SURE (sugar responsive) is a cis element in plant sugar signaling. The SURE element was reported first for potato, in which it confers sugar responsiveness to the patatin promoter. A SURE binding transcription factor has not been isolated. We have isolated a transcription factor cDNA from barley and purified the corresponding protein. The transcription factor, SUSIBA2 (sugar signaling in barley), belongs to the WRKY proteins and was shown to bind to SURE and W-box elements but not to the SP8a element in the iso1 promoter. Nuclear localization of SUSIBA2 was demonstrated in a transient assay system with a SUSIBA2:green fluorescent protein fusion protein. Exploiting the novel transcription factor oligodeoxynucleotide decoy strategy with transformed barley endosperm provided experimental evidence for the importance of the SURE elements in iso1 transcription. Antibodies against SUSIBA2 were produced, and the expression pattern for susiba2 was determined at the RNA and protein levels. It was found that susiba2 is expressed in endosperm but not in leaves. Transcription of susiba2 is sugar inducible, and ectopic susiba2 expression was obtained in sugar-treated leaves. Likewise, binding to SURE elements was observed for nuclear extracts from sugar-treated but not from control barley leaves. The temporal expression of susiba2 in barley endosperm followed that of iso1 and endogenous sucrose levels, with a peak at ‫ف‬ 12 days after pollination. Our data indicate that SUSIBA2 binds to the SURE elements in the barley iso1 promoter as an activator. Furthermore, they show that SUSIBA2 is a regulatory transcription factor in starch synthesis and demonstrate the involvement of a WRKY protein in carbohydrate anabolism. Orthologs to SUSIBA2 were isolated from rice and wheat endosperm.

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 94-amino acid N-terminal extension in the SBEIIb precursor. The (␤/ ␣) 8 -barrel topology of the ␣-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 ␣-1,4-bonded Glc moieties with rare ␣-1,6 branch points (for reviews, see Martin and Smith, 1995; Ball et al., 1996). Amylopectin is highly ␣-1,6-branched, with a complex structure of 10 6 to 10 8 M r and up to 3 ϫ 10 6 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 ␣-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(Sun et al., , 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 (Kossm...

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SUMMARYDeveloping car>opses of barley (Hordeum vulgare L. cv. Golf) were monitored for starch-branching enzyme activity for 1 month after anthesis. Homogenized caryopses fronn grains with the highest specific activity were used as starting material for purification of branching enzyme by FPLC chromatography. Several branching enzyme activity fractions were resolved. We have previously described the isolation of a 51/50 kDa protein with starch branching activity from one of these fractions. From three other fractions, we have now partially purified starch branching enzymes corresponding to forms I, Ila and lib.