Directory UMM :Data Elmu:jurnal:P:PlantScience:Plant Science_BioMedNet:241-260:
trends in plant science
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Promoters that respond to chemical
inducers
Christiane Gatz and Ingo Lenk
The introduction of foreign genes constitutes a powerful tool with which to study and
improve the genetic resources of plants. Transgene expression levels and expression patterns can be adjusted by combining the protein coding region with a suitable promoter. There
is a diverse spectrum of endogenous plant promoters and these are currently being broadened by the development of chimeric promoters that respond to otherwise inactive
chemicals. This range includes promoters that respond to inducers such as the antibiotic
tetracycline, the steroid dexamethasone, the copper ion, ethanol or the agrochemical RH-5992.
These chimeric promoters offer a range of options for transgene design for experimental and
field use.
T
he combination of biochemical, physiological and genetic
studies in genetically engineered transgenic plants has proven to be extremely useful for analysing biochemical and
developmental processes. Examples include the enhanced or ectopic expression of enzymes and regulatory proteins, and the expression of antisense RNA or dominant negative proteins that
reduce the amount of a gene product. The availability of a broad
spectrum of promoters that differ in their ability to regulate the
temporal and spatial expression patterns, dramatically increases
the application of transgenic technology.
In this context, promoters that are only activated in response to
a specific chemical are particularly valuable. In E. coli, an example of a promoter that provides such fidelity is the IPTG inducible
lac promoter, which is routinely used whenever expression of the
recombinant protein is going to interfere with growth. Similarly,
if a foreign gene product expressed in plants is going to interfere
with regeneration, growth or reproduction, an inducible promoter
is required. With such a tool, plants can be regenerated while the
promoter is inactive. Further analysis can then be performed after
activating expression of the transgene. When working with higher
plants, arguments other than the lethality of the gene product may
Box 1. Applications of chemically
inducible promoters
Basic research
• Expression of gene products that interfere with regeneration,
growth or reproduction.
• Expression of gene products at different stages of development.
• Differentiation between primary and secondary effects.
• Analysis of primary effects before homeostatic mechanisms
start to counteract.
• Clear correlation between induction of the transgene and occurrence of an altered phenotype.
Biotechnological applications
• Construction of a conditional male sterility system.
• Expression of transgenes that interfere with regeneration,
growth or reproduction (biofarming).
• Conditional expression of resistance genes as a means of pest
management to delay adaptive processes of the pathogen.
• Simultaneous induction of processes such as flowering and leaf
abscission.
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September 1998, Vol. 3, No. 9
favour the use of an inducible promoter over a constitutive one
(Box 1). Moreover, in addition to being a valuable means of elucidating gene function, chemically inducible promoters are increasingly relevant to the improvement of crops by genetic engineering.
Examples of biotechnological applications are listed in Box 1.
If only very low amounts of a gene product are required for a
cellular process, then the expression level of the promoter should
be close to zero in the absence of the inducer. In this case, high
expression levels in the presence of the inducer are not an essential requirement. In contrast, if only high amounts of a gene product are effective, residual activity in the absence of the inducer can
be tolerated, but it should be inducible to high levels in the presence of the inducer. Ideally, both features – very low expression
levels in the absence of the inducer, and high expression levels in
the presence of the inducer – should be combined in one system as
it is more broadly applicable. A further advance in use of a chemically inducible promoter is its use in combination with tissue specific promoters, so that gene expression can be restricted to a given
tissue at a specific time.
It is important that the chemical used is highly specific for the
target promoter: it should neither influence the expression of other
genes nor affect other cell functions. Uptake is another important
issue: once it is applied, either by foliar spray or root drenching,
the chemical should enter every cell. Additional requirements that
an ideal inducer should meet are given in Box 2.
To provide such tools, two different strategies are being adopted1.
First, plant promoters that respond to a given chemical have been
isolated: obviously, this concept will provide expression systems
that are not specific to the transgene alone. The second strategy
involves using regulatory elements from other organisms that
respond to chemicals that are not usually encountered by plants.
Box 2. Properties required for an ideal inducer
•
•
•
•
•
•
High specificity for the transgene, no toxicity.
High environmental compatibility.
Convenient application by foliar spraying or root drenching.
High efficiency at low concentrations and low use rates.
Low costs.
Availability of different derivatives with different properties:
inducing chemicals; inactivating chemicals; chemicals that
move systemically; chemicals that are confined to the site of
application; chemicals with different half-lives in the plant.
Copyright © 1998 Elsevier Science Ltd. All rights reserved. 1360 - 1385/98/$19.00 PII: S1360-1385(98)01287-4
trends in plant science
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O
C
Cl
SCH3
O
S
O
S
N
NH
C
NH2
O
N
Benzothiadiazole
Safener
(BTH)
N-(aminocarbonyl)-
2-chlorobenzenesulfonamide
HO
O
N
HO
OH
HO
OH
H
OH
NH2
OH
O
OH
O
O
F
O
Tetracycline
Dexamethasone
O
H
H
C
C
H
N NH
They appear to induce a smaller set of genes
as compared with BTH, most likely those
genes that encode enzymes responsible for
detoxifying xenobiotics. Several safenerinducible promoters have been cloned from
maize1. One of these, the In2-2 promoter,
has been fused to a reporter gene and tested
in transgenic Arabidopsis3. The promoter
was inactive in the absence of the safener
benzenesulfonamide (Fig. 1), and induction
occurred only in roots, apical meristems
and hydathodes. Induction was coupled to
retarded growth. After transfer of the plants
to safener-free medium, the promoter was
again inactivated and normal plant development was restored. This feature limits its
application to experiments that require only
transient expression in the above-mentioned
tissues. The In2-2 promoter might still be
valuable for controlling gene expression in
maize, as the expression pattern is more
favourable1. Also, the concentrations of
safeners required for induction do not affect monocots as severely as dicots.
Gene regulation by regulatory elements
from non-plant organisms
Organisms that are distant from plants in
evolutionary terms have developed mechanisms of gene regulation that respond to
H H
external signals that are not usually encountered by higher plants. Antibiotics, for
example, predominantly affect prokaryotes,
which in turn have evolved antibioticRH-5992
Ethanol
inducible resistance genes4. Lactose (or analogues such as IPTG) induces metabolic
Fig. 1. Molecules used for the chemical induction of transgene expression in plants.
genes in enteric bacteria5, but such processes are unlikely to affect plant genes.
Specific steroid hormones control animal
Both strategies have yielded useful tools that respond to a variety development by altering gene expression patterns6, but there is no
of unrelated chemicals (Fig. 1).
equivalent target molecule in plants. If the underlying regulatory
mechanisms involve the binding of only one regulatory protein to
Endogenous plant promoters
specific cis sequences, they can be engineered to work in plants,
A prominent example of a plant promoter that responds to chemi- thus yielding expression systems that respond to highly specific
cal treatment is the PR-1a promoter1. The PR-1a promoter, which chemicals.
drives expression of a defence gene, is normally induced upon
This strategy requires the expression of two genes in transgenic
pathogen attack, forms of oxidative stress and leaf senescence, but plants: the gene encoding the protein responsible for the reguit also responds to benzothiadiazole2 (BTH, Fig. 1). BTH can be lation (transcriptional repressor or activator) and the gene of interapplied by foliar spraying and is available under the trade name est, under the control of a suitable target promoter. This strategy
BION (Novartis). The PR-1a promoter has been used to drive is more complex than the use of endogenous plant promoters,
expression of the Bacillus thuringiensis d-endotoxin in transgenic which only require the transfer of the gene of interest under the
plants. Insect feeding damage was only inhibited in the chemically control of the inducible promoter. The use of heterologous regutreated plants. In those experiments, the alternative inducer iso- latory elements includes systems that respond to the antibiotic
nicotinic acid (INA) was used instead of BTH (Ref. 2). The PR-1a tetracycline (tc, Figs 2 and 3)7,8, the steroid dexamethasone (dx,
promoter can thus be used conditionally to express resistance Fig. 4)9, the copper ion (Fig. 5)10 or IPTG (Ref. 11). Here, we have
genes. As permanent expression of a resistance protein favours focused on their most recent applications, and the use of two novel
the adaptive processes of a pathogen, the PR-1a/BTH system might systems that respond to ethanoll2 and RH-5992 (I. Jepson, pers.
constitute a means of reducing these selection pressures to ensure commun.).
improved pest management. In this context, the fact that the chemical also induces a whole battery of other defence genes may thus The tetracycline-inducible promoter
be tolerated.
The concept of using a regulatory protein from a prokaryote to
For biotechnological applications, commercially certified chemi- control plant gene expression was first realized through the use of
cals are an attractive option for chemical regulation. Safeners are the bacterial repressor protein TetR, which binds to tet operator
agrochemicals that increase the tolerance of plants to herbicides1. DNA only in the absence of its inducer tc (Ref. 1). The TetR–tet
H
OH
O
September 1998, Vol. 3, No. 9
353
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Transactivator
Transcription initiation
complex
Transcription initiation
complex
Transactivator
CaMV
35S promoter tetR
CaMV35Spromoter
tetR
CaMV
35S promoter
CaMV35Spromoter
1
tetVP16
tetR
1
TetR
tTA
2
Transactivator
2
TetR
Gene X
Target promoter
Transcription initiation
complex
tTA
Gene X
No gene
expression
Target promoter
TetR/tc
complex
3
+ tc
Product X
Transactivator
3
Transcription initiation
complex
Gene X
+ tc
tTA/tc
complex
Target promoter
Gene X
Target promoter
4
Product X
4
No gene
expression
Fig. 2. The tetracycline-inducible expression system. (1) The Tn10
encoded Tet repressor (TetR, Mr 24 kDa) is synthesized under the
control of a strong constitutive promoter (e.g. CaMV 35S promoter).
(2) The target promoter (e.g. a modified CaMV 35S promoter)
contains three 19 bp tet operator sites that flank the TATA box.
By binding to these sequences, three TetR dimers interfere with
the assembly of the transcription initiation complex. As the
transcription initiation complex is stabilized by multiple protein–protein interactions, stringent repression requires high levels
of TetR. (3) The antibiotic tc binds to TetR with a high affinity,
abolishing its DNA-binding ability. (4) Under these conditions,
repression is relieved, leading to the synthesis of the gene product.
This system has been shown to work in tobacco, potato and tomato
plants, but not in Arabidopsis, which might not tolerate the amounts
of TetR needed for efficient repression.
Fig. 3. The tetracycline-inactivatable expression system. (1) A
fusion protein (tTA, Mr 38 kDa), consisting of TetR and the
acid activation domain of Herpes simplex protein 16 (VP16), is
expressed under the control of a strong promoter (e.g. CaMV 35S
promoter). (2) The target promoter contains several tet operators
upstream of a short DNA fragment encoding the TATA box.
Multiple binding sites guarantee strong activation owing to the
synergistic effects of multiple tTA proteins. In contrast to TetR
(Fig. 2), tTA does not compete with endogenous transcription
factors for access to the binding sites. Thus considerably less
amounts of tTA are needed compared with TetR. (3) The antibiotic
tc binds to the TetR moiety with high affinity, abolishing its DNAbinding ability. (4) Under these conditions, expression is efficiently
turned off. The system has been shown to work in tobacco and
Arabidopsis.
operator–tc interaction has now been engineered to regulate plant
gene expression (Fig. 2). When using b-glucuronidase as a reporter system, 500- to 800-fold induction is routinely observed, with
induced levels reaching the levels of the CaMV 35S promoter.
The benefits of the tc-inducible promoter were exploited recently
for the conditional overexpression of isopentenyl transferase (ipt),
an enzyme that catalyzes a rate-limiting step in cytokinin biosyn-
thesis13,14. Even small amounts of the ipt protein lead to cytokinininduced morphological changes. This makes the ipt gene an ideal
marker for determining how leaky a regulatory system is in its uninduced state. Untreated transgenic plants appear to be very similar to the wild type, with residual ipt activity being only visible by
the somewhat delayed senescence. Application of tc leads to inactivation of TetR, allowing the synthesis of ipt transcripts at far
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September 1998, Vol. 3, No. 9
trends in plant science
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Transactivator
Transcription initiation
complex
CaMV
35S promoter
CaMV35Spromoter
tf-gr
tetR
Transactivator
Transcription initiation
complex
CaMV
35S promoter
CaMV35Spromoter
1
ace
1
tetR
1
TF-GR
ACE1
HSP90
2
Gene X
HSP90–TF-GR
complex
Target promoter
No gene
expression
2
Gene X
3
Target promoter
+ Cu
No gene
expression
3
Transcription initiation
complex
+ dx
Gene X
Transcription initiation
complex
Gene X
Target promoter
4
ACE1/Cu
complex
Target promoter
4
TF-GR/dx
complex
Product X
Fig. 4. The dexamethasone-inducible expression system. (1) The
fusion protein TF-GR, consisting of a transcription factor (DNA
binding domain and transcriptional activation domain) and the
glucocorticoid binding domain, is expressed under the control of a
strong promoter (e.g. CaMV 35S promoter). (2) In the absence of
dexamethasone the activator is trapped by the formation of an
inactive complex with HSP90. (3) The binding of dexamethasone
mediates dissociation from HSP90 and allows binding of the
activator to a target promoter that contains multiple TF-GR binding
sites upstream of a short DNA fragment that encodes the TATA
box. (4) Transcription from the target promoter is induced. TF can
be any transcription factor that contains a DNA-binding domain and
an activation domain. Aoyama and Chua have used the DNAbinding domain of yeast transcription factor GAL4 and the acid
activation domain of Herpes simplex virus protein 16 (Ref. 9).
higher levels. Constitutive expression triggers a highly aberrant
phenotype, and induction with saturating amounts of tetracycline
(1 mg ml21) is lethal. By using lower amounts of tc (0.1 mg l21), it
proved possible to study the metabolic fate of de novo-synthesized
Product X
Fig. 5. The copper-inducible expression system. (1) Yeast transcription factor ACE1 is expressed under the control of a strong
promoter (e.g. CaMV 35S promoter). (2) In the absence of copper,
it cannot bind to its target promoter. (3) The binding of copper
induces a conformational change, enabling the ACE1 to bind to
specific cis sequences of the target promoter. (4) Transcription from
the target promoter is induced. For the ethanol-inducible system,
ACE1 is replaced by AlcR, and Cu is replaced by ethanol. However,
it is not clear whether ethanol directly binds to AlcR.
cytokinin in different parts of the plant14. In combination with
grafting experiments this system allowed an assessment of the
role of cytokinins as long-range root–shoot signals in the correlative control of apical dominance and sequential leaf senescence in tobacco. The experiments supported the hypothesis that
cytokinin is involved in paracrine signalling14. The system was also
used to obtain transgenic tobacco plants that express high levels of
oat arginine decarboxylase in their leaves15. These experiments
were carried out to investigate the action of specific polyamines in
different developmental processes. A study with transgenic potato
plants has demonstrated that successful antisense RNA experiments can also be performed using this promoter16. The tcinducible gene expression is also effective in both the leaves and
fruits of tomato17. Whether the plants expressing TetR still exhibit
an abnormal phenotype, as described before18, was unclear.
September 1998, Vol. 3, No. 9
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The ease with which the chemical is applied has to be addressed
when discussing chemically inducible promoters. Tetracycline is
most easily applied by infiltration into detached leaves14. When
plants are grown hydroponically, fresh tc has to be added every
other day. Some browning of roots is observed under non-sterile
hydroponic conditions. Soil drenching or application of tc to plants
cultivated in rockwool has also been reported to be successful15,18.
Alternatively, tc can be applied by repeated leaf painting, a procedure that is advantageous for the local induction of single organs
(K. Krupinska, pers. commun.).
The tetracycline-inactivatable promoter
The tc-inactivatable promoter8 is based on the same regulatory
elements as the tc-inducible promoter (see Fig. 3)1. It is especially
useful for the study of protein or RNA stability19: the expression
of a transgene can be specifically turned off, after which the decay
kinetics of the RNA or the protein can be analysed. In addition, it
is a valuable system if the regeneration of transgenic plants is the
only parameter affected by the transgene. In this case, regeneration
can be performed in the presence of tc. Subsequent analysis can be
performed without the chemical, so simplifying the experiments.
The original target promoter Top10 has been shown to be prone to
silencing8. Therefore reconstruction of the promoter is currently
being carried out in an attempt to reduce its susceptibility to this
epigenetic effect (C. Gatz and I. Lenk, unpublished).
control system should be used depends on the experiment: the
approach taken to clone target genes of AP-3 required the posttranscriptional control system because of the simultaneous use of
a protein synthesis inhibitor24.
By exploiting the same principle, but using domains from different proteins, a steroid-inducible promoter has been established
for transgenic maize. This system has been described in the context of an RNA extraction procedure by Pharmacia [Garnaat, C.W.
and Roth, B. (1997) Science Tools from Pharmacia Biotech. 2,
10–11]. The DNA-binding domain and the ligand-binding domain
were taken from the oestrogen receptor, whereas the activation domain was taken from the maize transcription factor C1. The 17aethylenylestradiol-inducible target promoter consists of oestrogen
response elements fused to a minimal plant promoter. By conditionally expressing the male fertility gene MS45 in a male-sterile background, male fertility was chemically controlled. Male sterility is
an important feature for the generation of hybrid seeds, and can be
achieved in the absence of the chemical. However, when fertile
lines are required then fertility can be restored by application of the
inducer.
Different dx treatments have been described: the addition to
medium in experiments using axenically grown plants; induction
of plants in hydroponic culture9; soil drenching23; and the local
treatment of leaves9 and flowers24.
Copper-inducible gene expression
Steroid-inducible promoters
Steroids such as the glucocorticoid dexamethasone (dx) are attractive options as chemical inducers because they exhibit high specificity for the transcriptional activator, the glucocorticoid receptor6.
Glucocorticoid-dependent transcription is based on the inhibitory
interaction between the heat shock protein HSP90 and the ligandbinding domain of the receptor that occurs in the absence of the
ligand20 (Fig. 4). Binding of the ligand leads to dissociation of the
receptor from HSP90 and thus to release of a transcriptional activator. Although the complete receptor protein does not work efficiently in transgenic plants21, the ligand-binding domain has been
shown to confer ligand-dependent activation of cis-located protein
domains. The glucocorticoid receptor binding domain has been
fused to different transcription factors: the maize transcription factor R (Ref. 21), the Arabidopsis transcription factor Athb (Ref. 22),
the flowering-time gene CONSTANS (Ref. 23) and the MADS box
factor AP-3 (Ref. 24). In these examples, the protein product to be
studied was directly controlled by the ligand-binding domain of the
receptor. This post-transcriptional regulatory system is very powerful. By expressing a steroid-regulated version of AP-3 in a suitable
mutant background, the immediate effects of AP-3 on the floral
mRNA population were analyzed by differential display while
secondary effects were blocked by a protein synthesis inhibitor24.
This system has also been modified to establish a dx-inducible
transcriptional control system9 that can be combined with any gene.
Such a system was generated by fusing the glucocorticoid receptor ligand-binding domain to a fusion protein that consists of the
DNA-binding domain of yeast GAL4 and the VP16 activation domain. The target promoter consists of six GAL4 binding sites upstream of the CaMV 35S promoter (Fig. 4). In transgenic tobacco
plants, luciferase activity was induced 100-fold by dx. Because
the background level in the absence of dx was arbitrarily set at 1,
it is unclear whether there is any detectable background activity.
Expression levels in the presence of inducers were not set in relation to the CaMV 35S promoter. Thus the data can be understood
as ‘proof of principle’, but more data, especially on the expression
levels, are required to obtain a good estimate of the efficiency of
the system. Whether the post-transcriptional or the transcriptional
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September 1998, Vol. 3, No. 9
In contrast to tc or dx, copper plays an important role in plant
metabolism: sufficient copper has to be present in the cell to drive
essential biochemical processes, but accumulation to high levels
leads to toxic effects. Nevertheless, a functional copper-inducible
expression system has been established in plants10. It is based on
control elements that regulate the expression of copper detoxification genes in Saccharomyces cerevisiae in response to elevated
copper concentrations. Regulation is mediated by the transcriptional activator ACE1, which binds to specific cis elements only
when coordinated with copper25. This simple mechanism was engineered into plants (Fig. 5). When the activator was driven by the
CaMV 35S promoter, a 50-fold induction of the target promoter
was observed with b-glucuronidase as a reporter enzyme. However,
expression from this construct in roots was constitutive owing to
plant activating sequence-1 left in the target promoter. As expected, elimination of this element abolished the background
activity26. Unexpectedly, the promoter appeared to have lost its
inducibility in leaves, although the data were not reported. This
‘root-specific’ copper-inducible system has been used to conditionally express the ipt gene in transgenic tobacco27. As mentioned
above, very low amounts of ipt lead to a visibly altered phenotype. Transgenic tobacco was morphologically identical to controls under noninducing conditions, indicating that the promoter
is not leaky in the absence of inducer. Following induction by
addition of copper, typical cytokinin effects, such as decreased
apical dominance and delayed senescence, were observed. These
changes are considered to be minor in comparison with the aberrant phenotype that elevated ipt levels normally elicit. These
minor effects support the notion that the promoter is not highly
induced in leaves. It is not clear whether the phenotypic effects
resulted from residual copper inducible gene expression in the
aerial parts of the plant or from the transport of cytokinins from
induced roots to the shoot.
By placing ACE1 under the control of the nodule-specific nod
45 promoter, the function of the system in an organ-specific manner was demonstrated26. In nodulated Lotus corniculatus plants,
which consisted of non-transformed shoots plus transformed hairy
root tissue, b-glucuronidase expression was specifically localized
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in nodules and not in roots after the addition of copper ions to the
nutrient solution. The nodule-specific system was used to express
antisense constructs of aspartate aminotransferase-P2 in transgenic L. corniculatus plants. When expression was induced, the
aspartate aminotransferase-P2 activity declined dramatically, and
a decrease in nodule asparagine concentration of up to 90% was
observed. Whether the inducing amounts of copper are specific to
the transgene only is not clear, and it is possible that detoxification
mechanisms are also activated.
Ethanol-inducible gene expression
A promising alternative to the systems described above has recently been established12. This novel promoter is based on the regulatory elements of the Aspergillus nidulans alcA promoter, which
is strongly inducible by ethanol. It is the most widely used promoter for overexpressing proteins in A. nidulans and other filamentous fungi, both for fundamental research and for applied
biotechnology28. The transcriptional activator AlcR, a DNA binding protein belonging to the C6 zinc binuclear cluster family,
binds to its target sequences within the alcA promoter when cells
are grown in the presence of ethanol or other inducers such as ethyl
methyl ketone29. The system was adapted for plants by placing the
alcR coding region under the control of the CaMV 35S promoter.
The target promoter contains the TATA box as well as upstream
sequences of the alcA promoter fused to position 223 of the CaMV
35S promoter12. When stably transformed into tobacco these constructs mediate ethanol-dependent expression of transgenes. It is
not known if the DNA-binding activity is directly or indirectly
affected by ethanol, but the principle of the regulation is similar to
that of the copper inducible promoter (Fig. 5). The promoter activity in the induced state is in the range of the CaMV 35S promoter.
As this system has only recently been developed, some questions
concerning the specificity of the inducer for the transgene, toxicity,
background levels (especially under anaerobic conditions) and induction levels over a longer time course have yet to be addressed.
As the system is put to greater use, the extent of its usefulness will
emerge.
The system has already been used to analyse carbon metabolism by conditionally expressing cytosolic invertase in transgenic
plants12. Constitutive expression of high levels of cytosolic invertase prevents plant maturation as chlorosis develops in the sink
leaves; plants conditionally expressing invertase grew normally
until induced with ethanol. Within four days of induction, the phenotype of the youngest leaves was severely affected.
Although spraying seems to work12, ethanol appears to be effective if taken up through the roots, either in hydroponic culture
using 0.1% ethanol, or by soil drenching with 1% ethanol. The
local induction of single leaves is not yet possible because ethanol
vapour induces the whole plant and its neighbours (U. Sonnewald,
pers. commun.). Caddick et al. are optimistic that the system
might function in field applications12. It seems feasible that efficient ethanol formulations or non-volatile inducers will be developed in the future. This would constitute a real breakthrough for
agricultural biotechnology as chemically inducible promoters are
still lacking. Neither the tc- nor the dx-inducible system are as
broadly applicable.
Insecticide-inducible gene expression
An important step towards the development of a chemically inducible promoter for applications in the field utilizes a novel chemically inducible expression system that responds to the already
certified agrochemical RH5992 (Fig. 1) (I. Jepson, pers. commun.).
The non-steroidal chemical RH5992 is effective as a lepidopteranspecific insecticide and acts by agonizing the effect of the insect
hormone ecdysone. The ecdysone receptor from Heliothis virescence was cloned by PCR, using degenerate oligonucleotides
directed against the DNA-binding domain of the animal steroid/
thyroid superfamily. A chimeric transcriptional activator was
assembled using the DNA-binding domain of the glucocorticoid
receptor, the VP16 activation domain and the ligand-binding domain of the H. virescence protein. Transcription from a suitable
target promoter was RH5992-responsive. Because the H. virescence
protein has not been studied as thoroughly as the mammalian
glucocorticoid receptor, any possible induction mechanism is still
highly speculative. Overall, the approach is very attractive because it uses a chemical that has already been tested for its environmental compatibility, but its practical effectiveness has yet
to be established.
Future prospects
It is feasible that chemically inducible promoters could be constructed using novel regulatory elements from organisms such as
E. coli, mammals, fungi and insects, but various questions remain
unanswered.
• How leaky are these promoters? Statistically relevant figures
based on either b-glucuronidase or luciferase activity have to be
presented; measurements close to background levels are often
difficult to quantify. The ipt gene, which gives rise to phenotypical changes even at very low expression levels, provides a
sensitive reporter system for these analyses.
• How do these promoters compare with the CaMV 35S promoter
in the activated state?
• How homogenous is induction in different organs?
• To what extent do the induction kinetics depend on the size of
the plant? Are lower leaves more rapidly induced than upper
leaves, or vice versa?
• How fast does the response decline after omission of the inducer, and how does this depend on the duration of induction?
• Is repeated application of the inducer necessary, even after long
exposure of the plants to the chemical?
• At what concentration is the inducer toxic? How does this compare with the dose–response curve of expression? Measurements made on photosystem II might be a good criterion for
evaluating the vigour or fitness of the plants treated with the
chemical.
• Does the chemical induce genes involved in the detoxification
of xenobiotics?
• Does the transcriptional activator cause adverse effects in the
plant? It has been reported that the VP16 domain might not be
tolerated in higher amounts30.
• How stable is gene expression, both somatically and meiotically?
A comparative analysis of the different promoters under standardized conditions, will help in the evolution of transgene design.
Several additional refinements can be added to these systems.
By combining activators with tissue-specific promoters it will be
possible to induce gene expression in a given tissue. One possible
development would be to generate a collection of activator plants
(e.g. tobacco and Arabidopsis) that express the activator protein
under the control of different tissue-specific or developmentally
regulated promoters. After crossing ‘activator plants’ with plants
encoding the gene of interest under the control of the target promoter (‘target plants’), gene expression will be inducible in the next
generation. In contrast to the recently described activator LhG4,
which consists of the Lac repressor and the GAL4 activation domain31, regulated activators will allow gene expression to be activated, or inactivated, by chemical inducers. Genomics will yield
ever more sequences of interest, the function of which might be
analysed by inducible expression.
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2 Gorlach, J. et al. (1996) Benzothiadiazole, a novel class of inducers of
systemic acquired resistance, activates gene expression and disease resistance
in wheat, Plant Cell 8, 629–643
3 De Veylder, L., Van Montagu, M. and Inzé, D. (1997) Herbicide safenerinducible gene expression in Arabidopsis thaliana, Plant Cell Physiol. 38,
568–577
4 Hillen, W. and Berens, C. (1994) Mechanisms underlying expression of Tn10
encoded tetracycline resistance, Annu. Rev. Microbiol. 48, 345–369
5 Beckwith, J.R. and Zipser, D. (1970) The Lactose Operon, Cold Spring
Harbor Laboratory Press
6 Evans, R.M. (1988) The steroid and thyroid hormone receptor superfamily,
Science 240, 889–895
7 Gatz, C., Frohberg, C. and Wendenburg, R. (1992) Stringent repression and
homogeneous de-repression by tetracycline of a modified CaMV 35S
promoter in intact transgenic tobacco plants, Plant J. 2, 397–404
8 Weinmann, P. et al. (1994) A chimeric transactivator allows tetracyclineresponsive gene expression in whole plants, Plant J. 5, 559–569
9 Aoyama, T. and Chua, N-H. (1997) A glucocorticoid-mediated transcriptional
induction system in transgenic plants, Plant J. 11, 605–612
10 Mett, V.L., Lochhead, L.B. and Reynolds, P.H.S. (1993) Copper controllable
gene expression system for whole plants, Proc. Natl. Acad. Sci. U. S. A. 90,
4567–4571
11 Wilde, R.J. et al. (1992) Control of gene expression in tobacco cells using a
bacterial operator–repressor system, EMBO J. 11, 1251–1259
12 Caddick, M.X. et al. (1998) An ethanol inducible gene switch for plants used
to manipulate carbon metabolism, Nat. Biotechnol. 16, 177–180
13 Motyka, V. et al. (1996) Changes in cytokinin content and cytokinin oxidase
activity in response to derepression of ipt gene transcription in transgenic
tobacco calli and plants, Plant Physiol. 112, 1035–1043
14 Faiss, M. et al. (1997) Conditional transgenic expression of the ipt gene
indicates a function for cytokinins in paracrine signaling in whole tobacco
plants, Plant J. 12, 401–415
15 Masgrau, C. et al. (1996) Inducible overexpression of oat arginine
decarboxylase in transgenic tobacco plants, Plant J. 11, 465–473
16 Kumar, A. et al. (1995) Potato plants expressing antisense and sense
S-adenosylmethionine decarboxylase (SAMDC) transgenes show altered
levels of polyamines and ethylene: antisense plants display abnormal
phenotypes, Plant J. 9, 147–158
17 Thompson A.J. and Myatt, S.C. (1997) Tetracycline-dependent activation of
an upstream promoter reveals transcriptional interference between tandem
genes within T-DNA in tomato, Plant Mol. Biol. 34, 687–692
18 Corlett, J.E., Myatt, S.C. and Thompson, A.J. (1996) Toxicity symptoms
caused by high expression of Tet repressor in tomato (Lycopersicon esculentum
Mill. L.) are alleviated by tetracycline, Plant Cell Environ. 19, 447–454
19 Gil, P. and Green, P.J. (1995) Multiple regions of the Arabidopsis SAUR-AC1
gene control transcript abundance: the 3′ untranslated region functions as an
mRNA instability determinant, EMBO J. 15, 1678–1686
20 Picard, D. (1994) Regulation of protein function through expression of
chimaeric proteins, Curr. Top. Biotechnol. 5, 511–515
21 Lloyd, A.M. et al. (1994) Epidermal cell fate determination in Arabidopsis:
patterns defined by a steroid-inducible regulator, Science 266, 436–439
22 Aoyama, T. et al. (1995) Ectopic expression of the Arabidopsis transcriptional
activator Athb-1 alters leaf cell fate in tobacco, Plant Cell 7, 1773–1785
23 Simon, R., Igeno, M.I. and Coupland, G. (1996) Activation of floral meristem
identity genes in Arabidopsis, Nature 384, 59–62
24 Sablowski, R.W.M. and Meyerowitz, E.M. (1998) A homolog of NO APICAL
MERISTEM is an immediate target of the floral homeotic genes
APETALA3/PISTILLATA, Cell 92, 93–103
25 Dameron, C.T. et al. (1991) A copper-thiolate polynuclear cluster in the ACE1
transcription factor, Proc. Natl. Acad. Sci. U. S. A. 88, 6127–6131
26 Mett, V.L. et al. (1996) A system for tissue-specific copper-controllable gene
expression in transgenic plants: nodule-specific antisense of aspartate
aminotransferase-P2, Transgenic Res. 5, 105–113
27 McKenzie, M.J. et al. (1998) Controlled cytokinin production in transgenic
tobacco using a copper-inducible promoter, Plant Physiol. 116, 969–977
28 Felenbok, M. (1991) The ethanol utilization regulon of Aspergillus nidulans:
the alcA–AlcR system as a tool for expression of recombinant proteins,
J. Biotechnol. 17, 11–18
29 Panozzo, C. et al. (1997) The zinc binuclear cluster activator AlcR is able to
bind to single sites but requires multiple repeated sites for synergistic
activation of the alcA gene in Aspergillus nidulans, J. Biol. Chem. 272,
22859–22865
30 Berger, S.L. et al. (1990) Selective inhibition of activated but not basal
transcription by the acidic activation domain of VP16: evidence for
transcriptional adaptors, Cell 61, 1199–1208
31 Moore, I. et al. (1998) A transcription activation system for regulated gene
expression in transgenic plants, Proc. Natl. Acad. Sci. U. S. A. 95, 376–381
Christiane Gatz* and Ingo Lenk are at the Albrecht von Haller
Institute of Plant Sciences, University of Goettingen,
Untere Karspuele 2, 37073 Goettingen, Germany.
*Author for correspondence (tel +49 551 397843;
fax +49 551 397920; e-mail cgatz@gwdg.de).
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358
September 1998, Vol. 3, No. 9
reviews
Promoters that respond to chemical
inducers
Christiane Gatz and Ingo Lenk
The introduction of foreign genes constitutes a powerful tool with which to study and
improve the genetic resources of plants. Transgene expression levels and expression patterns can be adjusted by combining the protein coding region with a suitable promoter. There
is a diverse spectrum of endogenous plant promoters and these are currently being broadened by the development of chimeric promoters that respond to otherwise inactive
chemicals. This range includes promoters that respond to inducers such as the antibiotic
tetracycline, the steroid dexamethasone, the copper ion, ethanol or the agrochemical RH-5992.
These chimeric promoters offer a range of options for transgene design for experimental and
field use.
T
he combination of biochemical, physiological and genetic
studies in genetically engineered transgenic plants has proven to be extremely useful for analysing biochemical and
developmental processes. Examples include the enhanced or ectopic expression of enzymes and regulatory proteins, and the expression of antisense RNA or dominant negative proteins that
reduce the amount of a gene product. The availability of a broad
spectrum of promoters that differ in their ability to regulate the
temporal and spatial expression patterns, dramatically increases
the application of transgenic technology.
In this context, promoters that are only activated in response to
a specific chemical are particularly valuable. In E. coli, an example of a promoter that provides such fidelity is the IPTG inducible
lac promoter, which is routinely used whenever expression of the
recombinant protein is going to interfere with growth. Similarly,
if a foreign gene product expressed in plants is going to interfere
with regeneration, growth or reproduction, an inducible promoter
is required. With such a tool, plants can be regenerated while the
promoter is inactive. Further analysis can then be performed after
activating expression of the transgene. When working with higher
plants, arguments other than the lethality of the gene product may
Box 1. Applications of chemically
inducible promoters
Basic research
• Expression of gene products that interfere with regeneration,
growth or reproduction.
• Expression of gene products at different stages of development.
• Differentiation between primary and secondary effects.
• Analysis of primary effects before homeostatic mechanisms
start to counteract.
• Clear correlation between induction of the transgene and occurrence of an altered phenotype.
Biotechnological applications
• Construction of a conditional male sterility system.
• Expression of transgenes that interfere with regeneration,
growth or reproduction (biofarming).
• Conditional expression of resistance genes as a means of pest
management to delay adaptive processes of the pathogen.
• Simultaneous induction of processes such as flowering and leaf
abscission.
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September 1998, Vol. 3, No. 9
favour the use of an inducible promoter over a constitutive one
(Box 1). Moreover, in addition to being a valuable means of elucidating gene function, chemically inducible promoters are increasingly relevant to the improvement of crops by genetic engineering.
Examples of biotechnological applications are listed in Box 1.
If only very low amounts of a gene product are required for a
cellular process, then the expression level of the promoter should
be close to zero in the absence of the inducer. In this case, high
expression levels in the presence of the inducer are not an essential requirement. In contrast, if only high amounts of a gene product are effective, residual activity in the absence of the inducer can
be tolerated, but it should be inducible to high levels in the presence of the inducer. Ideally, both features – very low expression
levels in the absence of the inducer, and high expression levels in
the presence of the inducer – should be combined in one system as
it is more broadly applicable. A further advance in use of a chemically inducible promoter is its use in combination with tissue specific promoters, so that gene expression can be restricted to a given
tissue at a specific time.
It is important that the chemical used is highly specific for the
target promoter: it should neither influence the expression of other
genes nor affect other cell functions. Uptake is another important
issue: once it is applied, either by foliar spray or root drenching,
the chemical should enter every cell. Additional requirements that
an ideal inducer should meet are given in Box 2.
To provide such tools, two different strategies are being adopted1.
First, plant promoters that respond to a given chemical have been
isolated: obviously, this concept will provide expression systems
that are not specific to the transgene alone. The second strategy
involves using regulatory elements from other organisms that
respond to chemicals that are not usually encountered by plants.
Box 2. Properties required for an ideal inducer
•
•
•
•
•
•
High specificity for the transgene, no toxicity.
High environmental compatibility.
Convenient application by foliar spraying or root drenching.
High efficiency at low concentrations and low use rates.
Low costs.
Availability of different derivatives with different properties:
inducing chemicals; inactivating chemicals; chemicals that
move systemically; chemicals that are confined to the site of
application; chemicals with different half-lives in the plant.
Copyright © 1998 Elsevier Science Ltd. All rights reserved. 1360 - 1385/98/$19.00 PII: S1360-1385(98)01287-4
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O
C
Cl
SCH3
O
S
O
S
N
NH
C
NH2
O
N
Benzothiadiazole
Safener
(BTH)
N-(aminocarbonyl)-
2-chlorobenzenesulfonamide
HO
O
N
HO
OH
HO
OH
H
OH
NH2
OH
O
OH
O
O
F
O
Tetracycline
Dexamethasone
O
H
H
C
C
H
N NH
They appear to induce a smaller set of genes
as compared with BTH, most likely those
genes that encode enzymes responsible for
detoxifying xenobiotics. Several safenerinducible promoters have been cloned from
maize1. One of these, the In2-2 promoter,
has been fused to a reporter gene and tested
in transgenic Arabidopsis3. The promoter
was inactive in the absence of the safener
benzenesulfonamide (Fig. 1), and induction
occurred only in roots, apical meristems
and hydathodes. Induction was coupled to
retarded growth. After transfer of the plants
to safener-free medium, the promoter was
again inactivated and normal plant development was restored. This feature limits its
application to experiments that require only
transient expression in the above-mentioned
tissues. The In2-2 promoter might still be
valuable for controlling gene expression in
maize, as the expression pattern is more
favourable1. Also, the concentrations of
safeners required for induction do not affect monocots as severely as dicots.
Gene regulation by regulatory elements
from non-plant organisms
Organisms that are distant from plants in
evolutionary terms have developed mechanisms of gene regulation that respond to
H H
external signals that are not usually encountered by higher plants. Antibiotics, for
example, predominantly affect prokaryotes,
which in turn have evolved antibioticRH-5992
Ethanol
inducible resistance genes4. Lactose (or analogues such as IPTG) induces metabolic
Fig. 1. Molecules used for the chemical induction of transgene expression in plants.
genes in enteric bacteria5, but such processes are unlikely to affect plant genes.
Specific steroid hormones control animal
Both strategies have yielded useful tools that respond to a variety development by altering gene expression patterns6, but there is no
of unrelated chemicals (Fig. 1).
equivalent target molecule in plants. If the underlying regulatory
mechanisms involve the binding of only one regulatory protein to
Endogenous plant promoters
specific cis sequences, they can be engineered to work in plants,
A prominent example of a plant promoter that responds to chemi- thus yielding expression systems that respond to highly specific
cal treatment is the PR-1a promoter1. The PR-1a promoter, which chemicals.
drives expression of a defence gene, is normally induced upon
This strategy requires the expression of two genes in transgenic
pathogen attack, forms of oxidative stress and leaf senescence, but plants: the gene encoding the protein responsible for the reguit also responds to benzothiadiazole2 (BTH, Fig. 1). BTH can be lation (transcriptional repressor or activator) and the gene of interapplied by foliar spraying and is available under the trade name est, under the control of a suitable target promoter. This strategy
BION (Novartis). The PR-1a promoter has been used to drive is more complex than the use of endogenous plant promoters,
expression of the Bacillus thuringiensis d-endotoxin in transgenic which only require the transfer of the gene of interest under the
plants. Insect feeding damage was only inhibited in the chemically control of the inducible promoter. The use of heterologous regutreated plants. In those experiments, the alternative inducer iso- latory elements includes systems that respond to the antibiotic
nicotinic acid (INA) was used instead of BTH (Ref. 2). The PR-1a tetracycline (tc, Figs 2 and 3)7,8, the steroid dexamethasone (dx,
promoter can thus be used conditionally to express resistance Fig. 4)9, the copper ion (Fig. 5)10 or IPTG (Ref. 11). Here, we have
genes. As permanent expression of a resistance protein favours focused on their most recent applications, and the use of two novel
the adaptive processes of a pathogen, the PR-1a/BTH system might systems that respond to ethanoll2 and RH-5992 (I. Jepson, pers.
constitute a means of reducing these selection pressures to ensure commun.).
improved pest management. In this context, the fact that the chemical also induces a whole battery of other defence genes may thus The tetracycline-inducible promoter
be tolerated.
The concept of using a regulatory protein from a prokaryote to
For biotechnological applications, commercially certified chemi- control plant gene expression was first realized through the use of
cals are an attractive option for chemical regulation. Safeners are the bacterial repressor protein TetR, which binds to tet operator
agrochemicals that increase the tolerance of plants to herbicides1. DNA only in the absence of its inducer tc (Ref. 1). The TetR–tet
H
OH
O
September 1998, Vol. 3, No. 9
353
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Transactivator
Transcription initiation
complex
Transcription initiation
complex
Transactivator
CaMV
35S promoter tetR
CaMV35Spromoter
tetR
CaMV
35S promoter
CaMV35Spromoter
1
tetVP16
tetR
1
TetR
tTA
2
Transactivator
2
TetR
Gene X
Target promoter
Transcription initiation
complex
tTA
Gene X
No gene
expression
Target promoter
TetR/tc
complex
3
+ tc
Product X
Transactivator
3
Transcription initiation
complex
Gene X
+ tc
tTA/tc
complex
Target promoter
Gene X
Target promoter
4
Product X
4
No gene
expression
Fig. 2. The tetracycline-inducible expression system. (1) The Tn10
encoded Tet repressor (TetR, Mr 24 kDa) is synthesized under the
control of a strong constitutive promoter (e.g. CaMV 35S promoter).
(2) The target promoter (e.g. a modified CaMV 35S promoter)
contains three 19 bp tet operator sites that flank the TATA box.
By binding to these sequences, three TetR dimers interfere with
the assembly of the transcription initiation complex. As the
transcription initiation complex is stabilized by multiple protein–protein interactions, stringent repression requires high levels
of TetR. (3) The antibiotic tc binds to TetR with a high affinity,
abolishing its DNA-binding ability. (4) Under these conditions,
repression is relieved, leading to the synthesis of the gene product.
This system has been shown to work in tobacco, potato and tomato
plants, but not in Arabidopsis, which might not tolerate the amounts
of TetR needed for efficient repression.
Fig. 3. The tetracycline-inactivatable expression system. (1) A
fusion protein (tTA, Mr 38 kDa), consisting of TetR and the
acid activation domain of Herpes simplex protein 16 (VP16), is
expressed under the control of a strong promoter (e.g. CaMV 35S
promoter). (2) The target promoter contains several tet operators
upstream of a short DNA fragment encoding the TATA box.
Multiple binding sites guarantee strong activation owing to the
synergistic effects of multiple tTA proteins. In contrast to TetR
(Fig. 2), tTA does not compete with endogenous transcription
factors for access to the binding sites. Thus considerably less
amounts of tTA are needed compared with TetR. (3) The antibiotic
tc binds to the TetR moiety with high affinity, abolishing its DNAbinding ability. (4) Under these conditions, expression is efficiently
turned off. The system has been shown to work in tobacco and
Arabidopsis.
operator–tc interaction has now been engineered to regulate plant
gene expression (Fig. 2). When using b-glucuronidase as a reporter system, 500- to 800-fold induction is routinely observed, with
induced levels reaching the levels of the CaMV 35S promoter.
The benefits of the tc-inducible promoter were exploited recently
for the conditional overexpression of isopentenyl transferase (ipt),
an enzyme that catalyzes a rate-limiting step in cytokinin biosyn-
thesis13,14. Even small amounts of the ipt protein lead to cytokinininduced morphological changes. This makes the ipt gene an ideal
marker for determining how leaky a regulatory system is in its uninduced state. Untreated transgenic plants appear to be very similar to the wild type, with residual ipt activity being only visible by
the somewhat delayed senescence. Application of tc leads to inactivation of TetR, allowing the synthesis of ipt transcripts at far
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September 1998, Vol. 3, No. 9
trends in plant science
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Transactivator
Transcription initiation
complex
CaMV
35S promoter
CaMV35Spromoter
tf-gr
tetR
Transactivator
Transcription initiation
complex
CaMV
35S promoter
CaMV35Spromoter
1
ace
1
tetR
1
TF-GR
ACE1
HSP90
2
Gene X
HSP90–TF-GR
complex
Target promoter
No gene
expression
2
Gene X
3
Target promoter
+ Cu
No gene
expression
3
Transcription initiation
complex
+ dx
Gene X
Transcription initiation
complex
Gene X
Target promoter
4
ACE1/Cu
complex
Target promoter
4
TF-GR/dx
complex
Product X
Fig. 4. The dexamethasone-inducible expression system. (1) The
fusion protein TF-GR, consisting of a transcription factor (DNA
binding domain and transcriptional activation domain) and the
glucocorticoid binding domain, is expressed under the control of a
strong promoter (e.g. CaMV 35S promoter). (2) In the absence of
dexamethasone the activator is trapped by the formation of an
inactive complex with HSP90. (3) The binding of dexamethasone
mediates dissociation from HSP90 and allows binding of the
activator to a target promoter that contains multiple TF-GR binding
sites upstream of a short DNA fragment that encodes the TATA
box. (4) Transcription from the target promoter is induced. TF can
be any transcription factor that contains a DNA-binding domain and
an activation domain. Aoyama and Chua have used the DNAbinding domain of yeast transcription factor GAL4 and the acid
activation domain of Herpes simplex virus protein 16 (Ref. 9).
higher levels. Constitutive expression triggers a highly aberrant
phenotype, and induction with saturating amounts of tetracycline
(1 mg ml21) is lethal. By using lower amounts of tc (0.1 mg l21), it
proved possible to study the metabolic fate of de novo-synthesized
Product X
Fig. 5. The copper-inducible expression system. (1) Yeast transcription factor ACE1 is expressed under the control of a strong
promoter (e.g. CaMV 35S promoter). (2) In the absence of copper,
it cannot bind to its target promoter. (3) The binding of copper
induces a conformational change, enabling the ACE1 to bind to
specific cis sequences of the target promoter. (4) Transcription from
the target promoter is induced. For the ethanol-inducible system,
ACE1 is replaced by AlcR, and Cu is replaced by ethanol. However,
it is not clear whether ethanol directly binds to AlcR.
cytokinin in different parts of the plant14. In combination with
grafting experiments this system allowed an assessment of the
role of cytokinins as long-range root–shoot signals in the correlative control of apical dominance and sequential leaf senescence in tobacco. The experiments supported the hypothesis that
cytokinin is involved in paracrine signalling14. The system was also
used to obtain transgenic tobacco plants that express high levels of
oat arginine decarboxylase in their leaves15. These experiments
were carried out to investigate the action of specific polyamines in
different developmental processes. A study with transgenic potato
plants has demonstrated that successful antisense RNA experiments can also be performed using this promoter16. The tcinducible gene expression is also effective in both the leaves and
fruits of tomato17. Whether the plants expressing TetR still exhibit
an abnormal phenotype, as described before18, was unclear.
September 1998, Vol. 3, No. 9
355
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The ease with which the chemical is applied has to be addressed
when discussing chemically inducible promoters. Tetracycline is
most easily applied by infiltration into detached leaves14. When
plants are grown hydroponically, fresh tc has to be added every
other day. Some browning of roots is observed under non-sterile
hydroponic conditions. Soil drenching or application of tc to plants
cultivated in rockwool has also been reported to be successful15,18.
Alternatively, tc can be applied by repeated leaf painting, a procedure that is advantageous for the local induction of single organs
(K. Krupinska, pers. commun.).
The tetracycline-inactivatable promoter
The tc-inactivatable promoter8 is based on the same regulatory
elements as the tc-inducible promoter (see Fig. 3)1. It is especially
useful for the study of protein or RNA stability19: the expression
of a transgene can be specifically turned off, after which the decay
kinetics of the RNA or the protein can be analysed. In addition, it
is a valuable system if the regeneration of transgenic plants is the
only parameter affected by the transgene. In this case, regeneration
can be performed in the presence of tc. Subsequent analysis can be
performed without the chemical, so simplifying the experiments.
The original target promoter Top10 has been shown to be prone to
silencing8. Therefore reconstruction of the promoter is currently
being carried out in an attempt to reduce its susceptibility to this
epigenetic effect (C. Gatz and I. Lenk, unpublished).
control system should be used depends on the experiment: the
approach taken to clone target genes of AP-3 required the posttranscriptional control system because of the simultaneous use of
a protein synthesis inhibitor24.
By exploiting the same principle, but using domains from different proteins, a steroid-inducible promoter has been established
for transgenic maize. This system has been described in the context of an RNA extraction procedure by Pharmacia [Garnaat, C.W.
and Roth, B. (1997) Science Tools from Pharmacia Biotech. 2,
10–11]. The DNA-binding domain and the ligand-binding domain
were taken from the oestrogen receptor, whereas the activation domain was taken from the maize transcription factor C1. The 17aethylenylestradiol-inducible target promoter consists of oestrogen
response elements fused to a minimal plant promoter. By conditionally expressing the male fertility gene MS45 in a male-sterile background, male fertility was chemically controlled. Male sterility is
an important feature for the generation of hybrid seeds, and can be
achieved in the absence of the chemical. However, when fertile
lines are required then fertility can be restored by application of the
inducer.
Different dx treatments have been described: the addition to
medium in experiments using axenically grown plants; induction
of plants in hydroponic culture9; soil drenching23; and the local
treatment of leaves9 and flowers24.
Copper-inducible gene expression
Steroid-inducible promoters
Steroids such as the glucocorticoid dexamethasone (dx) are attractive options as chemical inducers because they exhibit high specificity for the transcriptional activator, the glucocorticoid receptor6.
Glucocorticoid-dependent transcription is based on the inhibitory
interaction between the heat shock protein HSP90 and the ligandbinding domain of the receptor that occurs in the absence of the
ligand20 (Fig. 4). Binding of the ligand leads to dissociation of the
receptor from HSP90 and thus to release of a transcriptional activator. Although the complete receptor protein does not work efficiently in transgenic plants21, the ligand-binding domain has been
shown to confer ligand-dependent activation of cis-located protein
domains. The glucocorticoid receptor binding domain has been
fused to different transcription factors: the maize transcription factor R (Ref. 21), the Arabidopsis transcription factor Athb (Ref. 22),
the flowering-time gene CONSTANS (Ref. 23) and the MADS box
factor AP-3 (Ref. 24). In these examples, the protein product to be
studied was directly controlled by the ligand-binding domain of the
receptor. This post-transcriptional regulatory system is very powerful. By expressing a steroid-regulated version of AP-3 in a suitable
mutant background, the immediate effects of AP-3 on the floral
mRNA population were analyzed by differential display while
secondary effects were blocked by a protein synthesis inhibitor24.
This system has also been modified to establish a dx-inducible
transcriptional control system9 that can be combined with any gene.
Such a system was generated by fusing the glucocorticoid receptor ligand-binding domain to a fusion protein that consists of the
DNA-binding domain of yeast GAL4 and the VP16 activation domain. The target promoter consists of six GAL4 binding sites upstream of the CaMV 35S promoter (Fig. 4). In transgenic tobacco
plants, luciferase activity was induced 100-fold by dx. Because
the background level in the absence of dx was arbitrarily set at 1,
it is unclear whether there is any detectable background activity.
Expression levels in the presence of inducers were not set in relation to the CaMV 35S promoter. Thus the data can be understood
as ‘proof of principle’, but more data, especially on the expression
levels, are required to obtain a good estimate of the efficiency of
the system. Whether the post-transcriptional or the transcriptional
356
September 1998, Vol. 3, No. 9
In contrast to tc or dx, copper plays an important role in plant
metabolism: sufficient copper has to be present in the cell to drive
essential biochemical processes, but accumulation to high levels
leads to toxic effects. Nevertheless, a functional copper-inducible
expression system has been established in plants10. It is based on
control elements that regulate the expression of copper detoxification genes in Saccharomyces cerevisiae in response to elevated
copper concentrations. Regulation is mediated by the transcriptional activator ACE1, which binds to specific cis elements only
when coordinated with copper25. This simple mechanism was engineered into plants (Fig. 5). When the activator was driven by the
CaMV 35S promoter, a 50-fold induction of the target promoter
was observed with b-glucuronidase as a reporter enzyme. However,
expression from this construct in roots was constitutive owing to
plant activating sequence-1 left in the target promoter. As expected, elimination of this element abolished the background
activity26. Unexpectedly, the promoter appeared to have lost its
inducibility in leaves, although the data were not reported. This
‘root-specific’ copper-inducible system has been used to conditionally express the ipt gene in transgenic tobacco27. As mentioned
above, very low amounts of ipt lead to a visibly altered phenotype. Transgenic tobacco was morphologically identical to controls under noninducing conditions, indicating that the promoter
is not leaky in the absence of inducer. Following induction by
addition of copper, typical cytokinin effects, such as decreased
apical dominance and delayed senescence, were observed. These
changes are considered to be minor in comparison with the aberrant phenotype that elevated ipt levels normally elicit. These
minor effects support the notion that the promoter is not highly
induced in leaves. It is not clear whether the phenotypic effects
resulted from residual copper inducible gene expression in the
aerial parts of the plant or from the transport of cytokinins from
induced roots to the shoot.
By placing ACE1 under the control of the nodule-specific nod
45 promoter, the function of the system in an organ-specific manner was demonstrated26. In nodulated Lotus corniculatus plants,
which consisted of non-transformed shoots plus transformed hairy
root tissue, b-glucuronidase expression was specifically localized
trends in plant science
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in nodules and not in roots after the addition of copper ions to the
nutrient solution. The nodule-specific system was used to express
antisense constructs of aspartate aminotransferase-P2 in transgenic L. corniculatus plants. When expression was induced, the
aspartate aminotransferase-P2 activity declined dramatically, and
a decrease in nodule asparagine concentration of up to 90% was
observed. Whether the inducing amounts of copper are specific to
the transgene only is not clear, and it is possible that detoxification
mechanisms are also activated.
Ethanol-inducible gene expression
A promising alternative to the systems described above has recently been established12. This novel promoter is based on the regulatory elements of the Aspergillus nidulans alcA promoter, which
is strongly inducible by ethanol. It is the most widely used promoter for overexpressing proteins in A. nidulans and other filamentous fungi, both for fundamental research and for applied
biotechnology28. The transcriptional activator AlcR, a DNA binding protein belonging to the C6 zinc binuclear cluster family,
binds to its target sequences within the alcA promoter when cells
are grown in the presence of ethanol or other inducers such as ethyl
methyl ketone29. The system was adapted for plants by placing the
alcR coding region under the control of the CaMV 35S promoter.
The target promoter contains the TATA box as well as upstream
sequences of the alcA promoter fused to position 223 of the CaMV
35S promoter12. When stably transformed into tobacco these constructs mediate ethanol-dependent expression of transgenes. It is
not known if the DNA-binding activity is directly or indirectly
affected by ethanol, but the principle of the regulation is similar to
that of the copper inducible promoter (Fig. 5). The promoter activity in the induced state is in the range of the CaMV 35S promoter.
As this system has only recently been developed, some questions
concerning the specificity of the inducer for the transgene, toxicity,
background levels (especially under anaerobic conditions) and induction levels over a longer time course have yet to be addressed.
As the system is put to greater use, the extent of its usefulness will
emerge.
The system has already been used to analyse carbon metabolism by conditionally expressing cytosolic invertase in transgenic
plants12. Constitutive expression of high levels of cytosolic invertase prevents plant maturation as chlorosis develops in the sink
leaves; plants conditionally expressing invertase grew normally
until induced with ethanol. Within four days of induction, the phenotype of the youngest leaves was severely affected.
Although spraying seems to work12, ethanol appears to be effective if taken up through the roots, either in hydroponic culture
using 0.1% ethanol, or by soil drenching with 1% ethanol. The
local induction of single leaves is not yet possible because ethanol
vapour induces the whole plant and its neighbours (U. Sonnewald,
pers. commun.). Caddick et al. are optimistic that the system
might function in field applications12. It seems feasible that efficient ethanol formulations or non-volatile inducers will be developed in the future. This would constitute a real breakthrough for
agricultural biotechnology as chemically inducible promoters are
still lacking. Neither the tc- nor the dx-inducible system are as
broadly applicable.
Insecticide-inducible gene expression
An important step towards the development of a chemically inducible promoter for applications in the field utilizes a novel chemically inducible expression system that responds to the already
certified agrochemical RH5992 (Fig. 1) (I. Jepson, pers. commun.).
The non-steroidal chemical RH5992 is effective as a lepidopteranspecific insecticide and acts by agonizing the effect of the insect
hormone ecdysone. The ecdysone receptor from Heliothis virescence was cloned by PCR, using degenerate oligonucleotides
directed against the DNA-binding domain of the animal steroid/
thyroid superfamily. A chimeric transcriptional activator was
assembled using the DNA-binding domain of the glucocorticoid
receptor, the VP16 activation domain and the ligand-binding domain of the H. virescence protein. Transcription from a suitable
target promoter was RH5992-responsive. Because the H. virescence
protein has not been studied as thoroughly as the mammalian
glucocorticoid receptor, any possible induction mechanism is still
highly speculative. Overall, the approach is very attractive because it uses a chemical that has already been tested for its environmental compatibility, but its practical effectiveness has yet
to be established.
Future prospects
It is feasible that chemically inducible promoters could be constructed using novel regulatory elements from organisms such as
E. coli, mammals, fungi and insects, but various questions remain
unanswered.
• How leaky are these promoters? Statistically relevant figures
based on either b-glucuronidase or luciferase activity have to be
presented; measurements close to background levels are often
difficult to quantify. The ipt gene, which gives rise to phenotypical changes even at very low expression levels, provides a
sensitive reporter system for these analyses.
• How do these promoters compare with the CaMV 35S promoter
in the activated state?
• How homogenous is induction in different organs?
• To what extent do the induction kinetics depend on the size of
the plant? Are lower leaves more rapidly induced than upper
leaves, or vice versa?
• How fast does the response decline after omission of the inducer, and how does this depend on the duration of induction?
• Is repeated application of the inducer necessary, even after long
exposure of the plants to the chemical?
• At what concentration is the inducer toxic? How does this compare with the dose–response curve of expression? Measurements made on photosystem II might be a good criterion for
evaluating the vigour or fitness of the plants treated with the
chemical.
• Does the chemical induce genes involved in the detoxification
of xenobiotics?
• Does the transcriptional activator cause adverse effects in the
plant? It has been reported that the VP16 domain might not be
tolerated in higher amounts30.
• How stable is gene expression, both somatically and meiotically?
A comparative analysis of the different promoters under standardized conditions, will help in the evolution of transgene design.
Several additional refinements can be added to these systems.
By combining activators with tissue-specific promoters it will be
possible to induce gene expression in a given tissue. One possible
development would be to generate a collection of activator plants
(e.g. tobacco and Arabidopsis) that express the activator protein
under the control of different tissue-specific or developmentally
regulated promoters. After crossing ‘activator plants’ with plants
encoding the gene of interest under the control of the target promoter (‘target plants’), gene expression will be inducible in the next
generation. In contrast to the recently described activator LhG4,
which consists of the Lac repressor and the GAL4 activation domain31, regulated activators will allow gene expression to be activated, or inactivated, by chemical inducers. Genomics will yield
ever more sequences of interest, the function of which might be
analysed by inducible expression.
September 1998, Vol. 3, No. 9
357
trends in plant science
reviews
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Christiane Gatz* and Ingo Lenk are at the Albrecht von Haller
Institute of Plant Sciences, University of Goettingen,
Untere Karspuele 2, 37073 Goettingen, Germany.
*Author for correspondence (tel +49 551 397843;
fax +49 551 397920; e-mail cgatz@gwdg.de).
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