A Single Limit Dextrinase Gene Is Expressed Both in the Developing Endosperm and in Germinated Grains of Barley1

Abstract

The single gene encoding limit dextrinase (pullulan 6-glucanohydrolase; EC 3.2.1.41) in barley (Hordeum vulgare) has 26 introns that range in size from 93 to 822 base pairs. The mature polypeptide encoded by the gene has 884 amino acid residues and a calculated molecular mass of 97,417 D. Limit dextrinase mRNA is abundant in gibberellic acid-treated aleurone layers and in germinated grain. Gibberellic acid response elements were found in the promoter region of the gene. These observations suggest that the enzyme participates in starch hydrolysis during endosperm mobilization in germinated grain. The mRNA encoding the enzyme is present at lower levels in the developing endosperm of immature grain, a location consistent with a role for limit dextrinase in starch synthesis. Enzyme activity was also detected in developing grain. The limit dextrinase has a presequence typical of transit peptides that target nascent polypeptides to amyloplasts, but this would not be expected to direct secretion of the mature enzyme from aleurone cells in germinated grain. It remains to be discovered how the enzyme is released from the aleurone and whether another enzyme, possibly of the isoamylase group, might be equally important for starch hydrolysis in germinated grain.Starch is the major carbohydrate reserve in cereal grains, where it is located in the nonliving cells of the starchy endosperm and constitutes up to 60% of total grain dry weight (Aman et al., 1985). Starch consists of the essentially linear (134)-␣-glucan amylose, together with the branched (134,136)-␣-glucan amylopectin. The two polysaccharides are organized in semicrystalline starch granules, which in barley (Hordeum vulgare) grain contain 70% to 75% amylopectin and 25% to 30% amylose (for review, see MacGregor and Fincher, 1993).Following germination, the glucosyl residues of amylose and amylopectin are released to support seedling growth by the concerted action of ␣-amylases, ␤-amylases, debranching enzymes, and ␣-glucosidases. The debranching enzymes catalyze the hydrolysis of (136)-␣-glucosidic linkages in amylopectin or in (134,136)-␣-oligoglucosides released by ␣-amylases. Because the (136)-␣-glucosyl linkages in these oligoglucosides, which are also referred to as limit dextrins, are not hydrolyzed by ␣-or ␤-amylases, and because the action of ␣-glucosidase on branched oligoglucosides is relatively slow, debranching enzymes are considered to play a central role in the complete depolymerization of starch to Glc. The debranched oligosaccharides are susceptible to further hydrolysis by amylases and ␣-glucosidases (Lee et al., 1971).Starch-debranching enzymes have been divided into two groups based on differences in their substate specificities and action patterns (Lee et al., 1971). The first group includes the pullulanases (pullulan 6-glucanohydrolase; EC 3.2.1.41), endohydrolases capable of hydrolyzing (136)-␣-linkages in pullulan, a polysaccharide consisting of maltotriosyl residues linked by (136)-␣-linkages. The second group includes isoamylases (glycogen...

Significance of Light, Sugar, and Amino Acid Supply for Diurnal Gene Regulation in Developing Barley Caryopses

“…High expression levels in endosperm tissues were also found for the genes encoding SUSIBA2 (HvWRKY46) and limit dextrinase (HvLD), confirming earlier findings (Burton et al, 1999;Sun et al, 2003). The diurnally fluctuating transcripts for HvWRKY41 (Mangelsen et al, 2008) could mainly be detected in endosperm and pericarp tissues.…”

“…7). As reported earlier (Sun et al, 1998;Burton et al, 1999;Radchuk et al, 2009), the transcripts of HvSBEIIb and HvLD were mainly found in the endosperm and are thus likely to be involved in the biosynthesis of storage starch (Table I; Fig. 8).…”

Spatiotemporal Profiling of Starch Biosynthesis and Degradation in the Developing Barley Grain

“…Three SBE cDNAs were identified, each almost identical with the cloned SBEI (Mutisya et al, 2003) and SBEIIa and SBEIIb (Sun et al, 1998) transcripts. Three different ISA cDNA sequences, one full and two partial, were identified, while one partial sequence was homologous with PUL cDNA (Burton et al, 1999(Burton et al, , 2002a. We did not find another PUL gene (Kristensen et al, 1999) among the seed-specific ESTs, possibly because of its preferential expression during seed germination.…”

“…Starch granule initiation and growth are altered in a barley mutant lacking ISA activity (Burton et al, 2002a). The PUL gene is expressed in both the developing barley endosperm and the germinating caryopsis, indicating its potential function for starch modification during synthesis and for starch hydrolysis during germination (Burton et al, 1999;Dinges et al, 2003).…”

Changes in Germination and Malting Quality During Storage of Barley

“…3). Given that ␣-amylase and β-glucanase belong to a group of hydrolases synthesised by aleurone cells in response to gibberellin 12 , these increases are likely to be accompanied by increases in the production of other hydrolases synthesised by the aleurone in response to gibberellin, such as ␣-glucosidase 13 , limit dextrinase 7 and proteases 8 , all of which would contribute to improved malt performance within the brewery.…”