Journal of Plant Physiology 170 (2013) 211–219

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Journal of Plant Physiology

journal homepage: www.elsevier.com/locate/jplph

Functional Biotechnology

Coronatine, a more powerful elicitor for inducing taxane biosynthesis in Taxus

media cell cultures than methyl jasmonate
Miriam Onrubia b , Elisabet Moyano b , Mercedes Bonfill a , Rosa Ma Cusidó a , Alain Goossens c,d ,
Javier Palazón a,∗
a
Secció de Fisiologia Vegetal, Facultat de Farmacia, Universitat de Barcelona, E-08028 Barcelona, Spain
b
Departament de Ciències Experimentals i de Salut, Universitat Pompeu Fabra, E-08003 Barcelona, Spain
c
Department of Plant Systems Biology, VIB, B-9052 Ghent, Belgium
d
Department of Plant Biotechnology and Bioinformatics, Ghent University, B-9052 Ghent, Belgium

a r t i c l e i n f o a b s t r a c t

Article history: Coronatine is a toxin produced by the pathogen Pseudomonas syringae. This compound has received
Received 14 June 2012 much attention recently for its potential to act as a plant growth regulator and elicitor of plant secondary
Received in revised form metabolism. To gain more insight into the mechanism by which elicitors can affect the biosynthesis of
18 September 2012
paclitaxel (Px) and related taxanes, the effect of coronatine (Cor) and methyl jasmonate (MeJA) on Taxus
Accepted 19 September 2012
media cell cultures has been studied. For this study, a two-stage cell culture was established, in which
cells were first cultured for 14 days in a medium optimised for growth, after which the cells were trans-
Keywords:
ferred to medium optimised for secondary metabolite production. The two elicitors were added to the
Coronatine
Methyl jasmonate
medium at the beginning of the second stage. Total taxane production in the cell suspension was signif-
Taxus media icantly enhanced by both elicitors, increasing from a maximum level of 8.14 mg/L in control conditions
Cell cultures to 21.48 mg/L (day 12) with MeJA and 77.46 mg/L (day 16) with Cor. Expression analysis indicated that
Gene expression the txs, t13oh, t2oh, t7oh, dbat, pam, bata and dbtnbt genes were variably induced by the presence of
Paclitaxel the elicitors. Genes encoding enzymes involved in the formation of the polihydroxylated hypothetical
Biosynthetic pathway intermediate (TXS, T13OH, T2OH, T7OH) and the phenylalanoil CoA chain (PAM) were stronger induced
than those encoding enzymes catalysing the last steps of the Px biosynthetic pathway (DBAT, BAPT
and DBTNBT). Notably, although taxane accumulation differed qualitatively and quantitatively following
MeJA- or Cor-elicitation, gene expression induction patterns were similar, inferring that both elicitors
may involve distinct but yet uncharacterised regulatory mechanisms.
© 2012 Elsevier GmbH. All rights reserved.

Introduction Jasmonates (JAs) play many important roles in wound response

and secondary metabolite production in plants. Coronatine (Cor) is
A biotechnological approach to the production of the anticancer a phytotoxin produced by several pathovars of the plant bacteria
drug Px and other taxanes by means of Taxus spp. cell cultures has Pseudomonas syringae (Bender et al., 1999), and acts as a molecular
proven to be a good alternative to the use of whole plants, and mimic of the isoleucine-conjugated form of jasmonic acid (JA–Ile)
several companies are currently obtaining taxanes on an industrial (Katsir et al., 2008). Several studies have reported that Cor exerts
scale using cell cultures of different Taxus species. Nonetheless the its virulence effects by activating the host’s jasmonate signalling
generally low Px production in plant cell cultures requires the use pathway (Zhao et al., 2003) and plants insensitive to Cor, like the
of elicitors, notably methyl jasmonate (MeJA). As several studies Arabidopsis (Arabidopsis thaliana) coi1 mutant, exhibit resistance
have shown, MeJA dramatically enhances not only the produc- to Cor-producing strains like P. syringae (Zhao et al., 2003). The
tion of taxanes in cell cultures of different Taxus species (Onrubia actions of Cor include the induction of JA biosynthesis, impact on
et al., 2010) but also of other secondary metabolites of interest, phytohormonal signalling responses in tomato (Uppalapati et al.,
such as triterpene glycosides, lignans and alkaloids (Ionkova, 2009; 2005), and a wide range of biological functions, such as tendril coil-
Shohael et al., 2007; Goossens et al., 2003). ing, inhibition of root elongation, hypertrophy, chlorosis, secondary
metabolite production, ethylene emission, accumulation of pro-
teinase inhibitors and apoptotic cell death (Tamogami and Kodama,
2000; Yao et al., 2002; reviewed in Uppalapati et al., 2005).
∗ Corresponding author at: Secció de Fisiologia Vegetal, Facultat de Farmacia, Uni-
Cor seems to be a structural and functional analogue of jasmonic
versitat de Barcelona, Avda. Diagonal 643, E-08028 Barcelona, Spain.
Tel.: +34 934020267; fax: +34 934024093. acid (JA) and related signalling compounds such as MeJA and 12-
E-mail address: [email protected] (J. Palazón). oxo-phytodienoic acid (12-OPDA), the C18 precursor of JA and MeJA.

0176-1617/$ – see front matter © 2012 Elsevier GmbH. All rights reserved.
http://dx.doi.org/10.1016/j.jplph.2012.09.004
212 M. Onrubia et al. / Journal of Plant Physiology 170 (2013) 211–219

It consists of the polyketide coronafacic acid (CFA), which is a prod- 100 ␮M MeJA and 1 ␮M Cor. For analysis, three flasks were har-
uct of polyketide biosynthesis, and coronamic acid (CMA), a cyclised vested for each treatment at different time points: 0 h, 30 min, 1 h,
derivative of isoleucine. It has been shown that CFA mimics MeJA, 2 h, 4 h, 8 h, 12 h, 1 day, 2 days, 4 days, 8 days, 12 days, 16 days, 20
and that CMA or other aminoacids enhance toxicity. CFA acid has days and 24 days after elicitor treatment.
been found conjugated to other amino acids such as isoleucine, ser- Cell growth and viability were determined as previously
ine and threonine, and these conjugates also possess phytotoxic described (Expósito et al., 2010).
activity (Lauchli and Boland, 2003). JA and 12-OPDA control an
astonishingly large number of plant functions, notably the activa- Taxane determination
tion of secondary metabolism, by acting as signalling compounds in
plant defensive responses against several stress situations. Svoboda Taxanes were extracted from the culture media as described
and Boland (2010) have reported that the defense responses of (Bonfill et al., 2007) with some modifications. 20 mL of media was
plants to different external plant invaders are controlled by a suite mixed and vortexed during 2 min with 5 mL of dichloromethane
of phytohormones among which JA and JA–Ile play a major role. (DCM), followed by 1 h sonication at 25 ◦ C. Once the organic phase
As Cor resembles the JA–Ile conjugate their mode of action could was recovered, it was evaporated. Taxanes were extracted from
be similar. Importantly however, the chemical structure of Cor is freeze-dried cells with a microwave-assisted extraction protocol
more stable due to the rigid cis-orientation in its bi-cyclic skeleton, adapted from Talebi et al. (2004). 2 mL of methanol:water (9:1,
which does not permit transformation to a less active stereoiso- v/v) was added to 50 mg lyophilised material, heated for 8 min
mer or catabolism, and consequently may not be susceptible to in the microwave at 80 W, and filtered through nylon (0.50 ␮m
signal attenuation to avoid an exaggerated response, as do the filter, Maissa, Spain). The process was repeated twice, and both
natural compounds involved in the JA pathway formation (Heitz methanolic extracts were combined. After adding 4 mL of hex-
et al., 2012; Koo et al., 2011). This may explain the higher levels ane, the samples were centrifuged at 2500 × g for 20 min at room
of induction of secondary metabolism observed in plants/cell cul- temperature. The aqueous phase was recovered, mixed with 3 mL
tures treated by Cor as compared to those treated with ‘natural’ DCM:water (2:1, v/v) and vortexed until an emulsion of both
JAs. phases was obtained. After recovering the organic phase, the aque-
Whereas the effect of exogenous natural and synthetic JAs ous phase was vortexed again with 3 mL DCM:water (2:1, v/v).
(Hu et al., 2006; Qian et al., 2005) on secondary metabolite Finally, both organic extracts were combined and evaporated.
biosynthesis has been widely studied, there are relatively few All samples were resuspended in 500 ␮L methanol and filtered
reports on the action of Cor on secondary metabolite produc- prior to analysis (0.22 ␮m PVDF filters, Millipore, Billerica, MA,
tion. Tamogami and Kodama (2000) showed induced accumulation USA).
of some flavonoid phytoalexins when rice leaves were treated Quantification of Px and related taxanes was performed by
with different concentrations (0.05–0.4 mM) of Cor. The effect high performance liquid chromatography (HPLC) as described
of Cor on flavonoid production was greater than that of JA or (Richheimer et al., 1992). Criteria for identification included reten-
12-oxo-PDA (all at 0.5 mM concentrations). Haider et al. (2000) tion time, UV spectra, co-chromatography with standards and peak
showed the positive action of Cor and some structural analogues homogeneity by photo-diode detection when spiked with authen-
on benzo[c]phenanthridine alkaloid production in Eschscholzia cal- tic standards. The peak areas corresponding to the studied taxanes
ifornica cell cultures, although in these studies Cor had a lower from the samples, with the same retention time as authentic tax-
elicitor effect than MeJA and some analogues. The accumulation of anes, were integrated by comparison with an external standard
glyceollins, the phytoalexins of soybean (Glycine max L.), in soybean calibration curve. Px and related taxanes were provided by Hauser
cell suspension cultures has been studied after the addition of sev- Chemicals, Boulder (USA).
eral elicitors related with the JA biosynthetic pathway. JA and MeJA
showed weak phytoalexin-inducing activity when compared to an
early jasmonate biosynthetic precursor, 12-oxo-phytodienoic acid Quantitative real-time PCR (qRT-PCR)
(OPDA), or the bacterial phytotoxin Cor and certain 6-substituted
indanoyl-l-isoleucine methyl esters, which were all highly active RNA was isolated using the “RNeasy Mini Protocol for isola-
(Fliegmann et al., 2003; Lauchli et al., 2002). tion of total RNA from Plant cells and tissues and filamentous
No information about the effect of Cor elicitation on Px and fungi” (Qiagen, Germany). cDNA was prepared from 1 ␮g of RNA
related taxane production and the Px biosynthetic pathway in Taxus with SuperscriptII reverse transcriptase (Invitrogen, CA, USA) and
cell cultures is currently available. In this work, a selected Taxus qRT-PCR was performed with SYBR Green PCR Mastermix (Roche,
media cell line was elicited with 1 ␮M Cor or 100 ␮M MeJA at the USA) in a 384-well platform system (LightCycler® 480 Instrument,
beginning of the second culture stage, to compare the effects of Roche, USA). Gene specific primers were designed with Primer3
both elicitors on Px production. The expression levels of several software version 0.4.0 (Table 1) and the amplification efficiency
genes related with the biosynthesis of Px and related taxanes were of each primer pair was determined empirically by 10-fold serial
studied to shed light on the molecular changes that take place in dilutions of cDNA and calculated as described by Qiagen. Only those
elicited T. × media producer cells. primer pairs with an efficiency of over 0.8 were used. Expression
levels were normalised to the levels of the 18S from Taxus baccata.
For each gene, expression levels were indicated relative to those
Materials and methods at day 14 of culture in the growth medium (reference value = 1).

Plant material Statistics

The Taxus media TXS cell line (Expósito et al., 2010) was grown in Statistical analysis was performed with Statgraphics (Centu-
a two-stage culture as described previously (Cusidó et al., 2002a,b). rion XV) and Excel software. All the data are the indicated as the
Elicitors (MeJA and Cor; Sigma–Aldrich, St. Louis, MO, USA) were mean of 3 measurements ± SD. The multifactorial ANOVA analysis
added to the production medium prior to inoculation. All com- followed by the Tukey multiple comparison tests were used for sta-
pounds were filter-sterilised (0.22 ␮m sterile PES filters, Millipore, tistical comparisons. A p-value of <0.05 was assumed for significant
Billerica, MA, USA) and added to give the final concentrations of differences.
 M. Onrubia et al. / Journal of Plant Physiology 170 (2013) 211–219 213

Table 1
Sequences of the primers used to amplify the genes by quantitative real-time PCR.

Gene Primer sequence Amplicon size Reference – Acc. Number

18S Sense 5 -GTGCACAAAATCCCGACTCT-3 102 Onrubia et al. (2010)

Reverse 5 -GCGATCCGTCGAGTTATCAT-3

txs Sense 5 -TTCGCACGCACGGATACG-3 115 Onrubia et al. (2010)

Reverse 5 -TTCACCACGCTTCTCAATTCG-3

t13oh Sense 5 -GCCCTTAAGCAATTGGAAGT-3 100 AY866412 (T. × media)

Reverse 5 -CAGAGGAATGGCGTTTAGAG-3

t2oh Sense 5 -CGTGCCATTTGGAGGAGGGAGA-3 122 AY518383 (T. canadensis)

Reverse 5 -CGTGAGGGTCGATTGGCGTGTA-3

t7oh Sense 5 -GGTCCGCCCAAATTGCCAGAA-3 110 AY307951 (T. cuspidata)

Reverse 5 -CCCTGCAGAGCCCAAAAAACCT-3

dbat Sense 5 -AGTTGGATTTGGTGATCGAA-3 92 Onrubia (2012)

Reverse 5 -ATCCATGTTGCACGAGACTT-3
pam Sense 5 -CCCGGAGGCATGACGTGAAG-3 99 AY866411 (T. × media)
Reverse 5 -CGCCGTCTTCCGCCTTGC-3

bapt Sense 5 -TAAGCACTCTACAACAACAATGG-3 111 Onrubia et al. (2010)

Reverse 5 -GCATGAACATTAGTATCTTGATTCC-3

dbtnbt Sense 5 -CGGGGGGTTTGTTGTGGGATTA-3 104 Onrubia (2012)

Reverse 5 -TTAGCCTCTCCCCTCGCCATCT-3

Results (biomass obtained at the end of the experiment/inoculum biomass)

achieved by the control cultures and those treated with MeJA or Cor
Cell biomass and viability was 3.5, 2.1 and 3.0, respectively. The dry weight values obtained
during the time-course of the experiment corroborated the results
Two-stage suspension cultures of the TXS cell line were estab- indicated by the fresh weight (data not shown).
lished (Cusidó et al., 2002a,b). In the second stage the cells were Viability, determined as the percentage of living cells in relation
maintained for 24 days in the optimum medium for production and to the total cells, was also high, being on average 77%, 68% and
elicited or not with MeJA or Cor. MeJA was applied at 100 ␮M, the 80% for the control, MeJA- and Cor-treated cultures, respectively.
concentration previously found to be best for our culture system The fact that the fresh and dry weight results followed the same
(Bonfill et al., 2007). Cor was applied at 1 ␮M, which earlier stud- pattern suggests that the lower fresh weight of MeJA-treated cell
ies established as the optimum concentration for promoting taxane cultures was not due to osmotic changes but to a decrease in living
production in our TXS cell line with the least effect on biomass for- cells, which was also supported by the viability study.
mation (Onrubia, 2012). Samples were taken just before and during
this second stage of culture. Despite being in production media, Taxane production
growth was still observed in all three conditions (Fig. 1).
The capacity for biomass formation of Cor-elicited and Total taxane production (measured as the sum of 10-
unelicited cells was similar, except at the end of the experiment deacetylbaccatin III (DAB III), 10-deacetyltaxol (DAT), baccatin III,
when the former exhibited a reduced growth capacity whereas the cephalomannine and Px) increased significantly when cells were
latter continued to grow actively. The growth observed in MeJA- transferred from the growth medium to the production medium.
elicited cultures was lower than in the control and Cor-treated cells The highest taxane levels in the control cultures were observed in
throughout the experiment, especially after 12 days, when highly the second part of the second culture stage (8.8 mg/L at day 24)
significant differences were observed (p < 0.05). The growth index (Fig. 2). At the end of the culture, the production obtained was
almost 6 times higher than at the beginning.
Fresh Weight
800
C
700 MeJA
600 Cor
500
g/L

400
300
200
100
0
1h 2h 4h 8h 12h 1d 2d 4d 8d 12d 16d 20d 24d
Time

Fig. 1. Time courses of biomass production (expressed as g fresh weight/L) of the TXS
cell line cultured for 24 days in the production medium without elicitors (C, control) Fig. 2. Total taxane content (cell associated + extracellular) in the TXS cell line grow-
or with the addition of 100 ␮M methyl jasmonate (MeJA) or 1 ␮M coronatine (Cor). ing for 24 days in the production medium without (C) and with the addition of
In all cases, the inoculum consisted of 200 g/L of cells. Data represent the mean of 100 ␮M methyl jasmonate (MeJA) or 1 ␮M coronatine (Cor). Data are the mean of
three independent experiments ± SD. three independent replicates ± SD.
214 M. Onrubia et al. / Journal of Plant Physiology 170 (2013) 211–219

The time course of taxane production was similar in the MeJA- Table 2
Production of the minority taxanes (DAT: 10-deacetyltaxol; Ceph: cephalomannine),
and Cor-treated cells, but production in the latter was much
expressed as mg/L in the TXS cell line growing for 24 days in the production medium
higher throughout the culture period (Fig. 2). In both cases, taxane without (C) and with the addition of 100 ␮M methyl jasmonate (MeJA) or 1 ␮M
contents reached an early peak at day 4, but the overall maximum coronatine (Cor). Data are the mean of three independent replicates ± SD.
production in the Cor-treated cells occurred at day 16 (80.6 mg/L),
Treatments Days DAT Ceph
4 days later than in MeJA-treated cells (24.7 mg/L). The productiv-
ity achieved by the cultures under Cor elicitation corresponded to 1 Traces –
2 Traces Traces
5 mg L−1 day−1 . The total taxane profile obtained in this study for
4 0.32 ± 0.02 Traces
the control and treated cells agrees with the one observed by our 8 0.68 ± 0.08 Traces
Control
group in Taxus baccata cell cultures, also grown in a two-stage sys- 12 0.38 ± 0.05 Traces
tem, with or without the addition of MeJA (Palazón et al., 2003). 16 0.34 ± 0.05 0.50 ± 0.08
20 0.91 ± 0.08 1.22 ± 0.20
The increase of taxane production after MeJA elicitation is well-
24 1.01 ± 0.09 2.61 ± 0.31
documented (Bonfill et al., 2007; Cusidó et al., 2002a,b; Ketchum
et al., 1999; Yukimune et al., 1996). 1 – –
2 Traces –
The cell line studied showed a high capacity for taxane excre-
4 Traces –
tion from the producer cells to the medium, independent of elicitor 8 Traces –
MeJA
treatment. The percentage of taxane excretion, which equals the 12 – –
total taxanes in the medium divided by the total taxanes in the 16 0.75 ± 0.08 –
20 – 0.65 ± 0.07
medium plus cells, was extremely high: in control conditions it was
24 Traces Traces
over 91% until day 16, and subsequently over 50%. Under MeJA and
Cor treatment, more than 80% of total taxanes were excreted, with 1 – Traces
2 Traces Traces
maximum values reaching over 98% during the first days of culture.
4 1.65 ± 0.33 1.59 ± 0.33
The release of accumulated taxanes from the cells to the medium is 8 1.42 ± 0.20 2.58 ± 0.41
desirable, since it avoids toxic effects in the cells, enhances biosyn- Coronatine
12 1.12 ± 0.09 3.15 ± 0.43
thesis (Navia-Osorio et al., 2002), and also facilitates downstream 16 2.14 ± 0.31 5.91 ± 0.59
processing. 20 1.72 ± 0.23 4.74 ± 0.60
24 1.73 ± 0.24 3.85 ± 0.45
Regarding the qualitative accumulation of the determined tax-
anes (Fig. 3), the major one in the control and MeJA-treated cell
cultures during the first 8 days of culture was DABIII, which then
decreased until the end of the experiment. In this period, DABIII III peaked at day 16, achieving a level of 51 mg/L (Fig. 3), which
represented on average 78% and 52% of the total taxane contents in represented more than 63% of the total taxanes produced. In con-
the control and MeJA-elicited cell cultures, respectively. This was trol cultures, baccatin III was found in low or very low amounts
an expected result, since during the first days of culture, when the throughout the experiment.
level of total taxanes is at its lowest, the most abundant taxanes Px in the unelicited cultures increased with time, peaking at
are those without a lateral chain, especially DABIII, which is the day 20, when production was almost 4 mg/L (Fig. 3). The highest Px
precursor of baccatin III. This corresponded with the observation of contents in the MeJA-treated cultures, almost 10 mg/L, were found
Nims et al. (2006), who found DABIII to be the main taxane during at day 24. Px production in the Cor-treated cultures followed a
the first 7 days of culturing Taxus cuspidata cells, independently of different pattern, since two peaks were observed during the exper-
elicitation with 100 ␮M MeJA. In contrast, in Cor-treated cultures, iment. The first peak occurred very early, when at day 4 the Px
although the absolute levels of DABIII were not much lower than content reached 36 mg/L, and the second peak was at the end, when
in those under control and MeJA conditions, its content was very almost 13 mg/L Px was obtained (Fig. 3).
low relative to other taxanes (on average 7% of the total taxanes The content of DAT (Table 2) was not detectable or very low, both
considered). in the control and elicited cell cultures. Since no information on the
Baccatin III was the main taxane in the elicited cultures, espe- biosynthesis of DAT is available, it is difficult to interpret this result.
cially when the total production was at its highest. Cell cultures Cephalomannine (Table 2) was found in significant levels at day 24
treated with MeJA achieved a baccatin III production of 10.7 mg/L under control conditions and at day 16 under Cor elicitation. It is
at day 12 (Fig. 3), which corresponded to 43% of the total taxanes known that cephalomannine differs from Px in carrying a tigloy-
produced at that point. In cell cultures treated with Cor, baccatin lation instead of a benzoylation at position C3 of the lateral chain,
while both have baccatin III as a precursor. Although the main tax-
ane formed from baccatin III in Cor-treated cultures was Px, a small
amount of the very high levels of this precursor could have been
directed to the production of cephalomannine. Nevertheless, once
again it is difficult to provide a reliable explanation since the regu-
lation and characteristics of cephalomannine biosynthesis are not
yet understood. Nonetheless, DAT and cephalomannine are both
important taxanes since they can be used for the semisynthesis of
Px analogues.
When comparing the maximum levels of taxane production in
the three culture conditions, the content of baccatin III in the Cor-
treated cultures was 21.6- and 4.8-fold higher than in the control
and MeJA-treated cultures, respectively, whereas MeJA-elicitation
increased baccatin III production 4.5-fold compared to the control.
Px production under Cor treatment was 3.6- and 9.0-fold greater
Fig. 3. Taxane content (expressed as mg L−1 ) in the TXS cell line when maintained
for 24 days in the optimum medium for production complemented or not with than in MeJA-treated and control cultures, respectively.
elicitors. MeJA: 100 ␮M methyl jasmonate; Cor: 1 ␮M coronatine; DABIII: deacetyl Also worth emphasising is the high baccatin III level accu-
baccatin III; BIII: baccatin III. mulated in the Cor-treated cultures (52 mg/L) at day 16, when
 M. Onrubia et al. / Journal of Plant Physiology 170 (2013) 211–219 215

TXS T5 OH TAT
Geranylgeranyl Taxa-4(5), 11(12)-diene Taxa-4(20),11(12)-dien-5 -ol Taxa-4(20),11(12)-dien-
diphosphate 5-yl acetate
T13 OH
T10 OH
T1 OH
Taxadien-5 -13 -diol
T2 OH
5 -acetoxytaxadien-1 , 5 -acetoxytaxadien- 5 -acetoxytaxadien-10 -ol
2 , 7 , 10 , 13 -hexaol T7 OH 10 -diol
T9 OH
TBT
Epoxidase
Oxomutase C9 oxidase DBAT
Hypothetical 10-Deacetyl baccatin III Baccatin III
polyhydroxylated
intermediate

PAM CoA transferase

-phenylalanine -phenylalanine -pheyilalanoyl-CoA BAPT

DBTNBT T2’ OH
Taxol 3’-N-debenzoyltaxol 3’-N-debenzoyl-2’-deoxytaxol

Fig. 4. The taxol biosynthetic pathway. In bold: the enzymes encoded by the studied genes.

the productivity rate reached 3.3 mg L−1 day−1 . This result is very (140 and 130 times higher than the reference value) (p < 0.05), it
promising since new Px-related compounds with improved effi- is notable that txs transcript levels peaked 3 days earlier in Cor-
cacy and less toxicity are currently being sought, and most of them treated cultures. We have previously shown that the txs gene is
are being obtained semisynthetically from the natural precursor highly induced in MeJA-elicited cell suspension cultures (Onrubia
baccatin III (Expósito et al., 2009). et al., 2010).
Taxadiene-13␣-hydroxylase (T13␣OH) is the enzyme that
Gene expression profiles hydroxylates taxa-4(20),11(12)-dien-5␣-ol to taxa-4(20),11(12)-
dien-5␣-13␣-diol (Jennewein et al., 2001). As shown in Fig. 5,
To investigate the mode of action of the elicitors in tax- transcript accumulation of this gene was significantly higher in Cor-
ane biosynthesis and the relationship between gene expression treated cultures than in those supplemented with MeJA (p > 0.05).
and the pattern of taxane production, the expression of genes At the same time, both cultures presented significantly higher t13oh
encoding enzymes involved in Px biosynthesis was profiled mRNA levels than unelicited cultures (p > 0.05). Expression under
by qRT-PCR. The studied genes were: txs (encoding taxadiene MeJA began 1 h after elicitation, increasing until day 4, and under
synthase) and t13˛oh (encoding taxadiene 13␣-hydroxylase), Cor it increased from 1 h to day 2, decreasing thereafter. Results
both involved in early synthetic steps; t7ˇoh (encoding taxane obtained by Nims et al. (2006), indicate that the expression of this
7ˇ-hydroxylase), t2˛oh (encoding taxane 2␣-hydroxylase) and gene increases during the first hours after MeJA treatment. In our
dbat (encoding 10-deacetylbaccatin III-10␤-O-acetyltransferase), case, although RNAm accumulation of the t13oh gene was evident
which control intermediate synthetic steps; and pam (encoding after 1 h of elicitation, the highest level was achieved after 4 days.
phenylalanine aminomutase), bapt (encoding baccatin III-3-amino- The difference is probably due to the use of different cell lines and
13-phenylpropanoyltransferase) and dbtnbt (encoding 3 N-benzoyl culture systems.
transferase), which are involved in the last synthetic steps (Fig. 4). It is known that there is a branch point after the biosynthesis
Gene expression was determined from 1 h to 4 days after elic- of taxa-4(20),11(12)-dien-5␣-ol, which can be the substrate either
itation. Subsequent transcript accumulation is not shown in the for an acetylation at C5OH and a subsequent hydroxylation at C10,
figures because we observed that the highest expression and induc- or for an hydroxylation at the C13 of the taxane skeleton, giving the
tion thereof occurs in this early period (Expósito et al., 2010; taxa-4(20),11(12)-dien-5␣-acetoxy-10␤-ol or taxa-4(20),11(12)-
Onrubia et al., 2010). Similar results have been observed by other dien-5␣-13␣-diol, respectively (Fig. 4). Although the expression
research groups (Hu et al., 2006; Nims et al., 2006). of genes involved in the metabolic branch leading to taxa-
The expression of the TXS gene, which controls the first commit- 4(20),11(12)-dien-5␣-acetoxy-10␤-ol (TAT and T10ˇOH genes) has
ted step of the Px biosynthetic pathway (Hezari et al., 1995), was not been studied here, the high levels of the t13oh gene observed
greatly enhanced by the presence of elicitors in the culture medium in our TXS cell line could indicate that, after elicitation with MeJA
(Fig. 5). The accumulation of txs transcripts under MeJA-treatment and Cor, Px biosynthesis proceeds mainly through the step catal-
was detectable 1 h after elicitation, and reached a maximum at days ysed by the T13␣OH enzyme. Nims et al. (2006) also suggested a
1–4, decreasing thereafter (data not shown). At day 4, txs transcript preference for the T13␣OH-side of the branch pathway in elicited
levels were 5.2 times higher in the MeJA-treated cultures than in the T. cuspidata cell cultures, which confirms metabolic profiling data
control. Under Cor, txs transcript levels clearly increased after 1 h (Ketchum et al., 2003) showing that precursor flux leading to Px is
and peaked at 24 h after elicitation, decreasing thereafter, although via the 5␣, 13␣-diol through T13␣OH, rather than the 5␣-yl-acetate
remaining high until day 4. Maximum txs transcript levels in the derived from the alternative branch controlled by the TAT enzyme.
Cor-elicited cultures were 4.8 times greater than in the control, also In the intermediate steps of Px biosynthesis, the enzymes
at 24 h (Fig. 5). Although values were similar under both elicitors taxane-2␣-hydroxylase (T2␣OH) and taxane-7␤-hydroxylase
216 M. Onrubia et al. / Journal of Plant Physiology 170 (2013) 211–219

(T7␤OH) catalyse the hydroxylation of the 2C and 7C positions of respectively 17.4 and 10.3 times the reference value. Levels of dbat
the taxane skeleton, respectively (Chau and Croteau, 2004; Chau transcripts in MeJA-treated cell cultures were already very high at
et al., 2004) (Fig. 4). The two corresponding genes showed similar 12 h, and continued increasing until day 1, decreasing again there-
expression patterns, which were comparable to that of the t13oh after to values similar to those observed at 12 h. A similar trend
gene. was observed after treatment with Cor but in this case the high-
The expression of the dbat gene, corresponding to the enzyme est value was observed at day 2. The highest values observed in
for the transformation of 10-deacetylbaccatin III into baccatin III MeJA- and Cor-supplemented cultures were 5 and 3 times higher,
(Walker and Croteau, 2000) (Fig. 4), peaked at day 1 in the unelicited respectively, than in the control (Fig. 5).
cultures (3.6 times higher than the reference value). Elicitation with The pam gene, encoding the enzyme responsible for the forma-
MeJA resulted in a higher induction of the gene than with Cor, tion of ␤-phenylalanine from its isomer ␣-phenylalanine (Walker

Fig. 5. Gene expression in the TXS cell line during the first 4 days in the production media complemented or not with the elicitors methyl jasmonate (MeJA, 100 mM) or
coronatine (Cor, 1 mM). Y-axis: gene expression relative to that of the expression level in cultures maintained for 14 days in the growth medium; X-axis: time of culture,
14 days in growth medium, posterior time points in production medium with or without treatment. TXS, taxadiene synthase; T13˛OH, taxadiene 13␣-hydroxylase; T2˛OH,
taxane 2␣-hydroxylase; T7ˇOH, taxane 7␤-hydroxylase; DBAT, 10-deacetylbaccatin III-10-O-acetyltransferase; PAM, phenylalanine aminomutase; BAPT, baccatin III-3-amino,
13-phenylpropanoyltransferase; DBTNBT, debenzoyltaxol N-benzoyl transferase.
 M. Onrubia et al. / Journal of Plant Physiology 170 (2013) 211–219 217

et al., 2004) (Fig. 4), is considered to be the first gene involved in the conditions. We have previously shown (Expósito et al., 2010) that
biosynthesis of the lateral chain of Px. The expression of this gene, although TXS controls the first committed step in the Px biosyn-
in control cells, remained very low throughout the 4 days consid- thetic pathway, it is not a limiting enzyme involved in taxane
ered. In the elicited cultures (Fig. 5) two expression peaks were production, as taxadiene is not a limiting substrate in Px formation.
observed. The first occurred already 1 h after elicitation, with pam Hezari et al. (1997) suggested that taxadiene did not accumulate for
transcript levels in the MeJA- and Cor-treated cultures being 43 and longer than 2 days to any appreciable level in Taxus cell cultures,
63 times higher, respectively, than the reference value. The second indicating a rapid conversion of this metabolite by downstream
peak, which was the maximum pam transcript level achieved, was reactions.
observed at day 4 under MeJA and day 2 under Cor treatment, being The studied hydroxylases participate at different levels in
52 and 93 times higher, respectively, than the reference value, and the formation of the hypothetical polyhydroxylated intermediate
29 and 53 times higher than in the control. It is notable that Cor (Croteau et al., 2006), which is the substrate for the formation of
was almost twice as effective as MeJA for the induction of the pam DABIII and baccatin III. In our study, the three genes encoding these
gene. enzymes were the most strongly activated by the elicitors. It is also
In the final steps of the metabolic pathway (Fig. 4), the enzyme worth noting that the highest transcript levels occurred two days
baccatin III 13-O-(3-amino-3-phenylpropanoyl) transferase (BAPT) earlier under Cor than MeJA. This might suggest that the taxane sub-
(Walker et al., 2002) is responsible for binding the lateral chain to strates were available earlier for the downstream metabolic steps
baccatin III, leading to taxanes with ␤-phenylalanine as a lateral in the Cor-treated cells and explain the higher taxane accumulation
chain. The enzyme N-benzoyl transferase (DBTNBT) (Long et al., in these cultures.
2008) is involved in the last metabolic step leading to Px. Control Of the three genes, pam, bapt and dbtnbt, involved only in
cultures grown in the production medium presented only a slight the biosynthesis of taxanes with a phenylisoserine lateral chain
increase in the expression of the two corresponding genes over the attached to the C13 of baccatin III, the PAM gene was the most
4 sampled days (Fig. 5). When comparing the elicitor treatments, induced by the addition of Cor to the culture medium, probably
the highest expression level was observed for both genes under resulting in a high amount of ␤-phenylalanine, which would not
MeJA, being 1.3 times higher than the maximum under Cor. How- limit the lateral chain formation and consequently the biosynthe-
ever, Cor caused an earlier activation, as shown in Fig. 5 (days 1–2 sis of Px in the Cor-treated cultures. However, when total taxane
and 4, respectively). production was at its peak (day 16), the average baccatin III accu-
mulation was 2.3 times higher than the average sum of lateral
chain-bearing taxanes during the same period. This result indicates
Discussion a limited transformation of baccatin III into Px or lateral chain-
bearing taxanes, which might be due to the relatively low induction
The highest expression levels of the studied genes were of the bapt and dbtnbt genes by Cor (in terms of fold induction rel-
observed during the first hours/days after elicitation, however the ative to the reference value). It is also notable that although total
highest taxane production was generally achieved later. Similar taxane production was higher in the Cor-treated cells, the propor-
results have been found for other pathways (Pauwels et al., 2009). tion of Px was higher in MeJA-treated cultures. This could reflect
Nims et al. (2006) reported that a few days after their expression, the aforementioned enhanced levels of bapt and dbtnbt in the MeJA
the transcripts of several genes are no longer found in the cells, cultures, particularly at the end of the experiment.
indicating that enzyme activity may persist long after their cog- Our results have shown that all the studied genes were induced
nate mRNAs are absent from the cell. Nevertheless, our MeJA- and to a variable extent by the presence of the elicitors MeJA (100 ␮M)
Cor-treated TXS cell cultures showed a significant increase in Px and Cor (1 ␮M). The elicitors enhanced the expression of genes
and baccatin III production only 4 days after elicitation, although involved in the formation of both precursor parts of the Px
the highest level was obtained at days 12 and 16, respectively. molecule, the polyhydroxylated precursor of Px and the pheny-
Although the differences in the expression level of the stud- lalanoil CoA chain, but had a more limited effect in the last two steps
ied genes in the MeJA- and Cor-treated cell cultures were not of the Px biosynthesis pathway (Figs. 4 and 5). Clearly, the elicited
very remarkable, the total taxane production in these cultures cultures showed different taxane patterns and gene expression pro-
differed considerably, suggesting that the elicitors may involve dis- files, suggesting differing action mechanisms, even though Cor is
tinct but as yet uncharacterised regulatory mechanisms. Patil et al. structurally similar to the active jasmonate JA–Ile. Previous studies
(2012), working with MeJA-elicited T. cuspidata cell cultures of dif- carried out by Weiler et al. (1994) report that 1 ␮M Cor conditioned
ferent cell aggregate size, reported that differences in expression more accumulation of indole alkaloids in Rauwolfia serpentina cells
of several genes involved in paclitaxel biosynthesis were minor than 100 ␮M MeJA. However, the same authors observed that the
compared with differences in taxane accumulation, The authors content of benzophenacetridine alkaloids as a function of Cor con-
concluded that additional factors need to be uncovered before Px centration and the time course of response was about 50–100-fold
productivity can be fully optimised. lower than for MeJA in E. californica cell cultures. Katsir et al. (2008)
In our case, a possible explanation for the increased produc- have shown that Cor is more efficient than MeJA and JA–Ile in stim-
tion of taxanes in the Cor-treated cultures is that this elicitor is ulating the JA pathways. Uppalapati et al. (2005) demonstrated that
less toxic than MeJA, since the cell viability study showed a higher a 10,000-fold higher concentration of MeJA was necessary to have
percentage of living cells under Cor, especially towards the end of the same effect as Cor on anthocyanin content in tomato.
the culture period. A greater number of Px-producing cells in the Although it has been shown that both JA–Ile and Cor have
Cor-treated cultures would thus be responsible for the increased the COI1 protein as a receptor (Katsir et al., 2008), the different
taxane production. Alternatively, Uppalapati et al. (2005) showed response in MeJA and Cor-treated cell cultures might be explained
that Cor induces an accumulation of proteinase inhibitors and con- by the different signal transduction steps downstream of the
sequently, enzymes involved in the Px biosynthetic pathway would perception machinery. This could explain the different taxane-
have a more persistent activity in cell cultures treated with this accumulation behaviour in Cor- and MeJA-elicited cultures. Several
elicitor, resulting in higher taxane contents than in the control or authors studying the action mechanism of Cor, MeJA and other
MeJA-treated cultures. chemical analogues (Svoboda and Boland, 2010; Tamogami and
As indicated above, the expression of the txs gene in MeJA- Kodama, 2000; Uppalapati et al., 2005) have reported that the
or Cor-treated cell cultures was much higher than in control recognition process of these elicitors is very complex, so while
218 M. Onrubia et al. / Journal of Plant Physiology 170 (2013) 211–219

they might be recognised by several members of the same fam- Expósito O, Bonfill M, Moyano E, Onrubia M, Mirjalili MH, Cusidó RM, et al. Biotech-
nological production of taxol and related taxoids: current state and prospects.
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We thank the Technical Science Service from Barcelona Uni- Koo AJ, Cooke TF, Howe GA. Cytochrome P450 CYP94B3 mediates catabolism and
versity for their assistance. This research has been supported by inactivation of the plant hormone jasmonoyl-l-isoleucine. Proc Natl Acad Sci
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two grants from the Spanish MEC (BIO2011-29856-C02-01) and Lauchli R, Boland W. Indanoyl amino acid conjugates: tunable elicitors of plant
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