Purification, characterization and gene structure of (1→3)‐β‐glucanase isoenzyme GIII from barley (Hordeum vulgare)

Abstract

A new member of the barley (1 +3)-P-glucan glucanohydrolase family of enzymes has been purified from extracts of germinated grain and young seedlings by fractional precipitation with ammonium sulphate, ion-exchange chromatography, chromatofocussing and gel-filtration chromatography. The enzyme, which has been designated (1 +3)-P-glucanase isoenzyme GIII, is a basic protein with an apparent molecular mass of 32 000 Da. Oligosaccharide products released by the enzyme during hydrolysis of the (1+3)-P-glucan, laminarin, indicate that the enzyme is an endohydrolase. A 2349-bp fragment of barley genomic DNA has been isolated and identified as the gene encoding the (1 +3)-P-glucanase isoenzyme GIII. The open reading frame encoding the isoenzyme is interrupted by a single intron of 180 bp that splits a codon in the putative signalpeptide region. Northern-blot analyses with gene-specific probes indicate that the (1 +3)-P-glucanase isoenzyme GI11 mRNA accumulates in developing leaves; no mRNA transcripts were detected in the aleurone or scutellum of germinated grain, or in mature vegetative tissues. Although plant (1 +3)-Pglucanases are generally classified as 'pathogenesis-related' proteins, the physiological function of the barley (1 +3)-P-glucanase isoenzyme GI11 is unclear.During the germination of barley, hydrolytic enzymes depolymerize the starch and storage-protein reserves of the starchy endosperm into low-molecular-mass products that are translocated to the embryo where they support seedling growth. The hydrolases originate principally in the alcurone and scutellum, although some pre-exist in the starchy endosperm of ungerminated grain [I]. The most abundant enzymes in germinated barley include a-amylases, P-amylases, (1 + 3,l -.4)-p-glucanases, xylanases, carboxypeptidases and endopeptidases [2 -81. These enzymes participate in the degradation of storage polymers or in the removal of cell walls that separate the secreted hydrolases from their substrates within the cells of the starchy endosperm.High levels of (1 +3)-P-glucan endohydrolase activity are also found in the endosperm of germinated barley [9, lo], but the function of these enzymes is not so obvious. It has been suggested that they may participate in cell-wall degradation through an ability to hydrolyse regions of contiguous (1 +3)-fi-linked glucosyl residues in wall (1 +3,1-+4)-P-glucan [ll], but no regions of adjacent (1 +3)-P linkages have been conclusively demonstrated in this polysaccharide [I] and purified (1 + 3)-P-glucanases are unable to hydrolyse 40 "C water-soluble barley (1 +3,1+4)-B-glucans [12]. Another potential function of the (1 -+3)-~-glucanases in the germinating grain relates to the small deposits of (1 +3)-@-glucan that are scattered through the starchy endosperm 113 -151. The (1 +3)-Pglucanases might play a role in the recovery of glucose from these polysaccharides as an energy source for the young seedling. However, (1 +3)-b-glucanase levels are higher than those required to depolymerize these (1 +3)-P-glucan deposits. A possibl...

Purification and characterization of (1?3, 1?4)-?-glucan endohydrolases from germinated wheat (Triticum aestivum)

“…Immediately adjacent to the NH2-terminal lie residue of the mature protein is a (Val-Glu-Ser) amino acid sequence which is conserved at the signal peptide cleavage site in barley (1 --. 3, 1 --.4)-fl-glucanases [29] and is similar to the sequences observed in barley (1--.3)-fl-glucanases [ 16,32]. The coding region for the mature enzyme encodes a polypeptide of 306 amino acid residues (Fig.…”

“…Bound proteins were eluted with a linear 0-0.5 M NaC1 gradient in the same buffer. Fractions containing (1--+3, l~4)-flglucanase were concentrated and desalted by ultrafiltration and applied to a 0.5 cm x 70 cm chromatofocusing column containing PBE94 (Pharmacia-LKB) equilibrated in 25 mM triethylamine, pH 10 [32]. Bound proteins were eluted with 9~o polybuffer 96 (Pharmacia-LKB).…”

Extracellular β-1,3-glucanases are induced during early somatic embryogenesis in Cichorium

“…From six tryptic peptides the amino acids could be unambiguously identi®ed and compared with SWISS-PROT data base. The identi®ed sequences, in total covering 63 amino acids, shared 36±57% identity with plant b-1,3-endoglucanases from Arabidopsis thaliana (Uknes et al 1992), Nicotiana plumbaginifolia (Castresana et al 1990), Nicotiana tabacum (Ori et al 1990), Lycopersicon esculentum (Van Kan et al 1992) and Hordeum vulgare (Wang et al 1992 ; Fig. 3).…”

“…2C) and at least two 38-kDa extracellular conditioned-medium proteins on a 2D-PAGE gel (data not shown). Protein extracted from leaf explants developing somatic embryos were separated by SDS-PAGE and subjected to immunoblot analysis using P38-SH serum, to determine the relationship between 38-kDa extracellular proteins and (Uknes et al 1992), Nicotiana plumbaginifolia (Castresana et al 1990), Nicotiana tabacum (Ori et al 1990), Lycopersicon esculentum (Van Kan et al 1992) and Hordeum vulgare (Wang et al 1992). Common amino acids are indicated with asterisks the SER-38 proteins present in leaf tissues previously described (Hilbert et al 1992;Helleboid et al 1995).…”

Molecular evolution of plant β‐glucan endohydrolases

“…Screening both cDNA and genomic DNA libraries with oligonucleotide probes based on these amino acid sequences resulted in the isolation of six separate (l~3)-~-glucanase genes, the products of which have been designated isoenzymes GI-GVl (HCj et aL, 1989a;Wang et aL, 1992;Xu et al, 1992). An additional barley (1-~3)-~-glucanase gene has now been isolated (Malehorn et aL, 1993).…”