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Relative expression of genes of menthol biosynthesis pathway in peppermint

(Mentha piperita L.) after chitosan, gibberellic acid and methyl jasmonate
treatments

Article in Russian Journal of Plant Physiology · January 2017

DOI: 10.1134/S1021443717010150

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ISSN 1021-4437, Russian Journal of Plant Physiology, 2017, Vol. 64, No. 1, pp. 59–66. © Pleiades Publishing, Ltd., 2017.

RESEARCH PAPERS

Relative Expression of Genes of Menthol Biosynthesis Pathway

in Peppermint (Mentha piperita L.) after Chitosan, Gibberellic Acid
and Methyl Jasmonate Treatments1
F. Soleymani, H. Taheri*, and A. R. Shafeinia
Department of Agricultural Biotechnology, Ramin Agriculture and Natural Resources University of Khouzestan, Iran
*e-mail: hetaheri@ut.ac.ir
Received March 5, 2016

Abstract—Menthol as an important component of monoterpenes essential oil in peppermint (Mentha piperita

L.) is widely applied for medical and industrial uses. In this study, the effect of exogenous applications of chi-
tosan (200 mg/L), gibberellic acid (50 mg/L) and methyl jasmonate (300 μM) was investigated in the main
genes of menthol biosynthesis pathways within a 72 h time period using qRT-PCR. Transcript levels of most
genes were either unaffected or down-regulated following chitosan treatment relative to control plants.
Decreasing of geranyl diphosphate synthase (GDS) and limonene synthase (LS) genes transcript in chitosan
treatment could possibly be effective in reducing of limonene level. On the other hand, it seems that an
increase in menthone-menthol reductase (MMR) transcription level at 72 h under these treatments had a
positive role in increasing the amount of menthol in this plant. Since exogenous application of gibberellic acid
(GA3) down-regulated transcript levels of several genes involved in menthol biosynthesis, there is this expec-
tance that GA3 treatment might not have a prominent role in enhancing menthol yield via transcription reg-
ulation. Transcript level of the majority genes after methyl jasmonate treatment gradually increased and
reached the highest level at 72 h, therefore, it is possible that methyl jasmonate improves medicinal properties
of M. piperita.

Keywords: Mentha piperita, essential oil, exogenous application, monoterpenes, qRT-PCR and transcript level
DOI: 10.1134/S1021443717010150

INTRODUCTION thesis of monoterpens including menthol is restricted to

Monoterpenes belong to C10 isoprenoids that consti-
tute the major components of the essential oils of mint
family. Peppermint (Mentha piperita L.) is considered as
an experimental model system since the past several
decades because of its highly enriched sources of mono-
terpenes. Menthol is recognized as the most prominent
monoterpenes constituent in peppermint [1]. This valu-
able natural product has considerable economic impor-
tance due to its multitude aromatherapy and industrial
applications [2]. There are eight possible menthol ste-
reoisomers in which the main and stable form naturally
occurring is (–)- or l-menthol enantiomer [3]. Biosyn-
1 The article is published in the original.
Abbreviations: CHT—chitosan; DMAPP—dimethylallyl diphos-
phate; DXS—1-deoxy-D-xylulose-5-phosphate synthase;
FPP—farnesyl diphosphate; GDS—geranyl diphosphate syn-
thase; GGPP—geranyl geranyl diphosphate; GPP—geranyl
diphosphate; IDR—isopiperitenone reductase; IPP—isopente-
nyl diphosphate; L3H—cytochrome P450 (2)-limonene-3-
hydroxylase; LS—limonene synthase; MeJA—methyl jasmon-
ate; MEP—methyl erythritol phosphate; MFS—menthofuran
synthases; MMR—menthone-menthol reductase; MNMR—
menthone-neomenthol reductase; MVA—mevalonic acid; PR—
pulegone reductase.
highly specialized nonphotosynthetic secretory cells
derived from the epidermal layer called peltate glandu-
lar trichomes. Two other types of trichomes that are sit-
uated on peppermint leaves include capitate glands that
produce slight amounts of monoterpens and non glan-
dular unicellular hairs that are not able to synthesize
essential oils [4].
In spite of the fact that isoprenoids are a structur-
ally and functionally diverse group of natural prod-
ucts, all of them are derived from two C5 units namely
isopentenyl diphosphate (IPP) and its allylic isomer
dimethylallyl diphosphate (DMAPP). In plants, pre-
cursors for the biosynthesis of these isoprenes (C5) are
provided by cytosolic mevalonate and plastidial path-
ways [5]. Since the IPP cytosolic biosynthesis pathway
is blocked in peppermint, IPP is entirely synthesized
in plastid via methyl erythritol phosphate (MEP)
pathway [6]. Sequential elongation reactions with the
addition of one, two or three IPP units lead to the bio-
synthesis of geranyl diphosphate (GPP) (C10), farnesyl
diphosphate (FPP) (C15) and geranyl geranyl diphos-
phate (GGPP) (C20) which are the starting points of

59
60 SOLEYMANI et al.

7
1
OPP OPP 6 2 IPR IPI
IPP LS
GDS 5 3 L3OH NADPH
+ OH IPD O NADP+
PP 4
NADPH O O
+ O2 NADP+ NAD+
8 + H2O NADH
10 9

OPP geranyl (–)-trans- (–)-isopiperit- (–)-cis- (+)-pulegone

diphosphate (–)-limonene isopiperitenol enone isopulegone
DMAPP NADPH
+ O2
NADP+ NADPH

NADP +
+ H2O

OH OH
MMR MMR

(+)-isomenthol NADPH (–)-menthol NADPH

O
NADP+ NADP+

O (+)-menthofuran
MNMR MNMR O

(+)-isomenthone OH (–)-menthone
OH

(+)-neoisomenthol (+)-neomenthol

Fig. 1. The principal pathways for monoterpene biosynthesis in peppermint. The responsible enzymes are as follows: geranyl diphos-
phate synthase (GDS), (–)-limonene synthase (LS), (–)-cytochrome P450 (–)-limonene-3-hydroxylase (L3OH), (–)-trans-iso-
piperitenol dehydrogenase (IPD), isopiperitenone reductase (IPR), (+)-cis-isopulegone isomerase (IPI), (+)-menthofuran syn-
thase (MFS), (+)-pulegone reductase (PR), (–)-menthone:(–)-(3R)-menthol reductase (MMR) and (–)-menthone:(+)-(3S)-
neomenthol reductase (MNMR).

downstream pathways for the production of monoter-
penes, sesquiterpenes and diterpens respectively [7].
The major pathway for the biosynthesis of various
menthol isomers is conducted by eight enzymatic
reactions (Fig. 1). Plastidial geranyl diphosphate syn-
thase (GDS) condenses two C5 intermediate subunit
terpenoieds derived MEP pathway to produce acyclic
precursor GPP. The second specific step of the path-
way is cyclization of GPP by plastidial (–)-(4S)-limo-
nene synthase and thereby establishes limonene. Fol-
lowing hydroxylation at C3 and a sequence of redox
reactions on cyclohexanoid ring affords (–)-(1R, 3R,
4S) menthol [8].
An important side-product of menthol biosynthe-
sis pathway is menthofuran. This undesirable mono-
terpen is derived from C9 hydroxylation, cyclization
and dehydration of (+)-pulegone by an endoplasmic
reticulum-localized enzyme called menthofuran syn-
thase. Abiotic stress promotes the accumulation of
menthofuran and pulegone metabolites during leaf
expansion period [4]. Since few percent of these con-
stituents in commercially distilled peppermint oil are
considered as undesirable oil compartments, more
recent consideration has turned to engineer the meta-
bolic flux to interested compounds by up-regulation of
a rate-limiting steps and down-regulation of a side
route reaction to achieve the desired changes in essen-
tial oil composition and yield [9]. Alternatively, the
exposure of plants to stresses, including various elici-
tors or signal molecules such as chitosan, yeast extract
and plant hormones is an effective strategy for
enhancing the yield of pharmaceutical metabolites
[10]. Perception and transduction of elicitors signals in
plants leads to activation of transcription factors that
involved in regulatory of secondary metabolites bio-
synthetic genes [11].
Chitosan (ß-(1,4)-D-glucosamine polymer) is pro-
duced by the deacetylation of chitin and is localized in
the cell wall of pathogenic microorganisms [12]. It can
be recognized as a microbe associated molecular pat-
tern by the plant immune system and stimulates biosyn-
thesis of secondary metabolites through induction of
plant defense responses [13].
Gibberellins are recognized as compounds that
play an important role in the eliciting the biosynthesis
of secondary metabolites in plant cells. Gibberellins
are also reported to increase plant biomass and men-
thol content in M. piperita [14]. These signalling mol-
ecules belong to tetracyclic diterpene plant hormones
that are usually produced by plastidial GGPP derived

RUSSIAN JOURNAL OF PLANT PHYSIOLOGY Vol. 64 No. 1 2017

 RELATIVE EXPRESSION OF GENES OF MENTHOL BIOSYNTHESIS PATHWAY
from MEP pathway. In the MEP pathway, eight enzy-
matic reactions are used to yield gibberellic acid (GA)
from GGPP. Analysis of the expression of genes
involved in GA biosynthesis revealed new insights into
the regulation of GA concentration in plants [15].
Methyl jasmonate (MeJA) is derived from linolenic
acid by the octadecanoid pathway and induce in
response to pathogen attack or wounds that leads to
accumulation of ROS in plant cells [16]. It was also
reported that MeJA and its derivatives induce volatile
terpenoids in various plants [17].
Considering the pharmaceutical value of pepper-
mint essential oil, we investigated the effect of time-
course exogenous application of chitosan (CHT), GA3
and MeJA on M. piperita in term of their quantitative
transcript expression profiling analysis of monoter-
penes biosynthethic pathway. The present study will
help to figure out whether these elicitors affect ter-
penoid gene expression.
MATERIALS AND METHODS
Plant materials and growth conditions. This experi-
ment was carried out under natural light conditions in
the greenhouse of Ramin Agriculture and Natural
Resources University of Khouzestan, Iran. The rhi-
zomes of peppermints (Mentha piperita L.) were col-
lected from Pakanbazr Company, Esfahan, Iran. Then
these rhizomes were cut into pieces 10–15 cm and were
planted into pots. They were watered every day. Two
months-old uniform plants were selected for sampling.
CHT, GA3 and MeJA treatments and samplings.
CHT, GA3 and MeJA were purchased from Sigma-
Aldrich company (Germany). In order to treat the
plants, 200 mg/L CHT in 2% (v/v) acetic acid,
50 mg/L GA3 in distilled water and 0.3 mM MeJA in
2% (v/v) ethanol were separately sprayed on the sur-
face of the pepermint plants, while untreated pepper-
mint plants (control) were sprayed with only 2% (v/v)
acetic acid, distilled water and 2% (v/v) ethanol
respectively. Leaves from the untreated (control) and
treated peppermint plants were randomly sampled at
12, 24 and 72 h after treatment. For each sampling,
4 leaves under the second visible leaf from the apex
were harvested, frozen in liquid nitrogen, stored at
‒80°C immediately for RNA extraction.
RNA extraction, cDNA synthesis, primer design and
qRT-PCR reaction. Total RNA was isolated from mint
leaves using GeneAll® RiboEx™ kit (BioFrontier,
Korea) based on manufacturer’s protocol. The quality
of extracted RNA was checked by 1% agarose gel elec-
trophoresis. The first strand cDNA was synthesized
from 500 ng of total RNA, using Fermentas kit (Revert
Aid™ First Strand cDNA Synthesis Kit) according to
the manufacturer’s instructions.
Primer pairs of PR, MFS, LS, L3H, IDR, MNMR,
MMR and GDS genes were designed with online
primer Quest software according to the cDNA
RUSSIAN JOURNAL OF PLANT PHYSIOLOGY Vol. 64
61
sequences (PR (AY300163.1), MFS (AF346833.1),
LS (EU108697.1), L3H (AF124817), IDR (AY300162),
MNMR (DQ362936), MMR (AY288138) and GDS
(AF182828.1) of M. piperita, and actin (KM044035.1)
of M. spicata that was employed as an internal standard
(table). To ensure the specific amplification of
designed primers for these genes, PCR reaction was
performed using cDNA. Also another primer pair
(expecting to amplify a 1070 bp band) was designed for
isolation beta-actin gene of M. piperita using M. spi-
cata beta-actin sequence and then amplified band of
beta-actin primers was sequenced and annotated
sequence was submited to National Center of Biotech-
nology Information (NCBI).
The qRT-PCR was performed using HIFI SYBR®
Green kit (Iran) Master mix and Step One Plus®
(ABI, United States) machine under the following
conditions: 95°C for 30 s followed by 40 cycles 95°C
for 15 s, 52°C for 20 s and 72°C for 20 s. Relative
expression levels of the genes were calculated by
ΔΔCT. Relative Expression Software Tool (REST)®
software [18] was used to analyses of data. This exper-
iment was carried out with two biological and two
technical repeats.
RESULTS
After the total RNA was extracted, the 28S and 18S
ribosomal RNA bands in 1% agarose gel electrophore-
sis clearly reflect the good quality of extracted RNA
(Fig. 2a). The measured concentration of extracted
RNA and synthesized cDNA by NanoDrop® was
100–300 and 1500–3000 ng/μl respectively. Gradients
PCR was performed due to determine the most appro-
priate annealing temperature (52°C) (Fig. 2b). Also
the peppermint beta-actin gene was submited in
NCBI (KR082011).
Expression of Main Genes
in Menthol Biosynthetic Pathway
Considering the stimulant effects of elicitors on ter-
penoid biosynthesis, it is of interest to determine
whether these molecular signallings would induce an
increase of the expression levels of transcript of menthol
biosynthetic genes in peppermint. Accordingly, a set of
genes involved in the menthol biosynthetic pathway
were selected for qRT-PCR to analyze their expression
in chemical elicitors (CHT, GA3 and MeJA) treated
and control plants. The selected genes include GDS,
LS, L3H, IDR, PR, MFS, MNMR and MMR.
During time-course CHT elicitation, abundance
of GDS transcripts decreased remarkably to below the
control at 24 h (approximately 8 times lower than
those in the control). A significant drop in the expres-
sion levels of LS was also detected at 24 h compared
with corresponding control samples. The L3H mRNA
level increased strongly within 12 h of CHT elicitor
No. 1 2017
62 SOLEYMANI et al.

Sequence, melting temperature (Tm) and product size of act, GDS, LS, L3H, IDR, PR, MFS, MNMR and MMR primers
Gene Primer Sequence (5'-3') Tm, °C Product size, bp
act F TCCTGAGAGGAAGTACAGTGTC 62 108
R GACGGCCCAGATTCATCATAC 62
LS F TGACAGAGGTGTGGAAGAAG 62 113
R GTACATCAACTGCGCCATC 62
PR F GAAGCTGTGATCAACAACATGAG 62 126
R ACGAATTTGCTTTGGGATTAGC 62
MFS F TGACTGAAGCTCCTGGATTTG 62 111
R CCTTCCCTTCCGTGTGTATATG 62
MNMR F CAGAGGAGAAACTGGAGGAAG 62 115
R GCTGCTTTCGACACTTTGTAG 62
MMR F TCGGATCATAGCGCGAAAG 62 112
R AGCACCTTCAGCTTCACTTAG 62
GDS F TAGGGCAGCTCCATTGATTG 62 119
R AGAAAGGAGCATCATGTTTGTG 62
L3H F ATTTCGAGTTCGTCCCGTTC 62 129
R TCATTCCTTCCGCCAACTTC 62
IDR F CGAAGAAGTACCCGAGTTTCC 62 115
R TTCACCGGAACTTGAGCAG 62
Beta-actin F TGTCTGCGATAATGGAACTGG 62 1070
R ATTCATCATACTCCGCCTTAGC 62

treatment (15.2 times higher than that in the control).
Conversely, at 24 and 72 h expression changes were
not remarkable. While the level of transcripts of IDR
and PR were unaffected by CHT treatment, MNMR
(catalysing the conversion of menthone to neomen-
thol or isomenthone to isomenthol) transcripts level
decreased approximately 2.5 and 6 fold, at 12 and 24 h
posttreatment, respectively. Also MMR (catalysing the
conversion of menthone to menthol or isomenthone
to neoisomenthol) showed a decrease at 24 h and then
accumulated rapidly, reaching maximum levels at
72 h. The highest level of MFS mRNA was maintained
up to 12 h but declined rapidly thereafter, reaching its
lowest level by 24 h (Fig. 3).
Transcript levels changes of genes in plants exposed
to GA3 were slightly variable. The transcript level of
GDS was increased 6 fold than in the untreated control
at 12 h, whereas thereafter did not significantly change.
The expression level of LS, IDR, MFS and PR genes
didn’t show significant changes at all mentioned times
after treatment. The L3H mRNA level increased
whithin 12 h of GA3 elicitor treatment (5.2 fold higher
than that in the control) while at 24 and 72 h expression
changes were not remarkable. The transcript levels of
MNMR gene decreased to below the control at 12 h
(approximately 1.4 times lower than those in the con-
trol) as well as the expression of MMR notably reduced
(3 fold lower than those in the control) during 24 h fol-
lowing GA3 application (Fig. 4).
A different gene expression pattern in plants
exposed to MeJA was found. Transcript level of the
majority of monoterpenes biosynthetic pathway genes
gradually increased and reached the highest level at 72
h under MeJA treatment. Among them, mRNA level
of GDS, LS, L3H, PR and MFS genes were signifi-
cantly increased at 72 h after treatment (Fig. 5).

M
28S
200 bp
18S
1 2 3 4 5 6 7 8 9

(a) (b) 100 bp

Fig. 2. (a) – RNA samples on a 1% agarose gel. (b) – The amplified cDNA using specific primers of 1—GDS, 2—LS, 3—L3H,
4—IDR, 5—PR, 6—MFS, 7—MNMR, 8—MMR, 9—actin genes and M—size marker (1000 bp).

RUSSIAN JOURNAL OF PLANT PHYSIOLOGY Vol. 64 No. 1 2017

 RELATIVE EXPRESSION OF GENES OF MENTHOL BIOSYNTHESIS PATHWAY 63

17 12 h

Expression rate compared to control

CHT **
24 h
12
72 h
7 * **
ns ns ns ns ns
2 ns ns ns ns ns ns ns ns ns

–3 *
ns **
–8 * **
**
–13 *
GDS LS L3H IDR MFS PR MNMR MMR

Fig. 3. The relative expression rate of LS, GDS, L3H, IDR, MFS, PR, MNMR and MMR genes at 12, 24 and 72 h after applying
CHT treatment in M. piperita. **, * and “ns” indicate significant differences respectively at (P < 0.01), (P < 0.05) and non-sig-
nificant differences between control and CHT treated plants.

8 12 h
Expression rate compared to control

GA3
* 24 h
6 ns 72 h
4
ns
ns ns
2 ns ns ns ns ns
ns ns ns ns ns ns ns ns
0

–2 ns *
ns
ns
–4 ** ns *
GDS LS L3H IDR MFS PR MNMR MMR

Fig. 4. The relative expression rate of LS, GDS, L3H, IDR, MFS, PR, MNMR and MMR genes at 12, 24 and 72 h after applying
GA3 treatment in M. piperita. * and “ns” indicate significant differences respectively at (P < 0.05) and non-significant differences
between control and GA3 treated plants.

DISCUSSION activity was parallel with the reduction in chlorophyll

and carotenoid contents by GA3 application. Further-
Decreasing of GDS and LS genes transcript in
CHT treatment could possibly be effective in reducing more, the number and percentage of mono and ses-
of limonene level. It has been shown that time–course quiterpens – derived MEP pathway declined in
elicitation of CHT in basil did not lead to induce treated plants. This findings tightly has confirmed new
monoterpene limonene [13]. On the other hand, it insights into limiting the role of the MEP pathway in
seems that an increase in MMR transcription level at the synthesis of plastidic isoprenoids by exogenous
72 h under these treatments had a positive role in application of GA3 [21]. The current study also
increasing the amount of menthol. demonstrated that transcript accumulation for the
respective enzymes of early pathway steps remained
A regulatory role of DXS (catalyzing the first reac-
tion of the MEP pathway) in controlling the synthesis unaffected under GA3 treatment. One other interest-
of MEP-derived isoprenoids came from the analysis of ing result is elucidated that expression levels of genes
transgenic plants in which DXS was up-regulated. in later stages of oil biosynthesis, including MNMR
A distinct positive correlation between DXS transcript and MMR was down-regulated by GA3 treatment. This
levels and the synthesis of plastidic isoprenoids has result is consistent with finding in former study, which
been reported in Arabidopsis [19] and in M. piperita observed no substantial alterations in transcript levels
[20]. In vegetative cannabis plants, reduction in DXS of genes dedicated to the early steps of oil biosynthesis

RUSSIAN JOURNAL OF PLANT PHYSIOLOGY Vol. 64 No. 1 2017

64 SOLEYMANI et al.

10 12 h

Expression rate compared to control

MeJA * 24 h
8
72 h
6 ** *
4 ** ns
*
ns ns ns
2 ns ns ns ns
ns ns
ns ns ns ns
0

–2
ns ns ns
–4 *
*
–6 GDS LS L3H IDR MFS PR MNMR MMR

Fig. 5. The relative expression rate of LS, GDS, L3H, IDR, MFS, PR, MNMR and MMR genes at 12, 24 and 72 h after applying
MeJA treatment in Mentha piperita. **, * and “ns” indicate significant differences respectively at (P < 0.01), (P < 0.05) and non-
significant differences between control and MeJA treated plants.

(GDS, L3H, LS) in response to GA3 in M. arvensis genes mentioned above in M. piperita by extending the
[22]. A previous study also has reported the effect of elicitation period more than 72 h. This result is consis-
GA3 on the stimulation of trichome formation and tent with finding the former study, which observed
increases in its density and diameter on M. arvensis MeJA – elicited plants accumulated the highest levels
eventually resulted in increase oil yield [22]. Since the of terpenoid production in the longest time period
biosynthesis of gibberellins are derived from MEP after initiation of treatment in basil leaves [13].
pathway, Most of genes involved in the formation of Also in this study, an opposite trend was observed
bioactive GA, down-regulated by applying GA. Exist- in mRNA expression levels for PR and MFS genes only
ing evidences confirmed that GA biosynthesis is regu- at 12 and 24 h after treatment with CHT and MeJA
lated by feedback control [23]. This self-regulation While simultaneous changes were obseved in expres-
mechanism may interfere with biosynthesis of mono- sion of these genes at 72 h. A slight up-regulation of PR
terpens and other isoprenoids derived from MEP led to strong declining of transcript level of MFS gene
pathway. However, further investigations required to (24 h after CHT and MeJA treatments). In contrast,
understand the role of GA3 negative feedback mecha- PR expression was down-regulated when the tran-
nism in menthol biosynthesis. script levels of MFS prominently increased (12 h after
CHT treatment), which is in accordance with findings
Under MeJA treatment, genes involved in the reported by Mahmoud and Croteau [27]. Indeed, in
menthol biosynthesis pathway were transcriptionally this anticipated observation, menthofuran affected
activated in a coordinated fashion that seems tran- expression of pulegone reductase by acting as a week
scriptional regulation of these genes was controlled by competitive repressor of pulegone reductase. Trans-
a common transcription factor. Ma et al. [24] has genic plants overexpressing MFS or plants exposed to
characterized transcription factors that involved in abiotic stresses rose up the concentration of mentho-
artemisinin biosynthesis. Increased accumulation of furan. High levels of menthofuran confine expression
artemisinin in Artemisia plants by jasmonate was of plugone reductase to inhibit conversion to menthon
mediated by two AP2 family transcription factors [25]. and ultimately menthol [4, 28]. Conversely, decreased
It is noteworthy that genes encoding enzymes of JA menthofuran levels resulting from co-suppression of
biosynthesis were up-regulated by exogenous applica- MFS, up-regulate PR transcript level or enhance its
tion of MeJA [26]. Besides, mRNA expressions of mRNA stability with the consequence of reduction of
transcription factor genes implicated in regulation of pulegone content in peppermint oil. Consequently,
JA biosynthesis, simultaneously were enhanced after reducing oil levels of pulegone and menthofuran is
MeJA treatment [26]. This positive feedback regula- occurred by this single alteration and lead to increase
tory system for JA biosynthesis has probably led to rise oil yields and quality [27].
in transcript abundance of genes involved in monoter-
pene biosynthetic pathway. Interestingly, in current Although we quantified only the transcript abun-
research, a remarkable increase in transcript abun- dance of genes involved in monoterpenes-specific
dance of most genes involved in menthol biosynthesis enzymatic reactions in response to time-course elici-
initiated at 72 h after elicitation with MeJA, therefore, tation of CHT, GA3 and MeJA individually, further
it could be possible to enhance expression levels of the studies are required to shed more light on factors that

RUSSIAN JOURNAL OF PLANT PHYSIOLOGY Vol. 64 No. 1 2017

 RELATIVE EXPRESSION OF GENES OF MENTHOL BIOSYNTHESIS PATHWAY
regulate the composition of peppermint essential oils.
Catalytic properties of enzymes involved in monoter-
penes biosynthesis and metabolites constituents in
distilled oil should be compared in such elicitor-
treated plants coincidence with changes in relevant
transcript abundance. Recently, all efforts to engineer
glandular trichomes biology for enhanced essential oil
accumulation have shifted to identifying glandular tri-
chomes-specific promoters [29] and transcription fac-
tors that affect the initiation and development of such
specialized secretory structures [30]. More profound
studies on genes affecting trichome formation and
development could be the next step for a further
improvement yield of this valuable therapeutic agent.
ACKNOWLEDGMENTS
The authors would like to acknowledge the central
laboratory of Ramin Agriculture & Natural Resources
University of Khouzestan for providing the necessary
laboratory facilities.
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