Review TRENDS in Plant Science
Synthetic promoters: genetic control
through cis engineering
Mauritz Venter
Institute for Plant Biotechnology, Department of Genetics, Stellenbosch University, Private Bag X1, Matieland 7602, South Africa
Technological advances in plant genetics integrated
with systems biology and bioinformatics has yielded a
myriad of novel biological data and insights into plant
metabolism. This unprecedented advance has provided
a platform for targeted manipulation of transcriptional
activity through synthetic promoter engineering, and
holds great promise as a way to further our understand-
ing of regulatory complexity. The challenge and strategy
for predictive experimental gene expression is the accu-
rate design and use of molecular ‘switches’ and modules
that will regulate single or multiple plant transgenes in
direct response to specific environmental, physiological
and chemical cues. In particular, focusing on cis-motif
rearrangement, future plant biotechnology applications
and the elucidation of cis- and trans-regulatory mech-
anisms could greatly benefit from using plant synthetic
promoters.
Modifying promoters – the way forward?
One of the major challenges in a plant genetic engineering
program is to design a transformation-cassette that would
enable the precise control of transgene activity. The choice of
promoter, to confer constitutive, spatial and/or temporal
transgene expression, is one of the key determinants used
in plant biotechnology applications. In recent years, a wide
range of different promoters from plant, viral and bacterial
origin have been characterized and used extensively in
regulated transgene expression systems in plant cells [1–
3]. Several plant genetic engineering strategies have incorp-
orated the use of strong constitutive promoters in the study
of gene and transcription factor (TF) function as well as for
conferring transgene expression for crop improvement and
bio-pharmaceutical applications. The well-described cauli-
flower mosaic virus (CaMV) 35S promoter [4–7] confers
high-level gene activity and has been used most commonly
in plant transgene expression studies. The way forward for
the study and design of inducible transgene expression
cassettes was laid by research investigating the modifi-
cation of the 35S promoter using the core-region (essential
to initiate transcription), combined with dissected cis-regu-
latory elements, as well as early analysis of other dicot- and
monocot-promoters [8–15]. From these initial attempts it
has become apparent that the use of a synthetic and regu-
latory module that can be tuned, that is suited for a specific
application and driven by the core-transcriptional initiation
region of a constitutive promoter will prove invaluable in
Corresponding author: Venter, M. (mauritz@sun.ac.za).
Available online 9 February 2007.
www.sciencedirect.com 1360-1385/$ – see front matter ß 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.tplants.2007.01.002
Vol.12 No.
future genetic engineering programs. A few exciting studies
have described how changes in promoter architecture, and
the targeted design of cis-motif context can improve the
control of spatial and temporal gene activity, regulate
multiple transgenes, and overcome drawbacks such as
homology-dependent gene silencing in plant cells [16–20].
Results from these research efforts underscore the value of
using synthetic promoters to assist in elucidating synergis-
tic regulatory interactions, the role of individual cis-motifs
and in biotechnological applications. The scope of several
other important factors such as gene silencing and RNA
interference (RNAi), introns and splicing, transgene selec-
tion, different inducible systems and transformation tech-
nologies is too large to be covered in this review although
they all play major roles in plant inducible gene expression
studies. There is a considerable amount of data reporting on
the use of chimeric inducible systems including promoters
and transcription factors. In an attempt not to summarize
all the relevant literature, this review will focus specifically
on key examples highlighting previous and current strat-
egies for the accurate design of an inducible plant synthetic
promoter. An integrative design strategy for analysis and
putative prediction of gene expression is proposed that
incorporates transcriptomics, conserved cis-motif organiz-
ation and the use of intricate bioinformatic software.
Re-designing the regulatory code
Refined and targeted modification of promoter architecture
necessary for coordinated manipulation of gene activity
requires accurate deciphering of the regulatory frame-
work. The basics of cellular processes on a nucleotide
and protein level have been studied and defined, resulting
in a unified theory of eukaryotic gene expression and
regulation. Even with the significant understanding
gained from this fundamental research, major overlaps
and synergistic interactions at different levels of regula-
tion are still poorly understood. Studies in model organ-
isms focusing on the principles of transcriptional control,
promoters, gene expression and regulatory networks [21–
28] have emphasized the importance of combining regu-
latory data (cis-motifs and transcription factors) and gene
expression profiles to elucidate the underlying mechan-
isms governing genetic control. The construction of soph-
isticated in silico promoter models [29,30] has enabled
more accurate prediction of gene expression and/or associ-
ation (but not necessarily function). To predict desired gene
expression patterns in response to specific cues it is necess-
ary to understand combinatorial regulatory principles. A
ground-breaking study conducted in the model eukaryotic
 Review TRENDS in Plant Science
organism Saccharomyces cerevisiae (yeast) revealed how
conserved cis-motif-logic can be used for relatively accurate
prediction (73%) of a distinct expression pattern during a
specific condition [31]. Results of this study suggest that it
should be possible to design a synthetic promoter model
that could confer a particular expression pattern in a
biotechnological application dependent on the availability
of regulatory information. The strategy is simple. How-
ever, the complexity and synergistic interplay of a multi-
tude of regulatory mechanisms (although individually well
characterized) poses a significant challenge for future pro-
moter design. Given this complexity it is evident that the
integration of an improved understanding of genome-wide
regulatory mechanisms of complex, multi-cellular eukary-
otes and the power of systems biology, will serve as the
basis for synthetic and predictive biology [32,33]. In silico
predictions of gene regulatory events must ultimately be
validated experimentally. Synthetic promoters play a
major role in elucidating cis-motif logic (orientation,
strength–weight, position) to study gene regulation in vivo
[34,35] as well as advancing intricate design strategies of
expression cassettes to be used for metabolic pathway
engineering [16,36–39] and gene therapy applications
[40–43]. With the focus on plants, the accessibility of a
vast amount of genetic data that were produced as a result
of sequencing the whole-genomes of model organisms such
as Arabidopsis thaliana (thale cress) and Oryza sativa
(rice) have enabled the rapid identification of large sets
of promoter sequences. Recent years have also witnessed
much progress in understanding plant promoter architec-
ture and general TF assembly [44–47]. As a result of the
preceding factors, several promoters and TFs have been
selected for inducible transgene expression studies. Pro-
moters used in these studies include unmodified wild-type,
synthetic (with new combinations of cis-motifs from vari-
ous sources) and truncated (reduced to cis-motifs essential
for desired expression profile) modules, and are divided
into the following categories: (i) constitutive, (ii) inducible,
(iii) tissue- and (iv) developmental-stage specific. Most
promoters have a core- or minimal-region necessary to
initiate transcription. Established strategies for the iso-
lation of promoters using approaches for the large-scale
identification of candidate genes, expressed under a cer-
tain condition are effective but time-consuming. By con-
trast, the development of high-throughput technologies
for extensive identification of TF-binding sites (such as
chromatin immunoprecipitation assays – ChiPs) and
advances in plant TF-database assistance has proved
invaluable in characterizing native plant promoters
[48]. ChiP is a strategy that enables the rapid identifi-
cation of large sets of promoter regions using a specific TF
as a probe [49,50]. However, until now these strategies
have not been optimized for a wide variety of plant species.
In addition to fundamental cis-motif elucidation, other
factors considered to be stumbling blocks in applied plant
genetic engineering include expression of multiple trans-
genes, specific inducibility of gene expression and
homology-dependent gene silencing. The inability of con-
ventional wild-type plant promoters to address these hur-
dles has renewed interest in the development of synthetic
promoters.
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Vol.12 No.3 119
Even better than the real wild-type thing
A synthetic promoter designed to initiate transcription of
protein-encoding genes should adhere to the universal
requirements necessary for eukaryotic gene transcription.
Such a promoter is a stretch of DNA comprising a core-
promoter region and multiple repeats or combinations of
heterologous upstream regulatory elements (cis-motifs or
TF-binding sites). The core-promoter region (also known as
the minimal-region) usually contains a TATA-box necess-
ary for recruiting RNA polymerase II and the orchestrated
assembly of general transcription factors to form the pre-
initiation complex (PIC) [51]. The CaMV 35S core-promo-
ter is ideal for transcription initiation and has been used in
several plant promoter engineering strategies. The two
most frequently used strategies are (i) combinatorial
engineering of cis-motifs upstream of the core-promoter
and/or (ii) combined with bidirectionalization of a uni-
directional promoter (Figure 1). Synthetically organized
motifs include enhancers, activators and/or repressors that
bind to TFs expressed in response to specific conditions.
Promoters that respond to inducers can be regulated by (i)
environmental cues (i.e. light, cold and heat stress); (ii)
biotic and abiotic stress (pathogens, wounding, insects,
drought and salinity); (iii) hormones (i.e. ethylene, auxin,
abscisic and salicylic acid); and (iv) chemicals (i.e. tetra-
cycline, copper, estradiol and dexamethasone). The use of
synthetic promoters that allow for targeted inducibility is
of considerable interest for plant engineering strategies.
Non-plant transactivated- and chemical-inducible systems
(using transformation cassettes incorporating a core-pro-
moter and multimers of the upstream activation
sequences) have gained much attention, although not
emphasized in this discussion. Some of these systems have
proven highly flexible and could be used to either repress or
activate plant transgene expression. Comparative and in-
depth analyses of these systems have recently been
reviewed [52,53].
cis-Motif engineering
An in-depth study using synthetic promoters illustrated
the usefulness of combining TF knowledge to ‘cut and
paste’ pathogen-inducible cis-motifs [17,54]. Results from
this study showed that promoter inducibility and strength
varied depending on motif copy number and, more specifi-
cally, spacing of motifs (with the same core-sequence)
relative to the TATA-box. Moreover, one of the main obser-
vations in this detailed study revealed that promoter
activity was not necessarily enhanced with an increase
in motif copy number [17] and, in several instances, it was
shown that a single copy of a specific cis-motif was suffi-
cient for a pathogen-induced response. The functionality of
defense-related plant TF binding sites appears to be con-
served among different plant species [17]. This shared
regulatory function will assist in the future design and
application of pathogen-inducible promoters across the
borders of plant species [54]. There are still several com-
binatorial mechanisms of regulatory context and signaling
that are largely unknown, which prevents the optimal
design of a synthetic pathogen-inducible promoter. We
are still in the early stages of understanding plant–
pathogen interactions and it is evident that the use of
120 Review TRENDS in Plant Science
Figure 1. Simplified representation of a plant synthetic promoter. Core-region containing TATA-box (green block arrow) of a wild-type constitutive promoter, such as CaMV
35S, is used to drive transcription. A uni- or bidirectional synthetic promoter comprises the stretch of DNA containing multiple copies of a specific cis-motif (blue ellipses)
upstream of 35S core-promoter to bind transcription factors (red block arc) expressed in response to specific stimuli.
synthetic promoters will not only be valuable for genetic
engineering of disease resistant plants, but also contribute
to elucidating pathogen-induced motif function out of
native context [17,54]. Homology-dependent gene silencing
(HDGS) is of great concern in plant genetic engineering
strategies and is thought to be caused by multiple copies of
homologous transgene and promoter sequences. Trans-
gene silencing can occur on a transcriptional and post-
transcriptional level [55,56]. Investigations in Nicotiana
tabacum (tobacco) have shown that the use of synthetic
promoters with minimal sequence similarity could serve as
a valuable tool to overcome HDGS in plant transgenic
strategies [18]. Comparison was made between the ‘re-
shuffling’ (or swapping) of previously described functional
domains of 35S [7] and the incorporation of cis-motifs in a
synthetic stretch of DNA, both driven by 35S core-promoter
sequence [18]. Both strategies were evaluated in compari-
son to high-level expression of the wild-type CaMV 35S
promoter. Results from this study showed that domain
swapping led to a less efficient promoter activity compared
with insertion of cis-elements in a synthetic DNA-sequence
[18]. However, it is suggested that repetitive use of cis-
elements with identical core-sequences and homologous
intervening regions (within a functional domain) might
cause depletion of TFs, consequently reducing endogenous
gene expression [18]. Therefore, design strategies using
multimers of cis-motifs need to be optimized to achieve the
desirable inducibility of the transgene without compromis-
ing endogenous ‘house-keeping’ regulation in the plant cell.
An important study investigated individual and combina-
torial functions of light responding elements (LREs) in a
synthetic stretch of DNA driven by the core-promoters of
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Vol.12 No.3
the nopaline synthase (NOS) gene and CaMV 35S [15]. The
NOS core-promoter confers no basal transcriptional
activity, enabling an ‘ON or OFF’ phenotype to be used
and proved to be ideal for engineering inducible synthetic
systems [15]. Owing to limitations in our understanding
and shortfalls in current design strategies only a limited
number of studies have shed light on multimeric cis-motif
design.
Inducible transgene expression in both directions
In many plant biotechnological applications (e.g. metabolic
engineering or pathogen resistance) the advantages of
expressing multiple transgenes are not only advantageous,
but necessary. Gene-stacking, or pyramiding, remains a
challenge for crop improvement and several different sys-
tems and strategies have been reviewed [57,58]. There are
unique reports describing naturally occurring bidirectional
promoters in different organisms, expressing two genes
simultaneously [59–62]. A few key studies have focused on
the usefulness of modifying a unidirectional promoter to a
bidirectional promoter for specific biotechnological appli-
cations in plants (e.g. simultaneously expressing sense
and antisense transcripts to mediate gene silencing)
[16,63]. Comparative analysis of bi-directional promoters
in Vitis vinifera (grape) and Nicotiana tabacum (tobacco),
demonstrated higher reporter-gene expression efficiency
compared with a unidirectional expression system incor-
porating similar enhancer and core-promoter complexes
[64]. Furthermore, this study supported previous views on
how the formation of transcription machinery is more
efficient in a bidirectional mode compared with that in a
unidirectional regulatory structure. In addition to the
 Review TRENDS in Plant Science
above, recent investigations shed light on how the
synthetic cis-motif context (of a synthetically designed
activation module) and combinatorial interactions regu-
late gene expression and influence the stability of
transcriptional assembly on the TATA- box of the core-
promoter [19]. It appears that there is a fine balance
between complete occupancy of synthetic cis-motifs and
success-rate of complete transcriptional activation and
stability of the initiation complex assembled on the
TATA-box. This suggests that depletion of endogenous
TFs because of an excess of synthetic TF-binding
sites can cause transcriptional inactivation [18,19]. This
is one of the major challenges that could greatly limit
future promoter engineering strategies. Nevertheless,
advances in bioinformatics (incorporating plant TF-data-
bases) [65] combined with plant TF-function and accurate
in silico promoter models could assist in a more refined
strategy for designing synthetic promoters. A recent inves-
tigation successfully integrated computational assistance
and bidirectionalization to construct a synthetic transcrip-
tional unit for high-level reporter-gene expression in
response to specific elicitors, which yielded exciting results
[20]. The complete synthetic stretch of DNA, known as the
transcription activation module (TAM), was constructed
from regulatory sequences compiled from a database of
highly expressed plant genes [66]. Stepwise construction of
this TAM consisted of: (i) screening a computational data-
base for genes with high expression levels in plants and (ii)
identification of conserved TATA-box proximal regions and
motifs 500 base pairs upstream from the transcriptional
start site. Initial analysis showed that this TAM could
express the b-glucuronidase (GUS) reporter gene in stably
transformed tobacco (Nicotiana tabacum) at higher levels
compared with those expressed by the wild-type CaMV 35S
promoter [66]. As already discussed in this review, it has
been shown that the TAM could facilitate stable enhancer
complex (enhanceosome) assembly for accurate transcrip-
tional initiation [19]. The TAM was modified in a more
recent study to confer bidirectional expression of reporter
genes, b-glucuronidase (GUS) and green fluorescence
protein (GFP), and evaluated in transient and stably
transformed tobacco (Nicotiana tabacum) leaf discs [20].
Expression of both reporter genes was up-regulated in a
bidirectional mode in response to specific elicitors,
suggesting that enhanceosome assembly to form a stable
PIC on both TATA-boxes (modulated by a DNA-bending
sequence within the TAM) could initiate transcription in
both directions [20]. Specific design strategies for the
development of synthetic promoters are in their infancy.
The investigations described here, which were performed
by several different research groups, are some of the major
examples of the strategies being used in plant promoter
engineering, and could serve as important technical con-
tributions for future biotechnological applications as well
as in the elucidation of synergistic motif-TF interplay.
In silico analysis to assist in the grand design
The combination of high-throughput gene expression
profiles with promoter architecture and bioinformatics is
a powerful approach to compile data for the possible pre-
diction of coordinated gene expression during a specific
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Vol.12 No.3 121
condition [31,67,68]. In this section, a simplified
representation will be used to illustrate how this strategy
could facilitate a practical and refined design of a plant
synthetic promoter able to confer differential gene expres-
sion during defined conditions (Figure 2). Promoter regions
of gene-clusters co-expressed during a specific condition
contain conserved cis-motifs. The biggest challenge is to
dissect which conserved motif is associated directly with
the specific condition under investigation and which are
associated with co-expression [21]. The emergence of plant
TF-binding site database-assistance [65] has greatly facili-
tated large-scale plant promoter analysis. The three major
plant TF-binding site databases, PLACE [69], PlantCARE
[70] and TRANSFAC [71], are updated constantly and
provide a plethora of possible cis-motif combinations. To
analyze and/or construct accurate in silico promoter
models, it is essential to distinguish between over-
represented motifs and background ‘noise’. Several com-
putational methods have been developed for this purpose
[72–74], and assessments of the most prominent cis-motif
detection and/or classification methods and statistical tools
have been reviewed [75]. Identification of cis-regulatory
codes remain complex, particularly given that within a
promoter sequence of 500 to 5000 base pairs the core
sequence of a cis-regulatory motif can range between 4
to 10 base pairs. Gibbs sampling [76] and expectation
maximization (MEME) [77] are powerful methods for pre-
dicting over-represented motifs. There are numerous other
methods based on different operating principles [75] and
some are modified and/or improved versions combined
with statistical modeling techniques such as hidden Mar-
kov models (HMMs) [78]. Initial promoter sequence output,
using plant TF-database assistance, can be refined by
these probabilistic algorithms to select over-represented
cis-motifs in a multiple set of sequences upstream of co-
expressed genes. Furthermore, it should be possible to
identify universal or common cis-motifs that are synergis-
tically associated with cis-motifs of promoters that are
induced during different conditions [21]. Probabilistic
fine-tuning of promoter architecture is followed by con-
struction of a motif synergy map using design ‘rules’
AND-NOT-OR cis-motif logic. This simplified approach
accentuates a combination of previous studies conducted
in S. cerevisiae [21,31] and Arabidopsis [79] that could
assist in the design of one or several plant synthetic
promoters to facilitate a more accurate prediction of gene
expression in response to each specific condition (Figure 2).
Single copies of a cis-motif in a synthetic stretch of DNA
might be sufficient for a desired gene expression profile
[17]; however, the operation principles of the current com-
putational methods are more useful for identifying a clus-
ter of over-represented cis motifs in a contiguous segment
of DNA [75]. By contrast, conventional views regarding the
nature of combinatorial control appears to be inconsistent
with recent and exciting experimental evidence showing
that quantitative transcriptional control of the Drosophila
melanogaster (fruitfly) even skipped gene (eve) is operative
outside a compact arrangement of cis-motifs [80]. It is
evident that precise genetic control is governed by TF
binding specificity [80] and a complex network of regulat-
ory mechanisms on different levels (chromatin, histone and
122 Review TRENDS in Plant Science Vol.12 No.3

Figure 2. Combinatorial cis-motif engineering for the accurate design of synthetic promoters. (a) DNA microarrays enable large-scale identification of induced gene expression
(indicated by blue lightning bolt) in response to a specific condition (e.g. stress, chemical induction or plant development or growth). Upstream promoter regions (black line and
white block arrow) of gene clusters contain a multitude of conserved and non-conserved cis-motifs (black circles, triangles, rectangles and squares) [67]. (b) Probabilistic motif
detection algorithms (MEME and Gibbs sampling) combined with plant TF-database assistance enable the identification of conserved motifs (indicated by blue circles, yellow
rectangles, red triangles and green squares) within multiple promoter sequences associated with a particular condition [65,76,77]. (c) A cis-motif synergy map is constructed to
gain a holistic view of: (i) motif occurrence in relation to other motifs associated with a specific condition [21,23] and (ii) motif spacing relative to the TATA-box of the core-
promoter. Computational promoter modeling [29,30] based on the combined regulatory organization associated with different conditions and gene expression patterns enable
the use of a more refined synthetic design strategy. (d) Plant synthetic promoters are engineered by dissection of cis-regulatory context (crossed out elements are removed)
specific for induction of transgene activity in response to different conditions. Transcription is initiated by the 35S core-promoter sequence (indicated by green block arrow).

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 Review TRENDS in Plant Science
nuclear organization) [24] and over long genomic distances
[81,82]. Given that a single TF can bind to cis-motifs of
different genes, permitting combinatorial control [45,46],
the possibilities for future inducible expression systems
are exciting and challenging.
Concluding remarks
The goal of this review was to prioritize specific past and
more recent research investigations that have focused on
design strategies and applications of plant synthetic pro-
moters. It is evident that the complexity in dissecting cis-
regulatory architecture alone (where spacing, relative to
the TATA-box and other motifs, orientation, copy number
and function of the motif) poses a major challenge for
synthetic promoter design. Advances in bioinformatics
and a more holistic deciphering of regulatory processes
including plant TF networks and cis- and/or trans-syner-
gistic interactions, could greatly accelerate design strat-
egies for the construction of a synthetic promoter with
enhanced inducibility and selectivity. The well established
examples described have been discussed in the context of
relatively new strategies that serve as small ‘stepping
stones’ in the burgeoning and exciting field of plant syn-
thetic biology.
Acknowledgements
I thank Sue Bosch for constructive comments and critical review of this
manuscript.
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Plant Science Conferences in 2007

Gordon Research Conference – Epigenetics
5–10 August 2007
Holderness School, Plymouth, New Hampshire, USA
http://www.grc.org/programs.aspx?year=2007&program=epigen

Fourth International Symposium on Dynamics of Physiological Processes in Roots of Woody Plants

16–19 September 2007
Bangor, UK
http://www.joensuu.fi/metsatdk/gsforest/documents/Roots_Bangor.pdf

16th Biennial Australasian Plant Pathology Society Conference

24–27 September 2007
Adelaide, Australia
www.plevin.com.au/apps2007

www.sciencedirect.com