MJ elicit Taxol® _(generic

name paclitaxel) in Taxus canadensis suspension cultures.

Jasmonic acid and MJ act as signal compounds under some circumstances. Induction of proteinase inhibitors, defense genes, and secondary metabolism are noted from studies using a variety

of plants [1 – 3]. Application of jasmonic acid, its precursor, 12-oxo-phytodienoic acid, or MJ results in accumulation of phytoalexins in parsley [4] and rice [5]. In other species such as tomato, potato [6], or soybean [7], no phytoalexin accumulation is observed upon treatment with jasmonic acid. Many plant species tested in cell suspension cul-ture are elicited by exogenously supplied MJ with respect to the accumulation of secondary metabo-lites [2,8,9]. MJ induces rosmarinic acid biosynthe-sis in Lithospermum erythrorhizon cell suspension cultures [10] and shikonin, the red naphtho-quinone pigments of the root, as well as dihydroei-henofuran, an abnormal benzofuran metabolite [11]. Wounding-induced anthocyanin and flavonoid synthesis in petunia is enhanced by MJ [12]. Cooperative stimulation by ethylene and MJ of paclitaxel formation inTaxus cuspidata[13] and

T. canadensis [14] has been reported. Choi et al. [15] demonstrated elicitor and MJ acid treatments induce different genes of 3-hydroxy-3-methylglu-taryl-coenzyme A reductase as well as different * Corresponding author. Tel.: +1-970-4916122; fax: +

1-970-4911815.

E-mail address:[email protected] (J.C. Linden).

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types of antimicrobial isoprenoids using potato. The set of isogens and isoprenoids that are stimu-lated by elicitation is identical to that appearing after pathogen attack, whereas the set of isogens and isoprenoids stimulated by MJ is like those resulting from the wound response. These results indicate a defined role of jasmonic acid derivatives in the activation of the wound response, whereas defense responses are induced upon elicitation that simulates attack by some fungal pathogens [16]. Two types of b-1,4-linked glucosamine oligosac-charides, both potentially derived from the chitin cell walls of pathogenic fungi, act as potent elici-tors in suspension cultured plant cells. The first type, N-acetylchitooligosaccharides, induce phy-toalexin (momilactones and oryzalexins) forma-tion in rice cells even in the nanomolar range [17]. Inhibition studies with various other oligosaccha-rides show specificity of the binding site for oligosaccharides with degree of polymerization (DP) greater than or equal to that of N -acetylchi-tohexaose [18]. Using alkalinization of extracellu-lar medium as the assay, Felix et al. investigated a time and concentration-dependent saturation of chitin oligosaccharide surface binding sites on tomato suspension-culture cells and document desensitization of the primary defense response by repeated treatments with chitin oligosaccharides [19].

The second type of oligosaccharide used as elic-itor is derived from chitosan, the deacetylated form of chitin. Phytoalexin formation is not in-duced by this compound in the rice system [17], but chitosan is an active elicitor in other plant culture systems. Anthraquininone biosynthesis is stimulated inMorinda citrifoliaby both chitin and chitosan [10]. The degree of acetylation of chitin was found to be important in inducing defense responses. In actuality, the difference between chitin and chitosan is a continuum of the degree of

N-acetylation of the glucosamine residues in the polymer [20]. Chitosan elicitors induce formation of phytoalexins in legumes (soybean, chickpea, bean, alfalfa, pea) and solanaceous plants (potato, sweet pepper) [21].

Jasmonic acid arises in plants from linolenic acid via the octadecanoic pathway [22]. Rapid, but transient, synthesis of cis-jasmonic acid has been demonstrated in both whole plants and suspension cultures [8]. However, elicitor treatment as well as wounding leads to the induction of the jasmonic acid biosynthesis [2,23].

Co-mediation of oligosaccharides and MJ has been demonstrated for the induction of phy-toalexin in the rice system [5]. Exogenously ap-plied MJ increases production of momilactone A in elicited cells to levels higher than those elicited with N-acetylchitoheptaose alone. In suspension cultured cells of parsley, MJ potentiates elicitation of phytoalexins using a cell wall derived elicitor of

Phytophthora sojae (Pmg elicitor) [24]. These re-sults suggest MJ primes the parsley suspension cells in a time dependent manner to become more responsive to elicitation. Also using parsley sus-pension cultures, Ellard-Ivey and Douglas [25] show the elicitor response can be partially mim-icked by MJ pretreatment in expression of phenyl-propanoid genes.

Higher plants are suppliers of indispensable raw materials and drugs in the food and pharmaceuti-cal industries and of phytoalexins for plant de-fense. Paclitaxel is a plant-derived drug used in the treatment of breast, ovarian and lung cancers. Several papers now present results showing that MJ enhances paclitaxel production from several

Taxus species [13,26,27]. Chitin-derived oligosac-charides mimic the effects of elicitation from some pathogenic microbes. The addition of cell extracts and cultures filtrates of fungal cultures stimulate paclitaxel and other taxanes in Taxus sp. (RO1-M28) [28]. The contribution of this document is the study of the interaction of MJ with chitin and chitosan-derived oligosaccharides to stimulate pa-clitaxel production. Combinations of both com-pounds are used to analyze possible mutual influence. Experiments with T. canadensiscell cul-ture systems are described to more generally show co-mediated oligosaccharide and MJ elicitation may involve ethylene biosynthesis.

2. Experimental procedures

2.1. Plant materials and maintenance

The cell line is T. canadensis Marsh C93AD, kindly provided by the laboratories of M.L. Shuler (School of Chemical Engineering, Cornell Univer-sity, Ithaca, NY) and D.A. Gibson (USDA/ARS Plant Nutrition Laboratory, Ithaca, NY). The growth medium is according to Ketchum et al. [27]. The maintenance of cultures is carried out as follows: 10 ml of 14-day-old suspensions are

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trans-ferred by pipetting into 40 ml of fresh medium in 125-ml Erlenmeyer flasks. The culture flasks are capped with 28 mm i.d. Bellco silicone closures (Vineland, NJ) and kept in a New Brunswick Scientific (Edison, NJ) Model G-25 shaking incu-bator in the dark at 130 rpm and 25°C.

2.2. Elicitor preparations

Solutions of MJ (Bedoukian Research, Dan-bury, CT) are prepared in ethanol in such a way that constant volume aliquots are added to each well of the 12-well plate (see below) to make all suspensions 1% (v/v) in ethanol. A slight but reproducible increase in paclitaxel production be-cause of added ethanol has been noted [13]. At the same time as MJ addition, equal volumes of aqueous solutions of N-acetylchitohexaose (Seika-gaku, Tokyo 103, Japan), chitosan hydrolysate (AgriHouse Inc., Berthoud, CO) or suspensions of colloidal chitin are added to each well according to the experimental design. Preparation of col-loidal chitin is based on the methanesulfonic acid method of Hirano and Nagao [29] using practical grade chitin (Sigma, St. Louis, MO) that is milled to a 60 mesh flour. Concentrations are determined by the phenol – sulfuric acid method using N -acetylglucosamine as standard [30].

2.3. Characterization of oligosaccharide elicitors

The supernatants of the chitosan oligosaccha-ride preparations are analyzed using a Waters Associates Ultrahydrogel Linear Column (Med-ford, MA); eluant for this gel permeation chem-istry is 0.5 mM sodium sulfate in distilled water at 25°C flowing at 0.6 ml min−1_{. Overlaying the}

chromatogram of N-acetylchitohexaose with that of the chitosan hydrolysate allows identification of the degrees of polymerization of the oligosaccha-rides in the chitosan hydrolysate preparation based on retention times. The majority of material is pentasaccharide, and a lesser amount is tetrasac-charide. The quantity of oligosaccharide with DP 6 in the undiluted chitosan preparation is esti-mated as 8.0 mg ml−1_{, based on standard curve}

quantitation using a N-acetylchitohexaose stan-dard curve in the HPLC analysis. Since a 1000-fold dilution of the chitosan preparation was used in the 75 mg l−1 _{elicitation of the culture, the}

chitosan hexasaccharide concentration could be estimated to be 8 mg l−1_.

The degree of acetylation of the chitosan hy-drolysate is analyzed by proton NMR spectra obtained at the analytical service center in the Department of Chemistry at Colorado State Uni-versity. The – NH, – CH and – CH3proton

intensi-ties from 400 MHz spectra obtained from a 5 mM

N-acetylchitohexaose solution in 10% (v/v) D2O

are compared with those of the unknown chitosan hydrolysate solution. The relative methyl proton intensities from the chitosan hydrolysate are ap-proximately 20% as great as those from the fully acetylated N-acetylchitohexaose spectrum. The re-sults indicate that an average of 1 in 5 of the glucosamine residues in the chitosan hydrolysate is acetylated.

2.4. Experimental conditions

For cell culture studies of the interdependence of MJ with chitin and chitosan derived elicitors on formation of paclitaxel, experiments are conducted using 3.0 ml of 8-day-old suspensions in individual wells of Falcon (Lincoln Park, NJ) 12-well plates [31]. After additions of carbohydrate and MJ solu-tions to replicated wells according to the experi-mental design, the multi-well plates are individually sealed in their original paper wrappers and incubated in the dark at 25°C with 130 rpm shaking. Except in the kinetics study, samples are taken for analyses of paclitaxel, sucrose, glucose and fructose 13 days after elicitation.

2.5. Analysis of paclitaxel and sugars in cell culture media

Paclitaxel is measured on a Waters Associates Model 501 HPLC system equipped with a Waters Associates Model 486 UV detector at 228 nm using a Phenomenex Curosil G (250×4.6 mm) column. The mobile phase is acetonitrile – water (45:55) at 1.0 ml min−1_{. Extracellular paclitaxel is}

measured in 2.0 ml of a supernatant from each sample that is filtered through a 0.20 mm Gelman nylon filter [32]. Then, 150 ml of methanol is passed through the filter to desorb paclitaxel from the membrane and is collected into 0.2 ml vials for HPLC analysis. The analyzed data was qualified and quantified against standards (Hauser Chemi-cal, Boulder, CO), which were prepared in the same way as the samples. Verification of paclitaxel is based on UV absorption spectra and

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electron-spray mass spectrometer parent ion analyses. Su-crose, glucose and fructose in filtered, but unex-tracted, medium samples are analyzed using a Waters Associates Model 6000 HPLC system equipped with a Waters Associates RI detector using a BioRad HPX-87H column at 65°C with 0.008 N H2SO4 at 0.6 ml min−1.

2.6. Analysis of ethylene produced by cell cultures

These experiments are conducted such that eth-ylene is not provided to the cultures as in earlier work [13,14,33]; however, most of the cultures produce ethylene. For studies of ethylene produc-tion that results from elicitaproduc-tion with combina-tions of MJ and oligosaccharides, the silicon cap closures on 125 ml maintenance flasks (see above) are either covered with aluminum foil to impede diffusion of ethylene from the culture headspace or are replaced by serum caps. Following elicita-tion 21 days after culture transfer, ethylene con-centrations in culture headspace samples are determined and quantitated by GC as described previously [33]. In addition to final concentrations of 10 mM CaCl2 added to all flasks, various

amounts of MJ and N-acetylchitohexaose are transferred aseptically to the replicated 40 ml cul-tures. Headspace volume is 125 ml, from which small amounts of ethylene (0.1 ppm) could be reproducibly detected within 2 h by injecting 2.0 ml into the GC [33]. A hypodermic needle fitted with a sterile 0.45 m filter is inserted through the serum cap while the 2.0 ml headspace samples are

being withdrawn using a gas tight syringe. Such precautions to admit air to cultures in aluminum foil covered flasks is not necessary, but ethylene diffusion from the flasks is apparently significant because the ethylene headspace accumulation is only 0.001 of that in the experiment using serum capped flasks. Subsequent duplicate analysis from each flask was conducted daily. Ethylene accumu-lation is based on cell dry weight that is deter-mined at the end of the experiment.

3. Results

3.1. Elicitation experiments

This study is based on knowledge that evolu-tionarily conserved binding proteins are important in chitin oligosaccharide reception and subsequent signal transduction [34]. Formation of paclitaxel in suspension cultures ofT. canadensisby chitin- and chitosan-oligosaccharide preparations with and without application of MJ is studied in the follow-ing series of experiments.

3.1.1. N-acetylchitohexaose co-mediation with 100

mM MJ

Elicitation of theT. canadensiscell cultures with

N-acetylchitohexaose at 0.6 mg l−1 _{and greater}

exhibits very significant induction of paclitaxel only in the presence of 100mM MJ as co-mediator (Table 1). Paclitaxel concentrations are about 3-fold greater than the 100 mM MJ treatment-con-trols and nearly 15-fold greater than MJ-minus controls. The oligosaccharide alone is almost inef-fective in induction of paclitaxel production. From the data acquired in the presence of MJ, maximum accumulation of paclitaxel occurs at oligosaccha-ride concentrations greater than 0.6 mg ml−1_.

3.1.2. Co-mediation at se6eral MJ concentrations

using 0.6 mg l−1 _N_-_{acetylchitohexaose}

The optimal oligosaccharide concentration from Table 1 is used to study the dependence of the elicitation process on MJ concentration. These replicated results indicate nearly linear improve-ment of paclitaxel accumulation between 0 and 200 mM MJ (Fig. 1A). Inhibition of elicitation by MJ is always observed using concentrations greater than 200 mM MJ, regardless of the elicitor used ([33]; data not shown).

Table 1

Influence of methyl jasmonate (MJ) on the N -acetylchito-hexaose mediated elicitation of paclitaxel accumulation by

Taxus canadensissuspension cell culturesa

N-acetylchitohexaose Paclitaxel (mg l−1₎

(mg l−1₎

+100mM

−any added MJ MJ

0.05

0.0 0.34

0.07

0.3 0.57

0.07 1.02 0.6

– 1.03

1.2

0.07

3.1 0.97

0.90

6.3 0.08

a_{Maximum variation of duplicate data points from mean}_B

30%; experiments were repeated with similar results. ‘–’ indi-cates data not available.

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Fig. 1. Paclitaxel production inT.canadensis cell suspension cultures: relationships between methyl jasmonate (MJ) and chitin-and chitosan-derived glucans in elicitation. Variation of MJ concentrations in the presence of constant amounts of (A)

N-acetylchitohexaose (0.63 mg l−1_{); (B) chitosan hydrolysate (75 mg l}−1_{); (C) colloidal chitin (2.25 mg l}−1_{). Variation of elicitor}

concentrations (D)N-acetylchitohexaose; (E) chitosan hydrolysate; (F) colloidal chitin in the presence of 100 mM MJ.

3.1.3. Chitosan hydrolysate co-mediation with MJ

Similarly, application of various chitosan hy-drolysate concentrations with 100 mM MJ 8 days after culture transfer, reveals a dose dependence in paclitaxel concentrations (Fig. 1E). Compared to the treatment with only 100mM MJ added on day 8 (zero chitosan hydrolysate; Fig. 1E), the increase in paclitaxel accumulation is about 6-fold using a chitosan hydrolysate concentration of 75 mg l−1_.

The stimulation using 75 mg l−1 _{of chitosan}

hy-drolysate is evaluated as a function of concentra-tion of MJ (Fig. 1B). Under these condiconcentra-tions 200

mM MJ is considered optimal. However, as time passed the culture developed an intolerance to concentrations of MJ as great as 200 mM. There-fore, later experiments are conducted using 100

mM MJ.

3.1.4. Colloidal chitin co-mediation with MJ

The preparation of chitin that is presented to the cultures was a mixture of solids and very high

molecular weight chitin molecules. HPLC analysis of the supernatant does not detect soluble oligosaccharides. Data for the experiment using 100 mM MJ are given in Fig. 1F. Essentially, the 21-day production was stimulated only slightly, if at all, by 100 mM MJ at all of the colloidal chitin concentrations studied. Dependence on MJ in-creases between 0 and 200mM MJ (Fig. 1C) when evaluated using 2.25 mg l−1 _{of the chitin.}

3.2. Factorial design experiment

Three MJ concentrations (0, 50, 100 mM) and three N-acetylchitohexaose concentrations (0, 0.16, 1.6 mg l−1_{) are examined in a duplicated full}

factorial design experiment. The data from this work in Table 2 shows the best stimulation of paclitaxel production in replicates containing 1.6 mg l−1_{oligosaccharide and 100}_m_{M MJ elicitation}

concentration under the given culture conditions. Another treatment using 0.16 mg l−1 _{of the} _N

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-Table 2

Relationship of various concentrations of methyl jasmonate (MJ) and N-acetylchitohexaose on elicitation of paclitaxel accumulation by Taxus canadensis suspension cell culturesa

N-acetylchitohexaose Paclitaxel (mg l−1₎ ₍_m_M) _{(mg l}−1₎

0 0 0.16 (90.08)

0.52 (90.01) 0

0.16

1.60 0.58 (90.14) 0.43 (90.03)

0 50

3.09 (91.41) 50

0.16

1.60 0.32 (90.00) 0 100 1.04 (90.09) 0.55 (90.01) 100

0.16

100

1.60 4.05 (91.53)

a₉_{, variation of independent duplicate sample data points}

from mean).

acetylchitohexaose stimulates paclitaxel formation with the 50 mM MJ co-mediation. The concentra-tion-dependent sensitivity to MJ that was observed in this experiment is similar to that observed previ-ously between MJ and ethylene [13,14], the implica-tions of which are discussed below.

3.3. Kinetic studies

In the following kinetic studies MJ andN -acetyl-chitohexaose are added according to the following experimental design: A, control; B, 0.63 mg l−1

N-acetylchitohexaose; C, 100 mM MJ; D, 0.63 mg l−1 _N_{-acetylchitohexaose}₊₁₀₀ _m_{M MJ. Graphs}

labeled accordingly in Fig. 2 show the cultures without MJ addition are similar in terms of growth and sugar consumption. This is the case whether

N-acetylchitohexaose (oligo) is added (Fig. 2B) or not (Fig. 2A). Paclitaxel does not accumulate with

N-acetylchitohexaose elicitation alone (Fig. 2B).

Fig. 2. Kinetic profiles of growth, sugar uptake and paclitaxel production byT.canadensisin shake flask batch cultures elicited variously with methyl jasmonate (MJ) (100mM), andN-acetylchitohexaose (0.63 mg l−1_oligo): _{, growth;} _{, paclitaxel;}_"_,

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Fig. 3. Seven-day ethylene production kinetics by 40 mlTaxus canadensiscultures, which were in 125 ml Erlenmeyer flasks closed with serum caps, following elicitation as follows: , methyl jasmonate (MJ) (100 mM); , control; , combination of

N-acetylchitohexaose (37 mg l−1_{) and MJ (100}_m_M); _"_,_N_{-acetylchitohexaose (37 mg l}−1_).

MJ added on day 8 inhibits growth and sugar consumption (Fig. 2C,D). The growth yield (YX/S:

g cell dry weight g sugar consumed−1_{) drops from}

approximate 0.5 in treatments without MJ to 0.3 in treatments with MJ. Product yields (YP/S: mg

paclitaxel formed g sugar consumed−1_{) improve}

with MJ elicitation (Fig. 2C;YP/S=0.22), which is

only slightly less than when combined with N -acetylchitohexaose in this set of experiments (Fig. 2D; YP/S=0.24). These are compared to YP/S=

0.001 (Fig. 2A) from control andYP/S=0.01 from

N-acetylchitohexaose elicitation (Fig. 2B) after 23 days of paclitaxel accumulation. The apparent in-consistency of finding no greater productivity in the MJ and oligosaccharide experiment, in com-parison with those presented above in Tables 1 and 2, may be related to ethylene biosynthesis by the cultures and ethylene effects on signal trans-duction, as discussed below.

3.4. Ethylene production by cell cultures

Studies of ethylene production by cell cultures are conducted at the following final concentra-tions: A, control with appropriate replacements of medium in which elicitors are dissolved; B, 100

mM MJ; C, 37 mg l−1 ₍₂₈ _m_M) _N

-acetylchito-hexaose; D, 100mM MJ+37 mg l−1_N

-acetylchi-tohexaose. Data shown in Fig. 3 indicate rapid

accumulation of ethylene in each of the flasks during the first 4 days following elicitation. MJ causes the greatest accumulation of ethylene; N -acetylchitohexaose appears to inhibit ethylene biosynthesis both with and without MJ in that accumulation is less than that in the control. These results are comparable in terms of relative ethylene concentrations 4 days after elicitation with two such experiments in which the cell cultures that are not stoppered, but the accumulation was 1000-fold less (and at the limit of detection) because ethylene diffused through the silicon closures (data not shown). The results from the replicated stoppered flasks are shown, because the reproducibility of the experiment is better. The concentration of ethylene in the medium in equilibrium with headspace containing 10 ppm, calculated using Henry’s Law, is 0.067 mM. In the unstoppered flasks, from which measured ethylene concentra-tions were three orders of magnitude less, dis-solved ethylene concentrations are on the order of 0.1 nM at equilibrium. In a system for studying ethylene-induced chitinase, Boller et al. found ex-ogenously supplied ethylene at 1 ppm (presumed headspace concentration) was sufficient for half-maximal induction of chitinase activity and en-hancement of the endogenous ethylene formation [35].

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4. Discussion

Using T. canadensis suspension cell cultures, induction of paclitaxel formation by three chitin/

chitosan preparations together with or without MJ was examined. In several of the experiments stimulation of paclitaxel accumulation was ob-served only when MJ was applied at the same time as N-acetylchitohexasaccharide, colloidal chitin and chitosan hydrolysate. Variability ap-peared to be related to the condition of the re-spective cultures; cultures that were not producing even moderate levels of paclitaxel responded bet-ter to the combination of elicitors than did cul-tures that were in generally good condition. Stressed cultures may have been producing greater amounts of ethylene, which alone has been shown to influence paclitaxel accumulation [13]. High concentration of dissolved ethylene inhibit paclitaxel accumulation, as demonstrated using 25 and 50 ppm ethylene in cultures that were not also elicited with MJ [33]. MJ may be modulating the process by stimulating ethylene biosynthesis. The observations between MJ and oligosaccha-ride interactions made in the factorial design ex-periment are analogous to observations made previously between complex MJ and ethylene co-mediation of paclitaxel formation [33]. In those experiments 10 mM MJ stimulated paclitaxel for-mation in the presence of 5 ppm headspace ethyl-ene in equilibrium with the cell culture. However, 100 mM MJ was required to produce paclitaxel in flasks exposed to continuous flowing gas mixtures containing 10 ppm ethylene. Regulation of the ethylene concentration in the multi-well plates un-doubtedly was not as precise, and may not have been as consistent, as in shake flasks used previ-ously for the gas mixture optimization studies [13]. The kinetic and factorial design experiments were conducted using 100mM MJ when the sensi-tivity of the culture to MJ concentrations had possibly changed. Alternatively, the observed dif-ferences may depend on ethylene biosynthesis; elicited cultures may have accumulated levels of ethylene that became inhibitory to paclitaxel for-mation in some of the multi-well plate experi-ments. Because of the variability and importance of ethylene biosynthesis in such responses, ethyl-ene concentration should be monitored in large scale plant cell culture bioreactors. Concern about maintaining optimum ethylene and carbon diox-ide concentrations in plant cell bioreactors is

dis-cussed in the literature [36].

The concentrations MJ that inhibited elicitation decreased during the course of the study, as dis-cussed above. The reasons were possibly related to finding that MJ stimulates ethylene biosynthe-sis by the T. canadensis cell cultures. This finding is similar to other references in the literature. Treatment of tobacco, maize and bean petioles with a commercial fungal cellulase preparation, cellulysin, raises the level of endogenous jasmonic acid after 30 min and is followed by a transient emission of ethylene after 2 – 3 h [37]. Perhaps the responsible molecule(s) causing elicitation and ethylene formation in the cellulysin preparation is the same b-1,4-endoxylanase, for which recent re-ports that site-directed mutagenesis knocks out activity, but does not reduce ethylene biosynthesis stimulation when used as elicitor [38,39]. Rickauer et al. also consider jasmonate a phytohormone because it effects ethylene biosynthesis [40].

Various cellular responses, which are induced by oligosaccharide elicitors, relate expression of genes to pathogen-related reactions, including early membrane responses such as the changes in membrane potential, ion flux, oxidative burst, protein phosphorylation, and induction of jas-monic acid [21]. Ito et al. [18] have studied N -acetylchitooligosaccharide elicitor effects on transient ion fluxes through the plasma membrane in suspension rice cell cultures in conjunction with phytoalexin production. Using purified oligosac-charides, elicitation occurred only with oligomers with degree of polymerization greater than five. A gene called EAS that is responsible for sesquiter-pene cyclase activity was induced very rapidly using the fungal elicitor from Phythphthora para

-sitica var. nicotianaecell wall and MJ but was not induced at all using MJ alone [41]. Similar phe-nomena are reported by Felix et al. [19] using suspension cultured tomato cells, except that ef-fects from chitin oligomers of DP5=DP4 DP3DP2\DP1. While deacetylated oligomers were not active in rice, both chitin and chitosan derivatives in the presence of MJ act as modifiers of secondary metabolite production in the Taxus

system studied here. Roby et al. reported that chitin oligosaccharides, cell wall fragments derived from Colletotricum lagenarium and ethylene all required specific DNA sequences for induction of a reporter gene in Phaseolus 6ulgaris [41].

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Follow-ing treatment with oligosaccharins for which high affinity binding sites have been characterized: oligo-b-glucosides [42], oligochitosaccharides [18], a yeast N-glycan [43], b-1,4-linked galacturonate oligosaccharides with DP\10 [3,21] and reducing end- modifiedN-acetylchitooctasaccharides [44] all developed, responses termed ‘slow’ by Spiro et al. [44]. The slow response included induction of eth-ylene formation [44].

Halevy et al. [45] find changes in sensitivity to ethylene by the involvement of short chain fatty acids in petal senescence following pollination. Similarly, Saniewski et al. [46] have described rela-tionships of MJ action with fatty acids and sterols in elicitation processes. Kauss and coworkers dis-cuss sensitization of plant cell cultures and hypocotyls of etiolated cucumber seedlings (or seg-ments thereof) for induction of various local pathogen-related defense responses using partially acetylated chitosan [47], salicylic acid [48], fungal cell wall derived oligosaccharides [49], low molecu-lar weight lignin fragments [50] and benzothiadia-zole [49,51]. In analogy to human monocytes, the sensitized tissue recently has been called ‘primed’ [52]; in plant tissue, in particular that discussed in this paper, one might consider the possibility that priming is associated with ethylene accumulation in the tissue to a level critical for a given physio-logical effect. Most recently cutin monomers and surface wax constituents have been shown to me-diate ‘conditioning’ of hypocotyls of cucumber [53]. Conditioning refers to observations that hypocotyls are not elicitable immediately after cu-ticle abrasion, but become competent to elicitation within 1 or 2 h. Conditioning is improved in primed tissue when compared to non-primed con-trols [7,9,53]. Ethylene may be involved by height-ening tissue sensitivity to low levels of elicitor [54]. Wound dependent pathways are related to MJ action and presumably to MJ induced ethylene biosynthesis [55,56]. Elicitation causes emission of ethylene to be stimulated both in wild-type plants and in coi1-1 mutants ofArabidopsis[57]. Both the ethylene and jasmonate signaling pathways need to be triggered concomitantly and not sequen-tially, to activate PDF1.2 defense response. How-ever, functional ethylene and jasmonate signaling pathways are not required for growth responses induced by jasmonate and ethylene, respectively [57]. Sensitivity effects from interactions of MJ with downstream ethylene signal transduction

components and membrane-bound receptors re-main a possibility as well [58].

The greatest realization from this study has been learning the importance of ethylene in the elicitation process. Ethylene biosynthesis by this particular system in response to MJ or oligosac-charides is demonstrated. Otherwise, the effect of

N-acetylchitohexaose in co-mediation with MJ in only certain combinations and under certain cell culture conditions is difficult to understand. When ethylene is supplied to the headspace, more consis-tent glucan co-mediated elicitation of paclitaxel production, relative to controls is observed (data not shown). When the closure on the growth vessels are tight enough to keep ethylene produced by the plant cell from diffusing from the immedi-ate headspace of the culture without headspace mixture control, more inconsistent results are ob-tained (data not shown). While MJ stimulates ethylene formation, chitosan- and chitin-oligosac-charides used in this study inhibit ethylene biosyn-thesis by the plant cell culture. In certain of these cases, the ethylene concentration may effectively be great enough at the site of action in the cells to promote the MJ/ethylene co-mediation. Possible reasons for this and for observed inhibition of elicitation at high MJ concentrations has been presented earlier [13]. To address directly if ethyl-ene is causing the inconsistent results, experiments using N-acetylchitohexaose, MJ, and both to-gether with and without ethylene, should be con-ducted. A detailed study of these relationships is underway.

Acknowledgements

The work conducted at Colorado State Univer-sity was supported by a grant from the National Science Foundation Engineering Directorate Pro-ject BES-9702582. Work conducted by REU stu-dents, R.E. Johns and R.J. Granger under NSF grants EEC-9531731 and EEC-9820545, is ac-knowledged. M.L. Shuler (Cornell University) and D.M. Gibson (USDA/ARS at Ithaca, NY) are acknowledged for cell cultures. AgriHouse Inc. (Berthoud, CO) supplied the chitosan hydrolysate preparation. The extensive discussions about this manuscript with Axel Mitho¨fer at the Botanisches Institut der Universita¨t Mu¨nchen were especially helpful.

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J.C.Linden,M.Phisalaphong/Plant Science158 (2000) 41 – 51

46 Table 2

Relationship of various concentrations of methyl jasmonate (MJ) and N-acetylchitohexaose on elicitation of paclitaxel accumulation by Taxus canadensis suspension cell culturesa

N-acetylchitohexaose Paclitaxel (mg l−1₎ ₍_m_M) _{(mg l}−1₎

0 0 0.16 (90.08)

0.52 (90.01) 0

0.16

1.60 0.58 (90.14)

0.43 (90.03)

0 50

3.09 (91.41) 50

0.16

1.60 0.32 (90.00)

0 100 1.04 (90.09)

0.55 (90.01) 100

0.16

100

1.60 4.05 (91.53)

a₉_{, variation of independent duplicate sample data points} from mean).

acetylchitohexaose stimulates paclitaxel formation

with the 50

m

M MJ co-mediation. The

concentra-tion-dependent sensitivity to MJ that was observed

in this experiment is similar to that observed

previ-ously between MJ and ethylene [13,14], the

implica-tions of which are discussed below.

3.3. Kinetic studies

In the following kinetic studies MJ and

N

-acetyl-chitohexaose are added according to the following

experimental design: A, control; B, 0.63 mg l

−1

N

-acetylchitohexaose; C, 100

m

M MJ; D, 0.63 mg

l

−1

_N

_{-acetylchitohexaose}

₊

₁₀₀

_m

_{M MJ. Graphs}

labeled accordingly in Fig. 2 show the cultures

without MJ addition are similar in terms of growth

and sugar consumption. This is the case whether

N

-acetylchitohexaose (oligo) is added (Fig. 2B) or

not (Fig. 2A). Paclitaxel does not accumulate with

N

-acetylchitohexaose elicitation alone (Fig. 2B).

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N-acetylchitohexaose (37 mg l−1_{) and MJ (100}_m_M); _"_,_N_{-acetylchitohexaose (37 mg l}−1_).

MJ added on day 8 inhibits growth and sugar

consumption (Fig. 2C,D). The growth yield (

Y

X/S

:

g cell dry weight g sugar consumed

−1

_{) drops from}

approximate 0.5 in treatments without MJ to 0.3

in treatments with MJ. Product yields (

Y

P/S

: mg

paclitaxel formed g sugar consumed

−1

_{) improve}

with MJ elicitation (Fig. 2C;

Y

P/S

=

0.22), which is

only slightly less than when combined with

N

-acetylchitohexaose in this set of experiments (Fig.

2D;

Y

P/S

=

0.24). These are compared to

Y

P/S

=

0.001 (Fig. 2A) from control and

Y

P/S

=

0.01 from

N

-acetylchitohexaose elicitation (Fig. 2B) after 23

days of paclitaxel accumulation. The apparent

in-consistency of finding no greater productivity in

the MJ and oligosaccharide experiment, in

com-parison with those presented above in Tables 1

and 2, may be related to ethylene biosynthesis by

the cultures and ethylene effects on signal

trans-duction, as discussed below.

3.4. Ethylene production by cell cultures

Studies of ethylene production by cell cultures

are conducted at the following final

concentra-tions: A, control with appropriate replacements of

medium in which elicitors are dissolved; B, 100

m

M MJ; C, 37 mg l

−1

₍₂₈

_m

_M)

_N

-acetylchito-hexaose; D, 100

m

M MJ

+

37 mg l

−1

_N

-acetylchi-tohexaose. Data shown in Fig. 3 indicate rapid

accumulation of ethylene in each of the flasks

during the first 4 days following elicitation. MJ

causes the greatest accumulation of ethylene;

N

-acetylchitohexaose appears to inhibit ethylene

biosynthesis both with and without MJ in that

accumulation is less than that in the control. These

results are comparable in terms of relative ethylene

concentrations 4 days after elicitation with two

such experiments in which the cell cultures that are

not stoppered, but the accumulation was 1000-fold

less (and at the limit of detection) because ethylene

diffused through the silicon closures (data not

shown). The results from the replicated stoppered

flasks are shown, because the reproducibility of

the experiment is better. The concentration of

ethylene in the medium in equilibrium with

headspace containing 10 ppm, calculated using

Henry’s Law, is 0.067

m

M. In the unstoppered

flasks, from which measured ethylene

concentra-tions were three orders of magnitude less,

dis-solved ethylene concentrations are on the order of

0.1 nM at equilibrium. In a system for studying

ethylene-induced chitinase, Boller et al. found

ex-ogenously supplied ethylene at 1 ppm (presumed

headspace concentration) was sufficient for

half-maximal induction of chitinase activity and

en-hancement of the endogenous ethylene formation

[35].

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J.C.Linden,M.Phisalaphong/Plant Science158 (2000) 41 – 51

4. Discussion

Using

T

.

canadensis

suspension cell cultures,

induction of paclitaxel formation by three chitin

/

chitosan preparations together with or without

MJ was examined. In several of the experiments

stimulation of paclitaxel accumulation was

ob-served only when MJ was applied at the same

time

as

N

-acetylchitohexasaccharide,

colloidal

chitin and chitosan hydrolysate. Variability

ap-peared to be related to the condition of the

re-spective cultures; cultures that were not producing

even moderate levels of paclitaxel responded

bet-ter to the combination of elicitors than did

cul-tures that were in generally good condition.

Stressed

cultures

may

have

been

producing

greater amounts of ethylene, which alone has been

shown to influence paclitaxel accumulation [13].

High concentration of dissolved ethylene inhibit

paclitaxel accumulation, as demonstrated using 25

and 50 ppm ethylene in cultures that were not

also elicited with MJ [33]. MJ may be modulating

the process by stimulating ethylene biosynthesis.

The observations between MJ and

oligosaccha-ride interactions made in the factorial design

ex-periment are analogous to observations made

previously between complex MJ and ethylene

co-mediation of paclitaxel formation [33]. In those

experiments 10

m

M MJ stimulated paclitaxel

for-mation in the presence of 5 ppm headspace

ethyl-ene in equilibrium with the cell culture. However,

100 m

M MJ was required to produce paclitaxel in

flasks exposed to continuous flowing gas mixtures

containing 10 ppm ethylene. Regulation of the

ethylene concentration in the multi-well plates

un-doubtedly was not as precise, and may not have

been as consistent, as in shake flasks used

previ-ously for the gas mixture optimization studies

[13]. The kinetic and factorial design experiments

were conducted using 100

m

M MJ when the

sensi-tivity of the culture to MJ concentrations had

possibly changed. Alternatively, the observed

dif-ferences may depend on ethylene biosynthesis;

elicited cultures may have accumulated levels of

ethylene that became inhibitory to paclitaxel

for-mation in some of the multi-well plate

experi-ments. Because of the variability and importance

of ethylene biosynthesis in such responses,

ethyl-ene concentration should be monitored in large

scale plant cell culture bioreactors. Concern about

maintaining optimum ethylene and carbon

diox-ide concentrations in plant cell bioreactors is

dis-cussed in the literature [36].

The concentrations MJ that inhibited elicitation

decreased during the course of the study, as

dis-cussed above. The reasons were possibly related

to finding that MJ stimulates ethylene

biosynthe-sis by the

T

.

canadensis

cell cultures. This finding

is similar to other references in the literature.

Treatment of tobacco, maize and bean petioles

with a commercial fungal cellulase preparation,

cellulysin, raises the level of endogenous jasmonic

acid after 30 min and is followed by a transient

emission of ethylene after 2 – 3 h [37]. Perhaps the

responsible molecule(s) causing elicitation and

ethylene formation in the cellulysin preparation is

the same

b

-1,4-endoxylanase, for which recent

re-ports that site-directed mutagenesis knocks out

activity, but does not reduce ethylene biosynthesis

stimulation when used as elicitor [38,39]. Rickauer

et al. also consider jasmonate a phytohormone

because it effects ethylene biosynthesis [40].

Various cellular responses, which are induced

by oligosaccharide elicitors, relate expression of

genes to pathogen-related reactions, including

early membrane responses such as the changes in

membrane potential, ion flux, oxidative burst,

protein phosphorylation, and induction of

jas-monic acid [21]. Ito et al. [18] have studied

N

-acetylchitooligosaccharide

elicitor

effects

on

transient ion fluxes through the plasma membrane

in suspension rice cell cultures in conjunction with

phytoalexin production. Using purified

oligosac-charides, elicitation occurred only with oligomers

with degree of polymerization greater than five. A

gene called EAS that is responsible for

sesquiter-pene cyclase activity was induced very rapidly

using the fungal elicitor from

Phythphthora para

-sitica

var.

nicotianae

cell wall and MJ but was not

induced at all using MJ alone [41]. Similar

phe-nomena are reported by Felix et al. [19] using

suspension cultured tomato cells, except that

ef-fects from chitin oligomers of DP5

=

DP4

DP3

DP2

\

DP1. While deacetylated oligomers

were not active in rice, both chitin and chitosan

derivatives in the presence of MJ act as modifiers

of secondary metabolite production in the

Taxus

system studied here. Roby et al. reported that

chitin oligosaccharides, cell wall fragments derived

from

Colletotricum lagenarium

and ethylene all

required specific DNA sequences for induction of

a reporter gene in

Phaseolus

6 ulgaris

[41].

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Follow-ing treatment with oligosaccharins for which high

affinity binding sites have been characterized:

oligo-

b

-glucosides [42], oligochitosaccharides [18],

a yeast

N

-glycan [43],

b

-1,4-linked galacturonate

oligosaccharides with DP

\

10 [3,21] and reducing

end- modified

N

-acetylchitooctasaccharides [44] all

developed, responses termed ‘slow’ by Spiro et al.

[44]. The slow response included induction of

eth-ylene formation [44].

Halevy et al. [45] find changes in sensitivity to

ethylene by the involvement of short chain fatty

acids in petal senescence following pollination.

Similarly, Saniewski et al. [46] have described

rela-tionships of MJ action with fatty acids and sterols

in elicitation processes. Kauss and coworkers

dis-cuss sensitization of plant cell cultures and

hypocotyls of etiolated cucumber seedlings (or

seg-ments thereof) for induction of various local

pathogen-related defense responses using partially

acetylated chitosan [47], salicylic acid [48], fungal

cell wall derived oligosaccharides [49], low

molecu-lar weight lignin fragments [50] and

benzothiadia-zole [49,51]. In analogy to human monocytes, the

sensitized tissue recently has been called ‘primed’

[52]; in plant tissue, in particular that discussed in

this paper, one might consider the possibility that

priming is associated with ethylene accumulation

in the tissue to a level critical for a given

physio-logical effect. Most recently cutin monomers and

surface wax constituents have been shown to

me-diate ‘conditioning’ of hypocotyls of cucumber

[53]. Conditioning refers to observations that

hypocotyls are not elicitable immediately after

cu-ticle abrasion, but become competent to elicitation

within 1 or 2 h. Conditioning is improved in

primed tissue when compared to non-primed

con-trols [7,9,53]. Ethylene may be involved by

height-ening tissue sensitivity to low levels of elicitor [54].

Wound dependent pathways are related to MJ

action and presumably to MJ induced ethylene

biosynthesis [55,56]. Elicitation causes emission of

ethylene to be stimulated both in wild-type plants

and in coi1-1 mutants of

Arabidopsis

[57]. Both the

ethylene and jasmonate signaling pathways need

to be triggered concomitantly and not

sequen-tially, to activate PDF1.2 defense response.

How-ever, functional ethylene and jasmonate signaling

pathways are not required for growth responses

induced by jasmonate and ethylene, respectively

[57]. Sensitivity effects from interactions of MJ

with downstream ethylene signal transduction

components and membrane-bound receptors

re-main a possibility as well [58].

The greatest realization from this study has

been learning the importance of ethylene in the

elicitation process. Ethylene biosynthesis by this

particular system in response to MJ or

oligosac-charides is demonstrated. Otherwise, the effect of

N

-acetylchitohexaose in co-mediation with MJ in

only certain combinations and under certain cell

culture conditions is difficult to understand. When

ethylene is supplied to the headspace, more

consis-tent glucan co-mediated elicitation of paclitaxel

production, relative to controls is observed (data

not shown). When the closure on the growth

vessels are tight enough to keep ethylene produced

by the plant cell from diffusing from the

immedi-ate headspace of the culture without headspace

mixture control, more inconsistent results are

ob-tained (data not shown). While MJ stimulates

ethylene formation, chitosan- and

chitin-oligosac-charides used in this study inhibit ethylene

biosyn-thesis by the plant cell culture. In certain of these

cases, the ethylene concentration may effectively

be great enough at the site of action in the cells to

promote the MJ

/

ethylene co-mediation. Possible

reasons for this and for observed inhibition of

elicitation at high MJ concentrations has been

presented earlier [13]. To address directly if

ethyl-ene is causing the inconsistent results, experiments

using

N

-acetylchitohexaose, MJ, and both

to-gether with and without ethylene, should be

con-ducted. A detailed study of these relationships is

underway.

Acknowledgements

The work conducted at Colorado State

Univer-sity was supported by a grant from the National

Science Foundation Engineering Directorate

Pro-ject BES-9702582. Work conducted by REU

stu-dents, R.E. Johns and R.J. Granger under NSF

grants EEC-9531731 and EEC-9820545, is

ac-knowledged. M.L. Shuler (Cornell University) and

D.M. Gibson (USDA

/

ARS at Ithaca, NY) are

acknowledged for cell cultures. AgriHouse Inc.

(Berthoud, CO) supplied the chitosan hydrolysate

preparation. The extensive discussions about this

manuscript with Axel Mitho¨fer at the Botanisches

Institut der Universita¨t Mu¨nchen were especially

helpful.

(5)

J.C.Linden,M.Phisalaphong/Plant Science158 (2000) 41 – 51

References