Plant Gene 2 (2015) 2528

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Plant Gene
journal homepage: www.elsevier.com/locate/plant-gene

Transcriptional activation of sesquiterpene biosynthetic enzyme

-guaiene synthase gene in cell cultures of Aquilaria microcarpa
overexpressing cam1 and rac2 encoding calmodulin and Rac GTPase
Fumiya Kurosaki , Futoshi Taura
Laboratory of Medicinal Bio-resources, Graduate School of Medicine and Pharmaceutical Sciences for Research, University of Toyama, Sugitani, Toyama 930-0194, Japan

a r t i c l e

i n f o

Article history:
Received 4 February 2015
Received in revised form 4 March 2015
Accepted 5 March 2015
Available online 8 April 2015
Keywords:
Aquilaria microcarpa
Methyl jasmonate
Calmodulin
Rac GTPase
Transcriptional activation
-Guaiene synthase

a b s t r a c t
Expression level of a sesquiterpene biosynthetic enzyme gene, -GS, encoding -guaiene synthase of Aquilaria
microcarpa markedly elevated when cultured cells of the plant were treated with methyl jasmonate. The methyl
jasmonate-induced expression of -GS was, however, appreciably reduced in a dose-dependent manner in the
presence of N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide and NSC23766, the specic inhibitors for
calmodulin- and Rac GTPase-dependent cellular processes. Transformation of A. microcarpa cell culture by
cam1 and rac2, calmodulin and Rac GTPase genes isolated from the plant, triggered the transcriptional activation
of -GS even in the absence of methyl jasmonate. In contrast, A. microcarpa cultures overexpressing mutated rac2
of which translate is lacking in binding ability toward GTP did not show the signicant expression of -GS gene.
These results suggest that calmodulin and Rac GTPase proteins function as the important mediators in
jasmonate-induced activation of sesquiterpene biosynthesis in A. microcarpa.
2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).

The group of plant hormones called jasmonates, which include

jasmonic acid and its methyl ester methyl jasmonate (MJ), regulates defense responses of higher plants evoked by various environmental
stresses such as mechanical wounding and infection by pathogenic microorganisms (Creelman and Mullet, 1997; Zambounis et al., 2012;
Wasternack and Hause, 2013). It is also well known that the exogenous
application of jasmonates to plant cells enhances the accumulation of
various secondary metabolites (Gundlach et al., 1992; Lu et al., 2011).
The activation of plant secondary metabolism by jasmonates can be divided into two modes: i) the enhancement of biosynthetic activities
constitutively observed in plants; and ii) the induction of masked
biosynthetic abilities that occur only during unusual physiological processes. In response to wounding and/or microbial infection, Aquilaria
or some other woody plants develop heavy and avored resinoids in
the trunk, referred to as agarwood, which should not form under
healthy conditions (Ng et al., 1997; Ito and Honda, 2005). Although
agarwood is a highly valuable commodity used in incenses, perfumes
and traditional medicines, articial transformation of the woody tissues
Abbreviations: MJ, methyl jasmonate; W-7, N-(6-aminohexyl)-5-chloro-1naphthalenesulfonamide; RT, reverse transcription; CA, constitutively active; DN, dominant
negative.
Corresponding author at: Laboratory of Medicinal Bio-resources, Graduate School of
Medicine and Pharmaceutical Sciences for Research, University of Toyama, Sugitani
2630, Toyama 930-0194, Japan.
E-mail address: kurosaki@pha.u-toyama.ac.jp (F. Kurosaki).

of these trees to agarwood has been very difcult. However, Okudera

and Ito (2009) reported that the biosynthesis of sesquiterpenoids such
as -guaiene, -humulene, and -guaiene can be induced in a suspension culture of Aquilaria crassna or Aquilaria sinensis by incubating the
cells with MJ. In addition, we have recently isolated a sesquiterpene biosynthetic enzyme gene encoding -guaiene synthase (-GS, GenBank
accession no. KF800046) from A. microcarpa cell culture, and showed
that expression of the gene was remarkably activated by the treatment
of the cells with yeast extract or MJ (Lee et al., 2014). These observations
suggest that jasmonate-induced biosynthesis of sesquiterpene compounds in Aquilaria plants may be a suitable model to investigate the
mechanisms of hormone-induced activation of latent plant secondary
metabolism.
Recent studies demonstrate (Creelman and Mullet, 1997;
Wasternack and Hause, 2013) that jasmonates incorporated into plant
cells conjugate with isoleucine and the active form can associate with
a protein complex that functions as the receptor of this plant hormone
(Fig. 1). It has been also demonstrated that jasmonate signaling pathways ultimately lead to the degradation of ubiquitin-tagged proteins,
however, only very little is known regarding jasmonate-induced signal
transduction mechanisms in higher plant cells. We have reported
(Kasidimoko et al., 2005) that biosynthesis of the diterpene compounds
in Scoparia dulcis was markedly elevated by MJ treatment in vitro
and that this biosynthetic enhancement requires Ca2 +-inux into
the cytoplasmic space followed by the activation of an intracellular

http://dx.doi.org/10.1016/j.plgene.2015.03.004
2352-4073/ 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

26

F. Kurosaki, F. Taura / Plant Gene 2 (2015) 2528

COOR

Jasmonates
(R=H or CH3)

Jasmonoyl isoleucine
Ca2+

Rac GTPase
Protein complex
as receptor
Ubiquitin-proteasome
Ca2+
Calmodulin
Nucleus

Expression of biosynthetic
enzyme genes

Fig. 1. Hypothetical scheme of signal transduction processes in the transcriptional activation of biosynthetic enzyme genes involved in plant secondary metabolism by exposure to
jasmonates (Kenmotsu et al., 2013).

Ca2+-cascade. It has been also shown that calmodulin, a Ca2+-binding

protein, functions as a key mediator in the signal transduction mechanisms of MJ. In addition, we have recently demonstrated (Saitoh et al.,
2007; Shite et al., 2009; Mitamura et al., 2011; Kenmotsu et al., 2013)
that Rac GTPase, a plant-specic monomeric GTP-binding protein
(Zeng and Yang, 2000; Gu et al., 2004; Berken, 2006), would play important roles to evoke Ca2+-inux in MJ-signaling processes of higher plant
cells (Fig. 1). In the present study, we attempted to evaluate whether
calmodulin and/or Rac GTPase would participate in the early cellular

events involved in the transcriptional activation of -GS in

A. microcarpa cell cultures triggered by MJ. Levels of -GS transcription
in A. microcarpa cells transformed by calmodulin and Rac GTPase
genes were examined without the stimulation by MJ. We also tested
the effect of mutated genes of Rac GTPase on -GS expression in which
binding abilities of the translate proteins toward GTP were positively
and negatively engineered.
Changes in the transcriptional activity of -GS in A. microcarpa
cell cultures triggered by MJ was determined by reverse transcription (RT) PCR in the presence of N-(6-aminohexyl)-5-chloro-1naphthalenesulfonamide (W-7) and NSC23766, the specic inhibitors
for calmodulin- and Rac GTPase-dependent processes, respectively
(Kurosaki et al., 1987; Gao et al., 2004). In the parallel experiments,
cam2 gene (GenBank accession no. HM237344) constitutively
expressed calmodulin gene homologue of the plant (Kenmotsu et al.,
2010) was also amplied as the control. As shown in Fig. 2, enhancement of -GS expression in A. microcarpa in response to MJ stimulation
was markedly reduced by the treatment with 25 M and 100 M of
W-7 in a dose-dependent manner. Similarly, MJ-induced transcriptional
activation of -GS was appreciably inhibited in the presence of
NSC23766, and this set of the results appeared to be reproducible in repeated experiments. These observations suggest that calmodulin- and
Rac GTPase-dependent cellular processes are involved in transcriptional
activation of -GS in A. microcarpa cells triggered by MJ, and are consistent with our previous ndings that Rac GTPase would play roles in the
activation of Ca2 +-cascade in MJ-signaling processes of higher plant
cells (Kasidimoko et al., 2005; Kenmotsu et al., 2010; Kenmotsu et al.,
2013).
In the next experiment, we prepared transgenic cells of
A. microcarpa that overexpressing calmodulin and Rac GTPase genes,
and -GS transcription in these transformants was examined. We previously isolated two homologous Rac GTPase genes, rac1 and rac2
(GenBank accession nos. JF922914 and JF922915, respectively) from A.

Experiment 1
MJ -

Experiment 2
+
+
+

GS
cam2

MJ -

25 M 100 M

25 M 100 M

W-7

W-7

GS
cam2
25 M 100 M

25 M 100 M

NSC23766

NSC23766

Fig. 2. Effect of calmodulin and Rac GTPase inhibitors on MJ-induced transcriptional activation of -GS. Cultured cells of A. microcarpa were grown on Murashige and Skoog (1962) agar
medium supplemented with 3 M of 2,4-dichlorophenoxyacetic acid and 3 M of N6-benzyladenine at 26 C under darkness (Kenmotsu et al., 2010), and the expression levels of -GS
were measured semiquantitatively using RT-PCR. A. microcarpa cells (3-week-old, approximately 100 mg) were pre-incubated on the medium containing N-(6-aminohexyl)-5-chloro1-naphthalenesulfonamide (W-7) (BIOMOL Research Laboratory, 100 or 25 M) or NSC23766 (Calbiochem, nal concentration 100 or 25 M) for 4 days according to the method described
previously (Kenmotsu et al., 2013). MJ (100 M in 100-l aliquots) was then added to the cultures, and the cells were further incubated for 6 h. Then, total RNA was isolated from cultured
cells using the RNeasy Plant Mini Kit (Qiagen), and cDNA templates were generated by RT reaction. PCR amplication of -GS was carried out with the primer pair, 5-ATG TCT TCG GCA
AAA CTA GGT TCT GCC-3 as the forward and 5-TCA GAT TTC AAT AGC ATG ACG CAA CAA GGC-3 as the reverse primer (1644-bp product). As a control, cam2, a constitutively expressed
calmodulin gene homologue, was amplied in parallel with these reactions using the primers; forward 5-GAT GTG GAT GGT GAT GGG CAG ATC AAC T-3 and reverse 5-GAT CAG AAG CAA
ATG AAA TCG C-3 (298-bp product).

F. Kurosaki, F. Taura / Plant Gene 2 (2015) 2528

GFP
cam2
GFP
GFP cam1 rac2 CArac2 DNrac2
Wild type

Transgenic lines

Fig. 3. Transformation of A. microcarpa cell cultures by calmodulin, Rac GTPase and its mutated genes. Positively and negatively mutated rac2 genes, CArac2 and DNrac2, were prepared using QuikChange II Site-Directed Mutagenesis kit (Agilent Technologies), and
detail procedures were reported previously (Kenmotsu et al., 2013). The Agrobacterium
tumefaciens LBA4404 cells (Invitrogen) were transformed with the expression vectors according to the method described previously (Kenmotsu et al., 2013), and GFP expression of
non-transformed wild type and the transformed cells (GFP-transformant, cam1-, rac2-,
CArac2- and DNrac2-transformant co-expressing GFP) was conrmed by PCR using
5-ATG GTG AGC AAG GGC GAG GAG CTG TTC ACC-3 as the forward and 5-TTA CTT
GTA CAG CTC GTC CAT GCC GTG A-3 as the reverse primer (720 mer as the product, top
panel) with cam2 as the control (middle panel). Immunoblot analysis of GFP in
A. microcarpa cultures (bottom panel) was performed essentially according to the method
previously reported (Kurosaki et al., 1994). Partially puried samples were subjected to
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10% gel), and the separated
proteins were blotted onto Immobilon-P Transfer Membrane (Millipore) on a semi-dry
transfer cell (Transblot SD; Bio-Rad). After blocking with 2% (w/v) dry fat milk, the membrane was incubated with anti-GFP (Medical & Biological Laboratory), and, then, incubated with Protein A conjugated with horseradish peroxidase (Bio-Rad). After several
washings, primary antibody bound to GFP was visualized by color development reaction
using Peroxidase Stain DAB Kit (Nacalai Tesque).

J
M
MJ

- GS
cam2
GFP

Wild type

b
Relative expression level

microcarpa cell cultures (Kenmotsu et al., 2013), and found that rac2
translate might function in MJ-induced elevation of biosynthetic activity
of sesquiterpene compounds. In addition, we generated two rac2 mutants: a constitutively active (CA) and a dominant negative (DN)
forms deemed CArac2 and DNrac2, respectively. The CA (G15V) and
DN (D121A) mutant proteins permanently bind GTP or GDP, and are
expected to constitutively activate or block Rac GTPase-dependent signaling processes (Li et al., 1999; Zeng and Yang, 2000). A. microcarpa
cell culture was also transformed to overexpress cam1 (GenBank accession no. HM237343), a secondary metabolism-related calmodulin gene
homologue of A. microcarpa (Kenmotsu et al., 2010), and GFP was used
as a reporter gene for these transgenic cell lines; cam1, rac2, CArac2 and
DNrac2. A. microcarpa cell culture was transformed by Agrobacteriummediated procedures, and expression of GFP in the transformants was
conrmed by RT-PCR (Fig. 3, top panel). No signal was apparent for
the non-transformed control (wild type), however, the bands of GFP
with signicant intensities were observed for all the samples prepared
from transformed cells (only GFP, cam1 plus GFP as a reporter, rac2
plus GFP, CArac2 plus GFP and DNrac2 plus GFP). Although an attempt
was made to detect GFP protein in the transformed cell cultures under
ultraviolet light, no clear result was obtained, probably because of the
self-uorescence of the cultured cells employed in the present study.
Therefore, we performed an immunoblot analysis to visualize the translational product of the reporter gene (Fig. 3, bottom panel). The protein
bands of signicant intensities were observed for all the transformants
while no signal was apparent in wild type cells. These results strongly
suggested that A. microcarpa cell culture was accurately transformed
and overexpressed the respective genes to accumulate the desired
translates.
The transcriptional levels of -GS in the transgenic cell lines were determined by RT-PCR, and a typical result was shown in Fig. 4a. The expression of -GS in non-transformed cells (wild type) was observed
essentially only in MJ-treated cells, while a marked increase in -GS
transcription was observed even in the absence of MJ when cam1 was
overexpressed in the cell culture. Transformation by rac2 and CArac2

27

cam1

rac2 CArac2 DNrac2

Transgenic lines

1.2
1
0.8
0.6
0.4
0.2
0

MJ-

MJ+

Wild type

GFP

cam1

rac2

CArac2 DNrac2

Transgenic lines

Fig. 4. Expression levels of -GS in A. microcarpa cultures transformed with either cam1,
rac2, CArac2, or DNrac2. a, Transcriptional activities of -GS in cell cultures of
A. microcarpa (non-transformed wild type, GFP, cam1, rac2, CArac2, DNrac2) were determined by RT-PCR in parallel with cam2 as the control, and the typical result was shown.
b, Results obtained from four independent experiments were analyzed by ImageJ, and
the transcriptional activities of -GS were presented as the relative ratio (means and
standard deviations) in which the expression level of MJ-treated wild type cells was
taken as 1.0.

also induced considerable transcription of -GS without MJ, however,

the transgenic cells overexpressing negatively mutated DNrac2 showed
only very low expression. Four sets of the experiments were performed
independently, and intensities of the bands corresponding to -GS were
quantied by ImageJ (imagej.nih.gov/ij/). The results were expressed as
relative values by taking MJ-treated expression in wild type cells as 1. As
shown in Fig. 4b, marked transcription of -GS was induced in cam1,
rac2, and CArac2 mutants and the expression levels were almost comparable to MJ-treated wild type cells. In contrast, the expression of -GS in
GFP and DNrac2 mutants was very low as was non-transformed cells
without MJ treatment. These results clearly indicate that overexpression
of appropriate homologue(s) of calmodulin and Rac GTPase genes is capable of inducing -GS transcription in A. microcarpa cells without exposure to MJ.
The results obtained in the present study strongly suggest that calmodulin and Rac GTPase function as the important mediators in signal
transduction processes of MJ stimulation which leads to the induction
of -GS transcription, a sesquiterpene biosynthetic enzyme gene involved in latent secondary metabolism of A. microcarpa. The transcriptional levels of -GS in the transgenic cells overexpressing cam1, rac2,
and CArac2 appeared to be almost comparable to that of MJ-treated
wild type cells, and, in addition, obvious difference was not observed
between the cell lines transformed by rac2 and its positively mutated
gene CArac2. No direct evidence is available to explain this apparent discrepancy, however, it might be possible that GTP-bound active form of
Rac GTPase would function as an all or nothing switch to trigger the
transcription of -GS (Kawasaki et al., 1999; Li et al., 2001; Ono et al.,
2001). Accumulation of unphysiological concentration of rac2 translate
in A. microcarpa cells might perturb the equilibrium between GTPbound, GDP-bound and nucleotide-free forms of Rac GTPase protein,
and the concentration of GTP-bound form might reach to the sufcient
level to evoke the transcription of -GS gene.

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F. Kurosaki, F. Taura / Plant Gene 2 (2015) 2528

It would be assumed that jasmonate-signaling in higher plant cells

might, at least partly, share similar or common mechanism in a wide variety of plant species (Creelman and Mullet, 1997; Zambounis et al.,
2012; Wasternack and Hause, 2013). It might be possible, therefore,
that the engineering of jasmonate-signaling processes by transformation with appropriate genes would be applicable as a novel method to
the transcriptional activation of several biosynthetic genes involved in
masked secondary metabolism in various useful plants.
Acknowledgments
This work was supported, in part, by a research grant from the
Ministry of Education, Culture, Sports, Science and Technology in
Japan (no. 23590146 to FK) .
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