The two genes encoding starch-branching enzymes IIa and IIb are differentially expressed in barley

Abstract

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.

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.

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 α-helices and β-strands of the conserved (β/α)₈ barrel domain in α-amylases are indicated by open bars above the sequences. An additional α-helix conserved in SBEs (Martin and Smith, 1995) is labeled α0. 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.

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).

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.

Figure 5

Schematic representation of the barley sbeIIa and sbeIIb genomic clones. A, Upstream portions of the λ 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 λ 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).

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.

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).

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.

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.