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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

    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

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-transposon-like 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 94-amino acid N-terminal extension in the SBEIIb precursor. The (beta /alpha )8-barrel topology of the alpha -amylase superfamily and the catalytic residues implicated in branching enzymes are conserved in both barley enzymes.

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

Starch is a mixture of amylose and amylopectin, both of which are Glc polymers. Amylose is a mostly linear polymer of 200 to 2000 alpha -1,4-bonded Glc moieties with rare alpha -1,6 branch points (for reviews, see Martin and Smith, 1995; Ball et al., 1996). Amylopectin is highly alpha -1,6-branched, with a complex structure of 106 to 108 Mr and up to 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 alpha -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 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 glycogen-branching 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 5' 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
Top
Abstract
Introduction
Methods
Results
Discussion
References

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.

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, 5'-GGCGAGATGGCG-3'; primer 2, 5'-CCACGCGCCACCCAGAA-3'; primer 3, 5'-CAGTGATTGTTTCCGCA-3'; primer 4, 5'GCCTGCACAGAGAACTTGAT-3'; primer 5, 5'-CTCTTCAGGTGGATCATAAT-3'; primer 6, 5'-CCAAGTCGTCGCTCTCACC3'; primer 7, 5'-TGCCCCGCTGGATCGACGA-3'; primer 8, 5'-AGCAAGAACAGGAAGAAAAAGAGTGGGAAA-3'; primer 9, 5'-TAGTGAAACAGCGCTACAACTTGCAGCTAC-3'; primer 10, 5'-AGATTGGTAGGGGGCGGAGGGCGGATGCTA-3'; and primer 11, 5'CGAAATGAGGAGGCGCAGGGGGGTGTGCTA-3'.

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) lambda -ZAPII cDNA libraries were constructed from developing endosperm poly(A+) RNA. Approximately 2 × 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-N+ membranes (Amersham) and hybridized at 42°C with a solution containing 50% formamide, 6× SSC, 5× Denhardt's reagent, 0.5% SDS, 50 µg mL-1 salmon-sperm DNA, and 1 to 2 ng mL-1 DNA probe. The membranes were washed with 1× 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 5' cDNA ends of sbeIIa and sbeIIb were isolated by reverse-transcription PCR (Frohman et al., 1988; Sambrook et al., 1989). The poly(A+) 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 µg 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).

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 lambda -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 × 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, 6× SSC, 5× Denhardt's reagent, 0.5% SDS, 50 µg mL-1 salmon-sperm DNA, and 1 to 2 ng µL-1 DNA probe. Hybridization was performed at 42°C. The slides were washed at 42°C in 2× SSC with 40% formamide and counterstained with 4',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 (Gurr's Improved R66) as described by Linde-Laursen (1975).

Transcript Analyses

Barley RNA was electrophoresed and blotted onto nylon membranes (Hybond-N+, 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
Top
Abstract
Introduction
Methods
Results
Discussion
References

Isolation and Characterization of sbeIIa and sbeIIb cDNA Clones from Barley Endosperm

Two cDNA libraries were custom-made in lambda -ZAPII using poly(A+) 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 × 106 plaque-forming units resulted in 201 and 152 positive clones from libraries I and II, respectively. One-fifth 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 5' 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 5' 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 5' 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 5' primer for amplification of both sbeII cDNAs. Primer 4 (Fig. 1) was used as the 3' 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 accession nos. AF064560 and AF064561, respectively, and are also shown in Figure 1.


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  		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 5' PCR primer, and primer 4 as the 3' PCR primer. The overlapping regions (from the beginning of primer 4 to the respective vertical arrow) were sequenced. The 5' end gene-specific cDNA probes 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.

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 5' 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(A+) 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 5' end, where the sbeIIb cDNA is longer than the sbeIIa cDNA, and in the 3'-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.


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  		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 alpha -helices and beta -strands of the conserved (beta /alpha )8 barrel domain in alpha -amylases are indicated by open bars above the sequences. An additional alpha -helix conserved in SBEs (Martin and Smith, 1995) is labeled alpha 0. 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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  		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).

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 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 alpha -amylase superfamily and are predicted to contain a central catalytic alpha -amylase (beta /alpha )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 alpha -amylases and their postulated catalytic groups (Jespersen et al., 1993) are conserved in the barley SBEIIa and SBEIIb sequences. The SBEII-specific region between beta -strand 8 and alpha -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 protein-distance 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 5' 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 lambda -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).

5' End Mapping of the sbeIIa and sbeIIb Transcripts

By primer-extension analyses the 5' 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 5' ends of the sbeIIa transcript map to either a C, A, or G residue 447, 449, and 451 nucleotides upstream, respectively, of the translation start codon (accession no. AF064562).


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  		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.

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

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.


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  		Figure 5. Schematic representation of the barley sbeIIa and sbeIIb genomic clones. A, Upstream portions of the lambda  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 lambda  vector. B, BamHI; E, EcoRI; S, SalI. B, colonist1 insertion in barley sbeIIb. The upper panel shows a sequence comparison between the 5' 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 retro-transposon-like element colonist1 (Lutz and Genbach, 1996; accession no. ZMU90128).

The sequences of genomic clones g5 and g15 have been deposited in GenBank under the accession numbers AF064562 and AF064563, respectively. Analyses of the 5' 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 -146, -24, and -17 upstream of the transcription start point. In sbeIIa there is one GC box (CCGCCC) starting at position -608 and one (GGGCGG; homology at the opposite strand) starting at position -325 (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).

 
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  		Table I. Summary of putative regulatory sequences in the barley sbeIIa and sbeIIb promoter regions

Analysis of the Second Intron of sbeIIb

One major difference in the 5' 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 this intron significantly increases the degree of identity between the 5' 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 3' portion of the intron matches only poorly to the colonist1 sequence, suggesting that the sequence in intron 2 represents an inactive form of the retro-transposon 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 gene-specific 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.


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  		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.

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 Linde-Laursen (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).


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  		Figure 7. Fluorescence in situ hybridization of barley sbeIIa and sbeIIb to root-tip metaphase chromosomes. Barley chromosomes are displayed with a 1000× 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).

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, sbeIIb-hybridizing transcripts could only be detected in the endosperm (Fig. 8).


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  		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.

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).


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  		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 (a+b), 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 (a+b), sbeIIa, and sbeIIb probes, and around 1.5 kb for the paz1 probe.

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

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 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 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 expression 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.

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 production of amylose (Martin and Smith, 1995), one would postulate that the starch granule grows from an amylopectin-rich 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 Significance of the Long Second Intron in the sbeIIb Gene

The major structural difference in the 5' 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.

    FOOTNOTES
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 christer{at}biokemi.su.se; fax 46-8-153679.

   Received February 4, 1998; accepted June 1, 1998.

    ABBREVIATIONS

Abbreviations: GBSSI, granule-bound starch synthase I. SBE, starch-branching enzyme.

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Methods
Results
Discussion
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