Plant Physiol. (1999) 121: 225-236
Identification of cis-Acting Elements Important
for
Expression of the Starch-Branching Enzyme I Gene in
Maize
Endosperm1
Kyung-Nam Kim2 and
Mark J. Guiltinan*
Intercollege Graduate Program in Plant Physiology, The
Biotechnology Institute, and Department of Horticulture, The
Pennsylvania State University, University Park, Pennsylvania 16802
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ABSTRACT |
The
genes encoding the starch-branching enzymes (SBE) SBEI, SBEIIa, and
SBEIIb in maize (Zea mays) are differentially regulated in tissue specificity and during kernel development. To gain insight into the regulatory mechanisms controlling their expression, we analyzed the 5
-flanking sequences of Sbe1 using a
transient gene expression system. Although the 2.2-kb 5-flanking
sequence between 
2,190 and +27 relative to the transcription
initiation site was sufficient to promote transcription, the addition
of the transcribed region between +28 and +228 containing the first
exon and intron resulted in high-level expression in
suspension-cultured maize endosperm cells. A series of 5 deletion and
linker-substitution mutants identified two critical positive
cis elements, 314 to 295 and 284 to 255. An
electrophoretic mobility-shift assay showed that nuclear proteins
prepared from maize kernels interact with the 60-bp fragment containing
these two elements. Expression of the Sbe1 gene is
regulated by sugar concentration in suspension-cultured maize endosperm
cells, and the region 314 to 145 is essential for this effect.
Interestingly, the expression of mEmBP-1, a bZIP transcription
activator, in suspension-cultured maize endosperm cells resulted in a
5-fold decrease in Sbe1 promoter activity, suggesting a
possible regulatory role of the G-box present in the
Sbe1 promoter from 227 to 220.
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INTRODUCTION |
Starch, the major form of carbon and energy reserve in plants,
provides a major caloric source for the human population of the world
and is also an important industrial commodity. Although the pathway of
starch biosynthesis is not completely understood, there is no doubt
that it involves at least four groups of committed enzymes: ADP-Glc
pyrophosphorylase (EC 2.7.7.23), starch synthase (EC 2.4.1.21),
starch-branching enzyme (SBE; EC 2.4.1.28) and starch-debranching
enzyme (EC 2.4.1.41) (for review, see Preiss, 1991
; Martin and Smith,
1995).
SBEs catalyze the formation of amylopectin by introducing 
-1,6
branch points into the linear -1,4-linked Glc chains. The introduction of branches not only changes many of chemical and physical
properties of starch, but also facilitates starch synthesis by
increasing the number of nonreducing ends, the site of Glc addition by
starch synthases. Thus, SBEs are of crucial importance for the quantity
and quality of starch synthesized in the plant (Edwards et al., 1988).
In fact, mutations in Sbe genes of pea, maize (Zea
mays), and rice severely decreases the total starch content and
changes the ratio of amylose and amylopectin (Shannon and Garwood,
1984; Smith, 1988; Bhattacharyya et al., 1990; Mizuno et al., 1993).
Multiple forms of SBEs, differing in enzymatic and biochemical
properties, have been identified and characterized in various plants,
such as spinach (Hawker, 1974), pea (Matters and Boyer, 1981;
Smith, 1988), potato (Griffin and Wu, 1968; Khoshnoodi et al., 1993),
teosinte (Boyer and Fisher, 1984), rice (Mizuno et al., 1992; Nakamura
et al., 1992; Yamanouchi and Nakamura, 1992), and maize (Hodges et al.,
1969; Boyer and Preiss, 1978; Dang and Boyer, 1989). They were grouped
into two distinct families based on their structural relatedness and
named according to the prototypic family member from maize (Burton et
al., 1995; Gao et al., 1996). The SBEI family consists of maize SBEI,
rice SBEI, and pea SBEII; and the SBEII family encompasses maize SBEIIa
and SBEIIb, rice SBEIII, and pea SBEI.
Significant differences in the enzymatic properties between the SBE
families are well documented (for review, see Martin and Smith, 1995).
SBEs belonging to the SBEII family have lower affinity for amylose than
SBEI isoforms and prefer to use shorter glucan chains for further
branch formation. Another noteworthy difference between the SBEI and
SBEII families is that they are differentially regulated during seed
development. The SBEII family genes are expressed earlier than the SBEI
family members in developing seeds (Smith, 1988; Burton et al., 1995;
Gao et al., 1996), which may result in changes in the SBEI to SBEII
ratio. Since SBEI and SBEII have significantly different in vitro
catalytic properties (as mentioned above), such changes in the SBEI to
SBEII ratio may cause differences in the starch synthesized during
kernel development. During pea embryo development, changes in the SBE
isoform ratio was accompanied by transition in the branch lengths of
amylopectin (Burton et al., 1995).
SBEII has been further resolved in maize endosperm by chromatography on
4-aminobutyl-Sepharose into two fractions: SBEIIa and SBEIIb (Boyer and
Preiss, 1978). Although these two isoforms are similar in molecular
mass, amino acid composition, proteolytic digest map, and immunological
reactivity, they do have distinct properties (for summary, see Fisher
et al., 1996) and are encoded by different genes (Gao et al., 1997).
For example, SBEIIb is more active than SBEIIa in the branching of
amylopectin (Boyer and Preiss, 1978). Takeda et al. (1993) also showed
that these two SBEII isoforms have different optimum temperature and
specific activities in the branching linkage assay.
Isolation of the maize cDNAs encoding SBEI, SBEIIa, and SBEIIb has
allowed us to investigate the Sbe genes at the molecular level (Fisher et al., 1993, 1995; Gao et al., 1996, 1997). Gao et al.
(1996) showed that Sbe1 and Sbe2b are expressed
in a coordinate fashion with the granule-bound starch synthase and
ADP-Glc pyrophosphorylase, respectively, during maize endosperm
development. The finding that many genes involved in starch
biosynthesis are regulated by sugar availability (Muller-Rober et al.,
1990; Koch et al., 1992; Giroux et al., 1994; Salehuzzaman et al.,
1994; Fu et al., 1995b) suggests that they may share common regulatory
mechanisms controlling their expression. Therefore, knowledge of the
regulatory mechanisms for one of the starch biosynthetic genes aid in
the understanding of how the other genes are controlled in plants. Unfortunately, however, little is known about promoter elements, transcription factors, or molecular mechanisms involved in the regulation of starch biosynthetic genes.
To begin to explore these questions, we have recently isolated and
sequenced maize genomic DNA fragments containing the Sbe1 and Sbe2b genes (accession nos. AF072724 and AF072725,
respectively), and established the complete genomic organization of the
genes (Kim et al., 1998a, 1998b). We report in this study functional analysis of the Sbe1 promoter, which revealed DNA sequence
elements important for the high-level, sugar-responsive expression of
the Sbe1 gene in maize endosperm cells.
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MATERIALS AND METHODS |
Construction of Chimeric Plasmids
A transcriptional fusion of the Sbe1 promoter to a
luciferase (LUC) reporter gene was made as follows. A BamHI
restriction enzyme site was first created just before the translation
initiation site of the Sbe1 gene by PCR: The DNA sequence
between 253 and +27 of the Sbe1 gene was PCR-amplified
with PI-1 and PI-2 primers (Table I).
Four additional bases were included at 5-end of the primers to provide
for restriction enzyme sites at the ends of the PCR products for
subsequent cloning. The bases were chosen randomly by considering their
effect on Tm and on dimer and stem-loop formation of the primers.
Pfu DNA polymerase (Stratagene), which has proofreading
activity, was used to enhance the fidelity of PCR amplification.
(Pfu DNA polymerase was used for all of the following PCRs.)
Since an ApaI restriction enzyme site (GGGCCC) is located
immediately downstream of the 5 primer (PI-1) binding region of the
Sbe1 promoter, 203 to 198, the PCR product was digested with ApaI and BamHI. The resulting 236-bp
fragment was then cloned into pBluescript SK
(Stratagene) and sequenced to verify that no misincorporation had
occurred in the DNA sequence during the PCR amplification (all of the
following PCR products were sequenced). Next, the 236-bp fragment was
ligated to the 1,991-bp SalI-ApaI Sbe1
promoter fragment and cloned into the promoterless LUC plasmid (pLN)
cut with SalI and BamHI (promoterless LUC-NOS
gene in pUC119) (Montgomery et al., 1993), thereby creating plasmid
pKL101.
To construct a translational fusion of the Sbe1 promoter
containing the first exon and intron to a LUC reporter plasmid, the DNA
sequence between 253 and +228 was amplified with the PI-1 primer and
a 3 primer (PI-3) designed to anneal to the region just downstream of
the first intron of the Sbe1 gene. The 493-bp PCR product
was digested with ApaI and BamHI, and the
resulting 436-bp fragment was used to replace the
ApaI-BamHI fragment in pKL101. This construct was
called pKLN101. To make pKLM101, which contains the Sbe1
promoter with four exons and introns, the 236-bp ApaI and
BamHI fragment in pKL101 was replaced with the 1816-bp Sbe1 genomic DNA fragment.
The plasmid pKLNS101 was derived from pKLN101 by replacing the nopaline
synthase (NOS) 3 sequence with the native Sbe1 3-flanking sequence. To accomplish this, two primers, PI-4 and PI-5, were designed
to amplify Sbe1 DNA sequences containing the transcription stop signal and the polyadenylation site (from +5382 to +5780). A
419-bp PCR product was digested with SacI and
EcoRI, and the resulting fragment was then used for
substituting a 255-bp SacI-EcoRI NOS 3 sequence
in the pKLN101.
To create a series of 5 deletions in the Sbe1 promoter,
pKLN101 was first modified as follows: pKLN101 DNA was digested with HindIII and the resulting 7,190-bp fragment lacking the
452-bp HindIII fragment was gel purified. The fragment was
blunt ended by Klenow fill-in DNA synthesis and ligated with
SalI linkers. After complete digestion with SalI,
the DNA fragment was partially digested with BamHI to
isolate the 1,993-bp SalI-BamHI fragment, which
was then gel purified and cloned into pLN cut with SalI and
BamHI to produce pKLN101-1.
A series of 5 deletion mutants were made from the plasmid pKLN101-1
using an S1-nuclease-based system (Erase-a-Base, Promega) to produce
the 5 deletion series plasmids pKLN102 to pKLN107. All constructs were
sequenced with the pUC/M13 reverse primer to verify deletion end
points. For the 254 and 196 deletion constructs, two regions of the
Sbe1 promoter, 254 to 146 and 196 to 146, were
PCR-amplified by primer PI-6 and PI-8, PI-7 and PI-8, respectively.
Primers used in PCR to create the Sbe1-LUC constructs are
shown in Table II. Since each 5 primer,
PI-7 and PI-8, contains a HindIII restriction enzyme site,
and since a BstXI restriction enzyme site is located between
173 and 162, the PCR products were digested with HindIII
and BstXI and the resulting fragments were used to replace
the 2,047-bp HindIII-BstXI fragment of pKLN101.
Linker-Scanning Mutagenesis
A series of linker-scan mutations were introduced into the 60-bp
DNA region from 314 to 255 as described by Kunkel et al. (1987).
The HindIII-BamHI (314 to +235) fragment from
pKLN105, containing the DNA region to be altered, was subcloned into
the corresponding sites of a M13 mp19 vector to produce a
single-stranded template. To increase mutant recovery efficiencies, the
template was prepared from an Escherichia coli
dut ung
strain (CJ236) that allowed the incorporation of uracil into the newly
synthesized DNA. Next, a set of oligonucleotides with 10-bp mismatches
(Table II) were annealed to the template and extended with T7 DNA
polymerase. After the addition of T4 DNA ligase, the resulting
heteroduplexes were introduced into a wild-type E. coli
strain (MV1190) to generate mutated double-stranded DNAs. DNA
sequencing was performed to verify that the desired mutations were
correctly introduced and that no unintended mutations had occurred.
To create the mutated Sbe 1 promoter-LUC constructs
(pLS1-1 to pLS1-6), the HindIII-BamHI
fragment in pKLN105 was replaced by each mutated DNA sequence.
Particle Bombardment
Suspension-cultured cells of maize (Zea mays) endosperm
(inbred line A636), provided by J.L. Anthony (DEKALB Genetics
Corporation, Mystic, CT), were grown in 250-mL large-mouth Erlenmeyer
flasks containing 80 mL of Murashige and Skoog basal salt medium
(Murashige and Skoog, 1962) supplemented with 0.4 mg/L of thiamine, 2 g/L of Asn, and 30 g/L of Suc (Shannon and Liu, 1977). The culture was
maintained in the dark at 29°C on a rotary shaker (120 rpm) and
subcultured every 7 d by transferring a portion of the cell suspension into fresh medium.
For particle bombardment, about 600 mg (fresh weight) of actively
growing cells 3 d after subculture was evenly distributed over the
surface of a piece of filter paper (Whatman no. 4, 55 mm in diameter)
by vacuum filtration of 8 mL of suspension culture. The filter paper
bearing the cells was then placed over three layers of filter paper
(Whatman no. 4, 70 mm in diameter) moistened with 5 mL of the liquid
medium containing 12% (w/v) Suc, and positioned in the middle
of a 10-cm Petri dish.
Gold microcarriers (1.6-µm particle size, 60 mg) were washed three
times with 1 mL of 100% (w/v) ethanol and twice with 1 mL of
sterile de-ionized water, resuspended in 1 mL of sterile de-ionized
water, and dispensed in 50-µL aliquots (3 mg/50 µL). The
Sbe1 promoter-LUC constructs and a GUS reference plasmid
(pBI221, Jefferson, 1987) were coprecipitated onto the gold particles
as follows: under continuous vortexing, the following were added in
order to each 50-µL aliquot of gold particles: 5 µL of DNA (8 µg
of LUC reporter plasmid and 4 µg of GUS reference plasmid), 50 µL
of 2.5 M CaCl2, and 20 µL
of 0.1 M spermidine (free-base, tissue-culture
grade). The gold particles coated with DNA were pelleted in an
Eppendorf centrifuge at 10,000 rpm for 10 s, rinsed with 250 µL
of 100% (w/v) ethanol, and resuspended in 60 µL of 100%
(w/v) ethanol. Immediately after sonication, 8 µL of the DNA-coated gold particles was pipetted onto the center of macrocarriers (Bio-Rad) and dried in a low-humidity environment.
A He biolistic particle-delivery system (model PDS-1000, Bio-Rad) was
used for particle bombardment. The bombardment parameters optimized
included He pressure, gap distance (the distance from the power source
to the macroprojectile), and the target distance (the distance from
microprojectile launch site to the sample target). After optimization,
all bombardments were performed in a dimly lit room at 650 psi under a
vacuum of 26 inches of Hg, with a distance of 10 cm between the cells
and the barrel of the particle gun. Following the bombardments, the
Petri dishes were sealed with laboratory film and incubated in the dark
at 25°C for 24 h.
GUS and LUC Assays
The bombarded cells were harvested from the plates by vacuum
filtration, frozen in liquid nitrogen, and ground with a mortar and
pestle to a fine powder. The powder was then transferred into a
microfuge tube and extracted with cell-culture lysis buffer containing
300 mM Tris-P, pH 7.8, 2 mM DTT, 2 mM
1,2-diaminocyclohexane-N,N,N,N-tetraacetic acid, 10% (v/v) glycerol, and 1% (v/v) Triton X-100
(0.3 mL/g of tissue). Cell debris were pelleted in an Eppendorf
centrifuge at 14,000 rpm for 10 min at 4°C, and the supernatant was
split into two aliquots for assays of GUS and LUC activity.
For fluorometric GUS assays (Jefferson, 1987), 30 µL of the
crude extract was incubated at 37°C with 2 mM
4-methylumbelliferyl glucuronide in 0.3 mL of GUS assay buffer (50 mM NaPO4, pH 7.0, 10 mM
EDTA, 0.1% [v/v] Triton X-100, 0.1% [v/v] Sarkosyl,
10 mM 
-mercaptoethanol, and 20% [v/v]
methanol). After 0, 1, and 2 h of incubation, 0.1-mL aliquots were
removed and added to 0.9 mL of 0.2 M
Na2CO3 to terminate the
reaction. A fluorometer (model TKO 100, Hoeffer, San Francisco)
calibrated by setting a 100 nM methylumbelliferone to 1,000 fluorescence units was used to measure fluorescence of the product,
4-methylumbelliferone. For each sample, results of the GUS assay were
plotted on a graph of A405
(y axis) versus time in minutes, and the GUS activity was
expressed simply as the slope of the line. GUS activity from the
suspension-cultered maize endosperm cells that had been bombarded with
the naked gold particles (no DNA) was used as a control.
LUC activity was determined by measuring luminescence (Monolight 1500 luminometer, Analytical Luminescence Laboratory, San Diego) for 10 s after mixing 20 µL of cell extract with 100 µL of LUC assay
reagent containing 20 mM Tricine, pH 7.8, 1.07 mM (MgCO3)4Mg(OH)2·5H2O,
2.67 mM MgSO4, 0.1 mM
EDTA, 33.3 mM DTT, 270 µM CoA, 470 µM luciferin, and 530 µM ATP. LUC activity
from the maize endosperm cells that had been bombarded with the pLN was
used as a control. To correct for differences in sample variability and
transfection efficiency, the LUC activity in the light unit was
normalized with GUS activity, yielding the LUC to GUS ratio of each
sample.
Nuclear Extract Preparation
Maize kernels (inbred line B73) were harvested 30 d after
pollination and frozen in liquid nitrogen. Nuclear extract was prepared essentially according to the method described by Jensen et al. (1988).
Protein concentration was determined using a BCA protein assay kit
(Pierce) according to the manufacturer's instructions.
DNA Probe Preparation
The Sbe1 promoter region from 314 to 255 was PCR
amplified with a forward primer
(5-GGACTTACATAAAATAAAAAAAGGCA) and a reverse primer
(5-TGCTAAGCTTTCTGGGCCGATTGGCCTTTG), which contain
BamHI and HindIII restriction enzyme sites,
respectively, at their 5 ends (underlined). The PCR product was
digested with BamHI and HindIII, and the
resulting fragment was cloned into pBlueScript SK (Stratagene) cut with BamHI and
HindIII to create plasmid pRb4-1. The plasmid construct was
verified with DNA sequencing. For electrophoretic mobility-shift
assays, the DNA fragment was cut out from the plasmid pRb4-1 with
HindIII and BamHI, purified from agarose gels,
and end-labeled with [-32P]dCTP using the
Klenow fragment.
Electrophoretic Mobility-Shift Assay
The DNA-protein binding reaction was performed in 20 µL of
solution containing 0.5 ng of labeled probe, 10 µg of nuclear
protein, 1 µg of poly(dI-dC)·poly(dI-dC), 12% (w/v)
glycerol, 12 mM HEPES-NaOH (pH 7.9), 4 mM
Tris-Cl (pH 7.9), 60 mM KCl, 1 mM EDTA, and 1 mM DTT. After a 20-min incubation at room temperature, the
samples were loaded into a 4% (w/v) native polyacrylamide gel
that had been prerun at 4°C for 1 h at 150 V and electrophoresed
for 2.5 h at 150 V in Tris-Gly buffer at 4°C. Following
electrophoresis, the gel was dried in a gel dryer (Bio-Rad) and exposed
to Kodak x-ray film with two intensifying screens for 24 h.
Northern-Blot Analysis
Total RNA was isolated according to the method of Vries et al.
(1988) from suspension-cultured maize endosperm cells that had
been incubated for 24 h in the Murashige and Skoog basal salt medium supplemented with 0.4 mg/L of thiamine, 2 g/L of Asn, and various amounts of Suc from 0% to 15%. Northern-blot analysis was
performed as described in Gao et al. (1996). Radioactivity was detected
with a phosphor imager and quantified with the ImageQuant software
program (both from Molecular Dynamics). To correct for minor loading
errors between the lanes, the blot was washed at 95°C in a 0.1%
(w/v) SDS solution to remove the 32P-labeled
Sbe1 cDNA probe and rehybridized with a
32P-labeled tomato cDNA for 26S rRNA.
| |
RESULTS |
Transcribed Regions of the Sbe1 Gene Are Involved in
Gene Expression
To determine whether the 5 flanking sequence of the
Sbe1 gene has all of the DNA elements necessary to initiate
transcription, a 2,217-bp fragment upstream of the translation start
site (2,191 to +27) was fused to the LUC reporter gene in pUC119
(pKL101) as shown in Figure 1A. The
chimeric plasmid was then introduced into maize endosperm cells via
particle bombardment along with a reference plasmid containing the CaMV
35S promoter linked to a GUS gene (pBI221, Jefferson, 1987) to correct
for transfection efficiency. However, only very low levels of LUC
activity were detected relative to the other promoters described above.
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| Figure 1.
Effect of Sbe1 gene exons/introns
and 3 end on the level of LUC expression driven by the
Sbe1 promoter. A, Schematic diagram of chimeric
Sbe1 promoter-LUC constructs. Numbers indicate the
distance relative to the Sbe1 transcription start site.
Translation initiation starts at position +28. The light-gray boxes
indicate the Sbe1 promoter region. Angled lines indicate
exons and introns in the Sbe1 gene. White and black
boxes indicate LUC reporter gene and NOS 3 end sequences,
respectively. The striped box indicates the Sbe1 3
flanking sequence. B, Junction sequences between the
Sbe1 gene and LUC. The BamHI sites used
to join the two genes are underlined. The translation start site of
LUC is indicated by boldface letters. C, Expression
levels of the construct shown in A. LUC to GUS ratios were calculated
as described in ``Materials and Methods''. Each value represents the
average of four independent shootings. Error bars indicate
SE values.
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|
Since data have indicated that DNA sequences within transcribed regions
such as exons, introns, and 3 flanking regions are involved in the
expression of genes in either a qualitative or a quantitative manner
(Callis et al., 1987; Hamilton et al., 1992; Fu et al., 1995a; Ulmasov
and Folk, 1995), three different types of translational fusion
constructs were created to test the effect of downstream elements on
Sbe1 gene expression. First, the 5-flanking sequence, as
well as the first exon and intron of the Sbe1 gene (2,190
to +228), were fused in-frame to the LUC reporter gene to make pKLN101
(Fig. 1, A and B). Second, the NOS 3 sequence in the pKLN101 was
replaced with the Sbe1 3 flanking sequence (399 bp in
length), which contains the translation stop codon and polyadenylation
signal to create pKLS101. Finally, to determine whether an increase in
the number of exons/introns enhances gene expression, three more exons
and introns from the Sbe1 gene were added to the pKLN101 to
make pKLM101 (2,190 to +1,617).
The results of transient expression assays using the chimeric
constructs are shown in Figure 1C. Inclusion of the DNA sequence from
+28 to +228 containing the first exon and intron increased the level of
LUC expression by 14-fold, suggesting that the first exon and intron
region is required for high-level expression of the Sbe1
gene in maize endosperm cells. Since pKLN101 produced a fusion protein,
however, we cannot completely rule out the possibility that the
increase may have been due to changes in enzyme activity and/or
turnover rate caused by the added amino acid sequences. If the
additional amino acids have a negative effect, the enhancement of LUC
activity observed would be greater than 14-fold.
Replacement of the NOS 3 end in pKLN101 with the Sbe1 3
region did not have a significant effect on the level of LUC
expression, implying that the Sbe1 3 UTR does not have
indispensable control elements. However, it is still possible that the
region may be important for Sbe1 gene expression in other
cell types or inductive conditions.
Construct pKLM101 showed a slight reduction in LUC activity compared
with pKLN101, indicating that additional exons and introns had an
adverse effect on LUC expression in suspension-cultured maize endosperm
cells. The adverse effect could be explained by inefficient splicing
resulting from the introduction of multiple copies of the plasmid into
a single cell, or by formation of fusion protein consisting of the 5
end of SBEI and LUC, thus lowering LUC activity. Alternatively, it
could be due to the presence of negative cis elements in
this region.
5 Deletion Down to 314 Did Not Significantly Affect the
Sbe1 Promoter Activity
To identify promoter sequences critical for Sbe1
expression in maize endosperm cells, a series of 5 deletion mutants
were derived from pKLN101 as shown in Figure
2A. The activity of each 5 deletion
construct is presented in Figure 2B. Removal of the sequences to
1,332 caused a decrease in the level of LUC expression, while
deletion of an additional 422 bp, to 910, resulted in an increase in
the activity of the construct. This suggests that potential positive
and negative distal cis regulatory elements may be located
in the regions from 2,190 to 1,332 and from 1,332 to 910,
respectively. Further deletions down to 315 did not significantly affect the promoter activity, but a severe reduction in
activity was observed when an additional 169 bp, to 145, was deleted.
The 72 deletion construct produced a level of LUC activity slightly over background, showing that the minimal promoter is functional.
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| Figure 2.
Effect of 5 deletions on Sbe1
promoter activity. A, Schematic diagram of the 5 deletion chimeric
constructs. The thick black lines denote the Sbe1
promoter sequences. The numbers at left indicate deletion end points
relative to the transcription initiation site (+1) of the
Sbe1 gene. The light-gray boxes and the thin black
angled line represent the first exon and intron in the
Sbe1 gene, respectively. The white boxes indicate the
LUC gene. The black boxes denote NOS 3 end sequences. B, The relative
activity levels of the constructs shown in A. The relative activity
values are percentages of pKLN101 level. Each value represents the
average of six to eight independent experiments. Error bars indicate
SE values.
|
|
A 60-bp Region Is Critical for the Promoter Activity
To further delimit sequences essential for high-level expression
of the promoter, two additional 5 deletions with about 60-bp intervals
were created between 315 and 145. As shown in Figure 2, a deletion
to 255 (pKLN108) severely reduced the expression of the LUC reporter
gene, while a further deletion to 196 (pKLN109) did not further
reduce promoter strength. This indicates that a strong positive
regulatory element(s) is present in the 60-bp region between 315 and
255.
Linker-Scan Analysis Reveals Two cis Elements within
the 60-bp Region
Since the 5 deletion analyses indicated that the region of the
Sbe1 promoter from 314 to 255 is critical for promoter
activity, the 60-bp DNA fragment was further dissected by
oligonucleotide-directed in vitro mutagenesis, as described by Kunkel
et al. (1987). A series of six different substitution mutants,
designated pLS1 to pLS6, were created by altering the wild-type DNA
sequence of the Sbe1 promoter at 10-bp intervals. The
mutations were made by creation of transversion substitutions where
possible, while at the same time introducing restriction enzyme sites
for simplifying identification of the mutant forms.
The mutated constructs were tested for their promoter activity using
the transient assay system, and the results of the experiments are
shown in Figure 3. Mutations in the
regions from 314 to 305 and from 304 to 295, corresponding to
pLS-1 and pLS-2, caused a decrease in the Sbe1 promoter
activity to 60% and 72% of wild-type (pKLN105) expression,
respectively. The pLS3 construct showed almost the same level of the
LUC expression as the wild-type promoter, suggesting that the
nucleotides from 294 to 285 are not important for the promoter
activity in maize endosperm cells. However, a mutation in the pLS-4
region (284 to 275) decreased promoter activity to 40% of the
wild-type level. Also, other two mutants, pLS-5 and pLS-6, showed a
reduction of promoter activity to 55% and 50% of the wild-type
promoter, respectively.
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| Figure 3.
Linker-scan analyses of the 60-bp region in the
Sbe1 promoter. A, Schematic diagram of the linker-scan
constructs. DNA sequence of the 60-bp region in the Sbe1
promoter is shown to the right of the wild-type construct pKLN105. The
mutated bases in the linker-scan constructs are shown in lowercase
letters. Dashes represent the unaltered nucleotides. For an explanation
of the other symbols, refer to the legend to Figure 2. B, Relative LUC
activity levels of the constructs shown in A. The relative activity
values are percentages of construct 314 level. Each value represents
the average of four independent experiments. Error bars indicate
SE values.
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The 60-bp Fragment Interacts with DNA-Binding Proteins
Electrophoretic mobility-shift assays were performed to
investigate the possibility that a nuclear protein(s) might interact with the 60-bp Sbe1 promoter fragment from 314 to 255.
The 60-bp fragment was 32P end-labeled with
Klenow fill-in reaction and then incubated with nuclear extract
prepared from 30 DAP maize kernels demonstrated to highly express the
Sbe1 gene (Gao et al., 1996). As shown in Figure
4, two major shifted bands were observed
in the lane containing nuclear extract (lane 2) compared with the
control (lane 1). The bands were not detected after inclusion of
proteinase K in the binding reaction (lane 7), indicating that the
shifted bands represent DNA-protein complexes.
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| Figure 4.
Interaction of nuclear proteins from maize kernels
with the 60-bp Sbe1 promoter fragment from 315 to
255. The 60-bp fragment was radiolabeled and 1 ng of the probe was
incubated with 10 µg of crude nuclear proteins prepared from maize
kernels. After a 20-min incubation, the samples were electrophoresed in
a 4% (w/v) polyacrylamide gel at 4°C for 2 h. The gel
was then dried and autoradiographed at 80°C with an intensifying
screen. Lane 1, Control reaction without nuclear extract; lane 2, control reaction with nuclear extract; lanes 3 and 4, 10- and 100-fold
excess of the 60-bp unlabeled fragment, respectively; lanes 5 and 6, 10 and 100 ng of salmon-sperm DNA, respectively; lane 7, 4 µL of 1 mg/mL
of proteinase K. Bands reduced in mobilities are indicated as B1 and
B2.
|
|
Competition assays were conducted to determine whether the complexes
are due to the binding of sequence-specific proteins. Inclusion of 10- and 100-fold excess of the unlabeled 60-bp fragment in the binding
reaction significantly reduced formation of the complexes (lanes 3 and
4), while the same amount of nonspecific competitor DNA failed to
compete for binding (lanes 5 and 6). Thus, the complexes appear to be
the result of sequence-specific interactions between a nuclear
protein(s) and the DNA fragment, which is consistent with the
functional identification of this region as an important regulatory
element. Using six 60-bp fragments of linker-scan mutants (LS-1 to 6)
as competitors, we found that LS-1 did not affect the intensity of the
lower band, although the rest of the linker-scan mutants abolished its
formation (data not shown). This suggests that the lower band may be
the result of interactions between a trans-acting factor(s)
and the sequence ACATAAAATA, which is located within LS-1. All of the
linker-scan mutants reduced the intensity of the slower-migrating
complex to varying degrees (data not shown). Since LS-4, LS-5, and LS-6 were less effective competitors, wild-type sequences spanning these
regions (284 to 255) may be involved in formation of this complex;
however, binding may involve several overlapping regions in this
fragment.
Expression of the Sbe1 Gene Is Sugar Regulated
The SBEs are expressed in a coordinate fashion with the
granule-bound starch synthase and ADP-Glc pyrophosphorylase during maize endosperm development (Gao et al., 1996). The ADP-Glc
pyrophosphorylase gene (AGPase S) from potato and the genes encoding
granule-bound starch synthase and SBE in cassava plants have been shown
to be induced by an exogenous supply of sugars (Muller-Rober et al., 1990; Giroux et al., 1994; Salehuzzaman et al., 1994). This led us to
speculate that the Sbe1 gene in maize may also be regulated by the external sugar concentration.
To test this, suspension-cultured maize endosperm cells were incubated
in Murashige and Skoog medium containing different concentrations of
Suc, and their total endogenous RNAs were analyzed by northern-blot
hybridization. Suc was used in preference to other metabolizable
sugars, because it is known to be the major sugar unloading from the
pedicel tissue of maize kernels (Porter et al., 1985). The results are
shown in Figure 5. An increase in Suc
concentration from 0% to 9% elevated the Sbe1 mRNA level 2-fold, and at higher concentrations the increase was reduced. Hexoses
such as Glc, Fru, and myoinositol also increased the level of
transcript in a similar fashion (data not shown). However, L-Glc and PEG 200 at concentrations calculated to
have the same osmotic potential as a 9% Suc solution (263 mM) did not exhibit any effect, indicating that
the response is not an osmotic but a sugar-specific phenomenon. These
results suggest that, like other starch biosynthetic genes (Giroux et
al., 1994), expression of the Sbe1 gene in maize endosperm
cells is regulated by sugar availability. This metabolic feedback
mechanism may serve as a system to fine-tune the expression levels of
Sbe genes relative to the physiological status of a plant.
Shannon et al. (1996) showed that nonallelic starch mutants of maize
accumulating high levels of Suc in the endosperm contained increased
SBE activities compared with the control.
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| Figure 5.
Suc effect on the Sbe1 mRNA levels
in suspension-cultured maize endosperm cells. Total RNA was extracted
from cells incubated for 24 h in Murashige and Skoog medium
supplemented with different amounts of Suc from 0% to 15%. As osmotic
controls, L-Glc and PEG 200 (263 mM) were used
instead of 9% (w/v) Suc (263 mM). RNA gel blots
(10 µg per lane) were probed with the 32P-labeled
full-length Sbe1 cDNA, and quantified with a
phosphor imager. The Sbe1 mRNA levels were calibrated
with 26S rRNA levels to correct for minor loading errors among the
lanes. RL, Relative Sbe1 mRNA level. Each value is the
percentage of the Sbe1 mRNA level in 0% Suc.
|
|
Since we recently determined that two Sbe1 genes
(Sbe1a and Sbe1b) with divergent 5-flanking
regions exist in the maize genome (Kim et al., 1998a), it was necessary
to determine whether expression of the isolated Sbe1 gene
(Sbe1a) promoter responds to external Suc concentrations. To
test this, a gene not regulated by sugar concentration was required as
an internal control for the transient assay system. Since a CaMV 35S
promoter has been used as a control in other studies investigating the
Suc responsiveness of plant genes, the effect of Suc on the expression
of the CaMV 35S promoter-GUS chimeric gene (pBI221) in maize endosperm
cells was first investigated.
The plasmid pBI221 was bombarded into suspension-cultured maize
endosperm cells supplemented with 0% (w/v) Suc or 9%
(w/v) Suc medium and incubated at 25°C in the dark. After
48 h of incubation, the GUS activity and protein concentration
were measured from each sample to calculate specific GUS activity (data
not shown). The results showed that specific GUS activities of 9%
(w/v) Suc samples were almost 2.5-fold higher than those of 0%
(w/v) Suc samples, which is consistent with other reports
(Graham et al., 1994; Grierson et al., 1994). Since similar results
were obtained from a ubiquitin promoter (pACH18) and a 64 CaMV 35S
minimal promoter, which does not have an activation sequence
(as)-1, a binding site for the transcription factor TGA-1a
(Katagiri et al., 1989), it appeared that the elevated levels of
expression by the CaMV 35S and ubiquitin promoters in 9% (w/v)
Suc may have simply been a general phenomenon caused by an increase in
energy source rather than a sugar-specific effect. Therefore, we
reasoned that if the chimeric construct pKLN101 (the Sbe1
promoter-LUC) is sugar modulated, it will further enhance the level of
LUC expression beyond the general increase at the higher Suc
concentration.
As shown in Figure 6, after normalization
to GUS activity driven by the CaMV 35S promoter, the plasmid pKLN101
still showed approximately 2-fold greater LUC activity in 9%
(w/v) Suc medium than in 0% (w/v) Suc medium. This is
consistent with the result of the endogenous RNA analysis indicating
that the identified Sbe1 gene is regulated by sugar
availability. It also suggests that the nucleotide sequence containing
a 2.2-kb 5-flanking region and the first exon/intron of the
Sbe1 gene is sufficient to confer sugar responsiveness in
maize endosperm cells.
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| Figure 6.
Suc responsiveness of the Sbe1
promoter. Each construct was bombarded onto suspension-cultured maize
endosperm cells supplemented with 0% Suc (white bars) or 9% Suc
(hatched bars), and incubated for 48 h at 25°C in the dark. The
relative activity values are percentages of pKLN101 level in 0% Suc.
Each value represents the average of three independent experiments.
Error bars indicate SE values.
|
|
To delimit a region(s) necessary for the response, two deletion
constructs, pKLN105 and pKLN106, were also tested in the transient expression system (Fig. 6). Like pKLN101, pKLN105 (deletion end point
314) responded to a high Suc concentration (9%) by increasing LUC
expression by approximately 2-fold. However, pKLN106 (deletion end
point 145) showed similar levels of LUC expression in both low and
high Suc conditions. These results suggest that the region between
314 and 145 contains a cis-regulatory element(s)
necessary for the sugar response in maize endosperm cells. In addition, because the expression level was reduced in both Suc-treated and untreated cells, other regulatory elements may also reside in this
region.
Overexpression of mEmBP-1 Protein Represses the Sbe1
Gene Expression
The canonical G-box sequence, CCACGTGG (Giuliano et al., 1988),
was found in the 5-flanking sequence of the maize Sbe1
(228 to 221) as well as the rice Sbe1 gene (170 to
163). This evolutionary conservation suggests a possible role of the
G-box motif in the regulation of gene expression, although our 5
deletion analysis did not show it as an important regulatory element.
The G-box motif resides in the promoters of many plant genes,
responding to a variety of different environmental and physiological
stimuli, and is often associated with additional regions that act as
coupling elements, determining signal response specificity (Menken et
al., 1995).
Electrophoretic mobility shift analysis and DNase I footprint
analyses were performed to determine whether the G-box in the maize
Sbe1 promoter interacts with a G-box-binding protein in maize, mEmBP-1, which is a homolog of the wheat EmBP-1 (Guiltinan et
al., 1990) and is expressed during endosperm development (Carlini et
al., 1999). As expected, the analyses clearly showed that EmBP-1 interacts with the G-box sequence in vitro (data not shown). Since EmBP-1, a basic Leu zipper (bZIP) transcription factor, is implicated in ABA-induced Em gene expression in wheat (Guiltinan et
al., 1990), the data prompted us to ask two questions: First, is
Sbe1 gene expression regulated by ABA concentration? Second,
can the mEmBP-1 protein transactivate Sbe1 gene expression?
Transient expression assays failed to show a relationship between the
exogenous ABA concentration (1-100 µM) and
Sbe1 promoter activity in suspension-cultured maize
endosperm cells (data not shown), suggesting that the G-box in the
Sbe1 promoter is not ABA responsive; however, we cannot rule
out the possibility based on these data without further evaluation of
ABA levels and responses of endogenous genes in our assay system.
To address the second question, a chimeric construct containing the
CaMV 35S promoter fused to the full-length mEMBP-1 cDNA (35S-mEmBP-1)
was created and co-introduced with the plasmid pKLN101 (a full-length
Sbe1 promoter-LUC) into suspension-cultured maize endosperm
cells. We predicted that overexpression of mEmBP-1 protein would
enhance the LUC expression driven by the Sbe1 promoter, since mEmBP-1 is known as a bZIP transcription activator. Contrary to
the prediction, overexpression of mEmBP-1 protein actually resulted in
a significant reduction (5-fold) of the Sbe1 promoter activity, as shown in Figure 7. The
effect was apparently selective for the Sbe1 promoter, since
mEmBP-1 had little effect on expression of a LUC reporter gene linked
to the ubiquitin promoter (pACH18). Interestingly, substitution of the
G-box sequence (CCACGTGG) in pKLN105 with TTGAACTA did not cause a
reduction in promoter activity (data not shown), suggesting that the
G-box sequence is not required for high-level expression of the
Sbe1 gene in maize endosperm cells.
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| Figure 7.
Effect of mEmBP-1 overexpression on
Sbe1 promoter activity. Each reporter plasmid (4 µg of
Sbe1 promoter-LUC, pKLN101, ubiquitin-LUC, or pACH18)
and reference plasmid (CaMV 35S-GUS; pBI221) were coprecipitated onto
gold particles with (hatched bars) or without (white bars) 4 µg of
CaMV 35S-mEmBP-1. Suspension-cultured maize endosperm cells were
bombarded with the gold particles and incubated at 25°C for 24 h
in the dark. The relative activity values are percentages of the
pKLN101 or pACH18 levels without mEmBP-1 overexpression. Each value
represents the average of two independent shootings. Error bars
indicate SE values.
|
|
| |
DISCUSSION |
The expression pattern of the maize Sbe1 gene has been
investigated in almost all maize tissues (Gao et al., 1996). The
Sbe1 gene is constitutively expressed at a low level in
vegetative tissues while it is modulated during kernel development.
Sbe1 mRNA began to accumulate to high levels at the onset of
rapid starch deposition, especially in the endosperm. These findings suggest that the expression of Sbe1 is regulated by certain
factors that vary in concentration or activity during kernel
development.
As a step toward understanding regulatory mechanisms controlling
Sbe1 gene expression, we analyzed the Sbe1
promoter regions using a transient gene expression system. Transient
expression assays showed that expression driven by the maize
Sbe1 promoter greatly depends on the presence of the DNA
region spanning the first exon and intron of the maize Sbe1.
Addition of the DNA sequence (+28 to +228) containing the first exon
and intron of the Sbe1 gene into the transcriptional
chimeric construct (pKL101) increased reporter gene expression in
suspension-cultured maize endosperm cells up to 14-fold. Since such DNA
sequences containing transcriptional stimulating effects are useful in
investigations of gene expression in plant cells and for plant genetic
engineering, it will be necessary to determine whether the DNA sequence
has the ability to increase gene expression under the control of
other promoters.
There are several examples of plant genes that are regulated by DNA
sequences within the transcribed region (Callis et al., 1987; Bruce et
al., 1989; McElroy et al., 1990; Fu et al., 1995a). Among them, the
first exon and intron sequences of the maize Sh1 gene are
the best examples studied so far (Vasil et al., 1985; Maas et al.,
1991; Clancy et al., 1994). The Sh1 exon appears to have two
separate cis elements that act independently to increase gene expression via different mechanisms. One of the elements may
contain a novel promoter element that has the ability to interact with
transcription factors binding upstream. The other may act at the level
of translation efficiency or mRNA stability. The enhancing effect of
the Sh1 intron is likely the result of an increase in the
level of mature cytoplasmatic mRNA level, such as the maize
Adh1 first intron (Callis et al., 1987).
5-Deletion analysis of the maize Sbe1 promoter revealed
several cis-regulatory elements affecting promoter activity
in maize endosperm cells. Of special interest was the identification of the 60-bp positive element located in the region from 314 to 255
relative to the transcription initiation site. Further investigation of
the region using linker-scan analysis identified at least two separate
regions, 314 to 295 and 284 to 255, which are critical for gene
expression in maize endosperm cells.
Interestingly, as shown in Figure 8, the
314/295 region has striking similarity to the Suc-responsive
element (SURE-1) of the potato patatin-1 promoter (Grierson et al.,
1994), which has been shown to interact with a Suc-inducible nuclear
protein(s). Grierson et al. (1994) demonstrated that a 100-bp patatin-1
promoter fragment encompassing SURE-1 is sufficient to confer Suc
responsiveness. DNA sequences similar to the 314/295 region are
also found in the promoter regions of other sugar-inducible genes, such
as maize Suc synthase (Shaw et al., 1994), Arabidopsis -amylase
(Mita et al., 1995), and potato sporamin (Ohta et al., 1991) (Fig. 8). This finding, along with the sugar-enhanced expression of
Sbe1 demonstrated by northern-blot analysis (Fig. 5) and
transient expression assay (Fig. 6), strongly suggests that the
conserved sequences may be implicated in mediating sugar responsiveness of the Sbe1 gene. This was further supported by our recent
finding that the 314/196 region of the Sbe1 promoter is
sufficient to confer Suc responsiveness to the 64 CaMV 35S minimal
promoter (data not shown). Since high-Suc media were used for the
transient expression assays to maximize gene expression, it is
understandable that mutation of this region would decrease the level of
LUC expression. It remains to be determined whether other
Sbe genes are also sugar modulated. To date, we have not
detected sugar-dependent DNA-binding activity associated with the
Sbe1 promoter.
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| Figure 8.
Sequence comparison of
cis-regulatory regions found in sugar-modulated genes.
The maize Sbe1 promoter sequence from 314 to 294 was
aligned with the 5 sequences of the potato patatin-1
(PS20), sporamin-A1 (SPO), maize Suc
synthase (Sus1), and Arabidopsis -amylase
(Amy), all of which are modulated by sugar
concentration. The numbers indicate the positions of nucleotides from
the relevant transcription initiation sites. The vertical bars
represent conserved nucleotides, and dashes indicate gaps to maximize
alignment.
|
|
In potato and cassava plants, sugars have been shown to regulate the
expression of genes involved in starch biosynthesis (Muller-Rober et
al., 1990; Salehuzzaman et al., 1994). Our results demonstrated that
the maize Sbe1 is also modulated by sugar concentration. Such a sugar effect was not due to changes in the osmotic potential, because L-Glc and PEG, which are osmotically
active, did not affect Sbe1 gene expression. Recently, Jang
et al. (1997) provided evidence that hexokinase is involved in sensing
sugar concentration in higher plants, and sugar signaling mediated
through hexokinase is uncoupled from sugar metabolism.
Sequence comparison between the rice (Kawasaki et al., 1993) and maize
Sbe1 genomic DNAs (Kim et al., 1998a) revealed that the
5-flanking sequences proximal to the protein-coding regions are highly
divergent except for the canonical G-box sequences (CCACTGG), which are
located in similar positions relative to the corresponding
transcription initiation sites. This evolutionary conservation between
the species led us to postulate that the G-box may be involved in the
regulation of the Sbe1 gene expression, possibly in response
to one of the environmental or physiological stimuli, even though we
failed to show the importance of the G-box in Sbe1 promoter
activity using the 5 deletion analysis. It is possible that a
G-box-dependent mechanism controlling Sbe1 promoter activity
could not be appraised in our suspension-cultured endosperm cells. This
hypothesis is supported by the results showing interaction of the G-box
with mEmBP-1 protein in vitro and repression of the Sbe1
promoter activity by overexpression of mEmBP-1 (Fig. 7).
Additionally, the finding that disruption of the G-box sequence
(CCACGTGG) in pKLN105 did not cause a reduction in promoter activity
(data not shown) led us to speculate that the G-box and its binding
proteins are involved in down-regulation of the Sbe1 gene
expression rather than up-regulation. Although a specific role for the
G-box motif in Sbe1 gene expression has not been identified,
there is a possibility that the G-box in the Sbe1 promoter
may play a critical role under different environmental conditions or in
different tissues.
It has been noted that mutations decreasing starch accumulation in
maize endosperm also reduce storage protein synthesis, implying
possible interactions between these pathways (Barbosa and Glover, 1978;
Tsai et al., 1978). Giroux et al. (1994) showed that mutations
affecting synthetic events in one biosynthetic pathway affect the
expression of genes in both pathways, and demonstrated that the
expression of genes involved in starch and storage protein synthesis of
the maize endosperm are coordinately regulated. Elevation in sugar
concentration or alteration of the osmotic potential of the endosperm
was proposed to be a possible candidate for the primary signal
triggering this coordinate expression. In this context, knowledge of
the Sbe promoter elements and their associated regulatory
proteins may eventually lead to a better understanding of the
regulatory mechanisms controlling all of the starch biosynthetic genes
and the genes encoding storage proteins in maize endosperm.
| |
FOOTNOTES |
1
This work was supported by grants from Pioneer
Hi-Bred (to M.J.G., Charles D. Boyer, and Jack C. Shannon), from the
U.S. Department of Energy Bioscience Program (to M.J.G., Jack C. Shannon, and Donald Thompson; no. DE-FG02-96ER20234), and by the
Pennsylvania State University Agricultural Experiment Station (project
no. 3,303).
2
Present address: 451 Koshland, Department of
Plant and Microbial Biology, University of California, Berkeley, CA
94720.
*
Corresponding author; e-mail mjg9{at}psu.edu; fax 814-863-1357.
Received December 23, 1998;
accepted May 30, 1999.
| |
ACKNOWLEDGMENTS |
We thank Drs. J.L. Anthony and Albert Kriz from DEKALB Genetics
Corporation for providing the suspension-cultured maize endosperm cells, Dr. Jill Deikman for the pLN plasmid, and Dr. Peter Quail for
the pACH18 plasmid. We also thank Drs. Charles D. Boyer, Jack C. Shannon, Dane K. Fisher, and Ming Gao for their insight and support in
this project. The technical assistance of Kathy Wick is greatly
appreciated.
| |
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Abstract
Introduction
