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: [email protected]
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
Ta
ble
1E
xam
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so
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GT
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Po
llen
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d
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ssic
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ht
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(20
04)
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9T
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etu
m-s
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rsta
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-en
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cer
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uen
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Plant Mol Biol (2010) 73:363–378 365
123
Ta
ble
1co
nti
nu
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Nu
mb
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gen
esa
Pro
mo
ters
So
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toen
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-car
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(Zea
ma
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-car
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pec
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(b-C
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ten
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eto
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(ph
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pin
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pec
ific
idi
(iso
pen
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(b-c
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nst
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tE(g
eran
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syn
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(ph
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(ly
cop
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b-c
ycl
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aE
xcl
ud
ing
the
sele
ctab
lem
ark
erg
ene
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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