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Abstractβ-Amylase (α-1,4-glucan maltohydrolase; EC 3.2.1.2) is known to be an important enzyme in beer brewing. To identify the contribution of β-amylase in more detail, two β-amylase-deficient barley, ‘Backcrossed line (BCL)-Haruna’ and ‘Nittakei 81’, were used for the investigation of malt quality and brewing characteristics. ‘BCL-Haruna’ was bred by the backcross method for the introduction of the β-amylase-deficient trait from landrace barley (‘Tibet Violet 1’) to Japanese traditional malting barley ‘Haruna Nijo’. ‘Nittakei 81’ was bred by crossing between Japanese malting barley ‘Sukai Golden’ and ‘Tibet Violet 1’. With regards to the malting quality under 41.0% target cast moisture in micro-malting, the diastatic power (DP) and apparent attenuation limit (AAL) of ‘BCL-Haruna’ and ‘Nittakei 81’ were 408 oWK and 416 oWK and 12.4% and 14.8% lower than those of the control variety ‘Haruna Nijo’. Oligosaccharide analysis indicated that the maltose concentration of the ‘Nittakei 81’ wort was 19.7 g/L lower in comparison to a standard European malting barley ‘Propino’ wort, although the maltotetraose and maltopentaose concentrations of the ‘Nittakei 81’ wort were 5.6 g/L and 2.7 g/L higher than those of the ‘Propino’ wort. After fermentation, the alcohol concentration of the ‘Nittakei 81’ beer was 0.67% lower compared with the ‘Propino’ beer, and the extract and concentrations of maltotetraose and maltopentaose of the ‘Nittakei 81’ beer were 5.0 g/L and 2.5 g/L higher than those of the ‘Propino’ beer. The results indicate that the β-amylase-deficient barley has a unique malting quality, especially lower DP and AAL, which might induce inadequate starch degradation or fermentation. However, this trait caused no serious issues during the brewing process, suggesting the possibility of β-amylase-deficient barley as a unique brewing material including low-alcoholic and non-alcoholic beer production with a specific saccharide composition.

AbstractArabinoxylan is a major polysaccharide found in barley and wheat. As this polysaccharide has been reported to be involved in wort viscosity and filterability, as well as β-glucan, it has become increasingly important in beer brewing in recent years, despite the limited availability of a measurement methodology for arabinoxylan. In this study, we developed a simple method for determining arabinoxylan content in wort and beer products using a commercially available xylose determination kit. The results show a relationship between arabinoxylan and the viscosity of wort and beer products. These results were consistent with previous reports, suggesting that this simple method can be used to observe the behavior of arabinoxylan during malting and brewing.

Putative nudix hydrolase (NUDX) genes, which encode amino acid sequences showing homology with those of Arabidopsis NUDXs and conserve nudix motif, were identified from barley. The 14 deduced barley NUDXs (HvNUDX1-14) were classified into established subfamilies, except for 8-oxo-deoxyguanosine 5′-triphosphate (8-oxo-dGTP) pyrophosphohydrolase and mRNA decapping enzyme subfamilies, and three substrate-unknown subfamilies. Drought and UV-C stresses, respectively, up-regulated 7 and 4 HvNUDX genes, but some homologs of Arabidopsis NUDXs showed different responses to abiotic stress. HvNUDX12 gene, belonging to diadenosine tetraphosphates (Ap4A) pyrophosphohydrolase subfamily gene and up-regulated by UV-C, was expressed in Escherichia colicells. The recombinant protein showed 8-oxo-dGTP, Ap4A, and guanosine-3′,5′-tetraphosphate (ppGpp) pyrophosphohydrolase activities, and the suppression of the lacZamber mutation in a mutT-deficient E. colicells caused by the incorporation of 8-oxo-GTP into mRNA was prevented to a significant degree. These results suggest that barley NUDXs have unique constitution and response of NUDX to abiotic stress.

The gene expression and enzyme activity of superoxide dismutase, catalase, and ascorbate peroxidase in the space-grown barley were not significantly different from those of the ground-grown barley. Cu2+reducing and radical scavenging activities in an extract of the space-grown barley were lower than those of the ground-grown barley by 0.7 fold, suggesting that the space environment does not induce oxidative stress, and reduces antioxidant capacity in plants.

The yeast Saccharomyces cerevisiaeCdc7p/Dbf4p protein kinase complex was purified to near homogeneity from insect cells. The complex efficiently phosphorylated yeast Mcm2p and less efficiently the remaining Mcm proteins or other replication proteins. Significantly, when pretreated with alkaline phosphatase, Mcm2p became completely inactive as a substrate, suggesting that it must be phosphorylated by other protein kinase(s) to be a substrate for the Cdc7p/Dbf4p complex. Mutant Cdc7p/Dbf4p complexes containing either Cdc7-1p or Dbf4-1∼5p were also partially purified from insect cells and characterized in vitro. Furthermore, the autonomously replicating sequence binding activity of variousdbf4mutants was also analyzed. These studies suggest that the autonomously replicating sequence-binding and Cdc7p protein kinase activation domains of Dbf4p collaborate to form an active Cdc7p/Dbf4p complex and function during S phase in S. cerevisiae. It is shown that Rad53p phosphorylates the Cdc7p/Dbf4p complex in vitroand that this phosphorylation greatly inhibits the kinase activity of Cdc7p/Dbf4p. This result suggests that Rad53p controls the initiation of chromosomal DNA replication by regulating the protein kinase activity associated with the Cdc7p/Dbf4p complex.

The genetic improvement of enzymes important in the brewing process is one of the main goals of barley biotechnology. For the improvement of β-amylase thermostability in barley seeds, we have already constructed a mutant thermostable β-amylase gene, using site-directed mutagenesis and random mutagenesis to achieve the substitution of seven amino acids of the original barley β-amylase. This sevenfold-mutant barley β-amylase showed a thermostability increased by 11.6 °C compared to the original enzyme. In the present article, a thermostable β-amylase gene under the control of the barley β-amylase promoter was introduced to barley protoplasts, and fertile plants were generated from 9 independent transgenic lines. Subsequent analyses indicated that the thermostable β-amylase gene was expressed and β-amylase activity derived from both native and modified genes was detected in the seeds of 6 transgenic lines. The transgene was stably transmitted to progeny, and thermostable β-amylase was synthesized in T4seeds, demonstrating that our strategy is applicable for the improvement of seed quality for industrial utilization.

The 2.3 kb-DNA fragment containing the promoter region of the seed specific β-amylase gene was cloned from barley and sequenced. There were a TATA box and a CCAAT box-like sequence in the 5'upstream region of the β-amylase structural gene, but not an endosperm box-like sequence, which seems to be associated with the seed specific expression of the seed storage protein (prolamine) genes. In order to confirm the function of the cloned fragment as a promoter, the expression of the β-glucuronidase (GUS) gene connected to the downstream region of the cloned promoter was investigated. The results of the histochemical GUS assay showed that the cloned promoter was functional in the sub aleurone endosperm during the seed maturation of barley.

With the aim of the development of a culture method for efficient plant regeneration from barley (Hordeum vulgare L.) protoplasts, we examined several culture conditions for primary calli from immature embryos of cvs. Dissa and Igri, which were used for initiation of cell suspensions. Among the primary callus culture conditions tested, growth condition of donor plants had a great impact on these efficiencies; Igri protoplasts derived from embryos of plants grown in a greenhouse gave rise to albino plants and few green shoots while several cell lines originating from embryos of plants grown in a growth chamber (16h light, 12°C) yielded protoplasts developing into green plants. In contrast, cell suspensions were produced at higher frequencies from calli derived from embryos of greenhouse-grown Dissa plants. In Igri, increased levels of 2,4-dichlorophenoxyaceticacid (2,4-D) significantly reduced the efficiency of cell suspension establishment and plant regeneration from protoplasts was achieved only with suspension cells derived from calli induced at the lowest level (2.5 mg/l), while the effect of the 2,4-D concentration was not clear in Dissa. The developmental stage of immature embryos also affected the efficiency of cell suspension establishment, and the optimal embryo size was determined to be approximately 1mm in diameter. These results demonstrate the importance of callus induction conditions for successful barley protoplast culture.