There are four main types of promoters that can be used to regulate gene expression in plants: 1. Constitutive promoters direct constant expression in all tissues. The 35S and Ubi promoters are commonly used. 2. Tissue-specific promoters restrict expression to certain tissues like seeds, roots or fruits. 3. Inducible promoters are activated by stimuli like light, chemicals or stress. Examples include light-induced rbcs and heat-induced hsp promoters. 4. Synthetic promoters are assembled from elements of different origins to achieve a defined expression pattern. The 35S promoter has been modified by adding modules to increase its strength.

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PROMOTERS:
A promoter is a DNA sequence that can recruit transcriptional machinery
and lead to transcription of the downstream DNA sequence. The specific
sequence of the promoter determines the strength of the promoter (a
strong promoter leads to a high rate of transcription initiation).
In addition to sequences that "promote" transcription, a promoter
may include additional sequences known as operators that control the
strength of the promoter.
For example, a promoter may include a binding site for a protein that
attracts or obstructs the RNAP binding to the promoter. The presence or
absence of the protein will affect the strength of the promoter. Such a
promoter is known as a regulated promoter.
An input/output description of promoter function
Sometimes, we ignore the details of how a promoter works and think of a
promoter as a device that converts inputs into outputs. You can do this
when designing a multi-component system that includes promoters whose
activity must be regulated by other species in the system. A promoter can
be thought of as a device that outputs a certain number of transcribing RNA
polymerases per unit time. Promoters can have different numbers of
inputs. A constitutive promoter has no inputs. Technically, even a
constitutive promoter has inputs, such as the level of free RNA polymerase,
but we often assume that levels of free RNA polymerase are either
unchanging, or never be the limiting factor in transcription initiation. The
level of a repressor that negatively regulates a promoter is an input to a
promoter.
Types of promoters used to regulate gene expression
Promoters used in biotechnology are of different types according to the
intended type of control of gene expression. They can be generally divided
into:
1. Constitutive promoters: These promoters direct expression in virtually
all tissues and are largely, if not entirely, independent of environmental
and developmental factors. As their expression is normally not conditioned
by endogenous factors, constitutive promoters are usually active across
species and even across kingdoms.

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2. Tissue-specific or development-stage-specific promoters: These
direct the expression of a gene in specific tissue(s) or at certain stages of
development. For plants, promoter elements that are expressed or affect
the expression of genes in the vascular system, photosynthetic tissues,
tubers, roots and other vegetative organs, or seeds and other reproductive
organs can be found in heterologous systems (e.g. distantly related species
or even other kingdoms) but the most specificity is generally achieved with
homologous promoters (i.e. from the same species, genus or family). This
is probably because the coordinate expression of transcription factors is
necessary for regulation of the promoter's activity.
3. Inducible promoters: Their performance is not conditioned to
endogenous factors but to environmental conditions and external stimuli
that can be artificially controlled. Within this group, there are promoters
modulated by abiotic factors such as light, oxygen levels, heat, cold and
wounding. Since some of these factors are difficult to control outside an
experimental setting, promoters that respond to chemical compounds, not
found naturally in the organism of interest, are of particular interest. Along
those lines, promoters that respond to antibiotics, copper, alcohol, steroids,
and herbicides, among other compounds, have been adapted and refined to
allow the induction of gene activity at will and independently of other
biotic or abiotic factors.
4. Synthetic promoters: Promoters made by bringing together the
primary elements of a promoter region from diverse origins.
4 Different Plant Promoters in Gene Construct
Some of the plant promoters in gene construct are as follows:
1. Constitutive Promoters 2. Tissue-Specific Promoters 3.Inducible
Promoters 4. Synthetic Promoters.
As a rule, promoters of all structural genes that encode proteins are located
upstream of the start site, the site from which transcription begins. The
promoter determines (1) the level of expression, (2) the developmental

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stage and/or the tissues in which it will be expressed, and (3) the
physical/chemical factor by which the gene expression will be regulated.
A promoter suitable for gene expression in prokaryotes will not function in
eukaryotes, and vice-versa. Further, animal promoters are not suitable for
plants, and promoters that function well in dicots are usually much less
active in monocots, and vice-versa. Therefore, considerable thought has to
be given to the selection of an appropriate promoter for transgene
expression.
A variety of promoters are used to drive transgenes in plants, some of
which are listed in Table below.
These are essentially naturally occurring promoters belonging to the
following three broad groups: (1) constitutive promoters, (2) tissue-
specific promoters, and (3) promoters activated by specific
physical/chemical factors. In addition, number of (4) synthetic promoters
have been developed to achieve a defined regulation of the transgene
expression.

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Table: Some of the constitutive promoters used for driving the expression of
transgenes in different plant species.
1. Constitutive Promoters:
Genes driven by constitutive promoters are expressed in all the tissues and
during all developmental stages of the organism, and their expression is
largely unaffected by physical chemical stimuli. Some examples of such
promoters are 35S promoter, ubiquitin (Ubi) promoter, actinl (Act1)
promoter, nopaline synthase (nos) promoter, octopine synthase (ocs)
promoter, mannopine synthase (mas) promoter, etc.
The 35S, nos ocs, and mas promoters have been obtained from plant
pathogens, and were the first to be used; Ubil and Actl promoters are from
plant genes. In case of many constitutive promoters, the level of gene
expression in different tissues may show some variation, and some
promoters may respond to physical/chemical stimuli.
For example, Actl promoter contains elements that appear to negatively
regulate promoter activity in a tissue- specific manner, particularly in
roots. Similarly, Ubi 1 promoter shows some increase in promoter activity
in response to temperature stress.

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Use of constitutive promoters to produce transgenic plants offers
certain advantages:
(1) They can be used to drive scorable/selectable reporter genes, which are
critical for molecular biology studies, and for the development of
transgenic plants.
(2) Their use will be essential in the case of such proteins that are required
in all tissues and/or during all stages of plant development. Finally,
(3) They will be useful in driving genes encoding such transcription factors
that are involved in transcription regulation.
The 35S promoter is the most commonly used constitutive promoter in
dicot plants, but it does not work satisfactorily in most monocot species.
This promoter is ideal for driving marker genes. Some modifications of 35S
promoter show several-fold increase in their activity.
Maize ubiquitin gene (Ubil) promoter includes the first intron of this gene.
It is reported to function well both in monocots and dicots, and its activity
increases transiently in response to temperature stress. The promoter of
rice actin gene (Actl) shows strong constitutive activity in monocots. As in
the case of Ubil, the presence of first intron of the gene Actl is critical for
efficient function of the Actl promoter.
Summary
There are several advantages to using constitutive promoters in expression
vectors used in plant biotechnology, such as:
•High level of production of proteins used to select transgenic cells or
plants;

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•High level of expression of reporter proteins or scorable markers,
allowing easy detection and quantification;
•High level of production of a transcription factor that is part of a
regulatory transcription system;
•Production of compounds that requires ubiquitous activity in the plant;
and
•Production of compounds that are required during all stages of plant
development.
The first constitutive promoters used for the expression of transgenes in
plants were isolated from plant pathogens.
The search for other constitutive promoters has continued, especially to
identify control regions that are able to drive expression of transgenes in
monocots. In some monocots such as cereals, it has been found that
sequences present in 5' untranslated transcribed regions (e.g., introns) of
certain structural genes are essential for efficient gene expression. Thus,
promoters that work well in dicots, which lack introns, do not generally
work well in monocots.
2. Tissue-Specific Promoters:
Tissue-specific or organ-specific promoters enable the expression of
concerned genes in specific tissues/organs or during certain stages of
development. These promoters drive those genes, which are expressed
specifically in roots, tubers, vascular bundles, seeds, etc. (Refer Table
below)
For example, vicilin and PHA promoters are seed-specific promoters,
TA29 is a tapetum-specific promoter, and Bcpl is specific to both
tapetum and microspores. Although heterologous promoters, i.e.,
promoters from other species, can be used, it is preferable to use
homologous promoters, i.e., promoters from the same species.
Tissue-specific promoters are indispensable in such cases where it is
desired to limit the expression of transgene to a specific

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tissue/organ/developmental stage. For example, promoter TA29 has been
used to drive barnase gene in order to produce male sterility.
This gene encodes an RNAse, which causes cytotoxicity to the tissue in
which it is expressed. Since TA29 promoter is tapetum- specific, it limits
expression of barnase to tapetum cells leading to male sterility without any
adverse effect on other tissues of the transgenic plants.
Similarly, seed-specific promoters limit the expression of transgenes to
seed, and are ideal for driving genes encoding seed storage proteins.
Tissue-specific expression of transgenes would reduce the cost of
transgene expression to the plant. It would also reduce selection pressure
against the insect pest (in cases of trangenes confering insect resistance)
and, thereby, reduce the risk of development of resistance by the insect
pest.
Summary
As mentioned in the Introduction, there are promoters controlling gene
expression in a tissue-dependent manner and according to the
developmental stage of the plant. The transgenes driven by these type of
promoters will only be expressed in tissues where the transgene product is
desired, leaving the rest of the tissues in the plant unmodified by transgene
expression.
Tissue-specific promoters may be induced by endogenous or exogenous
factors, so they can be classified as inducible promoters as well.
Unlike constitutive expression of genes, tissue-specific expression is the
result of several interacting levels of gene regulation. As such, it is then
preferable to use promoters from homologous or closely related plant
species to achieve efficient and reliable expression of transgenes in
particular tissues.

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The purpose of this section is to present those patents directed to plant
tissue-specific promoters in broad terms:
•Root promoters: Root promoters that enhance or suppress the
expression of a linked gene in root cells. In addition, the invention
comprises methods for the identification and isolation of plant tissue-
specific promoters in general.
•Fruit promoters: A tissue-specific promoter includes fruit specific
promoters that control the expression of genes in mature ovary tissue of a
fruit and in the receptacle tissue of accessory fruits such as strawberry,
apple and pear. The genes driven by the promoters influence fruit
development and ripening.
•Seed promoters: University of California have granted patents and patent
applications drawn to seed-specific promoters in broad terms.

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Transcription cassettes having a seed-specific promoter and recombinant
molecules containing a seed-maturation promoter are part of the
inventions.
3. Inducible Promoters:
Promoters that are activated in response to a specific physical factor,
e.g., light, temperature, heat, cold, wound etc., or a specific chemical
compound are called inducible promoters or, sometimes, stimulus-
responsive promoters. These promoters, especially chemical- inducible
promoters, provide fine control on the regulation of gene expression.
A number of native plant gene promoters are stimulus responsive, e.g.,
Adhl promoter (responds to anaerobic condition), cab promoter and
rbcs promoter (respond to light), etc. (Refer above table). But chemical
inducible promoters are synthesized from promoter sequences of different
organisms.
It is important that chemically-regulated promoters should be derived from
such organisms that are as distantly related to plants as possible, e.g., from
bacteria like E. coli, yeast, Drosophila, and mammals.
A tetracycline- regulated system that can either activate (negative
regulation) or repress (positive regulation) transcription in the presence of
tetracycline has been developed.
Some examples of environment-responsive promoters are those of the
genes rbcs, Adhl, cab, hsp, etc. Nuclear gene rbcs encodes the smaller
subunit of RuBISCO (ribulose-1, 5-bisphosphate
carboxylase/oxygenase). This gene is expressed mainly in mature leaves
and, to a lesser extent, in stem and young leaf tissues, and its expression is
induced by light.
The – 166 to – 149 bp sequence of rbcS gene functions as light response
element (LRE) and is responsible for the expression of rbcS gene in
response to light. The alcohol dehydrogenase 1 (Adhl) gene of maize is
expressed only under anaerobic conditions due to the action of a
silencer sequence that suppresses expression in the presence of
oxygen.

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A number chemically regulated gene expression systems have been
constructed using genes and operator systems, preferably, from unrelated
organisms. Tetracycline-regulated system was the first such system to be
developed. Subsequently, several such expression systems were devised,
e.g., metal- regulated, steroid-regulated, plant hormone-regulated,
pathogenesis-regulated, wound-regulated, etc. expression systems.
Summary
As their name says, the activity of these promoters is induced by the
presence or absence of biotic or abiotic factors. Inducible promoters are a
very powerful tool in genetic engineering because the expression of genes
linked to them can be turned on or off at certain stages of development of
an organism or in a particular tissue.
This section presents a general view of promoters whose activity is
triggered by either chemical or physical factors. There are virtually
hundreds of inducible promoters that vary according to the organism
source and cells or tissues where they regulate gene transcription.
Inducible promoters are grouped as:
•Chemically-regulated promoters, including promoters whose
transcriptional activity is regulated by the presence or absence of
alcohol, tetracycline, steroids, metal and other compounds.
•Physically-regulated promoters, including promoters whose
transcriptional activity is regulated by the presence or absence of
light and low or high temperatures.
Chemically-regulated promoters
The activity of this class of promoters is modulated by chemical compounds
that either turn off or turn on gene transcription. As prerequisites, the
chemicals influencing promoter activity typically:
• should not be naturally present in the organism where expression of the
transgene is sought;

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• should not be toxic;
• should affect only the expression of the gene of interest;
• should be easy to apply or removal; and
• should induce a clearly detectable expression pattern of either high or
very low gene expression for their optimal use as modulators of gene
expression.
Physically-regulated promoters
Promoters induced by environmental factors such as water or salt stress,
anaerobic condition, temperature, illumination and wounding have
potential for use in the development of plants resistant to various stress
conditions. These promoters contain regulatory elements that respond to
such environmental stimuli.
Temperature-induced promoters include cold- and heat-shock-induced
promoters. In many cases, these promoters are able to operate under
normal temperature conditions, which vary according to the organism, but
when either cold or heat is applied, the promoters maintain activity. In
addition, expression can be enhanced by the application of higher or lower
temperature as compared to the normal temperature conditions. One of the
best studied eukaryotic heat-shock systems is the one found in
Drosophila (fruit fly).
4. Synthetic Promoters:
A promoter assembled by combining various primary elements required
for the defined promoter function may be referred to as a synthetic
promoter. The various elements used in such a promoter are, usually, of
diverse origin. Among these elements the TATA box, the transcription start
site or cap site, and the CCAAT consensus sequence are required for
accurate constitutive transcription. A synthetic promoter may be either
constitutive tissue-specific or inducible, depending on the functional
sequences used in its construction.
Attempts have been made to increase the activity of 35S promoter by
adding specific modules to the native promoter. Inclusion of an additional

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copy of the 35S enhancer (-208 to -46 bp), i.e., double 35S enhancer, caused
a 6-fold increase in 35S promoter activity.
Summary
As mentioned in the introductory information about promoters, a set of
minimum elements are required for an activity of eukaryotic promoter.
Among those elements are the TATA box, the transcription start site or
CAP site and the CCAAT consensus sequence, which is required for
accurate transcription.
From the sequences of these elements in diverse organisms, it is possible to
synthesize consensus sequences that may work across different organisms
and are not necessarily derived from a particular organism.
The group of patents under this section are directed to promoters whose
parts are synthesized as consensus sequences of the promoter elements
found in nature.