Volatile terpenoids: multiple functions, biosynthesis, modulation and manipulation by genetic engineering

Abstract

Terpenoids are structurally diverse and the most abundant
plant secondary metabolites, playing an important role in
plant life through direct and indirect plant defenses, by
attracting pollinators and through different interactions
between the plants and their environment. Terpenoids arealso signifcant because of their enormous applications in
the pharmaceutical, food and cosmetics industries. Due to
their broad distribution and functional versatility, eforts
are being made to decode the biosynthetic pathways and
comprehend the regulatory mechanisms of terpenoids. This
review summarizes the recent advances in biosynthetic
pathways, including the spatiotemporal, transcriptional and
post-transcriptional regulatory mechanisms. Moreover, we
discuss the multiple functions of the terpene synthase genes
(TPS), their interaction with the surrounding environment
and the use of genetic engineering for terpenoid production
in model plants. Here, we also provide an overview of the
signifcance of terpenoid metabolic engineering in crop pro -tection, plant reproduction and plant metabolic engineering
approaches for pharmaceutical terpenoids production and
future scenarios in agriculture, which call for sustainable
production platforms by improving different plant traits.

Full text

Vol.:(0123456789)
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Planta
DOI 10.1007/s00425-017-2749-x
REVIEW
Volatile terpenoids: multiple functions, biosynthesis, modulation
andmanipulation bygenetic engineering
FarhatAbbas1· YanguoKe1· RangcaiYu2· YuechongYue1· SikandarAmanullah3·
MuhammadMuzammilJahangir4· YanpingFan1,5
Received: 20 April 2017 / Accepted: 22 July 2017
© Springer-Verlag GmbH Germany 2017
also significant because of their enormous applications in
the pharmaceutical, food and cosmetics industries. Due to
their broad distribution and functional versatility, efforts
are being made to decode the biosynthetic pathways and
comprehend the regulatory mechanisms of terpenoids. This
review summarizes the recent advances in biosynthetic
pathways, including the spatiotemporal, transcriptional and
post-transcriptional regulatory mechanisms. Moreover, we
discuss the multiple functions of the terpene synthase genes
(TPS), their interaction with the surrounding environment
and the use of genetic engineering for terpenoid production
in model plants. Here, we also provide an overview of the
significance of terpenoid metabolic engineering in crop pro-
tection, plant reproduction and plant metabolic engineering
approaches for pharmaceutical terpenoids production and
future scenarios in agriculture, which call for sustainable
production platforms by improving different plant traits.
Keywords Volatile terpenoids· Multifunction·
Biosynthesis· Plant defense· Pollinator attractions·
Regulation· Genetic engineering
Introduction
Terpenoids are structurally diverse and the most abundant
group of floral volatiles, encompassing more than 40,000
individual compounds (Buckingham 2004; Muhlemann etal.
2014). A majority of these compounds are of plant origin
and have several biological functions in higher plants. Ter-
penoids are found in most plant species and are essential for
plant growth and development. They are the key components
of membrane structures (sterols C30), function as photosyn-
thetic pigments (carotenoids C40); abscisic acid (C15) and
gibberellins (C20) are phytohormones, and ubiquinones are
Abstract
Main conclusion Terpenoids play several physiological
and ecological functions in plant life through direct and
indirect plant defenses and also in human society because
of their enormous applications in the pharmaceutical,
food and cosmetics industries. Through the aid of genetic
engineering its role can by magnified to broad spectrum
by improving genetic ability of crop plants, enhancing
the aroma quality of fruits and flowers and the produc‑
tion of pharmaceutical terpenoids contents in medicinal
plants.
Terpenoids are structurally diverse and the most abundant
plant secondary metabolites, playing an important role in
plant life through direct and indirect plant defenses, by
attracting pollinators and through different interactions
between the plants and their environment. Terpenoids are
* Rangcai Yu
rcyu@scau.edu.cn
* Yanping Fan
fanyanping@scau.edu.cn
1 The Research Center forOrnamental Plants, College
ofForestry andLandscape Architecture, South China
Agricultural University, Guangzhou510642, China
2 College ofLife Sciences, South China Agricultural
University, Guangzhou510642, China
3 College ofHorticulture andLandscape Architecture,
Northeast Agricultural University, Harbin, Heilongjiang,
China
4 Institute ofHorticultural Sciences, University ofAgriculture,
Faisalabad, Pakistan
5 Guangdong Key Laboratory forInnovative Development
andUtilization ofForest Plant Germplasm, South China
Agricultural University, Guangzhou510642, China

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involved in mitochondrial electron transport. Many terpe-
noids (including mono-, sesqui- and diterpenes) are known
to be plant secondary metabolites which play fundamental
roles in plant–environment and plant-plant interactions (Yu
and Utsumi 2009; Dudareva etal. 2013).
Volatile terpenoids (isoprenes, monoterpenes and sesquit-
erpenes) constitute the largest class of plant volatile com-
pounds. They exhibit several carbon skeletons and extremely
variable in chemical structure yet share a common feature
of biosynthesis, which occurs in almost all plant organs,
including the roots, stems, leaves, fruits and seeds, but their
highest amounts are released predominantly from flowers
(Dudareva etal. 2013). Floral volatiles are lipophilic liquids
in nature, having high vapor pressure and low molecular
weight at ambient temperatures. These properties allow them
to freely pass through the cellular membranes for release
into the adjacent environment (Pichersky etal. 2006).
Among all terpenoids, mono- and sesquiterpenes are the
most commonly studied classes because of their extensive
distribution in the plant kingdom and their essential roles
in both human society and plants. Therefore, we will focus
more on these two terpene classes in this review article.
Terpenoids are synthesized from two inter-convertible C5
units: isopentenyl diphosphate (IPP) and its allelic isomer
dimethylallyl diphosphate (DMAPP). These five-carbon
units serve as substrates/precursors for the biosynthesis of
terpenoids. Based on their biosynthetic origin, floral volatile
organic compounds can be divided into three major classes:
terpenoids, benzenoids/phenylpropanoids and derivatives
of fatty acids, wherein terpenes constitute 55% of the plant
secondary metabolites, alkaloids 27% and phenolics 18%
(Muhlemann etal. 2014).
Terpenes play diverse roles in beneficial interactions
and in mediating antagonists among organisms (Das etal.
2013). They protect many plant species against pathogens,
predators and competitors (Hijaz etal. 2016). Furthermore,
herbivore-induced monoterpenoids act as airborne signals
to nearby plants in response to insect attack such as (E)-
β-ocimene (Arimura etal. 2004). Although terpenes are
mostly studied in the above-ground tissues, recently, their
novel function in the below-ground environment, as sign-
aling molecules, has been identified (Karban etal. 2014;
Delory etal. 2016). For example, β-caryophyllene, released
from the roots of maize plants against beetle (Diabrotica
virgifera) attack, acts as a volatile signal to attract predatory
nematodes, which will defend plants indirectly from further
damage (Rasmann etal. 2005). Similarly, the Hedychium
coronarium farnesyl pyrophosphate synthase gene shows a
quick response to herbivory and wounding and is involved
in floral biosynthesis (Lan etal. 2013). Terpenoids are also
beneficial for human beings. For example, limonene is exten-
sively used as a scent compound in cosmetic products (Brokl
etal. 2013). The potential anticancer and anti-inflammatory
compound zerumbone from shampoo ginger is a subject of
interest in pharmacological studies (Bertea etal. 2005; Yu
and Utsumi 2009).
Terpenoids are synthesized from two independent but
compartmentally separated pathways: the mevalonic acid
(MVA) and methylerythritol phosphate (MEP) pathways.
The MEP pathway is mainly responsible for the biosynthesis
of mono- and diterpenes, producing approximately 53 and
1% of the total floral terpenoids, respectively. Sesquiterpe-
nes are synthesized from the MVA pathway, contributing
approximately 28% of the total floral terpenoids (Muhle-
mann etal. 2014), as shown in Fig.1.
The commercial and ecological importance of terpenoids
has inspired rapid progress in floral scent engineering for
terpenoid production in model plants. The identification of
TPS genes, decoding the biosynthetic pathways and enzymes
involved in these pathways, has made the manipulation of
genetic engineering in plants extremely feasible (Dudareva
and Pichersky 2008; Yu and Utsumi 2009). Genetic engi-
neering can improve numerous input and output charac-
teristics in crops, including weed control through allelopa-
thy, pest resistance, increases in the aroma production of
fruits and vegetables (by altering the floral scent) and the
production of medicinal compounds (Aharoni etal. 2005;
Dudareva etal. 2013). Furthermore, model plants (Arabi-
dopsis, Tobacco) with altered terpenoid profiles can provide
useful information for exploring biosynthesis, regulatory
compounds and their ecological importance in plant–envi-
ronment interactions.
In this review, we emphasize the recent advances in
understanding the molecular mechanism of the biosynthetic
pathways and its regulation, function and manipulation of
genetic engineering of terpenoid production in model plants.
Here, we will focus on mono- and sesquiterpenoids.
Fig. 1 Pie chart showing the approximate constituents of the total
floral terpenoids—mono-, sesqui-, di- and other terpenes (like isopre-
noids hemi, tri-, and tetraterpenes). Monoterpenes dominate over half
of the pie chart, being operative in plastids and showing wide diver-
sity, while sesquiterpenes compose 1/3 of the pie chart and are dis-
tributed in the cytosol, endoplasmic reticulum and peroxisomes

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Multifunctionality ofvolatile terpenoids
Terpenoid metabolites are involved in various physiological
and ecological functions based on the differential expres-
sion profiles of terpene synthase genes found in response
to biotic and abiotic environmental factors throughout plant
development. As the largest class of natural plant products,
terpenoids possess different functions in mediating benefi-
cial and antagonistic interactions among organisms. They
protect many plant species, animals, and microorganisms
against pathogens, competitors and predators by transmitting
messages to mutualists and conspecifics regarding enemies,
the presence of food and mates (Gershenzon and Dudareva
2007). Terpenoids are also involved in the plant–plant
interaction, repelling pests and attracting enemies of pests
(Unsicker etal. 2009). Volatile terpenoids are involved
especially in attracting pollinators, seed dispersal, defense
against herbivores from both below- and above-ground (Ali
etal. 2012; Delory etal. 2016), plant–plant signaling and
protection against pathogens (Huang etal. 2012) (Fig.2).
Terpenoids asattraction forpollinators
Flowers employ various palettes for mediating the attrac-
tion of pollinators to ensure successful reproduction.
For pollinators, multisensory inputs (thermal, olfactory,
electromagnetic, visual) are necessary for locating breed-
ing sites and food. Many studies showed the role of plant
terpenoids in communication between plants and pollinators
(Baldwin etal. 2006). Terpenoids are a major cue for attract-
ing pollinators (animals, insects, mammals, birds and bats),
serving as vectors in pollen transfer (Abrol 2012; Farré-
Armengol etal. 2015) and many other miscellaneous func-
tions in plant biology and ecology (Degenhardt etal. 2009).
Floral scent is an important source of communication
between pollinators and flowering plants for their evolution,
particularly during long range communication (Dudareva
and Pichersky 2000; Farré-Armengol etal. 2013). It has
been proven that the information sent by the floral volatiles
depends on eliciting a distinct behavioral response and the
context and composition of their emission toward the respec-
tive pollinators. Long distance floral scent emission mostly
contributes to guiding pollinators to flowers, especially for
night-emitting plants, for which the production of scent
intensity is high to prevail over the low conspicuousness
of flowers under low illumination. For example, Sagittaria
latifolia and Petunia axillaris, pollinated by moths, emit
larger amounts of volatile terpenoids than day-emitting bee-
pollinated plants in the same genus, such as Silene dioica
and Petunia integrifolia (Waelti etal. 2008; Dudareva etal.
2013). Potential pollinators quickly identify and locate the
scented flowers, promoting association between pollinators
Fig. 2 A summary of volatile-
mediated interactions between
plants and their surrounding
environment. Plant–animal
interactions comprise the
attraction of seed dissemina-
tors and pollinators by fruit and
floral volatiles, the repellence/
attraction of herbivores and the
attraction of natural enemies of
aggressive herbivores in both
the rhizosphere and atmosphere.
The above-ground interactions
include priming or elicitation of
the defense responses of nearby
unattacked plants through
the leaves of the same plant.
Moreover, the below-ground
interactions consist of allelo-
pathic activity on the germina-
tion, growth and development of
competitive nearby plants. Ter-
penoid volatiles emitted from
roots and reproductive organs
also possess antimicrobial
activity, hence protecting the
plants against pathogen attack.
Furthermore, leaf volatiles (iso-
prenes) confer thermotolerance
and photoprotection

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and plants via individual ratios of general compounds or spe-
cial compounds (Wright and Schiestl 2009). Terpenoid vola-
tiles are emitted not only by petals but also as pollen odors,
such as β-ocimene, a universal attractant emitted by plants
that attracts long range pollinators (Knudsen etal. 2006),
such as bees, moths, butterflies and beetles (Muhlemann
etal. 2006; Okamoto etal. 2007), thus benefitting pollinator
foraging. They also have been found to attract bumblebees
and honey bees (Granero etal. 2005). Volatile terpenoids
attract certain pollinators by releasing pheromones; for
example, orchids employ floral scents that mimic a blend
of pheromones from female pollinators, triggering copu-
lation attempts by male pollinators with flowers (Bohman
etal. 2014). Tomato flowers in glasshouses mainly produce
four monoterpenes (p-cymene, α-pinene, (+)-2-carene, and
β-phellandrene); all of these are recognized as herbivore-
induced leaf volatiles, possessing toxic properties and func-
tioning in plant defense. The emission of the compounds
mentioned above is negatively correlated with bumblebee
visitation. Floral scents represent characteristic attributes of
plants pollinated via beetles and necrophagous flies, prov-
ing a clear link between specific floral scent chemistry and
certain pollinator guilds. The complex chemistry between
floral volatiles and pollinators is yet to be unraveled. The
increase in annual temperature is a potential threat to pol-
linators that affects their life cycle and flower visitation. The
excessive use of pesticides in field crops is a grave threat to
potential pollinators, which should be alleviated to improve
crop production.
Terpenoid volatiles: animmediate response inplant
defense
The plant kingdom shows two types of responses (direct
and indirect defense mechanisms) upon contact with herbi-
vores and pathogen and rodent attacks. The direct mode of
defense includes several physical structures such as thorns,
trichomes and accumulation of chemicals (phytochemicals)
having antibiotic activities. In most plant species, sesquit-
erpenes and diterpenes act as phytoalexins (Mumm etal.
2008). For example, in Gossypium species, gossypol and
its associated sesquiterpene aldehydes, derived from the
(+)-δ-cadinene precursor, provide inducible and constitu-
tive protection against pests and diseases (Townsend etal.
2005). Fourteen diterpene phytoalexins have been discov-
ered from Oryza sativa and divided into four types based
on structural distinctions: phytocassanes A–E, oryzalexin
S, momilactones A and B, and oryzalexins A–F. Numerous
volatile terpenoid compounds possess antifungal and antimi-
crobial activities invitro (Bakkali etal. 2008; Johnson and
Gilbert 2015), explaining the tissue-specific expression pat-
terns of their biosynthetic genes (Chen etal. 2004). Moreo-
ver, only some of them have been characterized due to their
role in defense against pathogens (Rosenkranz and Schnit-
zler 2016). (E)-β-Caryophyllene emitted from the stigma of
Arabidopsis flowers limited bacterial growth, while plants
lacking the secretion of this sesquiterpene showed a dense
population of bacteria on the stigma of the flower, result-
ing in reduced seed weight compared with the wild-type
plant. Huang etal. (2012) proved that (E)-β-caryophyllene
is important for plant fitness and functions in defense against
pathogenic bacteria.
The phenomenon “indirect defense response” refers to
the plants’ characteristics, which protect them against her-
bivore attacks by promoting the efficacy of herbivore’s natu-
ral enemies (Dicke and Baldwin 2010). This may include
releasing a blend of specific floral volatile scents that attract
predators of herbivores after being attacked (Kessler 2010;
Peñuelas etal. 2014)). In the model plant Arabidopsis,
females of parasitoids Cotesia marginiventris use TPS10
(a sesquiterpene) to track their lepidopteran host by utiliz-
ing the floral scent (Schnee etal. 2006). Arabidopsis has
been shown to emit two terpenoids, (3,S)-(E)-nerolidol and
its derivative (E)-4,8-dimethyl-1,3,7-nonatriene, when the
strawberry nerolidol synthase gene was introduced into
the plants, resulting in greater attraction of the predators
of predatory mites (Kappers etal. 2005). Caryophyllene,
emitted from the roots of Maize plants, is known to be an
herbivore-induced below-ground signal, which strongly
attracts entomopathogenic nematodes (Rasmann etal. 2005;
Delory etal. 2016). Similarly, Ozawa etal. (2000) demon-
strated that (Lotus japonicus) shoots infested with spider
mites released a blend of volatiles that attracted predators
of mites (Phytoseiulus persimilis). Terpenoids released from
Nicotiana attenuata affect the expression of several genes of
the adjacent conspecifics. Terpenoids released from the air
also play a significant role in plant defense against biotic and
abiotic stresses (Paschold etal. 2006; Unsicker etal. 2009;
Dudareva etal. 2013).
Plant terpenes emission could also be an internal signal
for plants to indicate the presence of herbivores and allow
the initiation of defense in neighboring tissues (Rosenk-
ranz and Schnitzler 2016). In the case of herbivore damage,
significant amounts of volatile terpene blends are emitted
from lima bean leaves to stimulate the adjacent leaves to
boost nectar secretion to attract herbivore enemies (Heil and
Bueno 2007). Developments in chemical ecology increas-
ingly confirm terpenoids’ defensive role. The above-men-
tioned examples strongly support the floral volatile terpe-
noids’ involvement in deterring unwanted floral visitors.
Many TPS genes from various crops have been identified
that showed defensive roles against herbivore and pathogen
attacks, but there is a need to explore them further. Poten-
tial TPS genes and their defensive roles can be enhanced
with the help of systems biology. Genetic engineering com-
bined with biotechnology and plant breeding is a valuable

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approach for enhancing the defensive role of TPS genes in
other crops that show less resistance toward pathogen and
herbivore attacks.
Volatile terpenoids: impact ontheagroecosystem
Volatile terpenoids have several important applications
in human society; the food and pharmaceutical industries
exploit their effectiveness and potential as flavor enhanc-
ers and medicines (Rosenkranz and Schnitzler 2016). The
most extensively used terpene by human beings is rubber, a
polyterpene composed of repeated isoprene subunits. Simi-
larly, terpenes fine important use as methanol, cleaners,
solvents, camphor, antiallergenic agents, pyrethrins (insec-
ticides), limonene, rosin, nepetalactone (in catnip), digitoxi-
genin, carvone and hecogenin (a detergent) (Croteau etal.
2000; Thimmappa etal. 2014). The antimicrobial activity
of terpenoids is the most significant because of the dras-
tic increase in bacterial resistance to antibiotics, which is a
growing cause of concern globally (Islam etal. 2003; Singh
and Sharma 2015). In livestock, the addition of terpenes
can replace conventional antibiotics, as they can slow down
the resistance of bacteria against antibiotics. Plant oils that
included terpenes in their composition showed promising
invivo bactericidal activity. Terpenoids also play key roles
in the clinical industry (Singh and Sharma 2015). Prabu-
seenivasan etal. (2006) proved that cinnamon oil showed
a broad range of activity against Pseudomonas aeruginosa.
Moreover, the terpene composition can vary markedly
among different species. Neolitsea foliosa (Nees) plant oil
contains caryophyllene (sesquiterpene), which is responsible
for its antibacterial properties (John etal. 2007). Terpenoids
have also been used in antibacterial soaps, household prod-
ucts and cosmetics due to their lipid organizational proper-
ties (Caputi and Aprea 2011).
The impact of terpenes on both nature and human appli-
cations is complicated; however, these compounds have a
significant impact on the agroecosystem and maintenance of
bio-diversity. For example, blueberry, apple, cucurbits and
canola are valuable to varying degrees from the insect pol-
linator point of view (Losey and Vaughan 2006; Tholl 2015)
and for self-incompatible dioecious crops. Floral scents
have also been unintentionally used to retain a few pollina-
tor attracting traits as a baseline, without which the crop
could not bear fruits. Jabalpurwala etal. (2009) observed
that self-incompatible Citrus grandis (pummelo) blossoms
emit larger quantities of volatile organic compounds than
other species such as lemon, orange and grapefruit flowers,
which comprise both female and male organs. Few experi-
mental studies have been conducted to evaluate the effects
of floral scents on crop pollination. For this purpose, bee
visitation was compared between Bacillus thuringiensis
(Bt) and wild-type (Bt)-expressing eggplant (Dudareva etal.
2013). Commercial bumblebees were strongly attracted by
Bt-eggplant instead of the wild-type in a glasshouse environ-
ment (Arpaia etal. 2011). The relationship between failure
in crop pollination and the composition of floral volatile
organic compounds is best demonstrated by alfalfa seed
production, which depends on pollination through honey-
bees that consistently showed low fidelity to alfalfa flow-
ers compared with other weedy plants and crops. To show
the link between crops and volatile terpenoid chemistry,
Morse etal. (2012) studied tomato production in a glass-
house, where imported bumblebees were regularly used as
pollinators, which showed a preference for other forage and
flowering plants outside of the glasshouse. The behavior of
bees toward tomato plants was attributed to the emission of
four monoterpenes (α-pinene, β-phellandrene, (+)-2-carene,
and p-cymene), which possessed toxic properties and were
useful in plant defense. Terpenoids are of utmost importance
in crop protection, plant reproduction, and future scenarios
in agriculture involving sustainable production platforms.
Terpenoids may have other mysterious functions in plants
that remain to be revealed.
Biosynthesis andsubcellular compartmentation
ofterpenoids inplants
Terpenoids are the largest class of plant floral volatiles,
encompassing 556 scent compounds. For the biosynthesis
of most terpenoids in plants, the simple five carbon unit
IPP and its allylic isomer DMAPP serve as the initial sub-
strate. IPP is the most frequent precursor of all terpenoids,
synthesized by two compartmentally separated and inde-
pendent pathways, the MVA and MEP pathways (Chen
etal. 2011). The MVA pathway comprises six enzymatic
reactions, carried out through step-by-step condensation of
three molecules of acetyl CoA, which then undergo reduc-
tion to the MVA pathway followed by two subsequent steps
of phosphorylation and decarboxylation, forming IPP as the
ultimate product (Lange etal. 2000; Tholl 2015). Similarly,
the MEP pathway involves seven enzymatic steps and begins
with the condensation of pyruvate and glyceraldehydes-
3-phosphate (G3P) (Nagegowda 2010; Muhlemann etal.
2014). IPP isomerizes to form DMAPP, which serves as a
substrate for hemiterpene biosynthesis or combines with one
IPP unit to produce geranyl diphosphate (GPP), catalyzed by
the GPP synthases (GPS). The condensation of one IPP with
one GPP molecule produces farnesyl pyrophosphate (FPP),
which is catalyzed in the presence of FPP synthases (FPS).
Similarly, GPP and FPP are precursors for the biosynthesis
of monoterpenes and sesquiterpenes, respectively (Vranová
etal. 2012), whereas prenyl diphosphates in the trans-con-
figuration are assumed to be the ubiquitous naturally occur-
ring substrates for terpene synthases. In wild-type tomato

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trichomes, Z, Z-FPP and two prenyl diphosphates, in the
cis-configuration, are also naturally occurring substrates for
the synthesis of sesquiterpenes catalyzed by FPP synthase
(zFPS). Similarly, iso-, mono- and sesquiterpenes synthases
convert DMAPP, GPP and FPP (or Z, Z-FPP) to isoprene,
monoterpenes and sesquiterpenes, respectively. Based on
their biosynthetic origin, all volatile organic compounds can
be divided into numerous classes, while several floral scent
volatiles are constituents of the terpenoid, phenylpropanoid/
benzenoid or fatty acid derivative groups of compounds, and
each volatile group is recognized by a few compounds that
produce a typical scent (Muhlemann etal. 2014).
Two different and independent pathways are involved
in the formation of five carbon isoprene building units, the
plastidial MEP and cytosolic MVA pathways; however,
the MEP pathway is also disseminated in the endoplasmic
reticulum and peroxisomes (Mahmoud and Croteau 2002;
Dudareva and Pichersky 2008; Lange and Ahkami 2013),
whereas these compartmentally separated isoprenoid bio-
synthetic pathways are interconnected by a metabolic “cross-
talk” (Flügge and Gao 2005; Orlova etal. 2009). Meva-
lonate-5-diphosphate is produced in Arabidopsis thaliana by
the MVA pathway through phosphorylation, and the entire
reaction is catalyzed by mevalonate kinase and phosphom-
evalonate kinase (Lluch etal. 2000; Yu and Utsumi 2009).
The MVA pathway in the peppermint glandular trichomes
is blocked at 3-hydroxy-3-methylglutaryl-CoA reductase,
which is an enzyme that controls the rate of the MVA path-
way as well as both mono- and sesquiterpenes biosynthesis
entirely based on the plastidial IPP. In the tobacco suspen-
sion cell line BY-2, the crosstalk between the MEP and
MVA pathways has been verified (Hemmerlin etal. 2003;
Dudareva etal. 2013), although the specific intermediate
has not yet been identified. Studies on snapdragon flowers
also indicate that the MEP pathway supplies IPP for the bio-
synthesis of monoterpenes and sesquiterpenes. Furthermore,
the trafficking of IPP occurs unidirectionally from the plas-
tid to the cytosol (Dudareva etal. 2005; Nagegowda 2010).
Moreover, both pathways co-operate with each other for a
while and provide the IPP precursor for the biosynthesis
of terpenoids as mentioned above. The chamomile sesquit-
erpenes are an adequate example showing clear metabolic
crosstalk between the two pathways in which both provide
IPP precursors for the production of certain terpenoids that
consist of 2 five-carbon isoprene units, which are derivatives
of the MEP pathway, while the third unit is formed by both
pathways (Towler and Weathers 2007). In the plastids, the
MEP pathway (Hsieh etal. 2008) results in the formation
of IPP and DMAPP from G3P and pyruvate. However, the
classical independent MVA pathway (Vranová etal. 2013)
is disseminated in the cytosol, peroxisomes and endoplasmic
reticulum (Pulido etal. 2012). The MVA pathway forms IPP
starting with acetyl-CoA condensation and is responsible for
the formation of most sesquiterpenes (C15), which account
for approximately 28% of all floral terpenoids. In snapdragon
flowers, the MEP pathway alone supports the biosynthesis
of sesquiterpenes (Dudareva etal. 2005), whereas the MVA
and MEP pathways are involved in the formation of ses-
quiterpenes in the roots and leaves of carrots (Hampel etal.
2005). Terpenoid biosynthetic pathways and their intracel-
lular compartmentation in plants are shown below in Fig.3.
The formation of the GPP precursor has been confirmed
to occur in plastids and is responsible for the biosynthesis
of monoterpenes as the final product, while sesquiterpenes
are produced in the endoplasmic reticulum, peroxisomes or
cytosolic compartments, which are also a site for FPP for-
mation. Nevertheless, two unlikely monoterpene synthases
of strawberry that generate several olefinic monoterpenes
and linalool lacked plastid targeting signals and were shown
to be targeted in the cytosolic compartment (Aharoni etal.
2004; Guirimand etal. 2012). Likewise, the overexpression
of the lemon basil α-zingiberene synthase gene in tomato
plants resulted in the production of a monoterpene in the
cytosol (Davidovich-Rikanati etal. 2008). Santalene and
bergamotene synthase (SBS) from tomato contain puta-
tive N-terminal plastid targeting peptides (Schilmiller
etal. 2009). Plant genomes have evolved to encode diverse
FPP synthase isoforms, which are present in the cytosol,
mitochondria, plastids or peroxisomes (Chen etal. 2011;
Thabet etal. 2012). Terpene biosynthesis occurs at specific
stages during plant development and within specific tissues
(Vranová etal. 2012).
Numerous TPS enzyme products, including isoprenes,
monoterpenes, sesquiterpenes and a few diterpenes in spe-
cialized metabolism, are volatile under the normal grow-
ing conditions (environmental and temperature) of plants.
To date, numerous flower-specific terpene synthases have
been characterized and isolated, as they are accountable
for the development of monoterpene linalool (A. thaliana,
Anthirrhinum majus, H. coronarium and Clarkia breweri)
(Nagegowda etal. 2008; Ginglinger etal. 2013; Yue etal.
2014), 1,8-cineole (Citrus unshiu, H. coronarium and Nico-
tiana suaveolens) (Roeder etal. 2007; Li and Fan 2007),
myrcene (Alstroemeria peruviana and A. majus) (Aros etal.
2012), E-(β)-ocimene (A. majus and H. coronarium) (Shi-
mada etal. 2005; Fan etal. 2003, 2007) and the sesquiter-
penes α-farnesene (Actinidia deliciosa and H. coronarium)
(Nieuwenhuizen etal. 2009; Yue etal. 2015), nerolidol (A.
chinensis and A. majus) (Green etal. 2011), valencene (Vitis
vinifera) (Lücker etal. 2004), germacrene D (V. vinifera,
Rosa hybrid and A. deliciosa) (Lücker etal. 2004; Nieu-
wenhuizen etal. 2009) β-ylangene, β-copaene, β-cubebene,
α-bergamotene (Cananga odorata var. fruticosa) (Jin etal.
2015) and β-caryophyllene (Ocimum kilimandscharicum,
Daucus carota) (Yahyaa etal. 2015; Jayaramaiah etal.
2016).

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Modulation ofplant terpenoid biosynthesis
Terpenoids are frequently synthesized and released at par-
ticular times from specific plant tissues. Ample evidence
explains the tempo-spatial TPS expression correlating with
the volatile terpenoid biosynthesis and emission, suggest-
ing that the volatile terpenoid biosynthesis is mostly regu-
lated at the transcriptional and post-transcriptional levels
(Tholl 2015). The regulation of plant terpenoid biosynthe-
sis is complicated and usually divided into two categories
during development, the temporal and spatial regulation, in
response to biotic and abiotic factors such as light intensity,
insect/pathogen damage, nutrients, humidity and tempera-
ture conditions (Van Poecke etal. 2001; Hakola etal. 2006).
Developmental, rhythmic andspatio‑temporal
regulation
The emission of particular volatile compounds into the
atmosphere depends on their rates of biosynthesis and emis-
sion. Numerous studies showed that terpenoid volatiles are
mostly synthesized de novo in a few special physical struc-
tures, such as oil glands (Gershenzon etal. 2000) and resin
ducts (Miller etal. 2005), which accumulate large quanti-
ties of terpenes. Biosynthesis occurs in the epidermal cells
of plant tissues, through which they can easily be released
into the atmosphere/rhizosphere (Kolosova etal. 2001; Chen
etal. 2004; Tholl and Lee 2011). Several conifers accumu-
late significant amounts of scent oleoresins in their spe-
cialized needles, bark blisters or resin ducts and sapwoods
(Zulak and Bohlmann 2010). The biosynthesis and regula-
tion of emission of plant terpenoids are mostly carried out
via petals, although other tissues, such as sepals, stamens,
pistils and nectaries, also contribute to the floral bouquet of
plant species (Farré-Armengol etal. 2013). Sometimes, the
regulation of terpenoids is induced via abiotic stresses, path-
ogens attack or feeding of herbivores (Vranová etal. 2012).
In Arabidopsis, mono- and sesquiterpene synthases are not
expressed in flower petals, and their expression is restricted
to nectaries, sepals, anthers and stigma (Tholl etal. 2005).
In the snapdragon flower, key volatile benzoid compounds,
the monoterpene myrcene, (E)-β-ocimene biosynthesis and
developmental regulation occur in the epidermal layer of
the upper and lower lobes of petals, controlled by the cir-
cadian clock following the diurnal rhythm (Kolosova etal.
2001; Muhlemann etal. 2014). Similarly, H. coronarium
promoters (PrHcTPS1 and PrHcTPS2) are involved in the
temporal and spatial regulation of HcTPS genes associated
with the biosynthesis of terpenoids (Li and Fan 2011; Li
etal. 2014). Likewise, cell-specific expression of the aroma
biosynthetic genes was also reported in C. breweri and
roses (Bergougnoux etal. 2007). In N. suaveolens, several
Fig. 3 An outline showing the
formation of iso-, mono- and
sesquiterpene biosynthetic
pathways in plants catalyzed
by different terpene synthases
and their intra-cellular com-
partmentation. The plastidial
MEP pathway starts with the
condensation of pyruvate and
GA-3P, which undergo different
reactions, producing iso-, mono-
and sesquiterpenes as their final
product. However, the MVA
pathway spans the cytosol,
peroxisomes and endoplasmic
reticulum and starts with the
condensation of acetyl-CoA,
which undergoes different reac-
tions, giving rise to mono- and
sesquiterpenes as the final prod-
ucts. The cross-talk between
the two pathways includes the
exchange of IPP between the
two compartments, the cytosolic
MVA and plastidial MEP
pathways, producing different
terpenes as end products (iso-,
mono- and sesquiterpenes)

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monoterpene nocturnal emissions from the stigma and petals
are an outcome of the transcriptional regulation of 1,8-cin-
eole synthase through the circadian clock (Roeder etal.
2007). Pre-revealed changes in the emission of terpenoids
that follow nocturnal, diurnal and circadian rhythms over the
lifespan of flowers may be associated with the pollination
of insects. In Artemisia annua, β-pinene emission fluctuates
with the day-night rhythm, which is usually higher in the day
compared with night (Lu etal. 2002; Tholl and Lee 2011).
The majority of terpene synthase genes belong to the TPSa
and TPSb sub-families, reaching peak expression accord-
ing to the bundle of accumulation of individual compounds,
whereas in the TPSg sub-family, only the linalool synthase
transcripts were observed during the ripening of berries.
Transcriptional andpost‑transcriptional regulation
During the floral lifespan, the gene expression is transcrip-
tionally regulated by more than one biochemical pathway.
The enzymes proficient in using numerous similar sub-
strates, such as acyltransferase and salicylicacidmethyl-
transferase (SAMT), supplied the precursors that controlled
the product type (Boatright etal. 2004). A relative investi-
gation on the regulation of monoterpenes and benzenoids
emission in snapdragon flowers showed that the orchestrated
emission of isoprenoids and phenylpropanoid compounds
were regulated upstream by individual metabolic pathways
(Muhlemann etal. 2012).
The formation of volatile flower terpenoids through
various independent pathways is not merely dependent on
the biochemical properties of the enzymes involved in the
biosynthesis but also on the contribution of the transcrip-
tional factors (TFs). Until now, only a few TFs had been
identified in the regulation of biosynthetic genes. Recently,
R2R3-type MYB TF, ODORANT1 (ODO1), isolated from
the petunia flower, was highly expressed in petal tissues
and was involved in the regulation of a major portion of
the shikimate pathway (Verdonk etal. 2005; Muhlemann
etal. 2014). EOBII (EMISSION OF BENZENOIDS II),
another R2R3-type MYB TF positively regulates the ODO1
that activates the biosynthetic gene (isoeugenol synthase)
promoter (Colquhoun etal. 2010). Similarly, the MYB4
transcriptional factor was a repressor of one of the enzymes
in the cinnamate-4-hydroxylase, phenylpropanoid pathway
and controlled the flux in petunia flowers (Colquhoun etal.
2011). Similarly, MYC2 activated two sesquiterpene syn-
thase genes (TPS11, TPS21) through the jasmonic acid (JA)
and gibberellic acid signaling pathway in A. thaliana during
inflorescence (Hong etal. 2012). In roses, the up-regulation
of terpenoid pathways occurred via the overexpression of the
PRODUCTION OF ANTHOCYANIN PIGMENT1 (PAP1)
transcriptional factor (Zvi etal. 2012). The enhancement in
the concentration of terpenes in response to different stresses
was usually connected to the increased transcriptional activ-
ity of a particular biosynthetic terpene gene (Nagegowda
2010; Nagegowda etal. 2010; Xi etal. 2012). In Medicago
sativa, pathogen attacks cause metabolic and transcriptional
changes in the plant cells (Suzuki etal. 2005).
Genetic engineering fortheproduction ofplant
terpenoids
Genetic engineering in plants/model plants for terpenoid
production is currently a dynamic research area. Metabolic
engineering offers a gigantic potential for enhancing plant
resistance to pests in agriculture and forestry due to the
abundance and contribution of secondary volatile metabo-
lites in altering the floral scent bouquets, improving fruit
quality, eradicating undesirable compounds and improving
plant defenses (direct and indirect defenses) for exploring,
the effect of changes in the volatile emission upon insect
behavior (Mahmoud and Croteau 2002; Capell and Chris-
tou 2004; Tholl 2015). Recent advances in the identification
of genes and enzymes responsible for volatile compound
biosynthesis have made metabolic engineering dramatically
realistic (Aharoni etal. 2006; Dudareva and Pichersky 2008;
Dudareva etal. 2013). In most studies, the overexpression of
TPS genes is a promising method for tackling these issues
by manipulating terpene formation in transgenic plants. The
first reported transgenic plants with modified monoterpene
profiles were petunia (Petunia hybrida) and mint species
(Mentha spp.) (Krasnyanski etal. 1999; Aharoni etal. 2005).
It has been demonstrated that upon herbivore attack, plants
emit a blend of volatiles, which is composed of more than
200 compounds that are directly responsible for deterring,
intoxicating or repelling herbivorous insects (Seybold etal.
2006). Alternatively, they can attract the parasitoids and nat-
ural predators of offending herbivores, resulting in securing
the plant from damage (Mercke etal. 2004; Degen etal.
2004; Dudareva etal. 2013). The transformation of petunia
with the (S)-limonene synthase gene from C. breweri results
in linalool production that repels aphids (Lücker etal. 2001).
The repellent/attraction properties of various terpenoid vola-
tiles in petunia flower were observed via silencing the bio-
synthetic genes individually responsible for the production
of scent compounds (Kessler etal. 2013). Another example
of floral scent engineering is the introduction of a maize
sesquiterpene synthase gene (TPS10) into Arabidopsis, lead-
ing to the production of a significant amount of various ses-
quiterpenes that are exploited by the female parasitoid wasp
C. marginiventris to navigate to their potential lepidopteran
host (Schnee etal. 2006; Delory etal. 2016). Manipulation
of genetic engineering for sesquiterpenes production seems
a challenging task and is less successful compared with that
for monoterpenes due to lack of suitable precursors.

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The manipulation of terpenoid biosynthesis is an effec-
tive strategy for improving the quality of floral scent, aroma
and flavor of fruits, herbs and vegetables (Pichersky and
Dudareva 2007; Dudareva and Pichersky 2008). The first
attempt to revolutionize the flavor of fruits was made
through genetic engineering of tomato plants by overexpres-
sion of the linalool synthase gene from C. breweri under the
control of the E8 promoter (fruit-specific promoter), which
improved the aroma of fruits that were originally discarded
by humans (Lewinsohn etal. 2001; Nagegowda 2010).
Plant metabolic engineering approaches
forpharmaceutical terpenoids production
A large amount of different terpenoids have been described
from which many of them are separated from different
medicinal plants, like Taxus chinensis, Salvia miltiorrhiza,
A. annua, Panax ginseng and Ginkgo biloba. Terpenoids
isolated from taxol, ginkgolides and artemisinin have great
effects on several diseases, such as paclitaxel derived from
taxol, a diterpenoid derived from Taxus brevifolia, a sig-
nificant anticancer agent. Structurally unique ginkgolides
diterpenoids families are highly particular platelet-activating
factor receptor antagonists (Lu etal. 2016). Similarly nowa-
days, a sesquiterpene (artemisinin) produced from A. annua
is the best therapeutic towards drug resistant and malaria
causing Plasmodium falciparum strains (Weathers etal.
2011). Several plants metabolic engineering approaches
holds a significant pledge to regulate the pharmaceutical
terpenoids biosynthesis based on Agrobacterium-mediated
genetic transformation. Effective strategies to enhance the
production of pharmaceutical terpenoids include the over-
expression of genes involved in the terpenoid biosynthetic
pathways and via suppress the competing metabolic path-
ways. Furthermore, regulating the relative transcription fac-
tors, primary metabolism and endogenous phytohormones
can also considerably boost up their yield. Combination
of all these approaches can help to increase the supply of
limited terpenoid drugs, ultimately minimizing the price of
expansive drugs and promote standards of people’s living.
Gene promoters
Gene promoters can be very useful in the manipulation of
genetic engineering, especially those promoters that drive
gene expression at a particular time and a specific place;
for example, cell types, tissues, and organs are interesting
targets for the genetic engineering of the metabolic pathway
(Benedito and Modolo 2014). To coordinate gene expres-
sion, promoters interact with the RNA polymerase under
certain conditions and are common to plant genetic studies
(e.g., actin, CaMV35S and ubiquitin). Specific promoters for
glandular trichomes are favorable tools for this purpose. For
example, the trichome-specific promoter of the cytochrome
P450 gene (CYP71AV1) from A. annua has been studied and
is a promising tool for genetic engineering of artemisinin
biosynthesis in the whole plant system.
Transcription factors
In organisms, gene expression is regulated via complex
regulatory networks, which are synchronized by transcrip-
tional factors (TFs). TFs are proteins that perform as hubs
or master regulatory genes through repressing or promoting
the RNA polymerase binding to specific promoter regions.
TFs can be used as powerful regulators of plant second-
ary metabolism, as most TFs can initiate the peculiar gene
expression coding for enzymes in biochemical pathways. In
A. annua, the artemisinin biosynthesis is positively regu-
lated via two TFs (AaERF1 and AaERF2) belonging to the
AP2/ERF family (Yu etal. 2012). Nevertheless, there is a
need to determine whether these TFs indirectly regulate the
glandular trichome development, expression of other genes
and membrane transport expression or directly regulate
them via promoting the expression of biosynthesis genes.
Similarly, another TF (AaWRKY1) was shown to activate
the expression of amorpha-4,11-diene synthase (ADS) and
others in the artemisinin pathway (Ma etal. 2017). These
findings also highlight the fact that TFs are dynamic tools
for manipulating the complete transcriptional networks
related to developmental or specific biosynthetic pathways.
This approach will help attain food plants with higher nutri-
tional values for both human and animal consumption. The
potential use of TFs in heterologous systems can boost the
synthesis of important secondary compounds.
Subcellular localization
Localization of genes (TPS) and appropriate prenyl diphos-
phate synthase in mitochondria or plastids is preferable
to cytosolic localization due to the tightening regulation
of cytosolic prenyl diphosphate pools. By introduction of
potent antimalarial drug (artemisinin) in tobacco via two
mega biosynthetic pathways into the nuclear genomes and
chloroplasts, resulted in enhanced level of artemisinin with-
out interrupting plant health. A comprehensive evaluation
was conducted by Wu etal. (2006) to evaluate the effect of
targeting different gene products to cytosol or plastids. The
most appropriate example is the accumulation of amorpha-
4,11-diene (ADS) and FPS in plastids, resulting in approxi-
mately a 5000-fold increase in ADS concentration in model
tobacco plants over another model plant (unchanged ADS
gene) having a cytosolic gene product (Wu etal. 2006).
Likewise, a mitochondrial-localized heterologous TPS gene
was first studied by Kappers etal. (2005), which revealed
that the overexpression of the FaNES1 gene from strawberry

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in the model Arabidopsis plant with an engineered ‘Met’
mitochondrial targeting sequence resulted in two new ter-
penoids involved in attracting predatory mites that poten-
tially aid in defense against herbivores. The potential use of
genetic engineering in the subcellular localization of TPS
genes provides new insights for further investigation.
Host organism selection
The selection of an appropriate host organism for genetic
transformation is important and should be done carefully.
To date, the study of plant metabolic engineering involves
transgenic plants expressing the heterologous gene relevant
for the accumulation of terpenoids using quick transforma-
tion procedures. Nevertheless, the production of engineered
terpenoids in model plants was very low, and the highest
accumulation levels reported were approximately 0.01%
of fresh weight biomass. However, diterpene production in
tobacco glandular trichomes is relatively high; the heterolo-
gous gene expression has not yet resulted in high production
novel diterpenoids. Therefore, high terpenoid accumulation
levels have been found for volatiles emitted from engineered
model plants. If the purpose of genetic engineering is to
adapt to the trophic interaction of plants with respect to
insects, then the terpenoid volatiles released from model
plants can have constructive effects against disease resist-
ance (Kappers etal. 2005; Yu etal. 2012). On the other
hand, volatilization is undesirable if terpenoid collection is
desired, which poses a strategic question for scientists. There
are limitations in genetic engineering for non-model plants
producing terpenoids, the main limitation being the low
efficacy of transformation as well as the lengthy period for
the regeneration of transgenic plants. Metabolic engineering
has been successfully employed in peppermint and lavender
plants to magnify the essential oil production (Lange and
Ahkami 2013).
Volatile terpenoids are widely used in different industries,
as mentioned above, for multiple uses by human beings.
Moreover, genetic manipulation for enhanced terpenoid
production in the future would be beneficial for human
beings. Much more work in this field is needed regarding
the manipulation of genetic engineering in flowering plants.
Genetic engineering techniques can revolutionize the use of
terpenoids in both plants and human beings.
Conclusion andprospects
Genetic engineering has greatly enhanced our understand-
ing of the function, biosynthesis, and regulation of vola-
tile terpenoids in the past two decades. Recent efforts have
demonstrated the viability of improving plant defenses
and enhancing the aroma quality of fruits and flower via
metabolic engineering. The information attained provides
new insights into terpenoid metabolism and will help in the
metabolic engineering of plants for attracting pollinators and
increasing the amount of the valuable products of volatile
terpenoids. Since plant defense mechanisms are beneficial
to agriculture, there is an increasing focus on terpenoids and
phytoalexins, which contribute to plant defense responses
and controlling pests, weeds and pathogens. Recently, plant
terpenoids have been exploited for biofuel development and
its by-products. Strategies in plant metabolic engineering
clutches a promising role to upregulate the pharmaceuti-
cal terpenoids contents in medicinal plants. Pharmaceutical
terpenoids production can be improved greatly via a series
of metabolic regulation in medicinal plants. In future, with
the constant development of metabolic engineering in plants,
more high value pharmaceutical terpenoids will be upregu-
lated. The spatial, temporal and transcriptional regulation of
terpenoid biosynthesis during the development and response
to environmental stresses is correlated to the physiological
or ecological functions, such as repelling herbivores, patho-
gens and attracting pollinators as well as natural enemies
of herbivores. Transcriptional and post transcriptional fac-
tors could also play important roles. Transcriptional factors
include a group or groups of significant regulatory genes,
which can perform key functions in genes that respond to
stress mechanism. The overexpression of TFs can activate
a group of loci, which function in a systematic approach for
reacting to unfavorable environmental conditions. Hence,
genetic engineering of transcriptional factors would be
a powerful technique for improving the genetic ability of
crop plants to address abiotic stresses. Transcriptional fac-
tors also play essential roles in signaling pathways under
temperature stress. On the other hand, the identification of
new TFs and overexpression cannot be neglected. Genomic,
metabolic and proteomic tools and validation of invitro cell
culture systems could be helpful in elucidating the terpenoid
biosynthesis regulatory mechanisms. Genetic engineering
plays fundamental roles in the improvement of crop plants
at every step, from the engineering of new biosynthetic path-
ways to altering the floral scent in scentless crops. Many
aspects of terpenoids in both plants and ecosystem are yet
to be elucidated.
Author contribution statement FA, YK, YY, SA, MMJ
design the manuscript. FA wrote the manuscript and RY
and YF revised the manuscript.
Acknowledgements This work was supported in part by the National
Natural Science Foundation of China to Yanping Fan (Grant nos.
30972026 and 31370694), a Specialized Research Fund for the Doc-
toral Program of Higher Education of China to Yanping Fan (Grant no.
20134404110016), and a Specialized Major Project of the Production-
Study-Research Collaborative Innovation of Guangzhou Science and

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1 3
Information Bureau to Yanping Fan (Grant no. 156100058). We would
like to say thank you to Mr. Yiwei Zhou (College of Forestry and Land-
scape Architecture), Dr. Umair Ashraf and Dr. Rashid Azad (College
of Agriculture) for their help in generating figures.
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