Plant Science 158 2000 41 – 51
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, CO
80523
, 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 of Taxus 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 200 mM 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 100 mM MJ and with 0.63 mg l
− 1
N-acetylchitohexaose and with 100 mM 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 www.elsevier.comlocateplantsci
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 in Taxus 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
:
jlindencvmbs.colostate.edu J.C. Linden. 0168-945200 - see front matter © 2000 Published by Elsevier Science Ireland Ltd. All rights reserved.
PII: S 0 1 6 8 - 9 4 5 2 0 0 0 0 3 0 6 - X
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 in Morinda citrifolia by 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. canadensis cell cul-
ture systems are described to more generally show co-mediated oligosaccharide and MJ elicitation
may involve ethylene biosynthesis.
2. Experimental procedures
