From the Graduate Institute of Life Sciences,
National Defense Medical Center, and Institute of Molecular Biology,
Academia Sinica, Taipei, Taiwan, Republic of China and the
§ Institute of Molecular Biology, Academia Sinica, Nankang,
Taipei 11529, Taiwan, Republic of China
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ABSTRACT |
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Expression of -amylase genes in both rice
suspension cells and germinating embryos is repressed by sugars and the
mechanism involves transcriptional regulation. The promoter of a rice
-amylase gene
Amy3 was analyzed by both loss- and
gain-of-function studies and the major sugar response sequence (SRS)
was located between 186 and 82 base pairs upstream of the transcription
start site. The SRS conferred sugar responsiveness to a minimal
promoter in an orientation-independent manner. It also converted a
sugar-insensitive rice actin gene promoter into a sugar-sensitive
promoter in a dose-dependent manner. Linker-scan mutation
studies identified three essential motifs: the GC box, the G box, and
the TATCCA element, within the SRS. Sequences containing either the GC
box plus G box or the TATCCA element each mediated sugar response, however, they acted synergistically to give a high level glucose starvation-induced expression. Nuclear proteins from rice suspension cells binding to the TATCCA element in a sequence-specific and sugar-dependent manner were identified. The TATCCA element
is also an important component of the gibberellin response complex of
the
-amylase genes in germinating cereal grains, suggesting that the
regulation of
-amylase gene expression by sugar and hormone signals
may share common regulatory machinery.
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INTRODUCTION |
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Sugar repression of gene expression is a fundamental and ubiquitous regulatory system for adjusting to changes in nutrient availability in both prokaryotic and eukaryotic cells. In microorganisms, glucose or other rapidly metabolizable carbon sources repress the expression of genes that code for enzymes related to the metabolism of other carbon sources. Our understanding of the mechanisms of sugar repression has been based largely on studies of microorganisms. In the case of Escherichia coli, a model to explain at the molecular level, the mechanism of sugar repression has been determined (1, 2). The mechanism of glucose repression in yeast is more complicated and is less understood than it is in E. coli (3-5). Studies using Saccharomyces cerevisiae mutants have revealed many of the components involved in the response to carbon catabolite repression (5), but it is still unclear how all of these components interact to regulate transcription. A universal signaling pathway which leads to the regulation of all glucose-repressible genes has yet to be determined.
As in microorganisms, sugar repression of gene expression also allows plant cells to cope effectively with changes in the carbon sources present in their environment. However, in multicellular plants, feedback repression by excess sugars provides an additional mechanism for maintaining an economical balance between supply (source) and demand (sink) for carbohydrate allocation and utilization among tissues and organs (6-8). Despite the fact that sugar repression of gene expression is likely a central control mechanism mediating energy homeostasis and carbohydrate distribution in plants, the molecular mechanism of sugar feedback regulation remains elusive. For example, sugar feedback regulation of genes involved in photosynthesis (9) and carbohydrate metabolism (10-12) has been shown to act at the level of gene transcription. A conserved glucose-sensing mechanism, via the action of hexokinase, has been observed between plants and microorganisms (13-15). However, the mechanism which connects the sensing and transmission of sugar signals and the repression of gene transcription in plants is mostly unknown.
The sugar-dependent repression of -amylase gene
expression provides an ideal model for studies on the molecular
mechanisms that mediate glucose repression in plants.
-Amylases are
endo-amylolytic enzymes which catalyze the hydrolysis of
1,4-linked glucose polymers that play an important role in the
degradation of starch and glycogen in higher plants, animals, and many
microorganisms.
-Amylases in plants are recognized as essential
enzymes whose major function is hydrolysis of starch stored in the
endosperm during germination of cereal grains. Expression of
-amylase genes in rice is found under different modes of
tissue-specific regulation: in the embryo of germinating seeds and in
cultured suspension cells, expression is activated by sugar deprivation
and repressed by sugar provision (8, 16-18); in the endosperm of
germinating seeds, expression is activated by gibberellic acid and
repressed by abscisic acid and osmotic stress (8, 19). Studies with
rice suspension cells have shown that
-amylase expression,
carbohydrate metabolism, and vacuolar autophagy are coordinately
regulated by sucrose levels in the medium (20). Both the transcription
rate and mRNA stability of
-amylase genes in cells increase in
response to sucrose depletion in the culture medium (12). Use of
transgenic rice carrying an
-amylase gene promoter-
-glucuronidase
(GUS)1 gene proved that the
regulation of
-amylase gene expression by sugars involves a
transcriptional control mechanism (10, 21, 22).
Sugar-dependent repression of
-amylase gene expression has also been observed in Aspergillus oryzae (23) and
Drosophila melanogaster (24) and the mechanism was shown to
involve transcriptional control (25, 26).
Rice -amylase isozymes are encoded by at least nine genes (7). By
using
-amylase gene-specific DNA fragments and nuclear run-on
transcription analysis, transcription of eight
-amylase genes was
shown to increase in response to sucrose starvation (27). A positive
correlation between the transcription rates and the steady-state
mRNA levels suggests that transcriptional regulation plays an
important role in the differential expression of individual
-amylase
genes. To date, studies on the transcriptional regulation of
-amylase gene expression in plants have mostly focused on the
hormonal regulation in germinating cereal grains (28-33). Since sugars
regulate the expression of
-amylase genes in germinating seeds as
well as in cultured suspension cells, studies on the mechanism of sugar
feedback regulation using rice suspension cells as a model system may
lead us to a better understanding of the mechanisms controlling
carbohydrate metabolism in higher plants. We chose
Amy3
as a model gene for this study because it constitutes approximately
60% of total
-amylase mRNAs in cells starved for sucrose (27)
and its expression in germinating embryo is regulated by sugars (8,
16). The
Amy3 promoter has been shown to mediate
sugar-dependent regulation of GUS reporter gene expression
in transgenic rice suspension cells (22).
In this report we developed a transient expression system using
protoplasts prepared from rice suspension cells to determine the
cis-acting sugar-responsive sequences in the
Amy3 promoter. The literature has revealed that, in the
case of studying regulation by environmental and physiological cues,
results obtained from transient assays often rapidly and efficiently
provide important information for further studies and reflect the
in vivo situation in planta. For example, studies
on auxin (34, 35) and gibberellin (28, 29, 32) response elements using
transient assays have led to the identification of interacting
transcription factors (28, 36). By conducting loss-of-function,
gain-of-function, and linker-scan mutation analyses, we defined the
minimal sequence in the
Amy3 promoter that drives high
level glucose starvation-induced expression. This minimal sequence
served as a transcriptional enhancer and converted a sugar-insensitive
promoter into a sugar-sensitive promoter. We also identified three
essential motifs, the GC box, the G box, and the TATCCA element, that
form the sugar response complex and act cooperatively in controlling
Amy3 expression. Finally, we demonstrated that nuclear
proteins from rice suspension cells bind to the TATCCA element in a
sequence-specific and sugar-dependent manner. To our
knowledge, this is the first identification of the functional sugar
response elements and the existence of interacting trans-acting factors
for sugar repressible genes in plants.
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EXPERIMENTAL PROCEDURES |
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Rice Cell Culture-- Suspension cell cultures of rice (Oryza sativa cv. Tainan 5) were propagated as described previously (17). Established suspension cells were subcultured every 7 days by transferring about 0.5 ml of cells into 25 ml of fresh liquid Murashige and Skoog medium (37) containing 3% sucrose in a 125-ml flask. Cells were cultured on a reciprocal shaker at 120 rpm and incubated at 26 °C in the dark.
Primer Extension Analysis--
Three 18-20-base gene-specific
oligonucleotides complementary to the signal peptide regions of
Amy7 and
Amy8, and the 5'-untranslated leader of
Amy3 (Fig. 1B) were synthesized and
used as primers. The primer extension analysis was performed according
to Sutliff et al. (38).
Plasmid Construction--
A 1.7-kilobase
SalI-EcoNI fragment, containing the promoter, the
5'-untranslated sequence, and 84 bp downstream of the translation start
site of Amy3 was end-blunted and cloned into the
ClaI site of pBSI-132, forming p3G-132II. pBSI-132 was
generated by insertion of a PvuII fragment (GUS coding
sequence-nopaline synthase gene (nos) terminator fusion)
from pBSI (21) into the HindIII site of pTRA132 (cauliflower
mosaic virus 35S RNA (CaMV35S) promoter-hygromycin B phosphotransferase
(hph) coding sequence-tumor morphology large gene
(tml) terminator fusion) (39) by blunt-end ligation. Plasmid p3G-132II was used as the progenitor for all constructs reported in
this paper. The sequences of oligonucleotides used in preparation of
the
Amy3 promoter constructs are listed in Table
I. Appropriate combinations of 5' and 3'
primers were used to generate different 5' deletions, internal
deletions, and other mutations by polymerase chain reaction (PCR). For
a series of 5'-deleted constructs, the PCR products were digested with
EcoRI and PstI and ligated into pBluescript KS+
(Stratagene) to generate p3.4, p3.5, and p3.6. These plasmids were
digested with KpnI and PstI and the promoter regions were ligated into the KpnI and
PstI-digested pLuc, forming p3Luc.3, p3Luc.4, p3Luc.5, and
p3Luc.6. pLuc was generated by insertion of a
SalI-BglII fragment (luciferase (Luc)
coding sequence-nos terminator fusion) from pJD312 (40) into
the SmaI site of pBluescript. For internal deletion
constructs, PCR was performed using the oligonucleotide-directed
mutagenesis as described by Picard et al. (41). In this
method, the 5' internal deletion primers were first paired with 3'
primers to generate short DNA fragments. The PCR products then served
as 5' primers and paired with 3' primers to generate internal deletion
fragments. These fragments were cloned into pLuc using the same
procedures for 5'-deleted constructs and generates p3Luc.7, p3Luc.8,
and p3Luc.9.
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Protoplast Isolation-- Three days after subculture, 25-ml of suspension cell culture was transferred to a 9-cm Petri dish. The Murashige and Skoog medium was removed and cells were washed once with CPW7.4 buffer (2 mM KH2PO4, 1 mM KNO3, 10 mM CaCl2/2H2O, 1 mM MgSO4/7H2O, 1 µM KI, 0.16 µM CuSO4/5H2O, 5 mM MES, and 0.4 M mannitol, pH 5.8) (43). Cells were incubated with 15 ml of protoplast isolation buffer (1% cellulase RS and 0.1% pectolyase Y-23 in CPW7.4 buffer) at 25 °C for 4 h with shaking at 50 rpm. After cell wall digestion, protoplasts were filtered through a 33-µm nylon mesh (Small Parts, Inc.), washed three times with CPW7.4 buffer by centrifugation at 100 × g for 5 min, and gently resuspended in 2 ml of CPW7.4 buffer. The protoplasts were layered on 5 ml of 0.6 M sucrose cushion in CPW7.4 buffer in a 15-ml Conical tube, and centrifuged at 40 × g for 10 min in a swing bucket rotor. Protoplasts at the top of sucrose cushion were collected and transferred to 10 ml of CPW7.4 buffer. Cells were then washed with electroporation buffer (0.14 M NaCl, 2.7 mM KCl, 0.7 mM KH2PO4, 4.2 mM Na2HPO4, 5 mM CaCl2, 0.4 M mannitol) (44), resuspended with the same buffer, and adjusted to 5 × 106 protoplast/ml.
Electroporation and Protoplast Culture--
Plasmid DNA was
transfected into rice protoplasts by electroporation. Each sample
containing 2 × 105 protoplasts in 0.4 ml of
electroporation buffer was mixed with 20 µg of test plasmid DNA, 5 µg of control plasmid DNA, and 50 µg of carrier (calf thymus) DNA.
The mixture was transferred to a cuvette and placed on ice for 10 min.
Electroporation was performed with a electroporator (BTX) with
conditions set at 1000 V/cm, 400 microfarads, and 186 . After
electroporation, the protoplasts were kept on ice for 10 min, then
mixed with 0.4 ml of electroporation buffer and 0.8 ml of 2 × modified Murashige and Skoog medium (containing 0.2 mg of
2,4-dichlorophenoxyacetic acid and 0.1 mg of kinetin per liter) plus
400 mM glucose or 400 mM mannitol and 5 mM glucose. The protoplasts were plated in a 3-cm Petri
dish and cultured 18 h at 26 °C in the dark.
GUS and Luciferase Assays--
Protoplasts (2 × 106) were collected by centrifugation for 10 s at
12,000 × g, resuspended in 0.3 ml of extraction buffer
(100 mM K2HPO4, pH 7.8, 1 mM EDTA, 7 mM -mercaptoethanol, 1% Triton X-100, and 10% glycerol), and vortexed for 10 s at high speed. The disrupted protoplasts were centrifuged at 12,000 × g and 4 °C for 5 min. Supernatant was collected and used
for GUS or luciferase activity assay. The enzyme activities in cell
extract could be maintained stable for at least 1 month at
80 °C.
Transformation of Tobacco--
DNA fragments containing the
Amy3-Luc-Nos chimeric gene were excised from plasmids
p3Luc.5 and p3Luc.6 with KpnI and SpeI and
ligated into the KpnI and XbaI sites of pBIN19
(47) to generate pA3Luc.5 and pA3Luc.6. pA3Luc.5 and pA3Luc.6 were
transferred into Agrobacterium tumefaciens strain EHA105
using an electroporation method described in the manufacturer's
instruction manual for the electroporator (BTX). The tobacco variety
Nicotiana tabacum L. cv. Petit Havana SR1 was used in this
study. Transgenic tobacco cell lines were obtained by transformation of
leaf discs with Agrobacterium according to the method of
Horsch et al. (48). Suspension cell cultures of the
transgenic tobacco were propagated as described previously (17).
Preparation of Nuclear Extract-- About 5 g (fresh weight) of rice suspension cells grown in the presence or absence of sucrose were pulverized in liquid nitrogen and homogenized in 200 ml of homogenization buffer (400 mM mannitol, 50 mM Tris-HCl, pH 7.9, 5 mM MgCl2, 1 mM EDTA, 0.1% bovine serum albumin, 0.1% Nonidet P-40, 5 mM dithiothreitol,and 1 mM phenylmethylsulfonyl fluoride). After this step, preparation of nuclear extract followed the procedures as described by Mitsunaga et al. (49).
Gel Mobility Shift Assay--
Oligonucleotides F1 through F5
were synthesized and their sequences are shown in Fig. 7A.
F2 used as probe was prepared by phosphorylation of the 5'-hydroxyl
terminal with T4 polynucleotide kinase and [-32P]ATP
(5000 Ci/mmol). DNA-protein binding reaction was carried out by
incubation of 0.02 ng of labeled F2 with 20 µg of nuclear extract in
a total volume of 20 µl of solution containing 17 mM Hepes, pH 7.9, 60 mM KCl, 7.5 mM
MgCl2, 0.12 mM EDTA, 17% glycerol, and 1.2 mM dithiothreitol, 0.5 µg of poly(dI-dC) (Pharmacia), and
3 or 10 ng (150- or 500-fold amount of probe, respectively) competitor
DNA. The assay mixture was incubated for 20 min at room temperature.
After this step, electrophoresis of the assay mixture and
autoradiography of gel followed the procedures as described by
Mitsunaga et al. (49).
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RESULTS |
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Sequence Analyses of the -Amylase Gene Promoters--
Although
the promoter regions of
Amy3(RAmy3D) (22),
Amy7(RAmy1A) (19), and
Amy8(RAmy3E) (10, 21) have been shown to
mediate sugar or hormonal regulation of GUS reporter gene expression in
transgenic rice, the transcription start sites of these
-amylase genes have not been mapped precisely. Here the transcription start site
of
Amy3 was mapped at 28 bp, and those of
Amy7 and
Amy8 were mapped 29 bp downstream
of the TATA box and designated as +1 (Fig.
1A). Inspection of the
promoter regions reveals two conserved sequence elements of 10 bp (TT
box) and 31 bp (GC box) (50) that are present in the
Amy3
and
Amy8 promoters but not in the
Amy7
promoter (Fig. 1B). The GA response element (28, 31) is
present in the
Amy7 promoter, but not in the
Amy3 and
Amy8 promoters. A G box sequence
(containing ACGT core) is located
141 to
132 of the
Amy3 promoter and
334 to
325 of the
Amy8 promoter. G box is present in the promoters of a
variety of genes that are responsive to several environmental and
physiological cues (51). Another sequence of 6 bp (TATCCA element) is
present in two copies in the
Amy3 promoter but only in
one copy in the
Amy7 and
Amy8 promoters.
Function of these elements in the sugar-dependent regulation of
-amylase gene expression has not been previously determined. We first chose the
Amy3 promoter as a model
for the following study.
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Deletion Analysis of the Amy3 Promoter--
We made a
transcriptional fusion of the 1-kilobase promoter region (900-bp
promoter plus 91-bp untranslated sequences) of
Amy3 to
the coding region of a luciferase gene (Fig.
2, p3Luc.3). This chimeric gene has been
stably transfected into rice suspension cells and found to confer
sugar-dependent repression of luciferase expression (data
not shown). To identify sequences in the
Amy3 promoter
that are involved in the sugar-dependent regulation, three
constructs containing progressive deletions at the 5' end of the
Amy3 promoter were made. The resultant constructs were then analyzed by transient expression in rice protoplasts (Fig. 2A). We found that 5 mM glucose could cause
starvation of rice protoplasts and 400 mM glucose is
normally required to maintain the osmolarity of rice protoplasts. The
protoplasts were therefore analyzed in 5 mM glucose plus
400 mM mannitol (starved) or 400 mM glucose
(non-starved) condition after transfection. The results show that
deletion to position
450 upstream of the transcription start site
(p3Luc.4) produced a dramatic drop in the level of glucose
starvation-induced expression. Expression was further reduced when
deletion was made to
274 (p3Luc.5). Despite the dramatic reduction in
the absolute level of expression caused by the two promoter deletions,
the fold induction of expression by glucose starvation was maintained
at a similar level. Deletion of the next 174 bp (to position
100)
(p3Luc.6) abolished the expression regardless of the concentration of
glucose.
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Functional Analysis of the Sugar Response Sequence in the Amy3
Promoter--
To determine whether the cis-acting
element(s) required for sugar-dependent regulation is
located within the region downstream of
274 of the
Amy3
promoter, fragments covering various regions between
274 and
82
were inserted upstream of a CaMV35S minimal promoter-AdhI-Luc fusion gene, as shown in Fig.
3. These constructs were then tested for
transcriptional activity in rice protoplasts. Expression of the basic
construct containing no
Amy3 promoter sequence (p35mALuc)
did not respond to glucose starvation. When the
274 to
82
(p3Luc.15) and
186 to
82 (p3Luc.18) fragments were fused upstream
of the 35S minimal promoter, high levels of glucose starvation-induced
expression of luciferase were observed. Fragment
274 to
176
contains three protein binding sequences, designated Box B1, Box B2, and
Box B3 (49). Each of the three boxes contain a conserved GCCG(G/C)CG
motif and have been proposed to be involved in
sugar-dependent regulation of
Amy3 promoter (49). Deletion of the promoter region containing the three boxes (p3Luc.18) resulted in a 30% increase of glucose starvation-induced expression, suggesting that this region may contain negative
cis-acting elements. Surprisingly, when fragment
186 to
82 was inserted in reverse orientation upstream of the 35S minimal
promoter (p3Luc.18R), expression of luciferase in response to glucose
starvation was as high as that with the promoter fragment inserted in
the correct orientation (p3Luc.18). These results demonstrate that the
region between
186 and
82 contains most, if not all, of the
cis-acting element(s) required to confer high level glucose
starvation-induced expression on the 35S minimal promoter. We have
designated this region as a sugar-response sequence (SRS).
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A 52-bp Fragment Containing the TATCCA Element Enhances
Transcriptional Activity--
Comparison of the luciferase activity
produced by p3Luc.40 and p3Luc.41 in Fig. 3 suggests that the 52-bp
fragment encompassing 133 to
82 enhances transcription. Mutation in
the TATCCA element within this fragment reduced transcription, which
further suggests that the TATCCA element is essential for enhancing
transcription. To demonstrate the function in enhancing transcription,
multiple copies of the 52-bp fragment were fused upstream of the 35S
minimal promoter and the luciferase activity was assayed. As shown in Fig. 4, duplication of the 52-bp fragment
resulted in the increase of starvation induced luciferase activity. The
increase became almost linear as more copies of the fragment were
added. Luciferase activity in non-starved cells also increased linearly
with additional copies of the 52-bp fragment, consequently, the fold
induction of the luciferase activity by glucose starvation was not
increased in parallel. The results suggest that the 52-bp fragment
enhances transcription of the minimal promoter regardless of the
glucose concentration.
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SRS Acts as a Transcriptional Enhancer in a Sugar-insensitive
Promoter--
The SRS conferred high level glucose starvation-induced
expression on the 35S minimal promoter in an orientation-independent manner, suggesting it may also function as a transcriptional enhancer. To confirm the enhancer function, SRS was inserted in one, two, and
three tandem copies in the EcoRI site (459 bp upstream of the transcription start site) of the rice Act1 promoter
(52). The wild type and the SRS-containing Act1 promoters
were fused upstream of Luc gene as shown in Fig.
5. These sugar response sequence
constructs were then tested for transcriptional activity in rice
protoplasts. Expression of the control construct (p35mALuc) was not
detected. Expression of the wild type Act1 promoter
(pActLuc) and the Act1 promoter containing one to three
copies of SRS was similar in high concentration glucose. Expression of
the Act1 promoter containing one copy of SRS (p3Luc.37+)
increased 2-fold as compared with that of the wild type Act1
promoter in the glucose-starved cells. Surprisingly, duplication of SRS
(p3Luc.37++) dramatically increased fold induction by glucose
starvation and the fold induction increased almost linearly as more
copies of SRS were added (p3Luc.37+++). The results demonstrate that
expression of the Act1 promoter becomes inducible by glucose
starvation if the promoter is inserted with multiple copies of SRS.
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Linker-scan Mutation Analysis of SRS in the Amy3
Promoter--
Understanding that SRS confers a high level glucose
starvation-induced expression on the Act1 and 35S minimal
promoters, the next step was to more precisely locate the
cis-acting elements involved in sugar responsiveness.
Various 10-bp fragments containing EcoRI sites (GAATTC) were
introduced into individual constructs to replace various regions within
SRS in p3Luc.18, thus generating constructs p3Luc.28 through p3Luc.36,
as shown in Fig. 6A. These constructs were then tested for luciferase activity and the results are
shown in Fig. 6B. All the linker substitutions had more or less effect on expression as compared with the wild type sequence. The
GC box can be further divided into three GC-rich subdomains designated
as GC1, GC2, and GC3 boxes. The GC2 and GC3 boxes each contain the
identical 9-bp sequence CCGACGCGG. Mutations in the GC2 box (p3Luc.29)
and GC3 box (p3Luc.30) resulted in 40 and 60% reduction of expression,
respectively. Two mutations in the duplicated TATCCA element (p3Luc.33
and p3Luc.34) caused dramatic reduction in the level of glucose
starvation-induced expression to 12 and 8% of the control (p3Luc.18),
respectively. Mutations in the G box (p3Luc.31) resulted in a 80%
reduction of expression. These results demonstrate that all of the
sequences within SRS are necessary, and that the GC3 box, the G box,
and the TATCCA element are the most important sequences for high level
glucose starvation-induced expression.
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Nuclear Proteins Binding to the cis-Acting Elements in
SRS--
Since the 52-bp fragment containing the TATCCA element
enhanced glucose starvation-induced transcriptional activity (Fig. 4)
and the TATCCA element is essential in conferring
sugar-dependent regulation (Fig. 6), we examined whether
nuclear proteins from rice suspension cells bind to the TATCCA element.
DNA fragments encompassing various regions of SRS were synthesized and
designated as F1 through F5 (Fig.
7A). These fragments were
assayed for their ability to interact with nuclear protein extract from
rice suspension cells grown in the presence or absence of sucrose. In
the gel mobility shift assay using F2, which contains the TATCCA
element, as the probe, two DNA-protein complexes (C1 and C2) were
observed regardless of whether the nuclear extract was from cells grown in the presence of sucrose (+S) (Fig. 7B, lane 2) or in the
absence of sucrose (S) (Fig. 7B, lane 3).
However, the band intensity between F2 and the nuclear extract was
5-fold higher for
S cells than that for +S cells. C1 and C2 were
competed out by a 500-fold amount of F2 itself (Fig. 7B, lanes 6 and 11) and a 150-fold amount of F5 which contains F2
(Fig. 7B, lanes 9 and 14).
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DISCUSSION |
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The GC Box, G Box, and TATCCA Element Act Synergistically in
Regulating Sugar-dependent Expression of a Minimal
Promoter--
Linker-scan mutation analysis identified the GC3 box, G
box, and TATCCA element within SRS as the most essential sequences to
confer high-level glucose starvation-induced expression. Fragment 186
to
122, which contains the GC box and G box (p3Luc.19) only, dramatically reduced the glucose starvation-induced expression of the
35S minimal promoter (Fig. 3). Such reduction was probably not simply
due to an alteration in the distance between these sequences and the
TATA box, because mutation of the TATCCA element within SRS (p3Luc.33
and p3Luc.34) without alteration in the distance still resulted in
significant reduction of glucose starvation-induced expression (Fig.
6). Apparently, the TATCCA element is an indispensable element for
high-level expression. The 52-bp fragment (
133 to
82), which
contains the duplicated TATCCA element only (p3Luc.40), shows no
induction in Fig. 3, but an 1.8-fold induction in Fig. 4. The
discrepancy was probably due to technical difficulty and variation in
preparation and electroporation of rice protoplasts from one experiment
to another, but the variation always remained below a factor of two in
three repeated experiments for each construct. Therefore, the
luciferase activity from p3Luc.40 is not significantly different
between cells grown with and without glucose and are close to
background. Duplication of the 52-bp fragment (p3Luc.41) recovered the
glucose starvation-induced expression, indicating that stable
interaction between trans-acting factor(s) and the TATCCA
element requires neighboring sequences around
133 and/or
82.
Mutations in either the downstream or upstream copy of the TATCCA
element (p3Luc.41 m1 and p3Luc.41 m2) reduced the starvation-induced expression which suggests both the sequence of TATCCA element and the
distance relative to TATA box are essential for expression.
The 52-bp Fragment Containing the TATCCA Element Enhances
Sugar-dependent Transcription--
It is interesting to
note that the absolute level of expression from more than four copies
of the 52-bp fragment containing the TATCCA element far exceeded that
from SRS regardless of the glucose concentration (Fig. 4).
Particularly, the 52-bp fragment can substitute for the GC box and G
box for conferring high level glucose starvation-induced expression.
Multiple copies of the 52-bp fragment enhanced transcription regardless
of the glucose concentration, which suggests that transcription factors
binding to the cis-acting element (possibly the TATCCA
element) is constantly present in the nucleus. More copies of the 52-bp
fragments may recruit more transcription factors to the promoter
region. The higher level of expression induced by glucose starvation
could be due to an increase in the abundance of transcription factors or in the affinity for binding between the cis-acting
element and the transcription factors under glucose starvation. On the other hand, the fold induction of luciferase activity from SRS under
glucose starvation was higher than that from multiple copies of the
52-bp fragment (Fig. 4), suggesting that an additional sequence(s) is
required for the sugar repression of gene expression. The additional
sequence(s) must be present between 186 and
133.
The TATCCA Element Enhances GA- and Sugar-dependent
Promoter Activities--
Examination of promoter sequences of nine
rice, nine barley, and five wheat -amylase genes that are available
in GenBank reveals that all except four of these genes have TATCCA
variants, and that they all contain a TATCCA element at positions
approximately 100 to 150 bp upstream of transcription start sites. This
observation suggests that the TATCCA element may play a role in the
regulation of
-amylase gene expression. Mutations of the TATCCA
element in the promoters of both barley high-PI
-amylase gene Amy
pHV19 (28) and low-PI
-amylase gene Amy32b (29) were found to lower expression to about 20% of maxima but maintained GA responsiveness. In
our study, mutation of the duplicated TATCCA element (p3Luc.33 and
p3.Luc.34) also reduced the
Amy3 promoter activity to 12 and 8%, respectively, of the wild type sequence (p3Luc.18) but maintained sugar responsiveness (Fig. 6). These results suggest that
the TATCCA element enhances transcription in concert with the hormone-
or sugar-regulated interactions on other sequences to promote the
transcription of
-amylase promoters in an environmental signal- and
tissue-dependent manner. The function of the TATCCA element, possibly with the involvement of its flanking sequences, seems
to operate somewhat differently between the GA- and
sugar-dependent regulatory systems, i.e. GA
control of expression exerted by the promoter fragments containing the
GA response element and TATCCA element from barley Amy pHV19 or rice
OSamy-c is orientation-dependent (28, 33),
whereas the sugar control of expression exerted by the promoter
fragment containing SRS from
Amy3 is
orientation-independent (Fig. 3).
SRS Converts a Sugar-insensitive Promoter into a Sugar-sensitive
Promoter--
The expression of the Act1 promoter-GUS gene
(data not shown) or Act1 promoter-Luc (Fig. 5,
pActLuc) in rice protoplasts is normally not significantly affected by
glucose. Insertion of SRS in the 459 position of the Act1
promoter alters the mode of regulation of this promoter by glucose.
Previously, the Act1 promoter deletion to nucleotide
459
has been shown to still display high activity in rice protoplasts (53),
suggesting that conversion of the promoter activity in response to
glucose is not simply due to disruption of the promoter sequence by
SRS. It is interesting to note that the absolute level of glucose
starvation-induced expression of the Act1 promoter
containing SRS was dramatically higher than that of the 35S minimal
promoter fused to SRS (compare Fig. 5 with Fig. 3), suggesting that SRS
may enhance the transcription of a promoter by a much higher magnitude
if the promoter has full function.
Nuclear Protein Factors Binding to the TATCCA Element and Its
Flanking Sequences in a Sequence-specific and
Sugar-dependent Manner--
An important step toward
understanding the molecular mechanism of sugar-dependent
regulation is the identification of trans-acting regulatory
proteins. By gel mobility shift assay, nuclear proteins from rice
suspension cells were found to specifically bind to the TATCCA element
and its flanking sequences (Fig. 7, B and C). The
protein factors were present in the nuclei of +S or S cells, which
suggests that the protein factors are constantly present in the
nucleus. Higher amounts of complex formations between the nuclear
proteins from the
S cells and the TATCCA element could be due to the
existence of higher amounts of transcription factors in the
S cells,
or the higher binding affinity of transcription factors with the TATCCA
element under sucrose starvation. Higher amounts of complex formations
between the nuclear protein(s) from the +S cells and the flanking
sequences of the TATCCA element can be similarly explained. However,
protein factors bind to the TATCCA element and its flanking sequences
may serve opposite functions, activator and repressor, respectively.
Although the data shown in Figs. 5 and 6 suggest that activators may be
involved in the sugar starvation-induced
Amy3 expression,
the possibility that the repressor is also involved in the sugar
repression of
Amy3 expression cannot be ruled out. This
possibility is supported by an observation that the protein synthesis
inhibitor cycloheximide enhanced transcription of the
Amy3 promoter (data not shown), probably through an
inhibition of repressor synthesis. As judged from the involvement of
multiple cis-acting elements in the sugar response and the
complexity of DNA-protein interaction patterns, the transcription
factors may be post-translationally modified, recruit additional
proteins, or exhibit specificity in their interaction with other
transcription factors.
Conclusion--
In the last decade, molecular mechanism of GA
regulation of -amylase gene expression have been extensively studied
and functional analyses have identified several promoter sequences
important for the GA response. Despite the fact that sugar also serves
as an essential signal in controlling the expression of
-amylase genes in cultured rice suspension cells (17), in germinating rice seed
(8, 16), and in germinating barley embryo (54, 55), to date the sugar
response sequence has been studied only for the rice
Amy3. In addition, although carbohydrate depletion induces expression of a variety of genes involved in photosynthesis, reserve mobilization, and export processes (56), the
cis-acting sugar response elements in the promoters of these
genes have not been precisely defined neither. A 20-bp sequence (at
position
90 to
70 upstream of the transcription start site) in a
Drosophila
-amylase gene promoter was shown to be
necessary for full activity of this promoter in transformed larvae
(25). However, this 20-bp sequence does not contain a GC box or G
box-like sequence or TATCCA element. Conserved cis-acting
elements have not yet been found among promoters of sugar repressible
genes in plants, Drosophila, and yeasts. Our studies have
initiated an important question as to whether the mechanisms through
which sugar regulates gene expression are conserved or diverged
throughout the evolution of different kingdoms.
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ACKNOWLEDGEMENTS |
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We thank Dr. Li-Fei Liu for providing rice suspension cells, Dr. Teh-hui Kao for critical review of this manuscript, and Lin-Tze Yu for technical assistance.
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FOOTNOTES |
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* This work was supported by a grant from Academia Sinica and National Science Council Grant NSC84-2311-B-001-026 of the Republic of China.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Current address: The Plant Laboratory, Dept. of Biology, University of York, P. O. Box 373, York YO1 5YW, United Kingdom.
To whom correspondence should be addressed. Tel.:
886-2-2788-2695; Fax: 886-2-2788-2695 or 886-2-2782-6085; E-mail:
sumay{at}ccvax.sinica.edu.tw.
1
The abbreviations used are: GUS,
-glucuronidase; bp, base pair(s); PCR, polymerase chain reaction;
CaMV35S, cauliflower mosaic virus 35S RNA; MES,
4-morpholineethanesulfonic acid; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; SRS,
sugar response sequence; Luc, luciferase.
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REFERENCES |
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