Plant Physiol. (1998) 118: 37–49
The Two Genes Encoding Starch-Branching Enzymes IIa and
IIb Are Differentially Expressed in Barley1
Chuanxin Sun, Puthigae Sathish, Staffan Ahlandsberg, and Christer Jansson*
Department of Biochemistry, The Arrhenius Laboratories, Stockholm University, S-106 91 Stockholm, Sweden
The sbeIIa and sbeIIb genes, encoding starch-branching enzyme
(SBE) IIa and SBEIIb in barley (Hordeum vulgare L.), have been
isolated. The 5* portions of the two genes are strongly divergent,
primarily due to the 2064-nucleotide-long intron 2 in sbeIIb. The
sequence of this intron shows that it contains a retro-transposonlike element. Expression of sbeIIb but not sbeIIa was found to be
endosperm specific. The temporal expression patterns for sbeIIa
and sbeIIb were similar and peaked around 12 d after pollination.
DNA gel-blot analysis demonstrated that sbeIIa and sbeIIb are both
single-copy genes in the barley genome. By fluorescence in situ
hybridization, the sbeIIa and sbeIIb genes were mapped to chromosomes 2 and 5, respectively. The cDNA clones for SBEIIa and
SBEIIb were isolated and sequenced. The amino acid sequences of
SBEIIa and SBEIIb were almost 80% identical. The major structural
difference between the two enzymes was the presence of a 94amino acid N-terminal extension in the SBEIIb precursor. The (b/
a)8-barrel topology of the a-amylase superfamily and the catalytic
residues implicated in branching enzymes are conserved in both
barley enzymes.
Starch is a mixture of amylose and amylopectin, both of
which are Glc polymers. Amylose is a mostly linear polymer of 200 to 2000 a-1,4-bonded Glc moieties with rare
a-1,6 branch points (for reviews, see Martin and Smith,
1995; Ball et al., 1996). Amylopectin is highly a-1,6branched, with a complex structure of 106 to 108 Mr and up
to 3 3 106 Glc subunits, making it one of the largest
biological molecules in nature. In the plant, starch is deposited as starch granules in chloroplasts of photosynthetic
tissues or in amyloplasts of endosperm, embryos, tubers,
and roots. In most plants, starch consists of 20% to 30%
amylose and 70% to 80% amylopectin. In photosynthetic
and nonphotosynthetic tissues the Glc moiety of ADP-Glc
is incorporated in the growing amylose polymer with the
help of starch synthases. The formation of a-1,6 linkages in
amylopectin is catalyzed by SBEs (EC 2.4.1.18). The final
structure of amylopectin is governed by the activities of
different SBEs, starch synthases, and a debranching enzyme (Ball et al., 1996).
SBEs exist as several isoforms in developing storage
tissues of maize, rice, pea (for review, see Martin and
Smith, 1995), barley (Sun et al., 1996, 1997), wheat (Morell
1
This work was supported by the European Union Biotechnology Program (no. FAIR-CT95-0568) and by the Foundation for
Strategic Research.
* Corresponding author; e-mail [email protected]; fax 46 –
8 –153679.
et al., 1997), potato (Larsson et al., 1996), and Arabidopsis
(Fisher et al., 1996). SBEs can be separated into two major
groups based on structural and catalytic properties. One
group, referred to as SBE family II or A (Martin and Smith,
1995), comprises SBEII from maize (Fisher et al., 1993; Gao
et al., 1997), wheat (Nair et al., 1997), and potato (Larsson
et al., 1996), SBE3 from rice (Mizuno et al., 1993), SBEI from
pea (Bhattacharyya et al., 1990), and SBE2 from Arabidopsis (Fisher et al., 1996). The other group, SBE family I or B
(Martin and Smith, 1995), comprises SBEI from maize
(Baba at al., 1991), wheat (Morell et al., 1997), potato (Kossman et al., 1991; Khoshnoodi et al., 1996), rice (Kawasaki et
al., 1993), and cassava (Salehuzzaman et al., 1992), and
SBEII from pea (Burton et al., 1995). In maize (Boyer and
Preiss, 1978; Gao et al., 1997) and Arabidopsis (Fisher et al.,
1996), it has been demonstrated that SBEII can be further
divided into two types, usually classified as SBEIIa and
SBEIIb, that differ slightly in catalytic properties.
The need for multiple isoforms of SBE in plants is not
understood and contrasts sharply to the single glycogenbranching enzyme found in bacteria and mammals. Most
likely, plants require different branching activities in different tissues and during different developmental stages of
sink tissues. Burton et al. (1995) showed that a shift from
SBEII to SBEII plus SBEI activity during pea embryo development was correlated with changes in the amylopectin
structure.
Little is known about the genomic arrangement for the
sbe genes and the structure of their promoters. In the
present work we undertook the isolation of cDNA and
genomic clones encoding two related SBEII forms from
barley (Hordeum vulgare L.) endosperm. The amino acid
sequences for the barley SBEII forms were analyzed and
their primary structures were compared with those of other
members of the SBEII family. We sequenced the 59 portions
of the two sbeII genes and characterized their promoter
regions. We determined the copy number for the sbeIIa and
sbeIIb genes and mapped their chromosomal location. Finally, we followed the tissue-specific and temporal expression of the genes.
MATERIALS AND METHODS
Plant Material
Barley (Hordeum vulgare L. cv Bomi) plants were grown
in soil under a 16-h photoperiod at 18°C/12°C day/night
temperatures.
Abbreviations: GBSSI, granule-bound starch synthase I; SBE,
starch-branching enzyme.
37
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38
Sun et al.
Plant Physiol. Vol. 118, 1998
Figure 1. Alignment of barley sbeIIa (IIa) and sbeIIb (IIb) cDNA sequences. Sequences were aligned and displayed using the
programs PileUp and Pretty (Genetics Computer Group). Identical nucleotides are indicated by solid black boxes. The
nucleotide sequences downstream of the vertical arrows were obtained from the cloned cDNAs. Sequences upstream of
primer 4 were obtained from reverse-transcription PCR. For both sbeIIa and sbeIIb cDNA amplification, primer 5 was used
for first-strand cDNA synthesis, primer 1 as the 59 PCR primer, and primer 4 as the 39 PCR primer. The overlapping regions
(from the beginning of primer 4 to the respective vertical arrow) were sequenced. The 59 end gene-specific cDNA probes
(Legend continues on facing page.)
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sbeIIa and sbeIIb Genes in Barley
DNA Clones and Oligonucleotides
The pea SBEI cDNA clone was a kind gift from Drs. Cathie
Martin and Alison M. Smith (John Innes Centre, Norwich,
UK). The maize SBEIIb cDNA clone was constructed by
Fisher et al. (1993) and was a kind gift from Dr. Andreas
Blennow (Lund University, Sweden). The following oligonucleotides were used: primer 1, 59-GGCGAGATGGCG-39;
primer 2, 59-CCACGCGCCACCCAGAA-39; primer 3, 59CAGTGATTGTTTCCGCA-39; primer 4, 59GCCTGCACA
GAGAACTTGAT-39; primer 5, 59-CTCTTCAGGTGGAT
CATAAT-39; primer 6, 59-CCAAGTCGTCGCTCTCACC39; primer 7, 59-TGCCCCGCTGGATCGACGA-39; primer
8, 59-AGCAAGAACAGGAAGAAAAAGAGTGGGAAA-39;
primer 9, 59-TAGTGAAACAGCGCTACAACTTGCAGCT
AC-39; primer 10, 59-AGATTGGTAGGGGGCGGAGGGCG
GATGCTA-39; and primer 11, 59CGAAATGAGGAGG
CGCAGGGGGGTGTGCTA-39.
Construction and Screening of Barley cDNA Libraries
Barley RNA was isolated by the method described by
Logemann et al. (1987). Two custom-made (Clontech, Palo
Alto, CA) l-ZAPII cDNA libraries were constructed from
developing endosperm poly(A1) RNA. Approximately 2 3
106 plaque-forming units were screened for each library.
Pea SBEI cDNA and maize SBEIIb cDNA were used as
probes. Plaques were lifted onto Hybond-N1 membranes
(Amersham) and hybridized at 42°C with a solution containing 50% formamide, 63 SSC, 53 Denhardt9s reagent,
0.5% SDS, 50 mg mL21 salmon-sperm DNA, and 1 to 2 ng
mL21 DNA probe. The membranes were washed with 13
SSC and 0.1% SDS at 65°C and exposed to radiographic
film (Fuji, Tokyo, Japan). Hybridizing plaques were purified by successive rounds of screening.
Reverse-Transcription PCR
The 59 cDNA ends of sbeIIa and sbeIIb were isolated by
reverse-transcription PCR (Frohman et al., 1988; Sambrook
et al., 1989). The poly(A1) RNA was isolated from endosperm 10 d after pollination using an mRNA purification
kit (QuickPrep Micro, Pharmacia). The first-strand cDNAs
were produced by reverse transcription as described by
Frohman et al. (1988) using 0.1 mg of RNA, 25 pmol of
primer 5, and 5 units of murine reverse transcriptase (Pharmacia). Amplification was carried out according to the
standard protocol (Sambrook et al., 1989), and consisted of
35 cycles of denaturation (95°C for 1 min), annealing (40°C
for 2 min), and extension (72°C for 2 min). Amplified
products were isolated and cloned into the pMOSBlue
T-vector (Amersham).
39
Construction and Screening of the Barley Genomic
DNA Library
Barley genomic DNA was isolated by using the standard
protocol (Sambrook et al., 1989). The DNA was partially
digested with Sau3AI and size-fractionated. A barley
l-EMBL3 genomic DNA library was custom made (Clontech) from the DNA. The protocol for screening was similar
to that for cDNA library screening. Approximately 2 3 106
plaque-forming units were screened.
Subcloning and DNA-Sequence Analysis
Positive cDNA and genomic clones were subcloned according to the method of Sambrook et al. (1989). A 4.4-kb
EcoRI sbeIIa and 3.4-kb BamHI and 1.1-kb SalI sbeIIb
genomic DNA fragments were subcloned into the
pGEM-3Z vector (Promega). Plasmid DNAs were sequenced on both strands at ProGene AB (Uppsala, Sweden)
using a DNA sequencer (ALF, Pharmacia). Sequences were
analyzed with a sequence analysis package (Genetics Computer Group, Madison, WI).
DNA Gel-Blot Analysis
Barley genomic DNA was digested with EcoRI, electrophoresed on agarose gels, and analyzed by DNA hybridization (Sambrook et al., 1989).
Fluorescence in Situ Hybridization
The fluorescence in situ hybridization protocol was modified from the method described by Schwarzacher et al.
(1989). Root tips and chromosomes were prepared from
2-d-old seedlings. The genomic DNA probes were labeled
with Cy3-dCTP (Amersham) and applied to hybridization
solution to make a final concentration of 50% formamide,
63 SSC, 53 Denhardt9s reagent, 0.5% SDS, 50 mg mL21
salmon-sperm DNA, and 1 to 2 ng mL21 DNA probe.
Hybridization was performed at 42°C. The slides were
washed at 42°C in 23 SSC with 40% formamide and counterstained with 49,6-diamidino-2-phenylindole (Sigma).
Preparations were analyzed on an epifluorescent microscope (Zeiss). Photographs were taken with color film
(Kodak Gold, ASA400). Final computer images were prepared with Adobe Photoshop. Following fluorescence in
situ hybridization, the hybridizing probes were stripped
off from the slides as described by Heslop-Harrison et al.
(1992). C-Banding analysis of the chromosomes was produced by staining the slides with Giemsa (Gurr9s Improved
R66) as described by Linde-Laursen (1975).
Figure 1. (Continued from facing page.)
employed for genomic clone isolation, and DNA and RNA gel-blot analyses are indicated by broken lines, and were
constructed by PCR amplification using primers 1 and 2 for sbeIIb and primers 1 and 3 for sbeIIa. Translation start and stop
codons and putative polyadenylation signals are indicated by asterisks, black bars, and hatched bars, respectively, above the
sequence for sbeIIa and below the sequence for sbeIIb.
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Copyright © 1998 American Society of Plant Biologists. All rights reserved.
40
Sun et al.
Plant Physiol. Vol. 118, 1998
Figure 2. Alignment of the primary structure of SBEII from higher plants. Sequences were aligned and displayed as described
in Figure 1. Identical amino acids are indicated by solid black boxes and similar amino acids by gray boxes. The Pro-rich
motif is indicated by a black bar under the sequences. The predicted positions of a-helices and b-strands of the conserved
(Legend continues on facing page.)
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sbeIIa and sbeIIb Genes in Barley
Transcript Analyses
Barley RNA was electrophoresed and blotted onto nylon
membranes (Hybond-N1, Amersham). The membranes
were hybridized with different cDNA probes. Probe labeling, hybridization, washes, and autoradiography were performed as described for the screening of the cDNA libraries. Primer extension was carried out as described by
Mohamed et al. (1993).
RESULTS
Isolation and Characterization of sbeIIa and sbeIIb cDNA
Clones from Barley Endosperm
Two cDNA libraries were custom-made in l-ZAPII using
poly(A1) RNA isolated from developing barley endosperm
10 d (library I) and 12 d (library II) after pollination. The
libraries were screened with heterologous probes made
from cDNA for maize SBEIIb and pea SBEI. Initial screening of 2 3 106 plaque-forming units resulted in 201 and 152
positive clones from libraries I and II, respectively. Onefifth of the positive clones were randomly selected and
purified. Restriction mapping showed that all clones
grouped into two distinct classes. The largest clones of each
cDNA class were partially sequenced. Sequence analysis
confirmed that both cDNA clones belonged to the sbe2 class
and that they represented two different genes. Comparison
with the sbeII cDNA sequences from maize and rice revealed that the inserts in both barley clones were truncated
at the 59 end. Extensive attempts to obtain full-length
cDNAs by library screening failed, so we instead used a
reverse-transcription PCR technique (Frohman et al., 1988;
Sambrook et al., 1989) in an attempt to amplify the 59 ends
for the respective cDNA clones.
Inspection of the cDNA nucleotide sequences for maize
sbe2b (Fisher et al., 1993) and rice sbe3 (Mizuno et al., 1993)
revealed a 12-nucleotide region at the 59 end where the two
genes shared a high degree of identity. Based on this conserved sequence (GGCGAGATGGCG), we constructed
primer 1 (Fig. 1) as the 59 primer for amplification of both
sbeII cDNAs. Primer 4 (Fig. 1) was used as the 39 primer for
both cDNAs. As expected, reverse-transcription PCR amplification with these primers yielded two different-sized
products. The PCR products were subcloned, and sequencing demonstrated that they each matched one of the cDNA
clones. Comparative sequence analyses showed that one
clone was closely related to maize sbe2b, so this clone is
referred to as barley sbeIIb and the other clone is called
barley sbeIIa. The sequences of the sbeIIa and sbeIIb cDNA
clones that were completely determined on both DNA
strands have been deposited in GenBank under the acces-
41
sion nos. AF064560 and AF064561, respectively, and are
also shown in Figure 1.
The open reading frames of sbeIIa and sbeIIb were 2202
nucleotides (positions 7–2208 in Fig. 1) and 2487 nucleotides (positions 7–2493 in Fig. 1) long, respectively. Thus,
the open reading frame of the sbeIIb cDNA is longer than
that of the sbeIIa cDNA by 284 nucleotides. Alignment of
the sequences shows that this difference is due to an extension at the 59 end of the sbeIIb cDNA coding region
(positions 7–291 in Fig. 1). Sequences identical to the consensus polyadenylation signal AATAA were found for
both the sbeIIa and sbeIIb cDNAs (positions 2499–2503 and
258–2586, respectively, in Fig. 1) upstream of the poly(A1)
tail. The cloned sbeIIa and sbeIIb cDNAs share a high degree
of identity, nearly 80% over a region that corresponds to
the open reading frame of the sbeIIa cDNA. The similarity
breaks down at the 59 end, where the sbeIIb cDNA is longer
than the sbeIIa cDNA, and in the 39-untranslated regions.
Sequence Analysis of Barley SBEIIa and SBEIIb
The deduced amino acid sequences of the sbeIIa and
sbeIIb cDNAs suggest that they encode polypeptides of 734
and 829 amino acid residues, respectively. This is within
the range of sizes reported for other cereal SBEII sequences:
799 residues in maize SBEIIb (Fisher et al., 1993), 825 in rice
SBE3 (Mizuno et al., 1993), 823 in one wheat SBEII (Nair et
al., 1997), and 729 in a second wheat SBEII (accession no.
U66376).
The primary structures of barley SBEIIa and SBEIIb were
aligned with those of other SBEs from the SBEII family (Fig.
2). All SBEII members share a high degree of amino acid
identity (90%–95%) in the central portion of their amino
acid sequences. The overall identity between barley SBEIIa
and SBEIIb is 78.3% (Fig. 3A). Apart from a few stretches of
sequence identity and similarity, the N termini of the SBEII
sequences are divergent. The barley SBEIIa sequence and
one of the wheat SBEII sequences (SBEIIa-1 in Fig. 2) are
considerably shorter at the N-terminal end compared with
the other SBEII sequences. The N terminus of barley SBEIIb
is 94 amino acid residues longer than that of barley SBEIIa.
Since SBEs are encoded by nuclear genes and imported
from the cytosol to amyloplasts or chloroplasts, the deduced amino acid sequences of barley SBEIIa and SBEIIb
should include an N-terminal transit peptide. Attempts to
sequence the N-terminal amino acids of barley SBEIIa and
SBEIIb showed that both N termini were blocked (Sun et
al., 1997). The hallmark for chloroplast transit peptides is
an amino acid composition with a high score of hydroxylated and positively charged residues and only a few carboxylated residues (Gavel and von Heijne, 1990). Such a
Figure 2. (Continued from facing page.)
(b/a)8 barrel domain in a-amylases are indicated by open bars above the sequences. An additional a-helix conserved in SBEs
(Martin and Smith, 1995) is labeled a0. Predicted catalytic sites with conserved amino acids are indicated by black bars and
asterisks, respectively, above the sequences. The barley SBEIIa and SBEIIb sequences were deduced from the cDNA
sequences. Sources of other SBEII sequences are as follows: wheat SBEIIa-1, accession number Y11282; wheat SBEIIa-2,
accession number U66376; maize SBEIIa, accession number U659480; maize SBEIIb, accession number L08065; rice SBE3,
accession number D16201; pea SBEI, accession number X80009; and Arabidopsis (Arabid) SBE2–2 and SBE2–1, accession
numbers U22428 and U18817, respectively.
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42
Sun et al.
Figure 3. Phylogenetic relationships between SBEII isoforms. A, Distance between deduced amino acid sequences of plant SBEII isoforms was determined by the program Distance (Genetics Computer
Group) using the Kimura protein-distance algorithm. The entire sequences shown in Figure 2 were used in the comparison. B, Dendrogram representation of the prediction in A. The dendrogram was
generated by the programs PileUp and GrowTree (Genetics Computer Group).
composition can be found in the first 55 amino acids of the
SBEIIb sequence. Based on these features, and by comparison with the determined N-terminal amino acids of the
mature maize SBEIIb (Fisher et al., 1993) and rice SBE3
(Mizuno et al., 1993), we postulate that the cleavage site for
the barley SBEIIb transit peptide is between Arg-55 and
Ala-56 (Fig. 2). Removal of a 55-amino acid-long transit
peptide from the barley SBEIIb precursor would produce a
mature SBEIIb with 774 amino acid residues and a computed molecular mass of 85 kD, which agrees with its
apparent molecular mass on SDS gels (90 kD; Sun et al.,
1997). For barley SBEIIa, no obvious transit peptide or
transit peptide cleavage site could be discerned.
Branching enzymes belong to the a-amylase superfamily
and are predicted to contain a central catalytic a-amylase
Plant Physiol. Vol. 118, 1998
(b/a)8-barrel domain (Jespersen et al., 1993). Both class I
and II SBEs conform to this topology (Martin and Smith,
1995). As is shown in Figure 2, the four sequences implied
in the active site of a-amylases and their postulated catalytic groups (Jespersen et al., 1993) are conserved in the
barley SBEIIa and SBEIIb sequences. The SBEII-specific
region between b-strand 8 and a-helix 8, corresponding to
the sequence P/EQXLPS/NGKF/II/VP (Burton et al.,
1995), is also present in the barley SBEIIs (Fig. 2).
From sequence alignments, it has been observed that the
presence of an N-terminal extension in SBEIIs is a structural feature that distinguishes them from SBEIs (Martin
and Smith, 1995). This extension, referred to as the
N-terminal arm, is predicted to be flexible and ends with
two or three Pro residues at its C-terminal region (Martin
and Smith, 1995). This Pro-rich motif is conserved in barley
SBEIIa and SBEIIb (Fig. 2). The length of the N-terminal
arm in SBEIIb is 89 amino acid residues, which is in line
with data from maize SBEIIb (62 amino acid residues), rice
SBE3 (76 residues), wheat SBEII (SBEIIa-2 in Fig. 2; 85
residues), pea SBEI (115 residues), and two Arabidopsis
SBEIIs (65 and 107 residues for SBE2–2 and SBEII-1, respectively; Fig. 2). The length of the SBEIIa N-terminal arm is
uncertain due to the difficulties in predicting the transit
peptide for SBEIIa.
The relatedness of the barley SBEIIa and SBEIIb with that
of other SBEIIs was determined using the Kimura proteindistance algorithm (Kimura, 1983; Fig. 3A). With this
method, which ignores gap positions, the highest degree of
amino acid identity to barley SBEIIa was found from the
two wheat SBEIIs (98.2% and 97.8%, respectively); barley
SBEIIb showed the highest level of identity with maize
SBEIIb (82.5%) and with wheat SBEIIa-1 and barley SBEIIa
(78.3%). A dendogram representation of the phylogenetic
relatedness between the different SBEII forms clusters barley SBEIIa with the two wheat SBEIIs and maize SBEIIa,
barley SBEIIb with rice SBE3 and maize SBEIIb, and Arabidopsis SBE2–1 and SBE2–2 with pea SBEI (Fig. 3B). The
phylogenetic analysis corroborates the grouping of barley
SBEIIb with maize SBEIIb and rice SBE3 as members of the
class SBEIIb, and barley SBEIIa as belonging to the class
SBEIIa.
Isolation of Genomic Clones for sbeIIa and sbeIIb
Gene-specific probes for the barley sbeIIa and sbeIIb were
constructed by PCR amplification of the divergent 59 cDNA
regions using the primers depicted in Figure 1. Primers 1
and 3 and 1 and 2 were used for sbeIIa and sbeIIb, respectively. Screening of a barley genomic library in l-EMBL3
yielded three clones that hybridized to the sbeIIa-specific
probe and two clones that hybridized to the sbeIIb-specific
probe. The five genomic clones were characterized by restriction mapping and DNA gel-blot analysis. Based on the
restriction patterns, the three sbeIIa clones represented one
sbeII gene and the two sbeIIb clones another sbeII gene (data
not shown).
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sbeIIa and sbeIIb Genes in Barley
5* End Mapping of the sbeIIa and sbeIIb Transcripts
By primer-extension analyses the 59 end of the sbeIIb
transcripts was mapped to a T residue 113 nucleotides
upstream of the translation start codon (accession no.
AF064563; Fig. 4A). The sbeIIa primer yielded three distinct
extension products (Fig. 4A). Thus, the 59 ends of the sbeIIa
transcript map to either a C, A, or G residue 447, 449, and
43
451 nucleotides upstream, respectively, of the translation
start codon (accession no. AF064562).
The activity of reverse transcriptase is known to be sensitive to the secondary structure of the RNA template
(Sambrook et al., 1989). Therefore, determination of transcription start sites by primer extension might give rise to
false start sites due to premature termination of reverse
transcription. To confirm the results shown in Figure 4A,
RNA gel-blot analyses with probes upstream and downstream, respectively, of the mapped transcription start sites
were carried out. Total RNA isolated from developing
barley endosperm hybridized to the downstream but not
the upstream probes (Fig. 4B), supporting the conclusion
from the primer-extension analysis.
Structure of the sbeIIa and sbeIIb Upstream Regions
Figure 4. Mapping of the transcription start sites for the barley sbeIIa
and sbeIIb genes by primer extension. A, Primer extension was
performed with antisense RNA primers 6 and 7, corresponding to
nucleotides 866 to 884 (accession no. AF064562) and 636 to 654
(accession no. AF064563) for sbeIIa and sbeIIb, respectively. Lanes
A, C, G, and T contained sequences produced by the same primers.
Extension products from the barley endosperm RNA are indicated by
arrows. The putative TATA boxes are lined on the right sides of
sequences. B, RNA gel-blot analysis with upstream and downstream
primers relative to the mapped transcription start site. Total RNA
from developing endosperm was probed with antisense RNA oligonucleotide primers 8 or 9, corresponding to nucleotides 734 to 763
and 764 to 793, respectively (accession no. AF064562) or with
primers 10 or 11, corresponding to nucleotides 439 to 468 and 469
to 498, respectively (accession no. AF064563). The sizes of the
hybridizing transcripts were approximately 2.9 kb.
Schematic representations of the upstream portion of the
18-kb-long clone g5, containing the entire barley sbeIIa gene
and the upstream portion of the 14-kb-long clone g15,
containing the entire barley sbeIIb gene, are shown in Figure 5A. A 4.4-kb EcoRI fragment of clone g5 and a 3.4-kb
BamHI plus 1.1-kb SalI fragments of clone g15 were subcloned and sequenced. We found that the EcoRI fragment
of g5 contained the first exon and intron plus the beginning
of the second exon of sbeIIa, and that the BamHI plus 1.1-kb
SalI fragments of g15 contained the first five exons and
introns plus the beginning of the sixth exon of sbeIIb (Fig.
5A). The canonical GT-AG rule applied to all six introns
sequenced.
The sequences of genomic clones g5 and g15 have been
deposited in GenBank under the accession numbers
AF064562 and AF064563, respectively. Analyses of the 59
flanking regions of the genes showed that a putative TATA
box could be located for the sbeIIa and sbeIIb genes at the
expected distance (within 30–40 nucleotides) from the
mapped transcription start site (Figs. 4A and 5A). Sequences indicative of two different general enhancer elements could also be found in both genes. One set contains
binding sites for the CAAT transcription factor. Two possible CAAT boxes (CATT) could be found in sbeIIa, starting
within 25 nucleotides upstream of the TATA box. In sbeIIb,
a possible CAAT box (CACT) was found at a similar position. Another set contains repeats of GC box regions with
the consensus motif CCGCCC, which serves as a binding
site for the activator Sp1. In sbeIIb there are three such
regions (CCGCCC) starting at positions 2146, 224, and
217 upstream of the transcription start point. In sbeIIa there
is one GC box (CCGCCC) starting at position 2608 and one
(GGGCGG; homology at the opposite strand) starting at
position 2325 (for a recent review on eukaryotic transcription activators, see Verrijzer and Tjian, 1996). In addition,
putative regulatory sequences can be found in the promoter regions of both sbeII genes (Table I).
Analysis of the Second Intron of sbeIIb
One major difference in the 59 upstream regions of the
barley sbeIIa and sbeIIb genes is the presence of the long
(2064 nucleotides) intron 2 in sbeIIb (Fig. 5A). Omission of
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44
Sun et al.
Plant Physiol. Vol. 118, 1998
Figure 5. Schematic representation of the barley sbeIIa and sbeIIb genomic clones. A, Upstream portions of the l genomic
clones g5 and g15, containing the barley sbeIIa and sbeIIb genes, respectively. The corresponding regions between sbeIIa
and sbeIIb are connected by broken lines. The putative TATA boxes and exons (e1–e6) are indicated. Asterisks denote sites
from the l vector. B, BamHI; E, EcoRI; S, SalI. B, colonist1 insertion in barley sbeIIb. The upper panel shows a sequence
comparison between the 59 region of sbeIIa with that of sbeIIb with the second intron omitted. The lower panel shows a
sequence comparison between an internal portion of the sbeIIb second intron and the upstream sequence of the retrotransposon-like element colonist1 (Lutz and Genbach, 1996; accession no. ZMU90128).
this intron significantly increases the degree of identity
between the 59 regions of sbeIIa and sbeIIb (Fig. 5B). Further
inspection of intron 2 in sbeIIb revealed that it contains a
nearly 700-nucleotide-long retro-transposon-like sequence
(Fig. 5B). The transposon element is of the colonist1 type
and has previously been detected in the maize gene for
acetyl-CoA carboxylase (Lutz and Gengenbach, 1996; accession no. U90128). The alignment in Figure 5B covers
approximately the first third of the colonist1 transposon,
which shares a 74% identity with the middle region of the
sbeIIb intron 2. The 39 portion of the intron matches only
poorly to the colonist1 sequence, suggesting that the se-
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Copyright © 1998 American Society of Plant Biologists. All rights reserved.
sbeIIa and sbeIIb Genes in Barley
45
Table I. Summary of putative regulatory sequences in the barley sbeIIa and sbeIIb promoter regions
Gene
Sequencea
sbeIIa
CATGCAC
2597 to 2590
RY repeat
ACGT
2555 and 2404
CAATTG
2184 to 2179
ACGT core in G-box-like
elements
E-box
TGATCT
2180 to 2175
WS motifs
CCAAT
CATGCA
CATGTG
AGATGT
2185
2157
2315
2397
Distal CCAAT elementb
RY repeat
E-box
WS motifs
sbeIIb
a
Start Position
Accession numbers AF064562 and AF064563.
to
to
to
to
2181
2152
2310
2392
b
Putative Motif
Reference
Thomas (1993) (Consensus
CATGCAT, Fujiwara
and Beachy, 1994)
Thomas (1993); de Pater
et al. (1994)
Thomas (1993) (Consensus
CANNTG)
Thomas (1993) (Consensus
TGATCT or AGATGT)
Nagata et al. (1993)
Thomas (1993)
Thomas (1993)
Thomas (1993)
Starch-inducible element.
quence in intron 2 represents an inactive form of the retrotransposon element. Computer searches of the Swiss-Prot
database revealed that the derived amino acid sequence of
the retro-transposon insertion was homologous to reverse
transcriptase (data not shown), which is conserved among
retro elements (White et al., 1994; Bennetzen, 1996).
Chromosome Analyses
To investigate the copy number of sbeIIa and sbeIIb in the
barley genome, DNA gel-blot analysis of EcoRI-digested
root-tip barley DNA was carried out using the genespecific probes used for isolation of genomic clones. The
two probes hybridized to one DNA fragment each (Fig. 6),
suggesting that sbeIIa and sbeIIb are each single-copy genes
in the barley genome. The signal for sbeIIa was rather weak
(Fig. 6), probably due to the short sbeIIa-specific probe.
Single copies of sbeIIa and sbeIIb are also consistent with the
outcome of the genomic DNA library screening, which
showed no heterogeneity within the pooled sbeIIa and
sbeIIb clones.
For physical mapping of the sbeIIa and sbeIIb genes by
fluorescence in situ hybridization, barley metaphase chromosomes were probed with the 4.4-kb EcoRI fragment of
sbeIIa and the 3.4-kb BamHI fragment of sbeIIb (Fig. 5A).
The two probes hybridized to different chromosomes (Fig.
7). The chromosome hybridizing to the sbeIIb probe exhibited a morphology characteristic of chromosome number 5
as described by Linde-Laursen (1978). The identity of this
chromosome was further confirmed by Giemsa staining,
which produced a C-banding pattern typical of what has
been found for chromosome 5 (Linde-Laursen, 1975). Similarly, comparison with the karyogram reported by LindeLaursen (1975) after Giemsa staining identified the chromosome hybridizing to the sbeIIa probe as number 2. In
addition, further upstream sequencing of genomic clone g5
harboring the sbeIIa gene showed that it contains the barley
5S-RNA gene, which has been localized to chromosome 2
(Kolchinsky et al., 1990).
Expression Patterns for the sbeIIa and sbeIIb Genes
Total RNA was isolated from endosperm, embryo, leaf,
or root and analyzed with the same sbeIIa- and sbeIIb-
specific probes as used for the isolation of genomic clones.
The results revealed that whereas sbeIIa-hybridizing transcripts were found in all tissues analyzed, sbeIIbhybridizing transcripts could only be detected in the endosperm (Fig. 8).
For analysis of temporal expression, barley endosperms
were collected 7 to 27 d after pollination and total RNA was
isolated. RNA gel-blot analysis was performed with the
sbeIIa- and sbeIIb-specific probes or with a PCR-amplified
probe for the paz1 gene, which encodes the storage protein
Z (Sørensen et al., 1989). The expression patterns for sbeIIa
and sbeIIb were similar, with a peak at around 12 d after
pollination (Fig. 9). Expression of the paz1 gene increased
up to or beyond 22 d after pollination and then sharply
declined, a pattern consistent with the results by Sørensen
et al. (1989).
DISCUSSION
The Barley SBEIIa and SBEIIb Isoforms Are Encoded by
Different Loci
The presence of two different SBEII isoforms, SBEIIa and
SBEIIb, has been reported in maize (Boyer and Preiss, 1978)
and rice (Yamanouchi and Nakamura, 1992) endosperm
and in Arabidopsis seedlings (Fisher et al., 1996). In maize
(Gao et al., 1997) and Arabidopsis (Fisher et al., 1996), the
two SBEII forms may be encoded by different genes. In the
present work we demonstrate that SBEIIa and SBEIIb in
barley endosperm are encoded by different genes located
on different chromosomes.
Barley SBEIIa and SBEIIb: a Structural Comparison with
Other SBE Forms
The catalytic properties of the SBEII and SBEI isoforms
differ, and it has been concluded that SBEIIs catalyze the
formation of amylopectin with shorter branch chains than
SBEIs (Smith, 1988; Guan and Preiss, 1993; for review, see
Martin and Smith, 1995). The four regions implicated in the
catalytic site of amylolytic enzymes (Jespersen et al., 1993)
are conserved in both the SBEII and SBEI families, as are
the catalytic groups identified within these regions (Burton
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46
Sun et al.
Figure 6. DNA gel-blot analysis of the barley sbeIIa and sbeIIb
genes. Genomic DNA was digested with EcoRI and probed with
gene-specific probes as depicted in Figure 1. Sizes of hybridizing
fragments are indicated.
et al., 1995; Martin and Smith, 1995). A fifth region, with
the reported consensus sequence P/EQXLPS/NGKF/
II/VP (Burton et al., 1995), is conserved in SBEIIs but not in
SBEIs and it has been inferred that this distinction between
the two families is a major reason for their different enzymatic activities (Burton et al., 1995; Martin and Smith,
1995). All five regions are conserved in the barley SBEIIa
and SBEIIb forms (Fig. 9). Analysis of the SBEII-specific
region in the nine SBEII sequences shown in Figure 2
suggested that the consensus sequence for this motif be
modified to pQXLpXGkvip, where uppercase letters indicate 100% identity at a position, lowercase letters indicate
less than 100% identity, and X denotes any amino acid.
Another structural feature of SBEII isoforms that separate them from the SBEI class is the presence of an
N-terminal extension, a domain characterized by a high
score of Ser residues and other amino acids with flexible
side chains that ends with a Pro-rich triplet toward the C
terminus. This “flexible arm” (Martin and Smith, 1995) is
also present in isoforms of starch synthases that are found
in the soluble phase of the amyloplast or tightly associated
with the starch granule, but is missing in starch synthases
that are strictly confined to the granule (for a review, see
Martin and Smith, 1995). It has been suggested that the
N-terminal arm might be responsible for the partitioning
behavior of starch synthases and SBEs (Burton et al., 1995;
Martin and Smith, 1995). There are no reports of SBEs being
exclusively bound to the starch granule. Barley endosperm
SBEI, SBEIIa, and SBEIIb were all isolated from the soluble
cell extract (Sun et al., 1997). In pea embryos SBEI and
SBEII can be isolated from both the soluble and the
granule-bound protein fractions (Denyer et al., 1993). The
same was reported for a SBEII form in wheat, barley,
maize, and rice endosperm (Rahman et al., 1995), and for
SBEIIb in maize endosperm (Mu-Forster et al., 1996). In
wheat, barley, and rice endosperm, on the other hand, SBEI
Plant Physiol. Vol. 118, 1998
was found only as a soluble form (Rahman et al., 1995).
Therefore, to our knowledge, there is as yet no conclusive
information regarding the physiological function of the
N-terminal arm of SBEIIs. The possibility that it determines
the degree or nature of the physical contact between the
enzymes and the starch granule or between SBEIIs and
starch synthases (Martin and Smith, 1995) is still feasible.
If the length of the N-terminal arm influences the interaction with the starch granule, then whereas SBEIIa catalyzes branch formations in the soluble phase, SBEIIb is
active more at the periphery and outer matrix of the granule. Such a scenario fits the available data on SBEI and
SBEII partitioning discussed above. Also, in the work of
Rahman et al. (1995), only one SBEII form could be detected
among the granule-bound proteins. Based on the size determined by protein gel-blot analysis, this SBEII form
should be SBEIIb. Therefore, SBEIIa might be predominantly soluble in these systems. Finally, a spatial differentiation in SBEII activity might explain the requirement for
two SBEII isoforms that exhibit the same temporal expres-
Figure 7. Fluorescence in situ hybridization of barley sbeIIa and
sbeIIb to root-tip metaphase chromosomes. Barley chromosomes are
displayed with a 10003 magnification. A, Seven pairs of metaphase
chromosomes stained with DAPI. B, Fluorescence in situ hybridization signals obtained with the sbeIIa genomic DNA probe on chromosome 2 (arrow). C, Fluorescence in situ hybridization signals
obtained with the sbeIIb genomic DNA probe on chromosome 5
(arrows).
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sbeIIa and sbeIIb Genes in Barley
47
Figure 8. Differential expression of the sbeIIa
and sbeIIb genes in various tissues of barley.
Total RNA was probed with gene-specific
probes as depicted in Figure 1. The sizes of the
hybridizing transcripts were around 2.9 kb.
sion pattern, at least as judged by steady-state transcript
levels (Fig. 9).
Expression of the sbeIIb Gene Is Endosperm Specific
Here we have shown that in barley, sbeIIb activity could
be detected only in endosperm, whereas sbeIIa transcripts
were found in endosperm, embryo, and vegetative tissues.
We do not yet know how (or if) the structure of starch in
barley endosperm and leaves differs. However, it is reasonable to assume that storage and transient starch should
differ in some aspects, and recent work by Tomlinson et al.
(1997) on pea supports this notion. We hypothesize that
barley utilizes the different composition of SBEII isoforms
in endosperm and leaves as one means of producing distinct amylopectin molecules.
tion of amylose (Martin and Smith, 1995), one would postulate that the starch granule grows from an amylopectinrich composition with short branches to an amylose-rich
structure containing amylopectin with a mixture of long
and short branches. As has already been pointed out by
Burton et al. (1995) and Martin and Smith (1995), such a
sequential formation of starch is consistent with analyses of
iodine-amylopectin complexes (Burton et al., 1995) and the
finding that the rate of amylose production increases during starch development (Shannon and Garwood, 1984).
The sbeII Genes Are Expressed Early during Barley
Seed Development
Steady-state levels of sbeIIa and sbeIIb transcripts in barley endosperm peaked at around 12 d after pollination,
which is about 1 week before maximum expression of the
barley sbeI gene (Jansson et al., 1997). This differential
expression of the barley sbeII and sbeI genes follows the
same pattern as reported for pea embryos (Burton et al.,
1995), maize endosperm (Gao et al., 1996), and, most likely,
rice endosperm (Mizuno et al., 1993). In wheat sbeII transcripts were shown to be abundant during the early stages
of kernel maturation (Nair et al., 1997). Mutant analyses of
wrinkled-seeded peas showed that SBEII activity precedes
that of SBEI activity (Smith, 1988; Bhattacharyya et al.,
1990). The maximum activity of SBEI largely coincides with
that of GBSSI, and the activity of SBEII with that of starch
synthase II (Dry et al., 1992; Mizuno et al., 1993; Burton et
al., 1995; Gao et al., 1996). Thus, SBEII and starch synthase
II might work in concert during the early stages of starch
granule formation, whereas the contributions of SBEI
and GBSSI activities have a later onset.
From in vitro investigations of purified maize SBEI and
SBEII, it could be demonstrated that SBEI transfers long
branches and SBEII short branches during amylopectin
synthesis (Guan and Preiss, 1993). Together with the assumption that GBSSI is responsible for the major produc-
Figure 9. Temporal expression of the sbeIIa and sbeIIb genes during
barley endosperm development. Total RNA was isolated from barley
endosperm on different days after pollination (d.a.p.). RNA was
probed with a cDNA fragment that recognized both sbeIIa and sbeIIb
transcripts, sbeII (a1b), or with cDNA probes specific for either
sbeIIa or sbeIIb (Fig. 1) or paz1 transcripts. The sizes for the hybridizing fragments were around 2.9 kb for the sbeII (a1b), sbeIIa, and
sbeIIb probes, and around 1.5 kb for the paz1 probe.
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48
Sun et al.
The Significance of the Long Second Intron in the
sbeIIb Gene
The major structural difference in the 59 portion of the
barley sbeII genes is the presence of the large second intron
in sbeIIb. There is ample evidence that introns affect gene
expression and contain regulatory cis elements in animals
(Raimond et al., 1995) and in plants (Callis et al., 1987).
Furthermore, it has been demonstrated that retro elements
might play a role in gene expression in plants (White et al.,
1994; Bennetzen, 1996). Intronic sequences similar to the
colonist1 element in barley sbeIIb were also found in two
other genes involved in sugar metabolism: the gene for
maize ADP-Glc pyrophosphorylase (Shaw and Hannah,
1992) and that for potato UDP-Glc pyrophosphorylase (accession no. U20345). Whether the second intron in barley
sbeIIb contributes to its regulation remains to be analyzed.
Ongoing experiments in our laboratory suggest that a region of the sbeIIb intron 2 (nucleotides 1774–1790; accession
no. AF064563) that share a high degree of similarity with
the B-box motif of the patatin promoter (Grierson et al.,
1994) might be involved in sbeIIb regulation.
Received February 4, 1998; accepted June 1, 1998.
Copyright Clearance Center: 0032–0889/98/118/0037/13.
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