Mutations in the gene of the Gα subunit of the heterotrimeric G protein are the cause for the brachytic1 semi-dwarf phenotype in barley and applicable for practical breeding.

BACKGROUND: Short-culm mutants have been widely used in breeding programs to increase lodging resistance. In barley (Hordeum vulgare L.), several hundreds of short-culm mutants have been isolated over the years. The objective of the present study was to identify the Brachytic1 (Brh1) semi-dwarfing gene and to test its effect on yield and malting quality. RESULTS: Double-haploid lines generated through a cross between a brh1.a mutant and the European elite malting cultivar Quench, showed good malting quality but a decrease in yield. Especially the activities of the starch degrading enzymes ß-amylase and free limit dextrinase were high. A syntenic approach comparing markers in barley to those in rice (Oryza sativa L.), sorghum (Sorghum bicolor Moench) and brachypodium (Brachypodium distachyon P. Beauv) helped us to identify Brh1 as an orthologue of rice D1 encoding the Gα subunit of a heterotrimeric G protein. We demonstrated that Brh1 is allelic to Ari-m. Sixteen different mutant alleles were described at the DNA level. CONCLUSIONS: Mutants in the Brh1 locus are deficient in the Gα subunit of a heterotrimeric G protein, which shows that heterotrimeric G proteins are important regulators of culm length in barley. Mutant alleles do not have any major negative effects on malting quality.

Morphology, Carbohydrate Distribution, Gene Expression, and Enzymatic Activities Related to Cell Wall Hydrolysis in Four Barley Varieties during Simulated Malting.

Many biological processes, such as cell wall hydrolysis and the mobilisation of nutrient reserves from the starchy endosperm, require stringent regulation to successfully malt barley (Hordeum vulgare) grain in an industrial context. Much of the accumulated knowledge defining these events has been collected from individual, unrelated experiments, and data have often been extrapolated from Petri dish germination, rather than malting, experiments. Here, we present comprehensive morphological, biochemical, and transcript data from a simulated malt batch of the three elite malting cultivars Admiral, Navigator, and Flagship, and the feed cultivar Keel. Activities of lytic enzymes implicated in cell wall and starch depolymerisation in germinated grain have been measured, and transcript data for published cell wall hydrolytic genes have been provided. It was notable that Flagship and Keel exhibited generally similar patterns of enzyme and transcript expression, but exhibited a few key differences that may partially explain Flagship's superior malting qualities. Admiral and Navigator also showed matching expression patterns for these genes and enzymes, but the patterns differed from those of Flagship and Keel, despite Admiral and Navigator having Keel as a common ancestor. Overall (1,3;1,4)-ß-glucanase activity differed between cultivars, with lower enzyme levels and concomitantly higher amounts of (1,3;1,4)-ß-glucan in the feed variety, Keel, at the end of malting. Transcript levels of the gene encoding (1,3;1,4)-ß-glucanase isoenzyme EI were almost three times higher than those encoding isoenzyme EII, suggesting a previously unrecognised importance for isoenzyme EI during malting. Careful morphological examination showed that scutellum epithelial cells in mature dry grain are elongated but expand no further as malting progresses, in contrast to equivalent cells in other cereals, perhaps demonstrating a morphological change in this critical organ over generations of breeding selection. Fluorescent immuno-histochemical labelling revealed the presence of pectin in the nucellus and, for the first time, significant amounts of callose throughout the starchy endosperm of mature grain.

A Chromogenic Assay Suitable for High-Throughput Determination of Limit Dextrinase Activity in Barley Malt Extracts.

Twenty-four malt samples were assayed for limit dextrinase activity using a chromogenic assay developed recently in our group. The assay utilizes a small soluble chromogenic substrate which is hydrolyzed selectively by limit dextrinase in a coupled assay to release the chromophore 2-chloro-4-nitrophenol. The release of the chromophore, corresponding to the activity of limit dextrinase, can be followed by measuring the UV absorption at 405 nm. The 24 malt samples represented a wide variation of limit dextrinase activities, and these activities could be clearly differentiated by the assay. The results obtained were comparable with the results obtained from a commercially available assay, Limit-Dextrizyme from Megazyme International Ireland. Furthermore, the improved assay uses a soluble substrate. That makes it well suited for high-throughput screening as it can be handled in a 96-well plate format.

Probing the structural details of xylan degradation by real-time NMR spectroscopy.

The biodegradation of abundantly available cell wall polysaccharides has recently received much attention, not least because cell wall polysaccharides are substrates for the human gut microbiota and for environmentally sustainable processes of biomass conversion to value-added compounds. A major fraction of cereal cell wall polysaccharides consists of arabinoxylans. Arabinoxylan and its degradation products are therefore present in a variety of agro-industrial residues and products. Here, we undertook to track the structural details of wheat arabinoxylan degradation with high resolution NMR spectroscopy. More than 15 carbohydrate residues were distinguished in the substrate and more than 20 residues in partially degraded samples without any sample cleanup. The resolution of a plethora of structural motifs in situ permits the readout of persisting structures in degradation processes and in products. Reaction progress was visualized for the biodegradation of arabinoxylan by different crude microbial enzyme preparations. The direct observation of structural details in complex mixtures containing arabinoxylan fragments is significant, as such structural details reportedly modulate the health-promoting functions of arabinoxylan fragments.

Distinct developmental defense activations in barley embryos identified by transcriptome profiling.

Proper embryo development is crucial for normal growth and development of barley. Numerous related aspects of this process--for example how the embryo establishes and sustains disease resistance for extended periods during dormancy--remain largely unknown. Here we report the results of microarray analyses of >22,000 genes, which together with measurements of jasmonic acid and salicylic acid during embryo development provide new information on the initiation in the developing barley embryo of at least two distinct types of developmental defense activation (DDA). Early DDA is characterized by the up-regulation of a specific set of genes around 20 days after flowering, including co-regulation of those for encoding 9-lipoxygenase and several oxylipin-generating enzymes, possibly leading to the formation of alpha-ketols. The same developmental phase includes an up-regulation of several defense genes, and indications of co-regulation of those for enzymes involved in the generation of phenylpropanoid phytoalexins. Late DDA is initiated prior to grain desiccation, around 37 days after flowering, with up-regulation of several genes encoding proteins with roles in antioxidant responses as well as a simultaneous up-regulation of several PR genes is notable. Throughout barley embryo development, there are no indications of an increased biosynthesis of either jasmonic acid or salicylic acid. Collectively, the results help explain how the proposed DDA enables protection of the developing barley embryo and grain for purposes of disease resistance.

Production of enzymatically active recombinant full-length barley high pI alpha-glucosidase of glycoside family 31 by high cell-density fermentation of Pichia pastoris and affinity purification.

Recombinant barley high pI alpha-glucosidase was produced by high cell-density fermentation of Pichia pastoris expressing the cloned full-length gene. The gene was amplified from a genomic clone and exons (coding regions) were assembled by overlap PCR. The resulting cDNA was expressed under control of the alcohol oxidase 1 promoter using methanol induction of P. pastoris fermentation in a Biostat B 5 L reactor. Forty-two milligrams alpha-glucosidase was purified from 3.5 L culture in four steps applying an N-terminal hexa-histidine tag. The apparent molecular mass of the recombinant alpha-glucosidase was 100 kDa compared to 92 kDa of the native barley enzyme. The secreted recombinant enzyme was highly stabile during the 5-day fermentation and had significantly superior specific activity of the enzyme purified previously from barley malt. The kinetic parameters Km, Vmax, and kcat were determined to 1.7 mM, 139 nM x s(-1), and 85 s(-1) using maltose as substrate. This work presents the first production of fully active recombinant alpha-glucosidase of glycoside hydrolase family 31 from higher plants.