Metabolic Engineering of Plant-Derived (E)-Beta-farnesene Synthase

7/29/2019 Metabolic Engineering of Plant-Derived (E)-Beta-farnesene Synthase

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Journal of Integrative Plant Biology 2012, 54 (5): 282299

Invited Expert Review

Metabolic Engineering of Plant-derived(E)--farnesene Synthase Genes for a Novel Type ofAphid-resistant Genetically Modified Crop Plants F

Xiu-Dao Yu1, John Pickett2, You-Zhi Ma1, Toby Bruce2, Johnathan Napier2, Huw D. Jones2

and Lan-Qin Xia1

1Institute of Crop Sciences (ICS), Chinese Academy of Agricultural Sciences (CAAS), Beijing 100081, China2Rothamsted Research, Harpenden, Hertfordshire AL5 2JQ, UK

Corresponding author

Tel(Fax):+86 10 8210 5804; E-mail: [email protected] Articles can be viewed online without a subscription.

Available online on 20 February 2012 at www.jipb.net and www.wileyonlinelibrary.com/journal/jipb

doi: 10.1111/j.1744-7909.2012.01107.x

Lan-Qin Xia

(Corresponding author)

Abstract

Aphids are major agricultural pests that cause significant yieldlosses of crop plants each year. Excessive dependence on insec-ticides for long-term aphid control is undesirable because of thedevelopment of insecticide resistance, the potential negative effectson non-target organisms and environmental pollution. Transgeniccrops engineered for resistance to aphids via a non-toxic mode ofaction could be an efficient alternative strategy. (E)--Farnesene

(EF) synthases catalyze the formation of EF, which for many pestaphids is the main component of the alarm pheromone involvedin the chemical communication within these species. EF can alsobe synthesized by certain plants but is then normally contaminatedwith inhibitory compounds. Engineering of crop plants capable of

synthesizing and emitting EF could cause repulsion of aphids and also the attraction of natural enemiesthat use EF as a foraging cue, thus minimizing aphid infestation. In this review, the effects of aphids onhost plants, plants defenses against aphid herbivory and the recruitment of natural enemies for aphidcontrol in an agricultural setting are briefly introduced. Furthermore, the plant-derived EF synthasegenes cloned to date along with their potential roles in generating novel aphid resistance via geneticallymodified approaches are discussed.

Keywords: Aphids; (E)--farnesene sythase; genetic-modified crops; terpenoid.

Yu XD, Pickett J, Ma YZ, Bruce T, Napier J, Jones HD, Xia LQ (2012) Metabolic engineering of plant-derived (E)--farnesene synthase genes fora novel type of aphid-resistant genetically modified crop plants. J. Integr. Plant Biol. 54(5), 282299.

Introduction

Aphids (Aphididae) are major agricultural pests that cause

significant yield losses of crop plants each year by inflict-

ing damage both through the direct effects of feeding and

by vectoring harmful plant viruses (International Aphid Ge-

nomics Consortium 2010). Annual worldwide crop losses due

to aphids are estimated at hundreds of millions of dollars

(Blackman and Eastop 1984; Oerke 1994; Morrison and Peairs

1998). Along with the application of nitrogen fertilizer and

elevation of atmospheric CO2 concentration, aphid infestation

becomes more serious (Awmack and Harrington 2000; Aqueel

C 2012 Institute of Botany, Chinese Academy of Sciences


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Engineering of Aphid-Resistant GM Crop Plants 283

and Leather 2011). For many crops, insecticides provide a

simple and effective strategy for aphid control. However, the

application of such chemicals is not desirable in the long

term, because of the development of insecticide resistance

(Sabater-Munoz et al. 2006) and the potential negative effects

on non-target organisms, and the need for more sustainable

agricultural practices with fewer external chemical inputs.

During a long period of co-evolution, plants and aphids

have established a complex interaction. On the one hand,

aphids have established complex life cycles involving extensive

phenotype plasticity with rapid population growth (Blackman

and Eastop 1984), and a very short generation time of as little

as 5 d (Morrison and Peairs 1998). They also have different

morphs: one genotype can generate winged or unwinged

forms depending on environmental stresses. Furthermore, they

can alternate between a parthenogenetic (a form of asexual

reproduction where growth and development of embryos occur

without fertilization of male and female gametes) morph with

asexual live-bearing females and a sexual morph with males

and egg-laying females during different seasons (International

Aphid Genomics Consortium 2010). On the other hand, plants

have also evolved delicate and complicated defense systems

comprising constitutive defense traits and defense pathways

induced uponaphid attack(Chen 2008). Conventional breeding

programs have been undertaken and considerable efforts have

been expended in the search for aphid resistance in small grain

germplasm worldwide (Stoger et al. 1999). However, due to the

complexity of plant-aphid interactions and the rapid develop-

ment of resistant pest biotypes (Stoger et al. 1999), outbreakof aphids causing substantial losses are reported regularly.

Breeders and growers are still struggling to find an efficient

strategy for aphid control in major crop plants. Development of

aphid-resistant crop plants through genetic engineering would

Table 1. Lectin genes have been engineered into the major crop plants for aphid control

Gene or gene products Transformed crops Targeted insects Reference

Lectins

Galanthus nivalis agglutinin Maize Corn leaf aphid Rhopalosiphum maidis Wang et al. 2005

Wheat Grain aphid Sitobion avenae Stoger et al. 1999

Wheat aphid Schizaphis graminum Xu et al. 2004

Grain aphid Sitobion avenae and

Rhopalosiphum padi Linnaeus

Liang et al. 2004

Galanthus nivalis agglutinin and

wheat-amylase inhibitor

Potato Peach-potato aphid Myzus persicae Gatehouse et al. 1996

Canavalia ensiformis lectin Potato Peach-potato aphid Myzus persicae Gatehouse et al. 1999

Pinellia ternata agglutinin and

cry1Ac

Wheat Wheat aphid Schizaphis graminum Rondani Yu and Wei 2008

Amaranthus caudatus agglutinin Cotton Cotton aphid Aphis gossypii Glover Wu et al. 2006a

Others

Trypsin inhibitor mti-2 Potato Peach-potato aphid Myzus persicae Saguez et al. 2010

be a good alternative strategy. To date, efforts on development

of aphid-resistant transgenic crops have mainly concentrated

on introduction of plant-derived lectin genes (Table 1), which

may potentially have harmful effects on aphid natural enemies

as well (Birch et al. 1999; Hogervorst et al. 2009).

Aphid populations are regulated by natural enemies such as

ladybird beetles and parasitoid wasps. Despite the effective-

ness of biological control, behavioral responses to the threat

of predation may allow aphids to persist as pests (De Vos

et al. 2010). For many pest species of aphids, avoidance

of predators involves the release of an alarm pheromone

comprising (E)--Farnesene (EF), a volatile sesquiterpene,

released from the cornicles (aka siphunculi) on the aphids

abdomen when attacked by its natural enemies (Bower et al.

1972; Pickett and Griffiths 1980; Dixon 1998). In this context,

EF could function either as a direct repellent or act as a

kairomone (chemical messengers emitted by organisms of

one species but benefit members of another species) for

attraction of natural enemies of aphids (Hatano et al. 2008).

Although application of EF to crop plants against pest aphids

would be an alternative approach, high oxidation rate of EF

has hampered the application of EF either in laboratory

tests (Yang and Zettler 1975) or directly on crops (Hille Ris

Lambers and Schepers 1978). Essential oils, which protect

EF from oxidation, have also been trialed in the laboratory

and field (Bruce et al. 2005), but the aphids are sensitive to

other minor components included, which reduce the repellent

response.

Many plant species produce EF, either constitutively orin response to herbivore damage (Crock et al. 1997; Mumm

et al. 2003; Harmel et al. 2007). However, most produce other

compounds that interfere with the response of EF (Dawson

et al. 1984) although some plants have evolved to avoid this


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284 Journal of Integrative Plant Biology Vol. 54 No. 5 2012

problem (Gibson et al. 1983). EF synthase genes, which

encode enzymes that convert farnesyl diphosphate (FPP)

to the acyclic sesquiterpene EF, have been isolated and

characterized from Douglas fir (Pseudotsuga menziesii) (Huber

et al. 2005), Yuzu (Citrus junos) (Maruyama et al. 2001),

sweet wormwood (Artemisia annua) (Picaud et al. 2005, Yu

et al. 2011) and black peppermint (Mentha piperita) (Crock

et al. 1997; Prosser et al. 2006) (Table 2). Studies of tritrophic

interactions involving aphids and their natural predators and

parasitoids have demonstrated that transgenic Arabidopsis and

tobacco plants expressing peppermint and sweetworm wood

EF synthase genes, respectively can repel aphids and attract

their natural enemies, thus minimizing aphid infestation (Beale

et al. 2006; Yu et al. 2011). This demonstrates a potentially

valuable strategy of using plant-derived EF synthase genes

for aphid control in economically important crops in an environ-

mentally benign way.

In this review, we briefly introduce the effects of aphids on

host plants, plants defenses to aphid herbivory and the recruit-

ment of natural enemies for aphid control in an agricultural

setting. Furthermore, the plant-derived EF synthase genes

cloned to date are discussed along with the strategies for

manipulating the EF synthetic pathway in crop plants and the

potential for generating novel aphid resistance via genetically

modified (GM) approaches.

Effects of Aphids on Host Plants

Aphids are major arthropod pests of agriculture worldwide;

the family Aphididae comprises more than 4 300 species,

damaging crops by sucking nutrients from the phloem and/or by

transmitting plant viruses. Unlike the majority of insects, aphids

can reproduce clonally and an aphids embryonic development

begins before its mothers birth (Goggin 2007). These traits

allow for rapid population growth of aphids in the field due to

their parthenogenetic lifestyle, short generation times, and the

fact that nymphs of certain aphid species can reach maturity in

as little as 5 d (Dixon 1988; Goggin 2007; De Vos et al. 2010).

Table 2. Plant-derived EF synthase genes isolated so far

Plant speciesGenbank

Referencesaccession no.

Douglas fir AY906867 Huber et al. 2005

Yuzu AF374462 Maruyama et al. 2001

Sweet wormwood AY835398; Picaud et al. 2005

GU294840; Yu et al. 2011

GU294841

Black peppermint AF024615; Crock et al. 1997

AJ86642 Prosser et al. 2006

Aphids damage their host plants by ingesting phloem sap

through narrow piercing-sucking mouthparts called stylets.

Successful phloem feeding requires overcoming a number

of phloem-related plant properties and reactions. The most

important hurdle is formed by the phloem wound responses,

such as coagulating proteins in the phloem sieve elements

of the plant and in the capillary food canal in the insects

mouth parts, i.e. the stylets (Tjallingii 2006). In order to actively

suppress the plant defense responses, aphids often initiate

probing of the leaf epidermis cells immediately upon landing.

Each probe takes less than one minute and involves stylet

penetration of the epidermis cell wall and membrane, injection

of saliva and ingestion of the cell contents, which causes

only minor damage to the host plant (Hogenhout and Bos

2011). During probing and feeding, aphids secrete two types

of saliva: gelling saliva, which is thought to protect stylets

during penetration, and water saliva, which is secreted into

various plant host cell types and phloem (Prado and Tjallingii

2007). Once prolonged feeding has been established, aphids

repeatedly inject watery saliva into plant cells and phloem

during salivation. In addition to having a protective function in

the form of polyphenol oxidases and other detoxifying enzymes,

aphid saliva likely contains factors that facilitate uptake of

phloem sap (Cherqui and Tjallingii 2000; De Vos and Jander

2009) and some salivary proteins share the same features

with plant pathogen effectors and therefore may function as

aphid effectors by perturbing host cellular processes (Bos

et al. 2010). Will et al. (2000) demonstrated that aphid saliva

also contains calcium-binding proteins and can inhibit calcium-dependent phloem occlusion, which is triggered by a calcium

flux in response to wounding. The ability to prevent sieve

tube plugging is an important adaptation that allows aphids

to remain at a single feeding site for hours at a time ( Goggin

2007).

Symptoms of aphid attack differ depending on the aphid

species and plant species in question. More common symp-

toms of aphid infestation include chlorosis, necrosis, wilting,

stunting, and malformation of new growth (Goggin 2007).

As far as cereal aphids are concerned, the Russian wheat

aphid (Diuraphis noxia Mordvilko) gives rise to leaf rolling

and discoloration, while the greenbug (Schizaphis graminum)gives rise to typical leaf discoloration and eventually necrosis

(Delp et al. 2009). In contrast, the bird cherry-oat aphid causes

no specific symptoms, except reduced plant growth at high

population densities (Delp et al. 2009). Meanwhile, Honek

and Martinkova (2004) found that the growth and population

density of the cereal aphid Metopolophium dirhodum (W.)

was mainly dependent on the quality of the host plant and

many other environmental factors are also known to affect the

fecundity and longevity of aphids (Aqueel and Leather 2011).

Furthermore, aphids also induce physiological changes in their

host plants. The pea aphid, for example, diverts nitrogen from


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the apical growth zones of alfalfa to nitrogen source tissues and

increases deposition rate of nitrogen in the middle part of the

infested tissue (Girousse et al. 2005). Infestations of grasses

by two species of aphids, greenbug and Russian wheat aphid,

increase the concentrations of amino acids and the propor-

tions of essential amino acids in the phloem sap of the host

plants (Sandstrom 2000). Greenbugs also greatly increase the

concentrations of free amino acids in the wheat leaves at the

infestation sites (Dorschner et al. 1987). Many of the symptoms

or the physiological changesof the host plants induced by aphid

infestation are clearly beneficial to aphids and deleterious to

their host plants. For example, during the compatible inter-

actions with Russian wheat aphid, susceptible wheat plants

react to the injection of aphid saliva by discoloration and rolling

of the leaves longitudinally around the main leaf vein to form

a tubular refuge that protects aphids from predators (Smith

et al. 2010); the discoloration can significantly reduce the

plant photosynthetic efficiency and results in weakened

plants with substantially lower grain yields (Smith et al.

1991).

Each year, worldwide crop losses due to aphids are esti-

mated at hundreds of millions of dollars (Blackman and Eastop

1984; Oerke 1994; Morrison and Peairs 1998). For example, in

20102011 crop seasons, 62.5% of the 26 million hectares of

the Chinese wheat growing area suffered severe aphid infesta-

tions (Xia et al. 2011). The major aphid species infesting wheat

in China are grain aphids (Sitobion avenae F.), greenbug (S.

graminum), Rhopalosiphum padiLinnaeus and Metopolophium

dirhodum Walker. Among them, grain aphids arethe more dom-inant and destructive species, affecting wheat production areas

in Yellow Huai and the Northern China Plain, the Southwest,

Northwest and the Middle Yangtze River regions (Zhang et al.

2009) and cause as much as 15 to 60% reduction in wheat

yield (Wang et al. 2011). Aphids also infest other agriculturally

important crops such as maize, cotton, oilseed and soybean

with the affected area estimated as 4.3, 4.7, 2.3 and 2.05

million hectares, respectively, resulting in yield reductions and

economic losses to farmers in spite of wide-spread use of

chemicals. The area of aphid infestation represents approxi-

mately 14%, 90%, 32% and 23% of the total growth area of

maize, cotton, oilseed and soybean, respectively, in Chinain the 2010/11 crop seasons (www.natesc.moa.gov.cn; the

statistical data of the affected acreages of soybean came from

personal communication with Professor Kunjun Zhao, China

Northeast Agricultural University).

Plant Defense Against Aphid Infestation

To defend against aphid attacks, plants have evolved a broad

range of defense mechanisms. These mechanisms can be

generalized into two categories: constitutive defenses and

inducible defenses (Chen 2008). Constitutive defenses include

physical and chemical barriers that exist before aphid attack.

For example, trichomes are modified epidermal cells found on

the surface of the aerial organs of most plants, which constitute

a physical barrier to aphid movement and settlement (Tingey

and Laubengayer 1981; Vargas et al. 2005); trichomes are

also involved in the secretion of large amounts of sucrose

esters, which play a major role in deterring the settling and

probing of aphids (Neal et al. 1990). In addition, some plant sec-

ondary metabolites, including phenylalanine ammonia-lyase

(PAL), polyphenol oxidase (PPO), peroxidase (POD) and

2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIM-

BOA) are considered important biochemical markers in cereal

plants resistance against aphids (Escobar et al. 1999; Han

et al. 2009). DIMBOA is the main hydroxamic acid aglucone

in wheat and maize extracts, and when added to artificial

diets, DIMBOA is deleterious to aphids (Argandona et al. 1983;

Escobar et al. 1999). Negative relationships were also found

between the DIMBOA content of wheat plants and the number

of aphids feeding on them (Niemeyer et al. 1989).

Relative to the constitutive defenses, inducible defenses

become activated upon aphid attack. Two types of inducible

defenses, direct defenses and indirect defenses, have been

observed in plants. Direct defenses indicate any plant traits

that by themselves affect the susceptibility of host plants to

insect attacks (Kessler and Baldwin 2001), including host cell

wall fortification, R gene-mediated hypersensitive responses

and anti-manipulation of hosts to limit food supply of aphids;

removing essential nutrients to reduce nutrient value supplied

for aphids and producing antibiotic chemicals (Goggin 2007;Chen 2008). Indirect defenses, on the other hand, include

plant traits that by themselves do not affect the susceptibility of

host plants, but can serve as attractants for natural enemies

of the attacking insect (Chen 2008). For example, certain

plants such as the aphid-infested potato specially emit EF and

the compound attracts egg-laying aphidophagous predators

Episyrphus balteatus (Harmel et al. 2007). Other more common

volatiles that are induced by aphid herbivory and attract natural

enemies include (Z)-3-hexenyl acetate (Yu et al. 2008), methyl

salicylate (Zhu and Park 2005) and cis-jasmone (Birkett et al.

2000; Diezel et al. 2011).

Interactions between Aphids and theirNatural Enemies

When attacked by predators or parasitoids, aphids secrete

sticky defensive droplets from the siphunculi, a pair of tube-like

structures on their dorsal surface (Kunert et al. 2005). These

droplets contain an alarm pheromone comprising a mixture of

compounds with EF as the predominant and, in some aphid

species, the only component (Bowers et al. 1972; Pickett and

Griffiths 1980; Kunert et al. 2005). EF can also function as

a kairomone in attracting aphid predators, including ladybirds


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(Nakamuta 1991; Zhu etal. 1999;Al Abassi et al. 2000; Mondor

and Roitberg 2000; Acar et al. 2001; Francis et al. 2004),

syrphids (Francis 2005a; Harmel et al. 2007; Verheggen et al.

2008b), lacewings (Zhu et al. 1999) and parasitoids (Dawson

et al. 1983; Micha and Wyss 1996; Du et al. 1998; Foster et al.

2005).

A large guild of parasitoids and predators in agro-ecosystems

are increasingly recognized as important sources of biocontrol

for invasive agricultural aphids. The species of aphid natural

enemies in certain regions are correlated with the type of agro-

ecosystem. For example, coccinellids, chrysopids, hemerobi-

ids, syrphids, braconids, aphelinids, and to a lesser extent,

nabids and anthocorids comprise the specialist natural enemy

fauna using cereal aphids in North America (Brewer and

Elliott 2004). Generalist predators are also predominant in the

natural enemy complex of soybean, among which, coccinellids

including the Harmonia axyridis Pallasand the anthocorid Orius

insidiosus (Say), have been shown to successfully suppress

pockets of Aphis glycines in soybean even when the overall

field densities were low (Fox et al. 2005; Desneux et al.

2006). The efficacy of biological control is highest during aphid

early colonization stages (Edwards et al. 1979; Chiverton

1986). The impact of generalist predators is thought to be

critically important in agro-ecosystems under organic farming

(Zehnder et al. 2007), where reduced pesticide application may

enhance densities of generalist predators, thereby potentially

strengthening their contribution to pest control (Hole et al. 2005;

Birkhofer et al. 2008).

Current Status of Development of GMCrops for Insect Resistance

Compared with conventional crop breeding programs, genetic

engineering of crop plants could not only widen the potential

pool of useful genes but also permit the simultaneous intro-

duction of several different desirable genes in a single event.

Ever since the first report of transgenic plants appeared in

1984 (Horsch et al. 1984), insect-resistant transgenic crops

have developed very fast and more than 20 different genes

conferring insect resistance have been genetically engineered

into the major crops. This progress has had a significantbeneficial effect on global agriculture, at least in terms of pest

reduction and improved quality (Gatehouse et al. 2011). On

22 October 2009, the Chinese Ministry of Agriculture issued

its first two biosafety certificates for commercial production of

two Bacillus thuringiensis endotoxin (Bt) rice lines (Chen et al.

2011), providing a broad and stable foundation for future growth

of GM wheat and other edible crop plants both in China and the

rest of the world (Xia et al. 2011). Although insect-resistant GM

crops such as Bt cotton, maize and soybean have had great

success in commercialization and planting in a global context,

less progress has been made in development of aphid resistant

GM crops. So far, efforts on development of aphid-resistant GM

crops have mainly concentrated on introduction of plant-derived

lectin genes (Table 1). Many plant lectins show a carbohydrate

specificity for glycoconjugates present in organisms (such as

viruses, micro-organisms, fungi, nematodes or phytophagous

insects) outside the plant kingdom, whereas these glycocon-

jugates (e.g., galactose, sialic acid) have low abundance or

are absent in plants (Vandenborre et al. 2011). The effect of

a broad spectrum of plant lectins has been tested on several

insect species, some of which are toxic to insects, especially

for aphids (Michiels et al. 2010). Up to now, more than four

plant lectin genes have been engineered for aphid-resistance

in crops. Among them, Galanthus nivalis agglutinin (GNA) has

been introduced into wheat (Stoger et al. 1999), maize (Wang

et al. 2005) and potato (Gatehouse et al. 1996), respectively.

Bioassay results showed that transgenic wheat plants from

lines expressing GNA at levels greater than 0.04% of total

soluble protein decrease the fecundity, but not the survival of

grain aphids (Stoger et al. 1999). Other lectin genes, Canavalia

ensiformis lectin (Gatehouse et al. 1999), Amaranthus cauda-

tus agglutinin (Wu et al. 2006a) and Pinellia ternata agglutinin

(PTA) (Yu and Wei 2008), have also been proved to be toxic

to aphids in transgenic plants. However, GNA could cause

adverse effects on predatory ladybirds and parasitoidsAphidius

ervi via aphids in the food chain (Birch et al. 1999; Hogervorst

et al. 2009), resulting in major concerns over the biosafety

of application of these lectin genes in agriculturally important

crops for use in aphid control. Therefore, other safe and moreeffective genes/genetic strategies for aphid control need to be

exploited. Recently, Pitino et al. (2011) developed the plant-

mediated RNAi technology for silencing a peach aphid salivary

gland-specific gene MpC002, which encodes a salivary protein

thatmay function as an aphid effector by perturbing host cellular

processes, and the gut-specific gene Rack-1; MpC002 and

Rack-1 expression were knocked down by up to 60% through

feeding on transgenic tobacco and Arabidopsis plants, and

silenced aphids produced less progeny, exemplifying the feasi-

bility of plant-mediated RNAi approach for aphid control. How-

ever, while MpC002 is an aphid gland-specific gene, Rack-1

is an intracellular receptor that binds activated protein kinaseG (PKG), an enzyme primarily involved in signal transduction

cascades (Rigas et al. 2003). Rack-1 protein is conserved

among plants and animals (Chen et al. 2006), suggesting

the application of this gene in genetic engineering of agri-

culturally important crop plants is also not feasible because

biosafety issues are of major concern. Therefore, although

the plant-mediated RNAi approach for pest control is currently

a hotspot, efforts are still needed to screen for the aphid-

specific genes necessary for effective aphid control using this

technology.


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Plant Biosynthesis of Terpenoids andtheir Roles in Pest Resistance

Terpenoids are major components of plant volatile blends

and play a predominant role in the attraction of enemies or

predators of herbivores (Kopke et al. 2008). All terpenoids are

either derived from the mevalonate pathway (MVA) or the 2-

C-methyl-D-erythritol 4-phosphate (MEP) pathway, which lead

to the formation of the C5 unit isopentenyl diphosphate (IPP)

and its allylic isomer, dimethylallyl diphosphate DMAPP, the

basic terpenoid biosynthesis building blocks (Mahmoud and

Croteau 2002). The sequential head-to-tail addition of IPP units

to DMAPP in condensation reactions initially yields geranyl

diphosphate (GPP, C10), farnesyl diphosphate (FPP, C15) and

geranylgeranyl diphosphate (GGDP), which are the precursors

of monoterpene, sesquiterpene and diterpene, respectively; in

most cases, biosynthesis of sesquiterpenes is assumed to take

place at the cytosol/endoplasmic reticulum boundary, whereas

monoterpene and diterpene biosynthesis are compartmental-

ized in plastids (Bohlmann et al. 1998) (Figure 1).

Some plant-derived terpenoids have been shown to act

as aphid feeding deterrents and are often toxic at higher

levels. The sesquiterpene p-benzoquinone perezone isolated

from Perezia adnata and even its non-natural derivatives ex-

hibited strong antifeedant activity against peach aphid (Bur-

Figure 1. Terpene biosynthesis pathway in plants (Cited from Bohlmann et al. 1998)

DMADP, dimethylallyl diphosphate; FPP, farnesyl diphosphate; G3P, glyceraldehyde-3-phosphate; IPP, isopentenyl diphosphate; GGDP,

geranylgeranyl diphosphate; GPP, geranyl diphosphate; MEP, 2-C-methyl-D-erythritol 4-phosphate pathway; MVA, the mevalonate pathway.

gueno-Tapia et al. 2008). Other sesquiterpenes farnesol and

bisabolene, and monoterpenes geraniol could significantly in-

hibit aphid settling in assays using apterous aphids Myzus

persicae and leaf discs embedded in agar (Gutierrez et al.

1997). -Citronellol, linalool and oils distilled from several

species ofArtemisia orAchillea millefolia (yarrow) also showed

aphid repellence (Halbert et al. 2009). Apart from acting

as feeding deterrents to insects, volatile terpenoids released

from different plant parts also act as host location cues for

insect natural enemies (Kappers et al. 2005) as well as play

crucial roles in pollinator attraction and interaction with the

surrounding environment (Weir et al. 2004; Nieuwenhuizen

et al. 2009). In a natural environment, caterpillar-damaged

maize-releasing EF repelled the corn leaf aphid Rhopalosi-

phum maidis (Bernasconi et al. 1998), and the trichomes of

wild potato Solanum berthaultii release EF, which acts as a

kairomone and repels aphids at short distances (Gibson and

Pickett 1983). Plant breeders have successfully obtained hybrid

potatoes bearing glandular trichomes from the wild potato,

which are well defended against infestation and colonization

by aphids (Tingey et al. 1982). However, some agronomically

important crop plants such as wheat, rice and others do not

produce this compound upon aphid infestation, and although

EF can also be synthesized by certain plants, in most cases,

it is often contaminated with inhibitory compounds.


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EF and its Potential Role in AphidControl

Aphids release alarm pheromone from the cornicles on their

abdomen when attacked by their natural enemies (Bower et

al. 1972; Pickett and Griffiths 1980; Dixon 1998). EF as the

main, and generally the only component of the aphid alarm

pheromone, that can interrupt aphid feeding and cause other

aphids in the vicinity to become agitated or disperse from

their host plant (Bowers et al. 1972; Pickett and Griffiths

1980; Wohlers 1981; Francis et al. 2005b). The quantity and

composition of aphid alarm pheromones varies with the aphids

morphological type and age (Gut and Oosten 1985). The

amount of EF released by aphid increases positively with

its body weight but the concentration declines exponentially

(Byers 2005). Aphids possess the OBP3 protein in their ol-

factory system, capable of detecting low concentrations of

the alarm pheromone EF (Qiao et al. 2009) and using EF

levels to assess their environment with regard to other aphids

(Verheggen et al. 2009). However, the release of EF is not

contagious, i.e. a non-stressed aphid receiving the alarm signal

does not release additional EF (Verheggen et al. 2008a;

Hatano et al. 2008). Furthermore, EF also plays a role in the

control of aphid morphological types, causing lower weights for

adult aphids and lower fecundity and prolonged development

when aphids are stimulated with EF during early instars

(Su et al. 2006). Therefore, aphid development and total off-

spring numbers can be negatively affected by EF perception

(Mondor and Roitberg, 2003).Generally speaking, EF can increase the proportion of alate

offspring (Kunert et al. 2005; Podjasek et al. 2005), while alate

aphids are usually more sensitive to EF than the apterae

ones and often leave host plants emitting EF (Crock et al.

1997), thereby resulting in lower aphid numbers and reduced

virus transmission in host plants. EF can also substantially

enhance the effectiveness of pesticides and mycoinsecticide

by increasing mobility of aphids (Griffiths and Pickett 1980;

Elagamy and Haynes 1992; Roditakis et al. 2000). Most impor-

tantly, EF can function as a kairomone for natural enemies

of aphids such as ladybirds (Nakamuta 1991; Zhu et al. 1999;

Al Abassi et al. 2000; Mondor and Roitberg 2000; Acar et al.2001; Francis et al. 2004), syrphids (Francis 2005a; Harmel et

al. 2007; Verheggen et al. 2008b), lacewings (Zhu et al. 1999)

and parasitoids (Dawson et al. 1983; Micha and Wyss 1996;

Du et al. 1998; Foster et al. 2005), and recruit these parasitoids

or predators for aphid control in an agricultural setting. Based

on this, continuous efforts have been devoted to the applica-

tion of synthetic EF in the field for aphid control in recent

years.

Application of synthetic EF to crop plants against pest

aphids is not feasible because it is a volatile hydrocarbon and is

not persistent in the environment. Furthermore, it is susceptible

to oxidation due to the presence of several double bonds in the

molecule (Qiao et al. 2009), has a short atmospheric life time,

only 11 min by the influence of OH radicals and 16 min by

the influence of ozone (Bouvier-Brown et al. 2009), and this

has hampered the application of EF either in laboratory tests

(Yang and Zettler 1975) or directly on crops (Hille Ris Lambers

and Schepers 1978). Attempts have been made to overcome

this problem by the synthesis of analogs, derivatives, or ex-

ploitation of natural essential oils. Some EF derivatives give

products that are more stable and less volatile, which inhibit

aphid settling and virus transmission, including the pesticide-

resistant aphids (Dawson et al. 1982, 1988). However, the

chemical synthesis of EF and its derivatives is complicated

and expensive, and application of these chemicals on a large

scale in the field is not cost-effective. EF isa component of the

essential oil of many plants (Flamini et al. 2003; Miyazawa and

Tamura 2007) and using essential oils with high EF content is

another potential option to counter this problem. In field plot

experiments, numbers of pea aphid (Acyrthosiphon pisum)

were significantly reduced when sprayed with the essential

oil from Hemizygia petiolata rich in EF (more than 70% of

the component was EF) (Bruce et al. 2005). However, this

strategy encountered problems with insufficient purity of EF.

Genetic engineering of crop plants to biosynthesize and emit

EF for aphid control could be a good alternative.

Characterization of EF Synthase GenesIsolated from Plants

EF can also be synthesized by certain plants. The genesencoding EF synthases, which convert farnesyl diphosphate

(FPP) to the acyclic sesquiterpene EF, have been isolated

and characterized from Douglas fir (Huber et al. 2005), Yuzu

(Maruyama et al. 2001), sweet wormwood (Picaud et al. 2005;

Yuet al. 2011) and black peppermint (Crock et al. 1997; Prosser

et al. 2006) (Table 2). For example, Crock et al. (1997) cloned an

EF synthase by random sequencing of a peppermint oil gland

library; the recombinant enzyme expressed in Escherichia

coli could convert FPP to EF (85%), (Z)--farnesene (8%),

-cadinene (5%) and other minor unidentified sesquiterpene

products, whereas only EF (98%) and (Z)--farnesene (2%)

were synthesized when Mn2+

was used as the co-factor.Cluster analysis (Figure 2A) and phylogenetic reconstruc-

tion analysis (Figure 2B) revealed that plant-derived EF or

sesquiterpene synthase genes from the same or relative plant

species were more closely related to each other than that of

their counterparts from distant species. Despite the remarkable

differences among sesquiterpene synthases, crystal structure

analyses revealed their three-dimensional similarities contain

highly-conserved amino acids. Among these there are three

important domains (Lesburg et al. 1997; Starks et al. 1997), i.e.,

an Asp-rich DDxxD motif in the middle part of sweet wormwood

EF synthase genes AaFS1 and AaFS2 isolated by Yu et al.

(2011) (corresponding to amino acids 327331 in AaFS1 with


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Figure 2. Sequence analyses of plant-derived EF synthase genes.


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(A) Comparison of the deduced amino acid sequences of plant-derived representative sesquiterpene synthases. 1, sweet wormwood EF

synthase AaFS1; 2, sweet wormwood EF synthase AaFS2; 3-5, EF synthase from Yuzu, black peppermint and Douglas fir, respectively;

6, 5-epi-aristolochene synthase from tobacco with known crystal structure. Amino acids identical in all six genes are marked in blue. Amino

acids identical in five genes are marked in green. The highly conserved DDxxD region is marked with a bar, solid circles indicate highly

conserved His, triangle indicates highly conserved Arg, and pentagram indicates highly conserved amino acid residues in J-K loop.

(B) Phylogenetic tree of the so far isolated plant-derived sesquiterpene synthase genes. The joint enrooted tree was generated using

MEGA4 by the neighbor-joining method. Bootstrap values were indicated at each branch. Sesquiterpene synthase 1 was under accession

no. AJ271793, Sesquiterpene synthase 2 was under accession no. AF304444, the GenBank accession no. of other genes were showed in

the Results section.

(C) The Gene Structure Display Server (GSDS) (http://gsds.cbi.pku.edu.cn/) output of the sweet wormwood EF synthase AaFS1 gene

structure. The exons, and introns are indicated by blue rectangles and green lines, respectively. 0, 1, 2 represent the intron phase.

(D) The GSDS output of the Asian peppermint EF synthase MaFS1 gene structure.

(E) The GSDS output of the Douglas fir EF synthase PmFS2gene structure.

the GenBank accession number of GU294840), two arginine

residue positions corresponding to AaFS1 at Arg290 and

Arg292, an aspartic acid residue Asp551 and a lysine residue

Lys561, respectively (Figure 2A). The DDxxD motif was involved

in the binding of the required divalent metal cofactor (Starks

et al. 1997). Based on the results of X-ray crystallographic

analysis of 5-epi-aristolochene synthase from tobacco, it was

suggested that Arg290 and Arg292 were located in A-C loop,

while Asp551 and Lys561 were located in J-K loop; the two loops

played an important role in binding with substrate (Starks et

al. 1997). At the same time, His, Cys and Arg residues have

been confirmed to be essential for sesquiterpene synthase

activity through inactivation experiments (Rajaonarivony et al.1992; Savage et al. 1995). Furthermore, five arginine residues

(Arg137,140,290,292,468) and one histidine (His103) were also highly

conserved among the compared sesquiterpene synthases

(Figure 2A).

The terpene synthase genes were divided into three classes

by comparison of intron/exon patterns (Trapp and Croteau

2001). Class I contained 1214 introns, while class II had nine

introns and class III six introns. Intron phases are defined as

relative positions of an intron within or between codons (Intron

phase 1 means that the intron occurs between the first and

second bases of a codon; phase 2 means that the intron occurs

between the second and third letters of a codon; phase 0 meansthat the intron occurs between codons), which is a conserved

character of eukaryotic gene structures, because any phase

change requires eithercompensatory double mutations or more

complex molecular mechanisms (Long and Deutsch 1999). So

far, among the isolated EF synthase genes, the AaFS1

gene with six introns fell into class III (Figure 2C), with its

intron number and phase identical to the Asian mint ( Mentha

asiatica) (E)--farnesene synthase genes MaFS1 (Figure 2D).

However, the Douglas fir EF synthase PmFS2gene had 11

introns (Figure 2E), and the black peppermint EF synthase

gene isolated by Prosser et al. (2006) had seven introns,

and neither fall into any of the categories mentioned above.

It remains unclear how the variations in intron number in these

plants affect the production of EF or other terpenes.

Metabolic Engineering of EF Synthesisin Crop Plants as a Potential ImportantAlternative Strategy for Aphid Control

Genetic manipulation of mono-, sesqui- and diterpenes in crop

plants could provide an alternative tool for improving pest aphid

control. For example, P450-suppressed transgenic tobacco

plants producing higher levels of diterpene cembratriene-olhad higher aphidicidal activity; in vivo assays with these plants

could greatly diminish aphid colonization (Wang et al. 2001).

Recombinant linalool/nerolidol synthase (FaNES1) catalyzes

the biosynthesis of the monoterpene alcohol linalool and its

sesquiterpene counterpart nerolidol; in dual-choice assays with

Myzus persicae, the FaNES1 transgenic Arabidopsis plants

significantly repelled the aphids (Aharoni et al. 2003).

Genetic engineering of crop plants to constitutively emit

EF for aphid control would be a good strategy (Beale et

al. 2006; Yu et al. 2011). Compared with the maize terpene

synthase 1 which could produce three sesquiterpenes: (E)-

-farnesene (26%), (E)--nerolidol (29%) and (E, E)-farnesol(45%) (Schnee et al. 2002), the EF synthase genes from

Douglas fir, Yuzu, sweet wormwood and black peppermint

produced only one major sesquiterpene, EF, indicating these

genes were good candidates for manipulating crop plants for

aphid control (Yu et al. 2011). Rothamsted scientists have

previously demonstrated that Arabidopsis can be genetically

engineered to emit extremely pure EF. These plants, ex-

pressing an EF synthase cloned from peppermint (Mentha

piperita), elicited potent effects on the behavior of the peach

aphid Myzus persicae (alarm and repellent responses) and its

natural enemy, the parasitoid Diaeretiella rapae (an arrestant


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response). This was the first example of plant genetic engineer-

ing to produce an insect pheromone and demonstrated that the

resulting emission affects behavioral responses at two trophic

levels such that the plants become more resistant to aphids

(Beale et al. 2006). Recently, the ICS, CAAS lab found that

overexpression of EF synthase genes AaFS1 or AaFS2

from sweet wormwood in tobacco plants also resulted in the

emission of pure EF (Figure 3). Behavioral studies involving

the peach aphids, and predatory lacewings (Chrysopa septem-

punctata) demonstrated that the transgenic tobaccoexpressing

AaFS1 and AaFS2 could repel peach aphids, but not as

strongly as expected, probably due to other deterrent plant

volatiles emitted by tobacco plants. However, AaFS1 and

AaFS2 lines exhibited strong, statistically-significant attrac-

tion to lacewings. Further experiments combining aphids and

lacewing larvae in an octagonal arrangement showed trans-

genic tobacco plants could repel aphids and attract lacewings,

thus minimizing aphid infestation (Yu et al. 2011). Therefore,

the EF-emitting transgenic crop plants may have practical

applications in agriculture as a result of not only repellence

Figure 3. Gas chromatography-mass spectra (GC-MS) profile of volatiles emitted by transgenic tobacco plants with EF synthase

gene from sweet wormwood (Adapted from our data published previously by Yu et al. 2011).

(A) The volatiles from non-transgenic control plants.

(B) The volatiles from transgenic plants. The compounds were as follows: 1, Tridecane; 2 and 6, siloxane contaminant; 3 and 5, unkown; 4,

Tetradecane; Peak A, EF.

(C) Mass spectrum of peak A.

(D) Mass spectrum of authentic EF.

to aphids and reducing aphids population growth, but also

recruiting the natural enemies of aphidsby increasing predation

on habituated aphids for aphid control (De Vos et al. 2010).

These findings demonstrate that genetic engineering of crop

plants to constitutively emit EF for aphid control is a feasible

and promising strategy.

However, the low sesquiterpene (including EF) production

of transgenic plants overexpressing sesquiterpene synthase

genes were observed in some studies, indicating that engi-

neering sesquiterpene production in plants is a more chal-

lenging task. For example, transgenic Arabidopsis plants over-

expressing the FaNES1 gene targeted to chloroplast emitted

the sesquiterpene nerolidol, but the expression level was

100 to 300-fold lower than that of linalool (Aharoni et al.

2003). Attempts to engineer sesquiterpene synthesis in to-

bacco have also been performed with a fungal trichodiene syn-

thase and only small amounts of the expected sesquiterpenes

could be detected (Hohn and Ohlrogge 1991). When another

sesquiterpene synthase, amorpha-4,11-diene synthase gene

from Artemisia annua, was transformed into tobacco, this only


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resulted in the production of amorpha-4,11-diene at level of

0.2 to 1.7 ng/d per g fresh weight (Wallaart et al. 2001).

Our previous study indicated that in tobacco transgenic lines

engineered with two sweet wormwood EF synthase genes,

the EF emission level ranged from approximately 1.55 to 4.65

ng/d per g fresh weight. This was lower than expected but

these transgenic lines still exhibited repellence to aphid and

attraction to lacewing (Yu et al. 2011). In summary, although

genetic engineering of crop plants to consecutively emit EF for

aphid control is a feasible and promising strategy, engineering

crop plants synthesizing significant amounts of EF remains a

challenge.

Challenges and Perspectives

The use of genetic engineering to produce novel cropplants emitting alarm pheromone for aphid control has two

important challenges. One is that the GM crop plants should

release high purity and sufficient EF to mimic the natural

aphid alarm response as well as increasing foraging by

aphid predators or parasitoids (Beale et al. 2006). A second

is the sub-cellular localization of both the precursor pool and

the introduced EF synthases. The third is to generate an

appropriate concentration of EF at a time when aphids

present a threat to the crop by using wound-inducible

promoters, which are underway at Rothamsted laboratory.

An important consideration for the use of EF to elicit an

alarm response in aphids is that it needs to be of very high

purity. EF is a main component of certain plant volatiles

and it has been shown that aphids are very sensitive to

even small amounts of EF but do not respond to EF in

the same way when it is released together with other plant

volatiles (Dawson et al 1984, Bruce et al. 2005) as men-

tioned briefly in the Introduction. For example, wild potato

Solanum berthaultii, releasing high quantities of EF, from

foliar trichomes, is more repellent to the green peach aphid

than the commercial varieties of Solanum, which produce

lower levels of EF along with some inhibitory compounds

such as (E)-caryophyllene (Dawson et al. 1984; Ave et al.

1987). In thiscontext, the EF synthase genes from Douglas

fir, Yuzu, sweet wormwood and black peppermint produced

only one major sesquiterpene, EF, indicating that these

genes were good candidates for manipulating crop plants

for aphid control (Yu et al. 2011).

In order to increase the amount of EF synthesized,

the following strategies may be considered. First, multi-

point metabolic engineering may be needed to increase

the flux through the pathways leading EF synthesis. The

availability of precursors plays an important role in plant

sesquiterpene engineering (Aharoni et al. 2006), so this

must be studied in the context of the whole pathway level,

including the precursor synthesis and supply, rather than

single-gene engineering strategies. HMGR (3-hydroxy-3-

methylglutaryl CoA reductase) catalyzes the irreversible

conversion of HMG-CoA to mevalonate, and FPP synthasecatalyzes the synthesis of FPP from IPP and DMAPP;

these two enzymes have been proposed to be key reg-

ulatory steps controlling isoprenoid metabolism (Figure 1)

(Bach 1995; Cunillera et al. 1997). Transgenic wheat plants

constitutively expressing both codon-optimized FPP syn-

thase gene from cow (Bos taurus) a n d EF synthase

gene from black peppermint emitted more EF than plants

constitutively expressing EF synthase alone (Pickett et al.

unpublished data by Rothamsted laboratory). Furthermore,

coupled overexpression of a patchoulol synthase (PTS)

with an avian FPP synthase gene increased sesquiterpene

patchoulol accumulation two- to five-fold higher than thatof plants harboring only PTS, and this could be further

augmented by another two- to six-fold by the concomitant

overexpression of a truncated HMGR (Wu et al. 2006b).

Second, foreign sesquiterpene synthases should be better

targeted to a suitable subcellular compartment. An FPP

synthase isoform existed in the mitochondria of Arabidopsis

(Cunillera et al. 1997), so Kappers et al. (2005) presumed

that FPP should be available in this cell compartment and

generated higher levels of nerolidol by targeting the straw-

berry FaNES1 gene to mitochondria. Besides, a chloroplast

form of FPP synthase exists in rice, wheat and tobacco

(Sanmiya et al. 1999), so the chloroplast could also be an

ideal compartment for sesquiterpene engineering in these

crop plants. Third, the sesquiterpene biosynthetic pathway

could also be redirected from its natural cytosolic location

to the plastids (indicated in Figure 1). Wu et al. (2006b)

generated transgenic tobacco plants producing high levels

of sesquiterpene by overexpressing both an avian FPP

synthase and an appropriate sesquiterpene synthase in

the chloroplast; the transgenic plants could increase the

synthesis of the sesquiterpenes patchoulol and amorpha-4,

11-diene more than 1 000-fold.

Farnesyl diphosphate is a ubiquitous isoprenoid inter-

mediate involved in sesquiterpene biosynthesis in the cy-

toplasm (Figure 1) (Crock et al. 1997), suggesting that

cytoplasm pools of FPP might be accessible to foreign EF

synthases. We formerly transferred AaFS1 and AaFS2

into tobacco under the control of a constitutive cauliflower

mosaic virus 35S promoter and observed relatively low EF

production, as well as non-linear correlation between the

levels of EF emission and AaFS1/AaFS2 expression

levels (Yu et al. 2011), indicating that the availability of the

precursor (FPP) supply might be a major limiting factor in

the biosynthesis of sesquiterpene in tobacco or other crop

plants.


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The supply of FPP for the biosynthesis of sesquiterpenes

probably depends on the crop plant species in question

and their physiological status. Compared with the higher

sesquiterpene levels in sweet wormwood and grand fir(Steele et al. 1998; Wallaart et al. 1999), the production

of endogenous sesquiterpene in tobacco was very low,

even undetectable (Chappell and Nable 1987; De Moraes

et al. 1998). This suggested that only a low amount of free

FPP could be used for the biosynthesis of sesquiterpene

in tobacco, and the foreign sesquiterpene synthase had to

compete with other endogenous ones for the limited supply

of FPP. However, Schnee et al. (2006) overexpressed the

maize tps10 gene in Arabidopsis plants to generate high

quantities of TPS10 sesquiterpene products, which were

identical to those released by maize, suggesting that FPP

in Arabidopsis plants was not limiting. On the other hand,the amount of FPP for sesquiterpene biosynthesis can also

be related to the plant physiological status. Sterols play a

structural role in membrane fluidity and are important for

plant vitality; FPP could be directed to the pathway of sterol

biosynthesis under normal conditions (Chappell and Nable

1987). Upon exposure to biotic stresses and suitable elici-

tors (e.g. fungus elicitor, herbivore elicitor, etc.), the produc-

tion of sesquiterpenes in tobacco can increase dramatically

at the expense of sterol production (Vogeli and Chappell

1988; De Moraes et al. 1998), indicating that the proportion

of FPP, which is partitioned for sesquiterpene biosynthesis

can be altered by elicitors. Indeed, the expression of FPP

synthase genes in wheat was greatly upregulated upon

aphid infestation, although different expression patterns

were observed in a tissue-specific manner (unpublished

data by ICS, CAAS lab).

At last, there can be phenotypic variation during the

manipulation of terpene and/or sesquiterpene synthesis

pathways and this might pose another challenge in ma-

nipulation of EF synthase genes in economically important

crop plants in which people desire GM plants with improved

aphid resistance without affecting other agronomic traits.

For example, although relatively low levels of EF were

emitted by AaFS1/AaFS2 transgenic tobacco plants, we

observed some phenotype differences compared with the

control, such as long leaf petiole, slim stem, small leaves

and more axillary buds (Yu et al. 2011). Interestingly, this

is not a single case because not only are the transgenic

tobacco plants expressing AaFS1 and AaFS2, but also

the plants expressing their counterparts from peppermint

exhibited the similar phenotype variation phenomena (un-

published data by ICS, CAAS laboratory). The reason might

lie in the fact that targeting EF synthase genes into the

cytoplasm of cells in tobacco plants might partly deplete the

limited FPPpool, which in turn affects the biosynthesisof ab-

scisic acid or other important metabolites, the same effects

as observed by other authors in engineering the terpene

synthesis pathway (Fray et al. 1995; Aharoni et al. 2003;Aharoni et al. 2006). For example, transgenic tomato plants

constitutively expressing the carotenogenic enzyme phy-

toene synthase showed a dwarf phenotype resulting from

the over-production of phytoene synthase, which converted

GGDP to phytoene and thereby diverted this intermediate

away from the gibberellin and phytol biosynthetic pathways

(Fray et al. 1995). Furthermore, overexpression of the FPP

synthase, a key enzyme in biosynthesis of sesquiterpenes

(Figure 1), in transgenic Arabidopsis plants led to a reduction

of IPP and DMAPP available for the biosynthesis of the

growth regulator cytokinin, which might have caused at

least in part the phenotype alteration observed (Masferrer etal. 2002). Moreover, Arabidopsis plants expressing a dual

linalool/nerolidol synthase (FaNES1) also showed retarded

growth compared with wild-type plants under the same

greenhouse conditions, and the phenotype was correlated

with levels of linalool production (Aharoni et al. 2003), and

transgenic potato plants that expressed FaNES1 produced

high levels of linalool, but also showed yellow and damaged

regions on their leaf blades (Aharoni et al. 2006). The

major explanation of this phenotype change might be due

to the reduction in the availability of substrates for other

metabolites produced in the plastids, which play an impor-

tant role in plant growth and development, including the

growth regulators gibberellin, abscisic acid and other vital

components such as carotenoids, chlorophyll, and quinones

(Aharoni et al. 2003).

However, these effects on phenotype variations seemed

to be species-dependent as transgenic Arabidopsis plants

transformed with peppermint EF synthase gene could

produce high levels of EF without any observed phe-

notypic variations (Beale et al. 2006). Thus the cost of

increasing sesquiterpenes level should be evaluated when

deciding the strategies for genetic engineering of EF

synthase gene in economically important crop plants. It

is worth mentioning that, by using the rice Rubisco small

subunit promoter (rbcS), which is reported to direct for-

eign gene expression specifically to leaves and other

green tissues (Huang and Lin, 2007), we successfully

generated EF synthase gene transgenic wheat plants by

Agrobacterium-mediated transformation method; so far, no

phenotype variations were observed in the progeny of trans-

genic lines except for improved aphid resistance, implying

there is sufficient substrate FPP in wheat green tissues

for synthesizing EF (unpublished data by ICS, CAAS

laboratory).


13/18


Acknowledgments

Some work mentioned in this review is partly funded by the

Research Initiative on Development of Disease and Insect

Resistance Transgenic Wheat Plants supported by the Chinese

Ministry of Agriculture (2008ZX08002001), Natural Science

Foundation of China (31171618) and the EU FP7 OPTICHINA

Project (266045). Rothamsted Research receives grant-aided

support from the Biotechnology and Biological Sciences Re-

search Council (BBSRC) of the UK.

Received 28 Nov. 2011 Accepted 9 Feb. 2012

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Metabolic Engineering of Plant-Derived (E)-Beta-farnesene Synthase