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 H
2
SO
4
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 elicitation 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 CaCl
2
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serum cap while the 2.0 ml headspace samples are being withdrawn using a gas tight syringe.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 of T. canadensis by 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
m M MJ
Elicitation of the T. canadensis cell cultures with N-acetylchitohexaose at 0.6 mg l
− 1
and greater exhibits very significant induction of paclitaxel
only in the presence of 100 mM 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
.
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 canadensis suspension cell cultures
a
N-acetylchitohexaose Paclitaxel mg l
− 1
mg l
− 1
+ 100 mM
− 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 meanB 30; experiments were repeated with similar results. ‘–’ indi-
cates data not available.
Fig. 1. Paclitaxel production in T. 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 100 mM 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 conditions 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 200 mM 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 mM MJ elicitation concentration under the given culture conditions.
Another treatment using 0.16 mg l
− 1
of the N-
Table 2 Relationship of various concentrations of methyl jasmonate
MJ and N-acetylchitohexaose on elicitation of paclitaxel accumulation by Taxus canadensis suspension cell cultures
a
MJ N-acetylchitohexaose
Paclitaxel mg l
− 1
mg l
− 1
mM 0.16 9 0.08
0.52 9 0.01 0.16
1.60 0.58 9 0.14
0.43 9 0.03 50
3.09 9 1.41 50
0.16 50
1.60 0.32 9 0.00
100 1.04 9 0.09
0.55 9 0.01 100
0.16 100
1.60 4.05 9 1.53
a
9 , 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 and N-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 mM 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 by T. canadensis in shake flask batch cultures elicited variously with methyl jasmonate MJ 100 mM, and N-acetylchitohexaose 0.63 mg l
− 1
oligo:
, growth; , paclitaxel; , sugar uptake.
Fig. 3. Seven-day ethylene production kinetics by 40 ml Taxus canadensis cultures, 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 mM; , N-acetylchitohexaose 37 mg l
− 1
.
MJ added on day 8 inhibits growth and sugar consumption Fig. 2C,D. The growth yield Y
XS
: 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
PS
: mg paclitaxel formed g sugar consumed
− 1
improve with MJ elicitation Fig. 2C; Y
PS
= 0.22, which is
only slightly less than when combined with N- acetylchitohexaose in this set of experiments Fig.
2D; Y
PS
= 0.24. These are compared to Y
PS
= 0.001 Fig. 2A from control and Y
PS
= 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 mM N-acetylchito- hexaose; D, 100 mM 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].
4. Discussion
