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Genetic Engineering, Volume 25. Edited by J.K. Setlow


Kluwer Academic/Plenum Publishers, 2003 (pp. 113-141).
FUNCTIONAL ANALYSIS OF PROMOTER ELEMENTS IN PLANTS
Slavko Komarnytsky and Nikolai Borisjuk
Biotech Center, Cook College
Rutgers University
59 Dudley Rd.,
New Brunswick, NJ 08901-8520
INTRODUCTION
Plant growth and development involve temporal and spatial expression oI the speciIic
genes subsets in response to various physiological and environmental Iactors mediated by
complex signal transduction pathways. A minimal gene regulatory network typically
consists oI an input signal receptor and a transduction pathway linking the extracellular
environment with intracellular targets; a core complex oI transacting regulatory proteins
and relevant cis-acting DNA sequences; and a subsequent molecular output (RNA and
protein) Irom the target genes. Generally, the result oI the activation oI such a regulatory
pathway is stimulation or repression oI expression oI the genes coding Ior structural,
metabolic, and behavioral capacities oI the plant cell. In addition, such networks oIten
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include dynamic Ieedback loops that provide Ior Iurther regulation oI the network
architecture and output.
Central to speciIic activation are DNA recognition sequences which interact with
basic transcription initiation complexes and numerous transcription Iactors. In plants,
these are usually 5'-Ilanking modules that include a core promoter, proximal regulatory
elements, and upstream enhancer sequences located close to the structural portion oI the
gene (1). Regulatory elements can oIten be positioned quite Iar Irom the transcription
initiation site (TIS) in mammalian genomes, whereas in yeast and plants they are located
within a Iew thousands base pairs oI the TIS (2).
The control regions oI plant genes may include multiple cis-acting elements that
contribute to the complex expression proIile oI that particular gene. Moreover, the same
transcription Iactors can act as activators or repressors depending on their concentration
and the presence oI interacting partner proteins (3). Earlier in the Genetic Engineering
series, GuilIoyle (4) published an excellent review Iocused on the basic structure oI plant
promoters and the conservation oI speciIic cis-acting elements within promoters that
respond similarly. While many oI these regulatory elements are well deIined today, there
is little logic apparent in the organization oI multiple regulatory elements, and even less
in the way that they interact to regulate gene expression. ThereIore, this chapter is aimed
at illustrating the diversity and intricacies oI plant regulatory sequences, and highlighting
how their interactions govern the structural and Iunctional interplay oI signal perception
pathways. We also attempt to provide a deeper insight into the regulatory Iunction oI AT-
rich sequences Iound in plant promoters.
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TRANSCRIPTIONAL ACTIVITY IN PLANT NUCLEUS
The eukaryotic nucleus contains three diIIerent classes oI RNA polymerases (pol).
RNA pol I is responsible Ior the transcription oI ribosomal DNA genes, and is active only
in the nucleolus region. RNA pol II transcribes all protein-encoding genes and several
small nuclear RNAs; while RNA pol III transcribes 5S ribosomal RNA genes, transIer
RNA genes, and some small nuclear RNAs. There is strong evidence that Iunctional
separation between all three major classes oI polymerases is Iurther enhanced by spatial
compartmentalization oI their activity in the diIIerent domains oI the eukaryotic nucleus
(5). Plant genes transcribed by RNA pol II typically contain common core elements
recognized by general transcription Iactors, and gene-speciIic DNA elements recognized
by regulatory Iactors, which in turn modulate the Iunction oI the general initiation Iactors
(6, 7).
The availability oI whole genome data and sophisticated microarray technology has
opened up new avenues Ior the analysis oI gene regulation and expression. Unraveling
the regulatory network(s) that inIluence expression oI a given gene or gene Iamily is
typically based on the a priori assumption that co-regulated genes usually have common
regulatory elements. Eunctional dissection oI the promoter region usually requires
identiIication and characterization oI a minimal promoter, location oI the putative binding
sites oI known and unknown transcription Iactors, and sorting out the eIIects oI distant
enhancer- and repressor-like modules. Local chromatin states, and the availability oI
scaIIold attachment regions and DNA methylation sites, may Iurther contribute to the
activity oI the 5`-Ilanking region.
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The primary strategy Ior the Iunctional analysis oI any promoter is a computer-
assisted scan over the entire sequence by a consensus approach, which relies on known or
conserved DNA elements. This is generally achieved by querying the sequence oI interest
against the reIerential databases using scanning soItware that does not require download
and installation (Table 1). This inIormation allows preliminary assessment oI promoter
strength, speciIicity, and regulation. Subsequently, a series oI wet -lab experiments must
be perIormed to dissect the individual and combinatorial activities oI the putative motiIs
both in vitro and in vivo (see below Ior recent reports and reIerences).
D promoter Iusion to a reporter gene (8)
D Iusion to a minimal promoter (9)
D transient or stable expression assay (10)
D promoter deletion and base substitution (11)
D insertional mutagenesis (12)
D linker-scanning mutagenesis (13)
D high-throughput SELEX-SAGE (14)
D one-hybrid yeast system (15)
D DNA-protein crosslinking (16)
D electrophoresis mobility shiIt assay (17)
D primer extension (18)
D DNase I Iootprinting (19)
D RNase protection assay (20)
D in vitro transcription systems (21)
CORE PROMOTER
Eukaryotic transcription is regulated by two main classes oI transcription Iactors:
general transcription Iactors (GTEs) and sequence-speciIic transcription Iactors (reviewed
in 22). GTEs bind to the core promoter close to the TIS and, together with RNA pol II
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assemble in an ordered Iashion to Iorm the pre-initiation complex. It has been suggested
that approximately 15 oI the genes oI Arabidopsis encode Ior transcription-related
proteins comprising more than 1500 transcription Iactors, 45 oI which are speciIic to
plants (23). About one-third oI all plant genes are expressed throughout the entire plant,
albeit at variable levels. Another one-third may have their products present in a Iew
organs, and the rest may belong to a unique organ as Iar as expression is concerned (24).
Core promoter elements are deIined as minimal DNA elements that are necessary and
suIIicient Ior accurate initiation oI transcription by RNA pol II in a reconstituted cell -Iree
system (6). Several major elements, acting independently or collectively, mediate the
direct binding oI the TEIID complex to a proper TIS (25). The Iirst element described as
regulating this process was a classical TATA box, TATA(A/T)A, located 25 to 30
base positions upstream oI TIS (26). However, subsequent studies suggested that AT-rich
sequences completely unrelated to the TATA-box stimulate transcription with equal or
increased eIIiciency (27). Eurthermore, although the Iirst step oI transcripti on initiation is
highly speciIic, TEIID also binds with high aIIinity to several TATA elements that do not
match the consensus sequence and is active in promoting transcription in vitro Irom these
elements (28). Another weakly conserved 'initiator element was described in the direct
vicinity oI the TIS (29). This element anchors the TEIID complex in the absence oI the
TATA-box, and may Iunction synergistically in TATA-mediated initiation (30).
Mammalian initiator elements share a consensus PyPyAN(T/A)PyPy core (31). A similar
pyrimidine rich initiator motiI has been described in the promoter oI the nuclear psaDb
gene encoding the Ierredoxin-binding subunit oI photosystem I, although the overall
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Irequency oI TATA-less promoters in non-photosynthetic plant genes is less than 10
(32).
Two more elements that contribute to the basic transcription initiation were described
recently. The 'downstream promoter element, (A/G)G(A/T)CGTG, located at position
30 bp downstream oI the TATA-less TIS, was able to direct transcription initiation in
Drosophila (33) and inIluence the activity oI the tungro bacilliIorm virus promoter in
transient expression assays in rice protoplasts (34). Einally, a Iorth class oI potentially
active core promoter elements with little consensus homology other than GC-rich
composition, has been reported in the mammalian system (35). Earlier, a homologous
conserved GC-rich element was described immediately upstream oI the TATA-box in the
barley pr1 promoter (36). These Iindings strongly suggest that even though most oI the
knowledge oI the core promoter elements was initially derived Irom yeast and
mammalian models, the major principles oI the regulation oI gene expression are
conserved in plants as well. A possible explanation Ior the observed di versity in the core
promoter elements could be their contribution to the combinatorial gene regulation,
including the restriction oI enhancer stimulatory capacity to a speciIic set oI promoters
(37).
Two more proximal regulatory elements, not universally required Ior the activity oI
the core eukaryotic promoters, when present, signiIicantly aIIect their initiation
Irequency. These sequences are located close to TIS (around 100 bp) and appear to
assist in the recruitment oI basic transcription Iactors to the TIS sites oI housekeeping and
TATA-less genes. The GC-box, GGGCGG, plays an important regulatory role in the core
promoter regions oI mammalian genes (38). Co-transIection oI expression cassettes
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together with the GC-box binding protein usually results in a Iour-, to ten-Iold increase in
GC-box-regulated promoter activity (39). GC-boxes have also been localized in the
upstream promoter regions oI many plant genes (40- 42). Another well conserved
element, the CCAAT-box, is oIten present at 80 to 150 bp upstream oI TIS, and may
operate cooperatively with other putative conserved motiIs (43). However, no uniIying
expression pattern Ior plant genes containing putative CCAAT elements has been
observed (44, 45). Moreover, multiple copies oI the genes coding Ior the subunits oI the
CCAAT-binding protein exist in Arabidopsis, suggesting the potential Ior multiple
alternative Iorms oI these complexes in plants (46).
UPSTREAM REGULATORY ELEMENTS
In plant promoters, all the necessary inIormation to direct proper gene expression is
generally located in a very compact region, less than 1000 bp upstream oI the TIS. Only
Iew plant promoters are known to be constitutively expressed in most plant cells. They
include promoters Irom housekeeping genes like ubiquitin (47) or actin (48). Interaction
oI the upstream regulatory elements with sequence-speciIic transcription Iactors (SSTEs)
determines the time, place and level oI activity oI all genes within a set oI highly
coordinated expression networks.
LIGHT-RESPONSIVE ELEMENTS (LREs)
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Higher plants have developed a complex biochemical system to perceive and respond
to light, which is based on three classes oI photoreceptors: phytochromes, blue-light
receptors, and receptors Ior UV-light. Light signals absorbed by these photoreceptors
regulate transcription oI a large number oI genes that encode proteins involved in
photosynthesis and various developmental processes through a complex signal
transduction cascade (49). It is generally believed that diIIerent light-activated pathways
target diIIerent LREs and transcription Iactors within the promoter region, but they may
also target common LREs (50). Extensive mutagenesis and deletion analysis oI the
promoter regions oI light-induced genes resulted in the identiIication oI a number oI cis-
acting elements involved in the control oI light-regulated transcription. Several oI these
motiIs, such as the GT1-box (GGTTAA), I-box (GATAAAGR), G-box (CACGTG) and
H-box, ACCTA(A/C)C(A/C), have been experimentally shown to be important
components in the light response. Although similar LREs are present in many diIIerent
promoters and are assembled in a variety oI combinations, no universal element has been
Iound in all photoregulated promoters. Some oI the LREs are present in promoters that
are not light-regulated (51). Eurthermore, the same LRE is Irequently Iound in promoters
with opposite light responsiveness, as was shown Ior the GT1-box (52). None oI the
LREs identiIied so Iar has been shown to conIer light responsiveness by itselI. Eor
example, while a tetramer oI the GT1-binding element was able to conIer light regulation
on a minimal (90 bp) 35S promoter containing the as-1 element, it Iailed to act in the
same way when combined with a shorter version oI the 35S promoter (46 bp) lacking
as-1 (53). It has also been shown that only artiIicial sequences composed oI paired
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combinations oI tetrameric repeats oI G- and GATA-boxes, or GT1- and GATA-boxes,
but not multimers oI a single motiI, can Iunction as LREs (54, 55).
These and many other experimental data resulted in a general hypothesis that plant
LREs are actually composite elements, i.e. combinations oI cis-regulatory sequences in
which their overall activity is the result oI synergetic interactions between cognate
transcription Iactors (56, 51). The modular component oI LREs is composed oI two
general elements, the light-speciIic element, and a coupling element. Light-speciIic
elements are targeted by transcription Iactors that are regulated by light. Coupling
elements bind protein Iactors that direct the light stimulus to transcription in a spatial or
temporal manner, or determine the relative strength oI the light-induced gene expression.
Numerous protein Iactors that bind to cis-elements containing GATA- and GT1-core
sequences have been identiIied and extensively characterized (51, 57). Although in many
studies clear diIIerences in speciIicities and suggestive changes in activity in response to
light were described, the conclusive Iunctions oI these DNA-binding proteins in light-
regulated transcription has yet to be determined (58). The only two transcription Iactors,
Ior which there is strong evidence suggesting their importance in light -mediated
regulation, are Arabidopsis Iactors HY5 (59) and PIE3 (60), which interact with the G-
box. The G-box-bound PIE3 was shown to speciIically bind phytochrome B Iollowing
light-stimulated conversion oI the photoreceptor to its biologically active Iorm. HY5 was
shown to be required Ior light induction oI the minimal light-responsive module CMA5, a
native 52-bp Iragment oI tobacco rbcS 8B promoter, which contains I- and G-box motiIs.
As HY5 itselI does not have an activation domain, it is assumed that it could aIIect
transcriptional activity through interaction with other Iactors (60).
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METABOLIC REGULATION
Hormones play a key role in regulating plant growth and development. Recent
evidence has suggested that sugars control gene expression and developmental processes
in a manner similar to classical plant hormones (61). The question is: how do relatively
simple molecules regulate a variety oI responses within an assortment oI cells, tissues,
and organs oI plants? The answer to this question requires an understanding oI hormone
and sugar perception and signal transduction as a part oI the metabolic regulation oI gene
expression in plants.
Auxin-Responsive Elements (AuxREs)
Auxin plays an important role in root Iormation, apical dominance, tropism, and
senescence at the organism level, while acting as a signal Ior division, extension, and
diIIerentiation inside the plant cell. Auxin-mediated responses at the gene level can be
detected as early as two to Iive minutes aIter auxin application (62, 63). The most
extensively studied auxin-responsive plant gene promoters are those Irom the pea PS-
IAA4/5 gene (64), the soybean GH3 gene (65, 66), and the soybean Small Auxin-Up
RNA, SAUR15A gene (67). These studies led to the identiIication oI the cis-acting motiIs
(G/T)GTCCCAT, within an AuxRE oI the pea PS-IAA4/5 promoter (64), and TGTCTC,
within the three small AuxREs oI the soybean GH3 promoter (65, 66). An AuxRE oI the
SAUR15A promoter contained both types oI these motiIs (67). A Iunctional study oI
AuxREs in the soybean GH3 promoter showed that the TGTCTC element requires a
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closely associated constitutive or coupling DNA element to Iunction as an AuxRE (66).
Similarly, TGTCTC and TGTCCCAT elements in the soybean SAUR15A promoter and
the pea PS-IAA4/5 promoter may also Iunction as composite AuxREs that contain
diIIerent coupling elements than those Iound in composite AuxREs oI the soybean GH3
promoter (63). Within the composite AuxREs, auxin-responsive elements repress the
transcription-stimulating activity oI the adjacent or overlapping constitutive element
when auxin level is low. When auxin level is high, the repression is released and the
composite element is activated (63). Depending on the nature oI the constitutive or
coupling elements oI diIIerent AuxREs, they could potentially conIer a variety oI tissue-
speciIic and developmentally regulated expression patterns (68).
Both TGTCTC and TGTCCCAT elements can Iunction as AuxREs in the absence oI
any coupling element when multimerized with appropriate spacing and orientation (64,
69, 70). Properly spaced and oriented, TGTCTC AuxREs are several Iold more active
than natural AuxREs (69, 70). A highly active palindromic repeat oI the TGTCTC
element was used as bait in a yeast one-hybrid system to identiIy the Iirst member oI the
auxin responsive Iactors, ARE1, Irom Arabidopsis (69). Both the ARE Iamily and the
Aux/IAA proteins have been identiIied as key regulators oI auxin-modulated gene
expression (71), however, the DNA-binding activity oI the Aux/IAA proteins has not
been conclusively demonstrated (72). Aux/IAA proteins are believed to regulate
transcription by modulating the activity oI AREs (63).
Another AuxRE that has received considerable attention is the ocs/as-1 element (73).
Originally Iound in the CauliIlower Mosaic Virus (CaMV) 35S promoter, activation
sequence-1 mediates both salicylic acid- and auxin-inducible transcription activation, and
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consists oI two imperIect TGACGTCA palindromes (74). Whereas the sequence can
deviate quite substantially Irom its consensus, the12 bp space between the two
palindromic centers is conserved in all osc/as-1 elements that respond to auxin (75). This
element was also detected in several plant promoters, including the auxin-inducible
tobacco Par genes (73, 76). A nuclear protein complex, ASE-1, that binds to the
TGACGTCA palindrome was identiIied by using electrophoretic mobility shiIt assay
(74). A number oI cDNAs encoding as-1-binding TGA proteins have been described in
tobacco (77, 75).
Gibberellin-Responsive Elements (GAREs)
The mechanism by which the gibberellic acid (GA) signal is perceived and
transduced in plants has been studied extensively using aleurone-speciIic expression as a
model (78). Analysis oI GA regulation oI ,-amylase promoters uncovered cis-acting
elements that are suIIicient Ior gibberellin responsiveness (79). These included the GARE
motiI (TAACA(A/G)A), TATCCAC-box, and pyrimidine box (C/T)CTTTT(C/T).
Eurther Iunctional analysis oI the barley ,-amylase gene promoter showed that
TAACA(A/G)A is very similar to the c-Myb consensus binding site. Subsequently, a
novel MYB-related clone (GAmyb) was isolated Irom a barley aleurone cDNA library.
When the ,-amylase promoter was Iused to the uidA reporter gene (GUS), GAMyb was
the sole gibberellin-regulated transcription Iactor required Ior transcriptional activation oI
the expression cassette in the absence oI GAs (80).
Similarly, synthesis oI EPB-1, a cysteine proteinase responsible Ior the degradation oI
seed endosperm storage proteins in barley, is induced by GAs and repressed by ABA.
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Eunctional analysis oI the EPB-1 promoter revealed that the GARE, a pyrimidine box,
and an upstream element are all necessary Ior GA induction. Constitutive expression oI
the GAMyb transcriptional Iactor, in the absence oI GA, led to the transactivation oI
EPB-1 expression in a dosage dependent manner with the highest level comparable to
that in Iully GA-induced tissue (81). Eunctional analysis oI another GA-regulated wheat
,-amylase promoter, ,-Amv2/54, has also indicated that three elements are essential Ior
GA-induced expression. They include the proposed GARE module and a new putative
element GATTGACTTGACC (82). It is interesting to note that the GA-responsive ,-
amylase gene was reported to be sugar-repressed in rice embryos, and that both GARE
and the pyrimidine box have been partially involved in sugar-induced gene repression
(83).
Abscisic Acid-Responsive Elements (ABREs)
Multiple ABREs are located upstream oI the ABA-induced genes. The G-box with
the sequence (C/G/T)ACGTGGCG, is probably the most studied element subject to ABA
regulation (84). Multiple tandem copies oI this module conIer ABA responsiveness when
Iused upstream oI the minimal 35S promoter (79). While the isolated G-box Iailed to
conIer ABA responsiveness to the minimal promoter, a complex module comprising the
G-box and a coupling element Iunctioned as an ABRE in vivo (85). Multiple ABREs
were identiIied in the 5` Ilanking region oI the wheat Em gene (86), and shown to be
activated in the presence oI VP1 transcriptional activator (87). Typically, the presence oI
VP1 enhances ABA induction oI late embryogenesis genes, but also suppresses
germination speciIic genes (88). As with auxins, organ- and species-speciIic activation oI
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ABA-responsive genes is achieved only by the cooperative action oI several cis-acting
elements (89).
Ethylene-Responsive Elements (EREs)
The best-known eIIect oI ethylene is the promotion oI Iruit ripening. Other notable
processes regulated by ethylene include seed germination, senescence, and responses to
stress Iactors such as Ilooding, wounding or pathogen attack (90). In climacteric Iruits
such as tomatoes, apples, bananas and avocados, the initiation oI ripening is associated
with a burst in ethylene biosynthesis, and signiIicant alteration in the expression proIile
oI many genes (91). A Iunctional ERE was identiIied in the tomato Iruit ripening genes
E4 and E8 (92, 93). Transcription oI E4 is rapidly activated by ethylene in both leaves
and Iruit, but E8 is activated by ethylene only in Iruit, and is additionally regulated by
separate Iruit ripening developmental signals. Sequences required Ior both ethylene
responsiveness and Iruit ripening regulation were identiIied within the 161 bp upstream
oI the E4 transcription start, and the sequence Irom 85 to 140 was shown to be
essential Ior ethylene-responsive gene transcription, but not suIIicient to conIer ethylene-
responsiveness to the minimal 35S promoter (94). It has been concluded that at least two
cooperative cis-acting sequences, an upstream (150 to 121) and a downstream (40 to
65) regulatory elements, are required Ior ethylene-responsive regulation oI E4 (94). A
nuclear protein that binds to the 5`-Ilanking regions oI both E4 and E8 genes, E4/E8BP,
was identiIied by gel shiIt assay and DNase I Iootprinting, and subsequently cloned (95).
A truncated version oI E4/E8BP-1 was shown to transactivate the E4 promoter in a
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transient assay, demonstrating that this DNA-binding protein can activate the E4
promoter to speciIically enhance gene transcription in vivo.
The senescence oI carnation Ilower petals involves an increased biosynthesis oI
ethylene. A 126 bp sequence within the promoter region oI the carnation glutathione-S-
transIerase gene was shown to be necessary and suIIicient Ior ethylene regulation during
petal senescence (96). A protein that interacts with that sequence was identiIied and
shown to have speciIic DNA-binding activity in pre-senescent petals, senescent petals
and petals treated with ethylene. DNase I Iootprinting deIined the DNA sequence
between 510 and 488 within the region speciIically protected by bound proteins. This
protein-protected region shares an 8-bp sequence A(T/A)TTCAAA with the protein
binding sequence oI the E4 promoter, raising the possibility that Ilower petal senescence
and Iruit ripening may have some common mechanisms Ior ethyl ene gene regulation
(93).
Sugar-Responsive Elements (SUREs)
Biochemical, molecular, and genetic experiments all support a central role Ior sugars
in the control oI plant metabolism, growth, and development, and have revealed
interactions that integrate light, stress, hormone signaling, and coordinate carbon and
nitrogen metabolism (61, 97). It has been proposed that sugar transporter-like proteins
and some extracellular sugar-binding proteins can serve as sugar sensors to perceive and
transmit sugar-mediated signals which alternatively may be triggered by hormones, light,
and other environmental stimuli that cross-talk with sugar signaling pathways (61, 98).
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Sucrose-responsive elements were Iound in the promoters oI genes coding Ior cereal
,-amylases (99, 100), potato tuber storage proteins (101), sweet potato vegetative storage
proteins (10, 102, 103), potato sucrose synthase (104), bean photosynthesis protein
RBCS2 (105), and the cucumber malate synthase (106). No conserved cis-acting element
(common to all sugar-regulated promoters) has been reported, indicating perhaps the
complex nature oI sugar signaling pathways. A sugar response sequence (SRS) Iound in
the promoter oI a sugar starvation-inducible rice amylase gene, Amv3, was shown to
conIer sugar responsiveness to a minimal promoter. The SRS contains three essential
motiIs: a GC-box, a G-box, and a TATCCA element, identiIied at 90 to 150 bp in all
promoters oI ,-amylase genes isolated Irom cereals (107). The TATCCA element bound
three structurally related rice OsMYBS proteins in yeast one-hybrid assays, and two oI
them, OsMYBS1 and OsMYBS2, were able to transactivate a TATCCA-containing
promoter in vivo (100). Analysis oI the --amylase promoter oI sweet potato, which is
induced by metabolizable sugars such as sucrose, glucose and Iructose, detected a
TGGACGC element that is important Ior sugar-inducible expression oI both sporamin
and --amylase genes (10).
Sucrose-responsive elements SURE1 (AATAGAAAA) and SURE2 (AATACTAAT)
were Iound in the promoter region oI patatin (103). A similar SP8 motiI (TACTATT)
was present in the promoters oI the sweet potato sporamin and --amylase genes (102,
103). The SP8 element speciIically binds protein Iactor SPE1, which is a negative
regulator that is transcriptionally repressed by sucrose (102). A putative homolog oI
SPE1 that encodes a WRKY domain transcription Iactor, has been also Iound in
cucumber and Arabidopsis (108). Both the W-box (wound-responsive) and G-box (ABA-
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responsive) elements have been reported in sugar-responsive amylase promoters (109,
110). Exogenous application oI ABA was shown to activate both the --amylase (111) and
--phaseolin promoters, the latter being modulated by externally supplied sucrose at the
same time (112). Together, these data provide strong evidence that sugar, hormone, and
deIense signaling may converge in the transcriptional control oI diverse promoters
through activation oI the W- and G-box elements.
ABIOTIC STRESS
Heat-Stress Responsive Elements (HSEs)
The heat-shock response is widely conserved in all living cells (reviewed in 113), and
heat stress transcription Iactors are the central proteins in this process. Despite their
general variability in sequence and size, their structure and promoter recognition
sequences are remarkably conserved among the eukaryotes. All oI them comprise an N-
terminal DNA-binding domain, the hydrophobic core oI which ensures the precise
location oI the central helix-turn-helix motiI at the HSE. This element contains a
repetitive pattern oI palindromic binding motiIs, nGAAnnTTCnnGAA (114), and plays a
major role in the heat-shock response in tomato (115), soybean (116), and sunIlower
(117). Eor example, expression oI the ascorbate peroxidase gene is oIten induced by both
oxidative stress and a subsequent heat-shock response, the latter resulting Irom the
accumulation oI hydroxyl and superoxide radicals, and hydrogen peroxide (118).
Sequence comparison oI the promoter regions upstream oI the ascorbate peroxidase gene
Irom pea (119) and Arabidopsis (120) revealed the presence oI only one region oI high
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homology that is located around the TATA box, and contains several sequence motiIs
characteristic oI the HSE identiIied in promoters oI all heat-shock-inducible genes. This
putative HSE was recognized by the tomato heat-shock Iactor in vitro, and contributes
partially to the induction oI the ascorbate peroxidase gene by oxidative stress (121).
Some plant genes use more than a HSE to regulate their thermo-induced expression. Eor
example, the soybean heat shock gene Gmhsp17.3-B is regulated via the classical HSE,
but Iull promoter activity requires additional sequences located upstream oI HSE.
Structural Ieatures within this putative enhancer region include a run oI simple sequences
which are also present upstream oI the HSE-like sequences oI other soybean heat shock
genes, and three perIect CCAAT boxes located immediately upstream Irom the most
distal HSE oI the promoter (122). While the AT-rich domain oI the 5' Ilanking region
was unable to direct transcription Irom the TATA box oI a truncated 35S promoter, heat -
inducible transgene activity was detectable when additional sequences Irom the native
promoter, containing three CCAAT boxes and a single HSE, were present in the
constructs.
Oxidative-Stress Responsive Elements
Increasing evidence indicates that H
2
O
2
Iunctions as a stress signal in plants,
mediating adaptive responses to various stresses by modulating expression oI many
genes, including those coding Ior antioxidant enzymes and modulators oI H
2
O
2
production (123, 124). A global microarray analysis oI gene expression in response to
H
2
O
2
showed that approximately 1 oI the Arabidopsis transcriptome is regulated by
H
2
O
2
. OI these genes, some were also regulated by various stimuli such as UV-light,
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elicitor treatment, and drought stress (125). Despite the Iact that H
2
O
2
-responsive
promoters were identiIied in that study, neither signaling pathway(s) nor transcription
Iactors or H
2
O
2
-regulatory DNA sequences have yet been isolated and characterized.
However, several promoter elements such as W-, G-, and H-boxes, and the ethylene-
responsive GCC element, which are present in the oxidative stress-responsive part oI
plant promoters, have been put Iorward as candidates Ior H
2
O
2
-responsible motiIs (126).
There is strong evidence that ethylene, ABA, SA, and calcium contribute to the
combinatorial regulation oI the response against to oxidative damage in Arabidopsis.
Both inhibitor and genetic data suggested that ethylene, SA, and ABA are actually used
by Arabidopsis in vivo to protect plant tissues against heat-induced oxidative stress (127).
The Iuture challenge will be to determine what signaling pathways these Iour components
are involved in, and to identiIy the other signaling components in these pathways.
Cold-, Drought-, and Osmotic Stress- Responsive Elements
When exposed to low temperatures, plant cells encounter three major problems:
changes in the spatial organization oI biological membranes, retardation oI biochemical
and chemical reactions, and alterations in the availability and status oI water. The latter
physiological state is oIten induced by the direct eIIect oI drought and osmotic stress as
well. Consequently, tolerance oI Ireezing and drought may have certain prospective
mechanisms in common, because the majority oI Ireezing injuries usually result Irom
Ireeze-induced dehydration oI plants. Several cold-responsive promoters were shown to
contain the dehydration response element (DRE) with a CCGAC conserved motiI (C-
repeat) repeated several times (128). The 5' region oI the cor15a gene oI Arabidopsis
20
harboring the C-repeat element between 305 and 78 base position is inactive, or very
weakly active, in most oI the tissues and organs oI plants grown at normal temperature
but becomes activated throughout most oI the plant in response to low temperature. Gene
Iusion experiments indicated that this region, in addition to imparting cold-regulated gene
expression, can impart ABA- and drought-regulated gene expression (129). More recent
eIIorts have deIined an Arabidopsis DRE transcription activator, CBE1 (130).
Mutation oI the core pentamer, CCGAC, oI two putative low temperature responsive
elements in the 5'-proximal region oI the winter Brassica napus cold-induced gene
BN115 resulted in complete loss oI low-temperature regulation by the promoter. This
indicates that the CCGAC sequence is critical to the low-temperature response in the
BN115 gene. In contrast, mutation oI two G-boxes, CACGTG, staggered between these
elements in the same region oI the promoter did not alter cold-inducible gene expression
(131). Similarly, a G-box in combination with an anaerobic response element is required
Ior cold stress induction oI the Arabidopsis aldehyde dehydrogenase promoter, but ABA
appears to play no role in cold-responsive expression oI this gene (132). Thus many oI
the changes in the gene expression that occur in response to low temperature and drought
appear to require ABA signaling via a second, independent signal transduction pathway.
Drought and cold stress inducible genes that are activated in this pathway usually contain
a potential ABRE, (T/C)ACGTGGG, in their promoter regions. A similar correlation can
be observed in the closely related Arabidopsis rd29A and rd29B genes. Even though
these genes are closely linked in the Arabidopsis genome, they are diIIerentially induced
under the conditions oI dehydration, low temperature, high salinity, or treatment with
exogenous ABA. It appears that rd29A has at least two cis-acting elements, one involved
21
in the ABA-associated response to dehydration, and the other induced by changes in
osmotic potential, and that rd29B contains at least one cis-acting ABRE that is involved
in ABA-responsive, slow induction (128). The maize rab28 gene has also been identiIied
as ABA-inducible in embryos and vegetative tissues, and it is activated by exposure to
water stress in young leaves. The proximal promoter region oI this gene contains a well -
conserved ABRE module. Transient expression assays in rice protoplasts indicated that a
134 bp Iragment (194 to 60) Iused to a truncated 35S promoter was suIIicient to conIer
ABA-responsiveness upon the gus reporter gene (133). These data suggests that complex
molecular responses to various dehydration- and cold-related stresses may be mediated
by both regulatory systems, where the ABA-signalling pathway does not induce an
immediate response, but plays an important role in prolonged, long-term adaptive
response to cold stress induced by dehydration oI plant tissues (134).
Anaerobic-Responsive Elements (AREs)
The low-oxygen response oI higher plants is complex and involves induction oI
speciIic gene sets. The primary target oI this response is a pathway leading to increased
expression oI the alcohol dehydrogenase gene (Adh) and ethanolic Iermentation (135).
The Adh gene Irom Arabidopsis can be induced by dehydration and cold, as well as by
hypoxia stress. Deletion mapping oI the 5' end and site-speciIic mutagenesis identiIied
Iour regions oI the promoter essential Ior expression under all three stress conditions
(136). The most critical region essential Ior expression oI the Adh promoter under
anaerobic conditions contained sequences homologous to the GT motiI, (T/C)GGTTT,
and the GC motiI, GCC(G/C)C, reported in maize (137). On the other hand, both G-box
22
regions close to the ARE did not aIIect expression under hypoxic conditions, but
signiIicantly reduced induction by cold stress and, to a lesser extent, by dehydration
stress (136). The Iunctional properties oI the ARE positioned in the maize Adh1 gene
have been analyzed using a transient expression assay in electroporated maize
protoplasts. ARE Iunctioned in both orientations, and the promoter activity under
anaerobic conditions was proportional to the number oI complete ARE sequences nested
in the Adh1 promoter (137). The MYB-related transcription Iactor, AtMYB2, was shown
to bind to two separate motiIs in the promoter oI the Arabidopsis Adh1 gene (138). When
driven by a constitutive promoter, AtMYB2 was able to transactivate Adh1 expression in
transient assays in both Arabidopsis and tobacco protoplasts, while mutation oI the GT-
motiI abolished binding oI AtMYB2 and caused a loss oI activity oI the Adh1 promoter.
BIOTIC STRESS AND WOUNDING
Wounding oI plants triggers a number oI diIIerent deIense reactions in order to
develop resistance throughout the plant. Like many other biological processes, pathogen
and wound-induced signal transduction pathways in plants oIten converge in the cell
nucleus (139). There is a large number oI known pathogen-inducible genes, and their
promoters are among the best studied in plants. Upstream regulatory sequences nested in
these promoters Iorm a complex combinatorial regulation network and respond to a
variety oI agents, including ethylene, salicylic acid (SA), jasmonic acid (JA), methyl
jasmonate (MeJA), ABA, and various bacterial and Iungal elicitors.
23
JA and MeJA are Iatty acid derivatives which are considered to be global signals oI
deIense gene expression since many deIense-related genes have been shown to respond to
jasmonates (140). Either pathogen elicitors or exogenous application oI JA activates de
novo synthesis oI phytoalexins (141). At the same time, JA and ethylene act
synergistically in inducing members oI the PR1 and PR5 gene Iamilies oI pathogenesis-
related proteins (142), and Arabidopsis plants impaired in JA perception or biosynthesis
are unable to mount appropriate deIense responses (143).
A MeJA-responsive region (JARE) has been identiIied in the promoter oI the vspB
gene which is stimulated by MeJA and sugars in soybean (144). A DNA domain that
mediates the MeJA response was localized between 535 and 585 bp, and contained a
G-box and a C-rich element. Similar regions containing the bZIP protein-binding
TGACG motiIs or G-boxes, are present in the promoters oI barley lipoxygenase 1 (145),
potato Pin2 proteinase inhibitor II (146), and nested in the as-1-like elements (147). More
recently, Menke and co-workers (148) described the elicitor-induced strictosidine
synthase (str) gene in Catharanthus that required JA as a second messenger. A 42 bp
region in the str promoter contained a GCC-box-like element, and was both necessary
and suIIicient Ior JA- and elicitor-responsive expression. Typically, the GCC box
commonly Iound in the 5' Ilanking regions oI ethylene-inducible deIense genes is
believed to be the core sequence Ior ethylene responsive transcription (149). The ERE
(ethylene-responsive element binding Iactor) proteins were Iirst isolated as GCC box
binding proteins Irom tobacco (150). ERE2 and ERE4 enhanced the GCC box-mediated
transcription oI a reporter gene in tobacco protoplasts, suggesting that they act as
transcriptional activators, while ERE3 Iunctions as a repressor (151). Over-expression oI
24
Pti4, a tomato transcription Iactor that belongs to the ERE Iamily, in transgenic
Arabidopsis plants induced a more than 2.5-Iold higher expression oI twenty eight PR
genes containing a GCC box (152). Ethylene is also required in the transduction pathway
leading Irom injury, and ethylene and JA seem to act together to regulate proteinase
inhibitor gene expression during the wound response (153). On the other hand, GCC box-
mediated transcription oI deIense genes does not always requires ethylene (154),
thereIore, it appears that minor variations in the core sequences oI the GCC-element
impart responsiveness to diIIerent environmental stimuli.
There is increasing evidence that W-boxes are a major class oI cis-acting elements
responsible Ior the pathogen inducibility oI many plant genes. The importance oI W-
boxes was illustrated recently by studies oI the Arabidopsis transcriptome during
systemic acquired resistance, SAR (155). The signiIicant over-representation oI W-box
motiIs, and their clustering, on PR1 subset gene promoters suggests that W-box binding
proteins, WRKY Iactors, are crucial in co-regulation oI these genes (156). Similarly, the
cognate W-box sequence was present in upstream regions oI all the SA-responsive
receptor-like protein kinase genes examined (157). Indeed, both GCC-like elements and
WRKY transcription Iactors have been implicated in gene expression in response to
wounding as well (158), suggesting that wound- and pathogen-induced signaling consists
oI networks with some shared components (159).
ORGAN-SPECIFIC EXPRESSION
Seeds
25
A number oI promoter elements including the RY-repeat motiI (CATGCATG),
ACGT motiI, E-box (CACCCTG), AACA motiI (AACAAACTCAATC), the GCN4
motiI (TGAGTCA), and the Prolamin-box (TGTAAAG) have been shown to be involved
in the seed-speciIic expression oI many seed storage protein (SSP) genes (160, 161). The
RY-repeat motiI is widely distributed in dicot and monocot SSP genes and comprises a
portion oI the 28-bp legumin box (162, 163). In a number oI studies it has been shown
that the sequence is important Ior expression oI SSP genes in coordination with other cis-
acting elements (164, 165). Deletion oI the RY repeat element not only dramatically
reduced expression directed by legumin or glycilin promoters in seeds but, in the case oI
the legumin LeB4 promoter induced expression in leaves (162). In a number oI plant
promoters the CATGCATG motiI interacts with the conservative B3-domain oI the
transcriptional activators JP1 oI maize (166), and fus3 and abi3 proteins oI Arabidopsis
(167, 168). Both the nucleotide sequence and the alteration oI purine and pyrimidine
nucleotides were shown to be essential Ior the activity oI the CATGCATG motiI (167).
The sequence TGTAAAG, commonly named the prolamin box (P-box), was initially
identiIied on the basis oI both its highly conserved nucleotide sequence and location (
300 bp) relative to TIS oI prolamin genes. It was recognized as a strong candidate Ior
coordinating the expression oI many SSPs, because it is present within the promoters oI
all zein genes in maize (169), as well as many SSP genes Irom related cereals. In many
prolamin genes, P-box and GCN4 motiIs are coupled with each other with only a Iew
nucleotides separating them. This tandem module is designated as the biIactorial
endosperm box (170). A third motiI, AACA, conserved in rice glutelin genes, is also
involved in controlling endosperm-speciIic expression (171, 172). These three promoter
26
motiIs are recognized by speciIic DNA binding proteins. The AACA motiI is recognized
by MYB proteins (173); the P-box is recognized by member oI the DOE class oI zinc
Iinger proteins, called PBE (174), and GCN4 is recognized by the bZIP protein Opaque-
2 and its homologues (175, 176). Recently, Wu et al., (177) perIormed an extensive
Iunctional analysis oI the rice storage protein gene promoter GluB-1, which contains
AACA, P-box, GCN4 and ACGT motiIs in its 197 bp promoter region. To gain insight
into the combinatorial interplay among these motiIs, a series oI constructs containing
various combinations and modiIications oI these motiIs was examined in transgenic rice.
Multiple copies oI GCN4 conIerred an endosperm expression pattern when Iused to the
minimal 35S-promoter Iollowed by the GUS-reporter gene, while tandem repeated copies
oI any oI the other three motiIs were unable to direct expression in transgenic rice plants.
The data indicate that the GCN4 motiI is essential Ior determining endosperm-speciIic
expression, whereas the AACA, ACGT and P-box contribute to the quantitative
regulation oI the GluB-1 gene.
Fruit
A number oI Iruit-speciIic genes that are activated during ripening have been isolated
Irom plant species with either climacteric or non-climacteric Iruits (178). Although Iruit-
speciIic promoters have been isolated and analyzed Ior a number oI species (179- 181),
tomato has long served as the primary model Ior the investigation oI Iruit - and ripening-
speciIic promoters (182-184). It has also served as a heterologous system to test the
Iunction oI putative promoter sequences isolated Irom other Iruit species, such as apple
(179) and pepper (180).
27
Eruit-speciIic promoters, such as tomato polygalacturonase (182) and E8 (183)
promoters, have attracted much interest because oI their practical use in the manipulating
Iruit metabolism and the production oI valuable pharmaceutical proteins such as
antibodies, and edible vaccines in genetically engineered Iruits (185, 186). However, the
detailed mechanisms oI Iruit-speciIic transcription are poorly understood, and many oI
the essential cis-elements have not been identiIied. Recently, a novel cis-acting element
that determines Iruit-speciIic, high-level expression oI cucumisin was identiIied and
Iunctionally characterized in melon (181). Gain-oI-Iunction experiments revealed that the
20-bp sequence, GACACGTGTCACAACCTAAT (which contains the perIect
palindromic G-box in its Iirst halI) includes a Iruit-speciIic enhancer element
(TGTCACA). Eour tandem repeats oI the Iull module in either orientation were suIIicient
to direct Iruit-speciIic expression when Iused to a minimal promoter. Stressing the
importance oI the TGTCACA motiI, gel mobility shiIt assays showed that the enhancer
element itselI, but not the G-box, was an essential target Ior a Iruit-speciIic protein
binding to that region oI the cucumisin promoter. No ethylene-responsive elements, such
as the GCC-box that is conserved in the promoters oI many ripening-induced genes
(150), have been Iound in the cucumisin promoter, indicating that its expression is
probably regulated in a developmental and organ-speciIic manner. This Iinding
demonstrates that cis-elements responsible Ior Iruit speciIicity could be successIully
separated Irom those that mediate ripening-associated developmental and ethylene-
mediated regulation.
Pollen
28
A number oI genes speciIically expressed during various stages oI pollen
development and germination have been isolated and characterized both Ior dicot and
monocot plants. The pollen-related genes are divided into early and late groups,
according to their maximal expression beIore or aIter pollen mitosis I, when there is a
transition Irom microspore development to pollen maturation (187). Promoter analysis oI
genes that are coordinately expressed during pollen development in tomato, tobacco and
maize revealed enhancer sequences and shared regulatory elements required Ior pollen-
speciIic transcription (8, 188, 189). Promoter deletion analysis in transgenic plants
demonstrated that relatively short proximal regions are required Ior developmentally
regulated expression in pollen, and in speciIic cell types oI the sporophyte. Cis-acting
regulatory elements oI three tomato late gene promoters LAT52, LAT59 (188, 190) and
LAT56 (191), tobacco genes g10 (8) and NTP303 (192) and the monocot pollen-speciIic
promoter ZM13 (189) Irom maize were characterized in detail using transient and stable
expression analysis. There is a striking architectural similarity between the promoters
Irom maize ZM13, tomato LAT52, LAT59 and the tobacco NTP303 gene. All oI them
contain a 30-32 bp module located 30-50 bp upstream Irom the TATA box responsible
Ior pollen-speciIic expression. In addition, short elements required Ior the enhancement
oI pollen-speciIic expression: the Q-element, AGGTCA, and an AAATGA motiI, have
been identiIied in the maize ZM13 and tobacco NTP303 promoters, respectively. The
maize Q-element was reported to positively modulate the expression oI the proximal
promoter region but showed no ability to cause expression in pol len on its own. A similar
enhancer-like, non-speciIic activity oI the short cis-elements has been reported Ior the
pollen-speciIic expression in tomato (190), Arabidopsis (193) and Brassica (194).
29
While many pollen-speciIic promoters are interchangeable among a wide variety oI
plant species, the 30-32 bp cis-elements essential Ior the expression in pollen show no or
little sequence similarities to each other (190, 193). Eor example, the tomato LAT52
promoter is active in several other dicot plants studied (191, 195, 196). Eurthermore, the
maize ZM13 promoter Iunctions well when stably transIormed into tobacco (197) and
Arabidopsis (189). This data suggest that there should be some conserved Ieatures in the
various pollen promoters that is not yet apparent, at least in terms oI consensus
sequences.
Extensive analysis oI the 100 to 72 bp region in the tomato pollen-speciIic LAT52
promoter revealed a core PBI motiI, TGTGGTT, a putative binding target Ior the GT-1
related transacting Iactors. Mutation oI the central GG residues in PBI reduced pollen-
speciIic expression approximately ten-Iold (188). The PBI motiI, together with two other
regulatory elements, GAAA and TCCACCATA, builds a strong pollen-speciIic
transcriptional activation unit. Eurther mutagenesis and Iunctional combinatorial analysis
demonstrated that PBIGAAA and GAAATCCACCATA Iunctional pairs could act in a
pollen-speciIic manner, while the PBITCCACCATA unit was not active. This data
together with the Iunctional analysis oI other pollen-speciIic promoters suggests the
interplay oI several transcription Iactors, one or more oI which is responsible in
establishing pollen-speciIic expression. Although several cDNAs encoding putative
transcription Iactors speciIically expressed in anthers and/or pollen have been isolated
(198-202), their Iunctional activity and target promoter cis-elements remain to be
characterized.
30
QUANTITATIVE TRANSCRIPTION ENHANCERS
An enhancer is deIined as a cis-acting module capable oI stimulating gene expression
when placed, in either orientation, upstream or downstream oI the gene. Eirst described in
animal viruses Ior their remarkable ability to dramatically increase gene expression upon
SV40 sequences acting in cis to the beta-globin gene (203), enhancers were shown to act
over considerable distances in the genome (204). While many enhancer-like sequences
oIten direct tissue-speciIic or regulated expression (see above), numerous plant genes
have been reported to include non-tissue-speciIic upstream regulatory elements with
quantitative enhancer-like qualities. Dean et al. (205) have reported that sequences
downstream oI the coding region contribute to quantitative diIIerences in expression oI
two petunia rbcS genes. Similar A/T-rich sequences have long been observed to direct
quantitative expression enhancement due to the potential combinatorial eIIect oI multiple
cis elements. Bustos et al. (206) have studied a 0.8 kb Iragment Irom the 5'-Ilanking
region oI a Erench bean beta-phaseolin gene. Gel retardation and Iootprinting assays
using nuclear extracts Irom immature bean cotyledons revealed strong binding oI the
nuclear proteins to an upstream region (628 to 682) that contained two inverted A/T-
rich motiIs. Eusion oI a 103-base pair Iragment, or a 55-base pair synthetic
oligonucleotide containing these motiIs, to a minimal 35S promoter/GUS cassette yielded
high gus expression in several tissues. In another report, a 33 bp double-stranded
oligonucleotide homologous to two AT-rich sequences located upstream (907 to 889,
and 843 to 826) to the TIS oI the soybean heat shock gene Gmhsp17.5E stimulated
transcription when placed 5' to a truncated (140) maize Adh1 promoter (207). Structural
31
Ieatures within this putative enhancer region included a run oI AT-rich sequences, and
three perIect CCAAT boxes located immediately upstream Irom the most distal HSE oI
the promoter (44). The A/T-rich positive regulatory region (444 to 177) oI the pea
plastocyanin gene promoter conIers enhanced gus expression in leaves oI transgenic
tobacco plants when Iused in either orientation to a minimal pea promoter (208).
In many cases, the enhancer-like activity oI the AT-rich sequences in the upstream
promoter module oI the gene can be related to the presence oI the matrix attachment
region (MAR) at this location. MARs appear to organize the eukaryotic genome into
large loops Iormed by the binding oI dispersed AT-rich sequences to non-histone proteins
oI the nuclear scaIIold (reviewed in 209). Individual DNA loops are likely to deIine
Iunctional units as well as topological domains, contributing to the regulation oI gene
expression and DNA replication in general. Comparison oI the large number oI the MAR
sequences showed no clear relationships among them and no strict consensus sequences
(210). However, they share the property oI being asymmetrically AT-rich and contain
dA/dT stretches which are responsible Ior a distinctively narrow minor groove to the
double helix (211). There is also a clear size requirement Ior interaction oI MAR
sequences with the nuclear scaIIold (Ior example, tight binding requires nearly 300 bp oI
AT-rich DNA in Drosophila (212). In addition to the DNA-benting properties, the AT-
rich regions readily become base-unpaired and are oI a great biological signiIicance to
the MAR Iunction (213). A set oI other cis-acting elements characteristic oI MARs
provide the binding sites Ior anchoring the core promoter and origin oI replication
complexes to the nuclear matrix (214). These include A- and T-boxes (210), bent DNA
motiI (215), topoisomerase II box (216), unwinding tract motiI (217) and autonomously
32
replicating sequences (218). In the intact plant nucleus, the MARs deIine individual loops
and can be isolated using low concentrations oI chaotropic agents to achieve selective
extraction under conditions in which precipitation artiIacts are minimized (219). The
distribution oI the diIIerent classes oI DNA within this continuum, with respect to the
predicted structural loops, reveals an interesting correlation: the long stretches oI mixed
classes oI highly repetitive DNAs are oIten segregated into topologically sequestered
units, whereas low-copy-number DNAs are positioned in separate loops (220). MARs
increase reporter gene expression both in stably transIormed plant cells (221) and
transgenic plants (222), and are widely used to minimize transgene silencing (reviewed in
223).
The non-random distribution oI MARs according to localization oI transcription
units, and their co-mapping with DNA sequences supporting the origin oI replication in
yeast (212) and pea (224), strengthens the idea that MARs play a role in replication
mechanisms as well. The potential relationship between anchorage oI Drosophila
ribosomal DNA to a nuclear substructure and replication mechanisms in the enhancer -
like upstream region oI the ribosomal DNA promoter have been suggested (225). Indeed,
the 520 to 80 AT-rich region immediately upstream oI the tobacco ribosomal DNA
promoter comprises a variety oI cis-acting elements with a typical quantitative enhancer-
like module. This ampliIication-promoting sequence (aps) signiIicantly increased the
copy number and expression activity oI the adjacent reporter genes when cloned in both
upstream and downstream position in regards to the expression cassette (226). Similarl y,
an 'upstream Sal repeats (USR) sequence located upstream Irom the ribosomal DNA
promoter oI Arabidopsis thaliana was tested Ior its inIluence on the in vivo activity oI
33
nearby protein coding genes. On average, the presence oI the USR element led to a Iour-
Iold increase in the expression oI a reporter gene (227).
CONCLUSIONS AND PERSPECTIVES
Over the last Iew years, a considerable body oI evidence has accumulated on the
structural organization and regulation oI plant promoters, many aspects oI which have
been previously reviewed (4, 24, 228). In this chapter, we have tried to summarize both
the well-known and newly described cis-active elements nested in the plant promoters,
deliberately emphasizing the most recent studies that illuminate their Iunctional activity
in the multi-level regulation oI plant gene expression.
The organization oI plant promoters Iollows the general structure common Ior other
eukaryotes: a 40 to 50 bp core promoter adjacent to the TIS, Iollowed by an upstream
region oI about 1 kb, containing the proximal and upstream cis-acting elements, and the
outside enhancer-like sequences (Eig. 1). While many plant core promoters share
combinations oI well-deIined elements such as TATA-box, initiator, DPE, and CCAAT-
box, their consensus sequences and spacing may vary signiIicantly, so that no two core
promoters are identical. ThereIore, the composition oI the transcription-initiation
complex that binds to the core promoter is variable, and this oIIers additional
opportunities Ior the regulation oI basic transcription (229-231). Nevertheless, the major
level oI transcriptional control is mediated by trans-acting Iactors binding to the upstream
regulatory elements. In Arabidopsis, the regulation oI genome expression requires the
products oI about 3,000 oI the predicted 25,498 genes, reIlecting the complexity oI this
34
transcription-regulation network (232). At any given time, a distinct set oI transcription
Iactors is available to Iorm the higher-order nucleoprotein complexes at the active
promoter (233). The composition oI individual trans-acting Iactors within these
complexes may oIten change when a speciIic signal is perceived, so that a given
transcription Iactor can play multiple roles, and aIIect multiple gene sets, depending on
its local concentration and availability.
It should be mentioned that one oI the most successIul strategies Ior Iunctional
characterization oI many cis-acting elements is the gain-oI-Iunction Iusion oI deIined
individual elements to a minimal plant promoter, thereby reducing the complexity oI the
expression proIile (9). Recent advances in the development oI stable in vitro transcription
systems based on rice whole-cell and tobacco nuclear extracts (234, 235), in combination
with a range oI available plant natural and synthetic promoters, provides Iurther
opportunities Ior the determination oI the molecular mechanisms underlying selective
gene expression in response to various signals that could be modeled in vitro.
Eunctional studies oI plant promoters have also given rise to a general concept that
upstream regulatory elements are composite and not individual, where each cis-acting
element contributes to the overall activity oI the module through synergistic interactions
between cognate transcription Iactors (52, 66, 82, 100, 136). Stimuli-speciIic elements
within a module are targeted by transcription Iactors that are regulated by signal -
perception pathways, while coupling elements bind protein Iactors that determine spatial,
temporal, or quantitative expression. Eor example, G-boxes serve as the coupling
elements in many modular regulatory units that respond to a variety oI chemical and
environmental signals. Such Ilexibility is achieved both by the diIIerent G-box Ilanking
35
sequences (236), and close interaction with immediate cis-and trans-acting Iactors. To
understand the interplay oI transcriptional networks in plants, we ultimately need to know
the expression patterns oI all trans-acting Iactors as well. IdentiIication and Iunctional
characterization oI the cis-acting element is no longer enough only thorough and
creative studies that can sort out the cross-talk oI pleiotropic or signal-speciIic eIIects in
mutants will deIine the physiological roles oI diIIerent signals and establish connections
between diIIerent pathways in the regulation oI plant gene transcription.
The practical importance oI better understanding the regulation oI plant promoters is
the potential to inIluence gene expression to manipulate plant metabolism (185), and
achieve compartmentalized production oI valuable pharmaceutical proteins Irom seeds
(237), leaves (238), roots (239), or Iruits (186).
AKNOWLEDGEMENTS
We wish to thank Peter Day, Michael Lawton, and Nir Yakoby Ior helpIul discussions
and critical reading, and Alison Garvey Ior her assistance in preparation oI the
manuscript.
36
Table 1. Online tools Ior structural and Iunctional analysis oI plant promoters.
Online Resource Description
PlantCARE
http://oberon.rug.ac.be:8080/PlantCARE/
ReIerential database with 435 plant cis-acting
elements describing 159 plant promoters.
PLACE
http://www.dna.aIIrc.go.jp/htdocs/PLACE/
Database oI cis-acting regulatory DNA
elements reported in vascular plants.
TRANSFAC
http://transIac.gbI.de/TRANSEAC/
Database on eukaryotic cis-acting regulatory
DNA elements and trans-acting Iactors.
Eukaryotic Promoter Database, EPD
http://www.epd.isb-sib.ch/
EPD is a specialized annotation database oI
eukaryotic promoters Irom EMBL.
Neural Network Promoter Prediction
http://www.IruitIly.org/seqtools/promoter.html
Prediction oI the putative promoters in
prokaryotic and eukaryotic DNA sequences.
Signal Scan
http://bimas.dcrt.nih.gov/molbio/signal/
Homology search Ior published cis-elements
based on TRANSEAC signal database.
MAR-Wiz
http://www.IuturesoIt.org/MAR-Wiz/
Detection oI putative matrix attachment regions
in eukaryotic DNA sequences.
37
Table 2. Common cis-acting elements involved in temporal and/or spatial regulation oI
gene expression in plants. Only transcription Iactors, Ior which there is strong evidence
suggesting their importance in gene regulation are included in this table.
Responses &LV-elements/Consensus 7UDQV-acting Factors
L
i
g
h
t
Light
GT-1 box, GGTTAA
I-box, GATAAAGR
G-box, CACGTG
H-box, ACCTA(A/C)C(A/C)
HY5 (59), PIE3 (60)
Auxin
TGTCTC motiI
TGTCCCAT box
osc/as-1 element
ARE1 (69)
ASE1 (74)
Gibberellin
TAACA(A/G)A element
TATCCAC element
(C/T)CTTTT(C/T) element
GAmyb (80)
Abscisic Acid G-box, CCACGTGG VIP1 (87)
Ethylene A(T/A)TTCAAA element E4/E8BP (95)
M
e
t
a
b
o
l
i
c

R
e
g
u
l
a
t
i
o
n
Sugars
TATCCA element
GC-box, GCC(G/C)C
G-box, CACGTG
SURE, (AA)TACTA(A/T)T
W-box, (T)TGAC(C/T)
OsMYBS (107)
SPE1 (102)
Heat GAATTC element HSE (121)
Oxidation
G-box, CACGTG
H-box, ACCTA(A/C)C(A/C)
W-box, (T)TGAC(C/T)
GCC element
Cold,
Drought
C-repeat, CCGAC
G-box, CACGTG
CBE1 (130)
Hypoxia
GT motiI, (T/C)GGTTT
GC motiI, GCC(G/C)C
AtMYB2 (138)
E
n
v
i
r
o
n
m
e
n
t
a
l

S
t
r
e
s
s
Pathogen,
Wounding,
Ethylene,
1A, SA
G-box, CACGTG
C-repeat, CCGAC
GCC element, AGCCGCC
W-box, (T)TGAC(C/T)
ORCA2 (148), ERE1 (150)
WRKY (155)
Seed-
RY motiI, CATGCATG
G-box, CACGTG
E-box, CACCCTG
AACA motiI
GNC4 motiI, TGAGTCA
P-box, TGTAAAG
EUS3 (167), ABI3 (168)
OsMYB5 (173)
Opaque2 (175)
PBE (174)
Fruit- TGTCACA motiI
D
e
v
e
l
o
p
m
e
n
t
Pollen- 32 bp motiI
38
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Fig. 1. Eundamental modules oI plant polymerase II promoters. Core promoter elements include TATA-
box, Initiator (INIT), and dowstream promoter element (DPE), and direct the proper positioning and
intiation oI transcription by RNA polymerase II and basic transcription Iactors (BTEs), including
TATA-binding protein (TBP) and TEIID. Proximal cis-elements (CAAT-, GCC-box) are present in many,
but not all plant promoters. Sequence-speciIic transcription Iactors (SSTEs) recognize gene-speciIic,
signal-responsive upstream regulatory elements. These elements are oIten modular and require copuling oI
a constitutive cis-element (e.g., G-box) and a signal-speciIic cis-element Ior a Iull-strength response.
Bending and other modiIications to the local DNA structure Iacilitate the cross-talk interaction between the
diIIerent modules and core promoter region, adding to the complex combinatorial regulation oI plant
promoters.
DPE INI TATA CAA GCC
RNA Pol
TFIID
BTFs
BTFs TBP
SSTF
SSTF DN
Proximal Elements
FLV1
Upstream Elements
G-box
G-box
FLV
FLV3
FLV2
Core Promoter

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