REVIEW

Promoter diversity in multigene transformation

Ariadna Peremarti • Richard M. Twyman • Sonia Gomez-Galera •

Shaista Naqvi • Gemma Farre • Maite Sabalza • Bruna Miralpeix •

Svetlana Dashevskaya • Dawei Yuan • Koreen Ramessar • Paul Christou •

Changfu Zhu • Ludovic Bassie • Teresa Capell

Received: 2 November 2009 / Accepted: 11 March 2010 / Published online: 31 March 2010

� Springer Science+Business Media B.V. 2010

Abstract Multigene transformation (MGT) is becoming

routine in plant biotechnology as researchers seek to gen-

erate more complex and ambitious phenotypes in trans-

genic plants. Every nuclear transgene requires its own

promoter, so when coordinated expression is required, the

introduction of multiple genes leads inevitably to two

opposing strategies: different promoters may be used for

each transgene, or the same promoter may be used over and

over again. In the former case, there may be a shortage of

different promoters with matching activities, but repetitious

promoter use may in some cases have a negative impact on

transgene stability and expression. Using illustrative case

studies, we discuss promoter deployment strategies in

transgenic plants that increase the likelihood of successful

and stable multiple transgene expression.

Keywords Promoter � Transgene �Multigene transformation � Transcriptional silencing �Constitutive � Spatiotemporal � Inducible

Introduction

Genetic engineering has increased our fundamental under-

standing of how plants function. By introducing new genes,

we can study how plants grow, develop, and defend them-

selves against pests, diseases and harsh environments, how

photosynthesis is controlled, and the basis of primary and

secondary metabolism (Slater et al. 2003). Genetic engineer-

ing can also be applied to generate hardier crops, more nutri-

tious food, and plants that produce chemical precursors, novel

oils, industrial enzymes and pharmaceuticals (Farre et al.

2010; Zhu et al. 2007; Ma et al. 2003). The transferred genes

play the most significant role in determining the phenotypes of

transgenic plants but like stage hands working hard behind the

scenes, the promoters that control transgene expression are

essential components that often get overlooked. Promoters

allow transgene expression to be regulated, restricted and fine-

tuned, delivering more precise control over the manner in

which phenotypes are expressed (Twyman 2003).

The promoters used in plant biotechnology are tradition-

ally divided into three categories—constitutive (active con-

tinuously in most or all tissues), spatiotemporal (tissue-

specific or stage-specific activity) and inducible (regulated

by the application of an external chemical or physical signal;

Potenza et al. 2004). However, all are based on similar core

sequences usually including an initiator and TATA-box as

well specific cis-acting motifs that bind to transcription fac-

tors (Box 1). Promoter sequences are the same in all plant

tissues whether the gene is expressed or not, so the activity of

a promoter depends on the availability and activity of the

transcription factors. Those binding to constitutive promoters

are available and active all the time, whereas those binding

to spatiotemporal and inducible promoters are themselves

rationed and made available only in certain tissues or

developmental stages, or in response to external signals.

A. Peremarti � S. Gomez-Galera � S. Naqvi � G. Farre �M. Sabalza � B. Miralpeix � S. Dashevskaya � D. Yuan �K. Ramessar � P. Christou � C. Zhu � L. Bassie � T. Capell

Departament de Produccio Vegetal i Ciencia Forestal, ETSEA,

Universitat de Lleida, Av. Alcalde Rovira Roure 191, 25198

Lleida, Spain

R. M. Twyman

Department of Biological Sciences, University of Warwick,

Coventry CV4 7AL, UK

P. Christou (&)

Institucio Catalana de Recerca i Estudis Avancats, Bellaterra,

Spain

e-mail: christou@pvcf.udl.es

123

Plant Mol Biol (2010) 73:363–378

DOI 10.1007/s11103-010-9628-1

In the early years of plant biotechnology, most trans-

genic plants contained two transgenes—one selectable

marker under the control of a constitutive promoter to

facilitate the selective propagation of transformed cells,

and a ‘primary transgene’ which could be under the control

of any sort of promoter and was intended to alter the plant’s

phenotype in a specific manner. MGT is now being

embraced as an approach to generate plants with more

ambitious phenotypes (Naqvi et al. 2009a). MGT allows

goals that were once impossible to be achieved—the

import of complex metabolic pathways, the expression of

entire protein complexes, the development of transgenic

crops simultaneously engineered to produce a spectrum of

added-value compounds (Zhu et al. 2007; Naqvi et al.

2009a). But each additional transgene requires its own

promoter, making it necessary to find different promoters

that achieve the same expression profile or use one pro-

moter multiple times in the same transgenic plant. In the

first case there may be a shortage of available promoters

with the desired expression profile, whereas in the second

case the deliberate introduction of repetitive sequences into

a transgenic locus has in some cases been linked with

undesirable negative effects on transgene expression and

stability (Mette et al. 1999, 2000; Mourrain et al. 2007).

Promoter diversity

The challenge of multiple coordinated transgene expres-

sion can be addressed using a promoter diversity approach,

where different promoters are used to drive different

transgenes with the same expression profile. This is

achievable with most expression strategies when the

number of transgene is small, but becomes increasingly

difficult as the number of transgenes increases due to the

lack of available promoters with suitable expression pro-

files. Table 1 provides examples where MGT has been

carried out with transgenes driven in a coordinated manner

by different promoters.

Constitutive promoters

Constitutive promoters show the most diversity because

there are two major sources—plant viruses and plant

housekeeping genes. Plant viruses have small genes that

are easy to define genetically, and small genomes that are

easy to manipulate in vitro, so many of the earliest con-

stitutive promoters were derived from plant viruses and are

still widely used today. The most prevalent of these is the

Cauliflower mosaic virus 35S promoter, which controls the

synthesis of the 35S major transcript (Odell et al. 1985;

Kay et al. 1987). The full-sized regulatory region for the

CaMV 35S major transcript is just under 3 kbp in length

(Odell et al. 1985). However, shorter cloned fragments are

active, and the typical CaMV 35S promoter used in plant

expression vectors is a 352-bp fragment spanning nucleo-

tides -343 to ?9 (Fang et al. 1989). The core promoter

containing the TATA box extends to position -46 and

confers basal activity. Full activity requires upstream pro-

moter elements, some of which are tissue-specific, which

Box 1 Plant promoters, the basics

The core promoters of protein-encoding genes in plants are similar to those found in animals. They often contain a TATA box positioned at

approximately position -25 relative to the transcriptional initiation site, with the consensus sequence TATAWAW. They may also possess

an initiator element, which is found immediately adjacent to the transcriptional start site and has the consensus sequence YYCARR. These

two elements help to position the general transcription factor TFIID, which contains the TATA-binding protein (TBP) and various TBP-

associated factors (TAFs). However, some promoters lack both a TATA box and an initiator, and the genes under their control tend to have

multiple transcriptional initiation sites reflecting an inability to position TFIID precisely. Other components of the transcriptional initiation

complex then assemble: TFIIA, TFIIB, RNA polymerase II (recruited to the complex by TFIIF), TFIIE and TFIIH. These proteins have been

isolated and functionally characterized in a number of plants, and appear closely related to their animal counterparts (Twyman 2003).

Adjacent to the core promoter, further motifs are clustered in what is known as the upstream promoter region, which extends 100–200 bp on

the 50 side of the transcriptional start site. These motifs bind transcription factors that interact with the transcriptional initiation complex and

facilitate its assembly, improve its stability or increase the efficiency of promoter escape (movement away from the promoter, into the gene

itself) once the transcriptional machinery sets off. Some of these transcriptional activators are constitutive while others may be cell-specific,

stage-specific or inducible by external signals. Therefore, although the DNA sequence of the promoter is the same in each plant cell, the

availability of the transcription factors that bind to individual motifs varies considerably, and this provides the main mechanism for

transcriptional regulation. As well as the upstream promoter, there are more distant cis-acting elements known as enhancers that fulfill a

similar function. In virus genomes, enhancers are often found quite near the gene and promoter, allowing the isolation of all regulatory

elements on a relatively short DNA fragment. In plant genomes, enhancers may be located many kbp away, and may be found upstream,

downstream or even within the introns of the genes they control. Because of their distant location relative to the core promoter, interactions

with the transcriptional initiation complex are achieved by looping out the intervening DNA. Other proteins that bind to the upstream

promoter disrupt the transcriptional initiation complex, either directly or by blocking the activity of transcriptional activators. These

transcriptional repressors also bind at more distant sites known as silencers. Transcriptional activators and repressors function in various

ways to influence the stability of the initiation complex. Some are DNA binding proteins while others interact at the protein level. Some may

form direct contacts with the initiation complex, or indirect contacts through a bridging complex called mediator, while others modify

chromatin structure or introduce bends or kinks into the promoter DNA to facilitate or disrupt other interactions. Some regulatory proteins

can act as either activators or repressors depending on promoter context and the other proteins that are present.

364 Plant Mol Biol (2010) 73:363–378

123

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Plant Mol Biol (2010) 73:363–378 365

123

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366 Plant Mol Biol (2010) 73:363–378

123

means that constitutive expression is achieved through the

additive effects of multiple tissue-specific motifs (Lam

et al. 1989). The region between nucleotides -90 and

-208 is an enhancer, which can increase promoter activity

in a linear fashion when up to four copies are present (Kay

et al. 1987). The enhancer can function in either orientation

and at different positions relative to the gene (Fang et al.

1989; Ohtsuki et al. 1998), and can also enhance the

activity of heterologous promoters to which it is attached

(Zheng et al. 2007).

Although widely used, the CaMV 35S promoter has

certain limitations such as its poor performance in mono-

cots, its suppression by feeding nematodes (Goddijn et al.

1993; Urwin et al. 1997) and the intellectual property

issues affecting its commercial deployment. For this rea-

son, alternative virus promoters with similar or improved

properties have been sought. Because the CaMV 35S

promoter has proven so successful, related caulimoviruses

have been the first port of call. Examples include promoters

from Figwort mosaic caulimovirus (FMV; Bhattacharyya

et al. 2002), Cassava vein mosaic virus (CsVMV; Verda-

guer et al. 1996), Cestrum yellow leaf curling virus

(CmYLCV; Stavolone et al. 2003), Mirabilis mosaic virus

(MiMV; Dey and Maiti 1999) and Peanut chlorotic streak

virus (PClSV; Maiti and Shepherd 1998; Bhattacharyya

et al. 2003). The CmYLCV promoter sequence is available

from Syngenta Biotechnology, Inc. for limited research

purposes, and has been used to express glyoxalase I (gly I)

to achieve salt tolerance in tobacco (Veena et al. 1999) and

mung bean (Bhomkar et al. 2008).

Badnaviruses are closely related to the caulimoviruses,

and they contain a strong constitutive promoter that tran-

scribes the entire genome. The Commelina yellow mottle

virus (CoYMV) promoter is active in tobacco (Medberry

et al. 1992), but more detailed studies have been carried out

in monocots where its activity is broad albeit not universal

(Torbert et al. 1998). The Sugarcane bacilliform virus

(ScBV) promoter is active in monocots (banana, corn,

millet and sorghum) and dicots (tobacco, sunflower, canola

and Nicotiana benthamiana) and has been shown to drive

high level gusA expression in transgenic banana and

tobacco plants (Schenk et al. 2001). A subcloned fragment

of the promoter was also shown to drive gusA expression in

sugarcane to at least the same level as achieved with the

corn Ubi-1 promoter (Braithwaite et al. 2004).

Subterranean clover stunt virus (SCSV) is a member of

the Circoviridae and has eight circular genome segments

each containing a promoter. Initial studies showed that the

promoters from segments four and seven were comparable

in strength to the CaMV 35S promoter in cotton, potato and

tobacco, and further analysis showed they were also suit-

able for expression in monocots, giving rise to the pPLEX

series of expression vectors (Schunmann et al. 2003a, b).

Plant housekeeping genes are another important source

of constitutive promoters, since housekeeping genes

encode proteins that are required by all cells for basic

functions such as core metabolism and maintenance of cell

structure and integrity. One major class of housekeeping

genes sees to the synthesis of cytoskeletal components, and

includes large families of genes encoding actins and tub-

ulins. The rice actin1 promoter drives strong transgene

expression in rice protoplasts transiently expressing gusA

(McElroy et al. 1990) and in most tissues of transgenic rice

plants (Zhang et al. 1991). Interestingly, McElroy and

colleagues showed that transient expression was abolished

in the absence of the first intron of the actin1 gene

(McElroy et al. 1990) and that the inclusion of this intron in

a chimeric CaMV 35S promoter enhanced its activity in

transgenic rice and corn plants by 40-fold (McElroy et al.

1991). In contrast, the first intron of the recently charac-

terized rice actin2 gene contains a negative regulatory

element whose removal is required for high-level promoter

activity; 2.6 kbp of upstream sequence contains all the 50

regulatory elements necessary for high-level constitutive

gusA expression in transgenic rice (He et al. 2009a). Other

actin promoters that have been used for constitutive or

near-constitutive expression include Arabidopsis ACT2

(An et al. 1996) and banana ACT1 (Hermann et al. 2001).

The ubiquitins are another highly conserved family of

housekeeping genes, some constitutively expressed (Ka-

walleck et al. 1993) others responsive to stress (Christensen

et al. 1992; Cornejo et al. 1993; Christensen and Quail

1996). For example, the Arabidopsis UBQ1 and UBQ6

promoters are active in all tobacco tissues, albeit at dif-

ferent levels (Callis et al. 1990), and the corn Ubi-1 pro-

moter is more than 10 times stronger than the CaMV35S

promoter in corn protoplasts when combined with the first

intron (Norris et al. 1993). Significant improvements in

activity have also been observed with three Arabidopsis

polyubiquitin promoters (UBQ3, UBQ10 and UBQ11) and

the potato ubi7 promoter (Garbarino et al. 1995).

Other housekeeping genes are involved in protein syn-

thesis and core metabolism. One example is NeIF4A-10, a

member of the tobacco eIF4A gene family—even a small

region of the promoter (to position -188) drives consti-

tutive expression in transgenic tobacco plants (Mandel

et al. 1995). Further characterization of the promoter

revealed a strong enhancer in the region -151 to -73 and

showed that an intron is not required to boost promoter

activity (Tiana et al. 2005). Other examples are the pro-

moters for genes encoding acetolactate synthase (ALS),

which catalyses the first step in the biosynthesis of the

branched chain amino acids leucine, isoleucine and valine,

and ACC synthase, which plays an important role in eth-

ylene synthesis. The Brassica ALS3 promoter is constitu-

tive and comparable in strength to the CaMV 35S promoter

Plant Mol Biol (2010) 73:363–378 367

123

(Baszczynski et al. 1997). VR-ACS1 is an auxin-inducible

ACC synthase gene from mung bean, whose promoter is

constitutive and 4–6 times more active than CaMV 35S in

Arabidopsis and tobacco (Cazzonelli et al. 2005). It is not

clear why the promoter is constitutive in a heterologous

background when it is tightly regulated in mung bean, but

this probably reflects the absence of a critical transcrip-

tional repressor in the transgenic plants.

With such a spectrum of diverse constitutive promoters

available it is perhaps surprising that there are so few

examples where different promoters have been incorpo-

rated into the same transgenic plant to drive multiple

transgenes (Table 1). In most of these examples, the CaMV

35S promoter has been used to regulate one transgene, with

the other(s) driven by a different viral or plant constitutive

promoter. However, there are cases where multiple pro-

moters other than the CaMV 35S promoter have been used

particularly in commercial varieties where the IP con-

straints surrounding the CaMV 35S promoter would make

commercial development more challenging.

Spatiotemporal promoters

Many different plant promoters have been described that

restrict expression to particular cells, tissues, organs or

developmental stages. Within this collection the number of

different promoters that can be used to drive coordinated

transgene expression (multiple transgenes with the same

expression profile) is somewhat more limited, and case

studies in which the promoter diversity strategy has been

applied are therefore fewer in number (Table 1).

Arguably the most abundant spatiotemporal promoters

are those that restrict transgene expression to seeds, and for

many reasons the seeds are often a favored target for

transgene expression, particularly if the goal of an experi-

ment is to force the accumulation of a heterologous product

that might interfere with vegetative growth at high con-

centrations or to improve the nutritional quality of seeds

used as staple foods. Many promoters have been identified

that target genes specifically to the seed, or to a particular

region of the seed. Storage proteins such as corn zein

(Schernthaner et al. 1988), rice glutelin (Leisy et al. 1989;

Takaiwa et al. 1991; Zheng et al. 1993), barley hordein

(Marris et al. 1988), rice prolamin (Qu and Takaiwa, 2004)

and wheat glutenin (Colot et al. 1987) have been rich

sources of seed-specific promoters, predominantly directing

expression to the endosperm (Wobus et al. 1995). Addi-

tional promoters have been shown to direct gene expression

to the embryo and aleurone (Opsahl-Sorteberg et al. 2004;

Qu and Takaiwa 2004; Furtado and Henry 2005).

The spatial and temporal activities of seed-specific

promoters reflect their peculiar combination of cis-acting

regulatory motifs (Onodera et al. 2001). The endosperm-

specific bZIP transcription factors O2 (Opaque-2; corn) and

SPA (storage protein activator; wheat) have been shown to

activate prolamin genes by binding to the GCN4-like motif

in the bifactorial endosperm box (Albani et al. 1997;

Schmidt et al. 1992). The GCN4 motif, a cis-acting ele-

ment that is highly conserved in the promoters of cereal

seed storage protein genes, plays a central role in con-

trolling endosperm-specific expression (Wu et al. 1998).

The barley endosperm-specific bZIP transcriptional acti-

vators BLZ1 and BLZ2 activate storage protein genes by

interacting with the GCN4 motif in barley B1-hordein

promoters (Vicente-Carbajosa et al. 1998; Onate et al.

1999), and the rice bZIP transcription factor, RISBZ1

works in a similar manner (Onodera et al. 2001). It has

been demonstrated that GCN4 and AACA motifs are

conserved in all glutelin gene promoters isolated thus far

(Takaiwa et al. 1996), and the GCN4 motif is also observed

in the PPK, SBE1 and 10, 13 (PG5a) and 16 kDa prolamin

promoters. This motif acts as a key element conferring

aleurone and subaleurone-specific expression (Wu et al.

1998; Qu and Takaiwa 2004).

Anther-specific promoters are also very useful because

they can be used to control male fertility, an important trait

in crop breeding. Numerous anther-specific and pollen-

specific promoters have been identified in a variety of

plants, including the TA29 promoter from tobacco (Kol-

tunow et al. 1990), the A9 promoter from Arabidopsis (Paul

et al. 1992) and the RA8 promoter from rice (Jeon et al.

1999). A cis-acting element found between 285 and

2,207 bp upstream of the TA29 gene confers strong, tape-

tum-specific expression and appears to work in heterolo-

gous settings, as it has been used to create male-sterile

tobacco and rapeseed plants through the expression of

Bacillus amyloliquefaciens barnase, a potently cytotoxic

ribonuclease (Mariani et al. 1990). Fertility can be restored

in F1 plants if female barnase transgenic parents are cros-

sed with males expressing the barnase inhibitor barstar

(Mariani et al. 1992), but the frequency of full restoration

can be low due to poor control over transgene expression.

Bisht et al. (2004, 2007) addressed this by enhancing and

augmenting the expression of barstar using a combination

of gene optimization, promoter engineering and MGT. The

wild-type and synthetic barstar genes were expressed using

either the TA29 and A9 promoters separately, or a chimeric

promoter combining elements from each. Although the

chimeric promoter was more active than either of the

individual promoters, the expression of different versions

of barstar using the two different tapetum-specific pro-

moters achieved the greatest frequency of restoration to

fertility.

Many case studies have been published in which

multiple transgenes are expressed in specific tissues by

combining the use of spatiotemporally-regulated and

368 Plant Mol Biol (2010) 73:363–378

123

constitutive promoters but there have been few examples of

studies in which different spatially-restricted promoters

have been used in the same plant. The barnase/barstar case

study described above is one example (Bisht et al. 2004,

2007). Another involved the use of five different promoters

to drive five transgenes in corn endosperm, i.e., corn

phytoene synthase 1, Pantoea ananatis phytoene desatur-

ase, Gentiana lutea lycopene b-cyclase, G. lutea b-caro-

tene hydroxylase, and Paracoccus sp. b-carotene ketolase,

controlled by the low molecular weight wheat glutenin,

barley hordein, rice prolamin, rice glutelin-1 and corn

c-zein promoters, plus the selectable marker gene under

constitutive control (Zhu et al. 2008). This study showed

how combinatorial transformation with multiple genes

could be used to generate a library of plants with different

phenotypes representing carotenoid biosynthesis. The dif-

ferent promoters ensured that, in any combination, it would

be possible to isolate plants without multiple copies of the

same promoter thus reducing the likelihood of transcrip-

tional silencing.

Inducible promoters

Arguably the most useful promoters in plants are those

responsive to the changing environment. Many different

inducible promoters have been identified in plants and

these generally fall into three categories—(1) those

responsive to endogenous signals (plant hormones); (2)

those responsive to external physical stimuli (abiotic and

biotic stresses); and (3) those responsive to external

chemical stimuli. Such promoters provide immense scope

for the precise regulation of transgene expression through

external control, ranging from the precise control of

transgene activation/inactivation in experimental settings

to the ability to activate transgenes on an agricultural scale

by the application of chemical sprays. Like constitutive

promoters, there are two major sources for inducible pro-

moters—endogenous genes and heterologous systems.

Inducible promoters have been combined with both con-

stitutive and spatiotemporal promoters in transgenic plants

but are generally not combined with each other, i.e., where

multiple genes need to be induced together it is standard

practice to place them under the control of the same pro-

moter. The inducible promoters most commonly used for

MGT strategies include hormone responsive promoters,

heat shock promoters and those responding to pathogens.

The most widely-used hormone-responsive promoters

are those induced by auxins, gibberellins and abscisic acid,

although promoters responsive to heterologous hormones

(from insects and mammals) are also useful because they

do not activate endogenous pathways. For example, Mar-

tinez et al. (1999) developed a hybrid system consisting of

the tobacco budworm ecdysone receptor ligand-binding

domain fused to the mammalian glucocorticoid receptor

DNA-binding domain and the VP16 transactivation

domain. The receptor responds to tebufenozide (an insec-

ticide better known by its trade name CONFIRM). Simi-

larly, Padidam et al. (2003) have developed a system that is

based on the spruce budworm ecdysone receptor ligand-

binding domain, and responds to another common insec-

ticide, methoxyfenozide (INTREPID). Another system

based on the European corn borer ecdysone receptor also

responds to this insecticide (Unger et al. 2002).

The application of exogenous auxins induces certain

genes within minutes so auxin-inducible promoters are

suitable for rapid responses. The cis-acting auxin response

elements in these genes comprise the motif TGTCTC adja-

cent to or overlapping a constitutive motif, such that the

auxin-response element is occupied and the constitutive

element repressed when the auxin concentration is low, and

the opposite occurs when the concentration is higher. Syn-

thetic auxin response elements can be engineered to contain

multiple TGTCTC repeats, and two domains from the pea

PS-IAA4/5 promoter can induce transcription from the

CaMV 35S basal promoter following auxin application

(Ballas et al. 1995). Additional auxin response elements

include the as-1/ocs-like element (TGACG(T/C)AAG(C/

G)(G/A)(C/A) T(G/T)ACG(T/C)(A/C)(A/C); Ellis et al.

1993). Auxin-response elements are often found combined

with response elements to other hormones, allowing com-

plex control. For example, the promoter of the brassinolide-

inducible BLE3 gene in rice also contains auxin response

elements which allow dual regulation by brassinolides and

IAA (Yang et al. 2006). Gibberellins also induce gene

expression rapidly, but in this case signaling is achieved

when the presence of gibberellic acid (GA) induces the

degradation of DELLA proteins, which bind to silencer

elements in GA-inducible genes and interfere with the

binding of transcriptional activators to GA response ele-

ments (GARE). In the absence of DELLA proteins, the GA-

regulated MYB transcription factor (GAMYB) binds to

GAREs and activates transcription (Sun and Gubler 2004;

Woodger et al. 2003). GA-regulated gene expression has

been studied exhaustively in the barley a-amylase genes,

revealing the core of the GARE to be a TAACAAA-like box

which can be repressed by abscisic acid (ABA). Skriver et al.

(1991) reported that when the GARE is fused as a tandem

repeat of six units it can confer GA responsiveness to a

CaMV 35S minimal promoter. Other studies have identified

further elements that lie a conserved distance from the

GARE and, in different combinations, help to modify the

expression profile. These elements combined with the

GARE constitute what is known as a GAR complex, which

can include the pyrimidine box (C/TCTTTT), the TATC-

CAC box, the CAACTC box and the Box1/O2S-like ele-

ment. Promoters that are inducible by ABA usually contain

Plant Mol Biol (2010) 73:363–378 369

123

one or more ABA response elements (ABREs), which may

include a G-box/C-box as well as MYC and MYB binding

sites. There have been many reports of promoters whose

activity has been modified by the incorporation of such

elements. Sequence analysis of the cotton late embryogen-

esis-abundant (LEA) D113 gene promoter (Luo et al. 2008)

revealed the presence of ABREs, a drought-response ele-

ment, and MYB and MYC sites; a 158 bp fragment was

shown to be sufficient for ABA induced reporter gene

expression. Some promoters can also be negatively regu-

lated by ABA. The corn HyPRP gene is specifically

expressed in immature embryos and its expression is

inhibited when ABA levels increase during maturation. A

construct containing 2 kbp of the HyPRP promoter joined to

the gusA gene allowed the repression of GUS activity in corn

and tobacco following the application of ABA. Two ACGT

elements at positions -260 and -93 bp in the promoter are

likely to mediate this effect (Jose-Estanyol et al. 2005).

Many metabolic genes need to respond sensitively to the

levels of specific metabolites to facilitate feedback regu-

lation loops that tie gene expression to metabolic balance.

Therefore, many promoters respond to the presence of

certain chemicals, and these can be extremely useful to

regulate transgene expression in plants because unlike

hormones they do not have pleiotropic effects on growth

and development. Sugar responsive promoters contain a

variety of response elements and these can confer sugar-

sensitivity on heterologous genes. For example, elements

from sporamin and amylase promoters have been studied in

detail and the minimal a-amylase 3 promoter makes the

normally constitutive rice actin1 promoter sensitive to the

presence of sugar (Lu et al. 1998). SURE (SUcrose

REsponsive) elements are found in sugar-inducible patatin

promoters (Grierson et al. 1994; Zourelidou et al. 2002)

and in the promoter of the VvTH1 gene, which encodes a

hexose transporter (Atanassova et al. 2003).

Plant defense genes often have promoters that are

inducible by chemicals (elicitors) produced by pests and

pathogens. Different combinations of trans-acting factors

are responsible for the elicitor-mediated activation of

defense related genes and numerous cis-acting elements

have been identified in the corresponding promoters. They

can be found as single or multiple copies and in different

combinations, allowing some genes to respond to more

than one elicitor, and particular pathogens to elicit distinct,

functionally-related sets of genes. The cis-acting elements

are highly conserved among different plant species so they

are very useful for making transgenes pathogen-inducible

in a range of heterologous settings. Individual elicitor

response elements appear to work well in a different pro-

moter context allowing a certain degree of ‘plug-and-play’

between promoters. The number of copies of each element

also appears to have an effect on the strength of induction,

thus promoters with one or two copies are best suited to

mediate pathogen-specific expression (Rushton et al.

2002). Many plants also respond to the physical aspects of

pest activity (wounding) by expressing a set of protective

genes, many of which are also inducible by jasmonate or

ethylene. Wound-inducible genes include those encoding

proteinase inhibitor II (pinII; Godard et al. 2007; Keinon-

en-Mettala et al. 1998; Keil et al. 1989; Xu et al. 1993), the

Agrobacterium tumefaciens enzymes nopaline synthase

(An et al. 1990) and mannopine synthase (Guevara-Garcıa

et al. 1993; Ni et al. 1996), and peroxidases such as rice

R2329 (Sasaki et al. 2007). The use of wound-inducible

promoters in transgenic plants shows that wound signaling

pathways are functionally conserved across taxonomic

groups (Wu et al. 1999; Yevtushenko et al. 2004).

Elevated temperatures result in the expression of so-

called heat shock proteins (HSPs), which protect plants

from the effects of heat by helping to stabilize proteins and

other cellular components thus conferring thermotolerance.

HSP genes have heat-inducible promoters that are also

strongly conserved across species. For example, the soy-

bean hsp17.3B promoter was used to drive gusA expression

in the moss Physcomitrella patens resulting in no GUS

activity at 25�C but increasing GUS activity at higher

temperatures (Saidi et al. 2005). Heat shock elements

(HSEs) were identified in the tomato ftsH6 promoter, and

were recognized by another HSP acting as a transcription

factor (heat shock factor, HSF; Sun et al. 2006). The heat

shock response may be universal or tissue specific. The

Arabidopsis GolS1 promoter confers universal GUS

activity in transgenic Arabidopsis plants following a tem-

perature shift (Panikulangara et al. 2004), whereas the

barley hspl7 promoter confers heat-induced expression

only in the xylem bundles of the stem and petioles—this

expression profile is conserved in transgenic monocot and

dicot plants (Raho et al. 1996).

Repetitious promoters and transcriptional

gene silencing

The promoter diversity approach discussed above is

employed to avoid repetitious use of the same promoter,

which can in some cases promote transcriptional gene

silencing through promoter methylation and inactivation.

This effect is usually constitutive but may be tissue-specific

(Kloti et al. 2002). However, there have been plenty of

reports describing transgenic plants carrying multiple

transgenes under the control of the same promoter yet

showing strong and stable expression. For example,

although Zhu et al. (2008) used five different endosperm-

specific promoters in corn to achieve the high-level

expression of carotenogenic genes, Naqvi et al. (2009b)

370 Plant Mol Biol (2010) 73:363–378

123

achieved strong expression of four genes in the same sys-

tem using the barley D-hordein promoter to control each

transgene, with no adverse effects. The literature suggests

that repetitious use of the same promoter is often suc-

cessful, so what are the exact limitations of using the same

promoter to control more than one transgene?

To answer this question, it is necessary to look more

closely at the reasons for gene silencing caused by repetitive

promoter use. In plants, transcriptional gene silencing

resulting from sequence homology in promoter regions is

correlated with increased promoter methylation (Kooter

et al. 1999) and appears to be driven by the production of

double-stranded RNA (dsRNA) matching the promoter

sequence (Mette et al. 2000). This has been demonstrated

by deliberately expressing dsRNA corresponding to the nos

promoter in transgenic plants carrying a second transgene

driven by the nos promoter (Mette et al. 1999) and by

constructing transgenic plants in which the transgene locus

triggers both transcriptional and post-transcriptional

silencing simultaneously, by producing dsRNA corre-

sponding to the promoter and transcribed sequences of

different target genes (Mourrain et al. 2007). The compo-

nents of this RNA-driven DNA methylation system have

been systematically sought and investigated, and include

RNA-dependent RNA polymerase 2 (RDR2) and Dicer-like

3 (DCL3; Xie et al. 2004), Pol IV (Herr et al. 2005; Kanno

et al. 2005; Onodera et al. 2005), Pol IVb/Pol V (Pontier

et al. 2005; Wierzbicki et al. 2008; He et al. 2009b), Arg-

onaute 4 (AGO4; Zilberman et al. 2003), chromatin

remodeling protein DRD1 (Kanno et al. 2004), and de novo

DNA methyltransferase DRM2 (Cao and Jacobsen 2002).

In the absence of deliberately-created promoter dsRNA,

the transcriptional silencing seen in some transgenic plants

carrying multiple copies of the same promoter appears to

arise from dsRNA produced serependitiously. The organi-

zation of a transgenic locus is difficult to control, and it is

therefore a common occurrence in both Agrobacterium-

mediated transformation and direct DNA transfer that the

juxtaposition of transgenes or fragments thereof can result

in the creation of hairpin promoter structures at the DNA

level that are transcribed into aberrant dsRNA species.

Such arrangements can be obvious and easy to detect, but

even where gross rearrangements are absent it is possible

that undetected ‘micro-rearrangements’ are present in the

transgenic locus, as observed by Mehlo et al. (2000) when

investigating the structure of a transgenic locus in corn

generated by direct DNA transfer. The siRNAs that trigger

RNA-dependent DNA methylation are just 24 bp in length,

so it is conceivable that inverted repeats of \50 bp could

be sufficient for transgene silencing, and such structures

would be undetectable using the coarse analysis methods

typically employed to study transgenic plants, such as

Southern blot hybridization.

There are at least four mechanisms by which promoter-

specific hairpin RNAs could be generated in transgenic

plants carrying different transgenes under the control of the

same promoter. In many cases, the creation of hairpin RNA

structures may be a consequence of the relative position of

intact transgenes, which is itself a reflection of the mecha-

nism of transgene integration. The organization of integrated

T-DNA sequences differs among Agrobacterium strains, but

a common feature of nopaline-type derivatives such as C58

is the preferential integration of T-DNA as dimers with an

inverted repeat configuration, linked either at the left or right

borders (Jones et al. 1987; Jorgensen et al. 1987). Where

cotransformation is carried out with two T-DNAs containing

different genes, the different T-DNAs often integrate as

heterodimeric inverted repeats, preferentially around the

right border (De Block and Debrouwer 1991). If the same

promoter is used for both genes, this would favor the for-

mation of hairpin structures that could be transcribed from

the opposite strand. The structure of loci generated by direct

DNA transfer is more variable, but inverted repeat structures

involving promoter sequences are not uncommon, allowing

the same silencing mechanism to operate (Kohli et al. 2003).

The second mechanism reflects the frequent occurrence

of spontaneous transgene rearrangements (truncation,

fragmentation, recombination) which can be thought of as

‘collateral damage’ occurring to DNA during the transfor-

mation process due to the activities of nucleases and repair

complexes that facilitate the integration of exogenous DNA.

There have been few systematic studies of this phenomenon

in Agrobacterium-mediated transformation, but Afolabi

et al. (2004) and Zhu et al. (2006) found that nonintact

T-DNAs were present in [70% of transgenic rice lines, in

most cases reflecting loss of the mid to right border portion

of the T-DNA. Similarly, Rai et al. (2007) found that about

50% of rice plants transformed with a T-DNA containing

the phytoene synthase (psy) and phytoene desaturase (crtI)

genes showed evidence of rearrangements, and in the

majority of cases the rearrangements occurred in the crtI

expression cassette, which was adjacent to the right T-DNA

border. Rearrangements involving the left border are often

characterized by the insertion of variable-size regions of

filler DNA, which may be derived from the T-DNA

sequence or from plant genomic DNA, providing further

scope for the creation of hairpin structures (De Buck et al.

1999; Kumar and Fladung 2000, 2002).

A third mechanism for silencing triggered by repetitive

promoter usage reflects the structure of the promoter itself.

Rearrangements affecting the promoter of a transgene may

be more common than expected due to the presence of

specific recombinogenic sequences, such as the imperfect

palindromic structure that is present in the CaMV 35S

promoter (Kohli et al. 1999). This sequence has the ability

to adopt a cruciform secondary structure, which may

Plant Mol Biol (2010) 73:363–378 371

123

stimulate recombination events. Notably, many other pro-

moters contain palindromic sequences of variable length

within 100 bp of the transcription start site, often because

they bind dimeric transcription factors, so it is possible that

promoters may be disproportionately involved in transgene

rearrangement events, a hypothesis that will need to be

examined through the detailed analysis of transgenic pop-

ulations carrying multiple copies of other promoters.

Evidence from many transformation experiments indi-

cates that there is no simple correlation between transgene

copy number and expression level, with the exception of

certain carefully controlled experiments using boundary

elements. In some cases, higher copy numbers have sup-

pressed overall expression levels (e.g., Cannell et al. 1999;

Spencer et al. 1992) whereas in others higher copy numbers

have enhanced expression (Stoger et al. 1998; Gahakwa

et al. 2000). Where suppression effects have occurred, it has

been suggested that ‘‘runaway expression’’ resulting in the

generation of aberrant RNAs lacking polyadenylate tails

has triggered potent silencing through the post-transcrip-

tional silencing pathway (Schubert et al. 2004). From the

prospect of promoter usage, this means in a practical sense

that multiple copies of the same promoter can appear in a

transgenic locus because of the presence of multiple copies

of the same transgene, so why should it matter if the pro-

moter is also represented with different transgenes? A

potential answer to this question comes from experiments

involving the use of the CaMV 35S promoter for large-scale

screening, and reveals a fourth mechanism that may trigger

transcriptional silencing. The CaMV 35S promoter and

enhancer have been used as a random insertional mutagen

to hyperactivate adjacent genes and generate gain-of-func-

tion phenotypes. It has been noted that such screens using

T-DNA cassettes containing the enhancer elements from

the CaMV 35S promoter return a lower than expected fre-

quency of morphological mutants (Chalfun et al. 2003).

Detailed analysis revealed a correlation between the num-

ber of T-DNA insertion sites, the methylation status of the

enhancer sequence and enhancer activity. All plants con-

taining more than a single T-DNA insert were methylated

on the enhancer and its activity was reduced, with the

amount of methylation and the reduction of enhancer

activity correlating with the number of T-DNA copies,

particularly those with right border inverted repeats (Chal-

fun et al. 2003). Even so, methylation was still detected at a

lower frequency in plants without right border inverted

repeats suggesting other triggers were active in these lines.

Alternative solutions

Research into the structure and activity of plant promoters

has allowed the identification of specific cis-acting elements

with defined functions, so the next generation of promoters

is likely to comprise a mix and match of cis-acting elements

that confer upon transgenes exactly the properties required

by the experimenter. To a certain extent this has already

been achieved through the use of heterologous recombinant

promoters such as the inducible promoters based on insect

hormones (see above), but the key disadvantage of these

systems is that they require the corresponding trans-acting

factors to be imported, a problem that would not be

encountered when using recombined endogenous elements

to generate bespoke plant promoters. In some cases, the aim

is simply to increase the activity of an endogenous pro-

moter, which can be achieved in the case of the CaMV 35S

promoter by duplicating its enhancer elements, a strategy

that also works when these elements are imported into other

promoters. Chimeric promoters have also been engineered

using sequences from the CoYMV and CsVMV major

transcript promoters as activating sequences to augment the

CaMV 35S promoter, resulting in higher activity compared

to CaMV 35S in stably transformed tobacco plants and

higher activity compared to corn c-zein in corn endosperm

(Rance et al. 2002). In other cases, it is desirable to con-

strain promoter activity in some way, which has been

achieved with synthetic auxin response elements that make

heterologous promoters more auxin-sensitive. In such cases,

the TGTCTC element is placed either as tandem or inverted

repeats with appropriate spacing, making the promoters up

to 10 times more sensitive to exogenous auxins (Guilfoyle

1999). The ‘super c-zein promoter’ contains a duplication

(region -444 to -174) from the corn endosperm-specific

27 kDa c-zein promoter, making it more active in endo-

sperm tissue (Marzabal et al. 1998). This has been used to

increase the expression of the bacterial genes crtB and crtI

resulting in greater accumulation of b-carotene (Aluru et al.

2008). Many plant promoters can also be enhanced by

including an intron (e.g., Vain et al. 1996; Mitsuhara et al.

1996; Fiume et al. 2004; Chiera et al. 2007).

Can this strategy of modulating promoter structure be

used to reduce promoter duplication in MGT? Several

reports have described the creation of artificial promoters

specifically to avoid repetition. For example, Sawant et al.

(2001) have described a strategy in which the sequences of

the strongest plant promoters were compared, to facilitate

the design of ideal artificial consensus promoters. By

combining the ideal minimal cassette (TATA box, initiator

and Kozak sites) with the ideal upstream activator, they

were able to develop a 450-bp synthetic promoter that was

active in cotton leaves, potato tubers and cabbage stems,

and was stronger than the CaMV 35S promoter in trans-

genic tobacco plants. In theory, several such artificial pro-

moters could be developed and used in combination, or with

native promoters. Bhullar et al. (2003) have developed two

alternative approaches to avoid promoter homology, one in

372 Plant Mol Biol (2010) 73:363–378

123

which functional cis-acting elements are embedded in dif-

ferent synthetic DNA sequences, and another in which the

functional cis-acting elements of different promoters with

the same activity are swapped to create non-homologous

chimeras. A set of modified CaMV 35S promoters was

developed and tested in transgenic tobacco plants, showing

that the chimeric promoters were generally weaker than the

native CaMV 35S promoter but that promoters constructed

from cis-elements embedded in a synthetic DNA sequence

were generally comparable in strength.

Most promoters used in plant biotechnology are unidi-

rectional, but bidirectional promoters are becoming

increasingly useful for MGT experiments as they allow the

simultaneous expression of two gene products. In many

promoters, only the core promoter sequence needs to be

oriented with respect to the coding sequence of the gene

to ensure transcription in the correct direction. Often,

upstream promoter elements can be present in either ori-

entation, and enhancer elements enjoy even more freedom

in terms of position and orientation. If a minimal promoter

consisting of a TATA box and initiator is duplicated and

placed facing outwards, cis-acting elements between the

minimal promoters can be used to drive transcription in

two directions simultaneously. The duplicated minimal

CaMV 35S promoter has been used for bidirectional tran-

scription in several different species (Xie et al. 2001;

Zhang et al. 2008) and can also be combined with other

promoters or synthetic response elements to make bidi-

rectional chimeric regulatory complexes (Chaturvedi et al.

2006). Xie et al. (2001) fused the minimal CaMV 35S

promoter to the constitutive PClSV promoter on one side

and the JA-sensitive OPR1 promoter at the other, and fused

a minimal promoter derived from the senescence-specific

SAG12 gene to the 50 end of the CaMV 35S promoter.

Naturally bidirectional promoters have been reported in

plants, including those directing the oleosin and methionine

sulphoxide reductase genes in canola (Keddie et al. 1994;

Sadanandom et al. 1996), the Arabidopsis cab1 and cab2

genes (chlorophyll a/b-binding protein; Mitra et al. 2009)

and the rice Ocip1 and Ocpi2 genes (chymotrypsin prote-

ase inhibitor; Singh et al. 2009). Bioinformatic analysis of

Arabidopsis, rice and poplar genome sequences has iden-

tified several putatively paired genes whose promoters may

be useful in the future for bidirectional gene expression

constructs (Dhadi et al. 2009). The different promoter

strategies discussed above are summarized in Fig. 1.

Conclusions and outlook

MGT is here to stay and in the future it will be desirable,

even essential, to increase the number of simultaneously

introduced transgenes still further (Naqvi et al. 2009a).

Currently, the creation of transgenic plants with stable

transgene expression relies on a healthy dose of luck, with

the best performing plants being selected from large pop-

ulations, the remaining plants being discarded if they fail to

live up to expectations. As MGT experiments become more

ambitious, the recovery rates will start to suffer from the

law of diminishing returns. How many plants will need to

be screened to find a line that stably expresses 10–20 or

more different transgenes? This is a realistic outlook for the

forthcoming decade, and in order to achieve such ambitious

objectives it will be necessary to minimize the likelihood

of transgene silencing triggered by the repetitive use of the

same promoter sequence. This will be addressed by a

number of strategies, including promoter diversity (facili-

tated by the discovery and characterization of new pro-

moters with relevant expression profiles), the use of

synthetic and modified promoters to reduce the extent of

homology between transgenes, and the use of bidirectional

promoters to reduce the promoter to transgene ratio

(a)

(b)

(c)

(d)

(e)

(f)

Fig. 1 Strategies for promoter deployment in nuclear multigene

transformation. a Repetitious use of the same promoter is a

convenient approach that often works effectively, but some research-

ers have found that it increases the risk of homology-dependent

transcriptional gene silencing. The following alternative approaches

may therefore be chosen instead. b Promoter diversity involves the

use of different promoters for each transgene, although this depends

on availability and becomes more difficult with larger numbers of

transgenes. c Synthetic consensus promoters can be developed from

the conserved features of native promoter sequences, and can add to

the repertoire of available promoters (Sawant et al. 2001). d Chimeric

promoters comprising the functional segments of native promoters

can be prepared by domain swapping (e.g., Bisht et al. 2004) or by

importing specific functional motifs from one promoter into another

(e.g., introducing the CaMV 35S enhancer or actin1 intron into

heterologous promoters to increase activity, or introducing hormone

response elements into constitutive promoters to make them induc-

ible). e The functional cis-elements of natural promoters can be

embedded in a synthetic DNA sequence (e.g., Bhullar et al. 2003).

f Bidirectional promoters can halve the number of promoters required

for MGT. Strategies d, e and f can be combined in different ways to

generate functionalized unidirectional and bidirectional chimeric

promoters with potentially limitless diversity

Plant Mol Biol (2010) 73:363–378 373

123

(Fig. 1). Novel approaches, such as the use of episomal

constructs like plant artificial chromosomes will also play a

role in the future, although for the time being such systems

are limited to a few model species and are technically

demanding.

Acknowledgments Work in our laboratory is supported by the

Ministry of Science and Innovation, Spain (BFU2007-61413 and

BIO2007-30738-E), a European Research Council Advanced Grant

(BIOFORCE) to PC, and the CONSOLIDER Agrigenomics program

funded by MICINN, Spain.

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