Mutations in starch BRANCHING ENZYME 2a suppress the traits caused by the loss of ISOAMYLASE1 in barley.

KEY MESSAGE: The hvbe2a mutations restore the starch-deficient phenotype caused by the hvisa1 and hvflo6 mutations in barley endosperm. The genetic interactions among starch biosynthesis genes can be exploited to alter starch properties, but they remain poorly understood due to the various combinations of mutations to be tested. Here, we isolated two novel barley mutants defective in starch BRANCHING ENZYME 2a (hvbe2a-1 and hvbe2a-2) based on the starch granule (SG) morphology. Both hvbe2a mutants showed elongated SGs in the endosperm and increased resistant starch content. hvbe2a-1 had a base change in HvBE2a gene, substituting the amino acid essential for its enzyme activity, while hvbe2a-2 is completely missing HvBE2a due to a chromosomal deletion. Further genetic crosses with barley isoamylase1 mutants (hvisa1) revealed that both hvbe2a mutations could suppress defects in endosperm caused by hvisa1, such as reduction in starch, increase in phytoglycogen, and changes in the glucan chain length distribution. Remarkably, hvbe2a mutations also transformed the endosperm SG morphology from the compound SG caused by hvisa1 to bimodal simple SGs, resembling that of wild-type barley. The suppressive impact was in competition with floury endosperm 6 mutation (hvflo6), which could enhance the phenotype of hvisa1 in the endosperm. In contrast, the compound SG formation induced by the hvflo6 hvisa1 mutation in pollen was not suppressed by hvbe2a mutations. Our findings provide new insights into genetic interactions in the starch biosynthetic pathway, demonstrating how specific genetic alterations can influence starch properties and SG morphology, with potential applications in cereal breeding for desired starch properties.

Characterization of a near isogenic barley line with high grain ß-amylase activity reveals a separation in the tight co-regulation of B-hordeins (Hor2) with endosperm-specific ß-amylase (Bmy1).

GSHO 2096 is a near isogenic barley line with extremely high grain ß-amylase activity, a desirable trait in the malting and brewing industry. High levels of grain ß-amylase activity are caused by a surge in endosperm-specific ß-amylase (Bmy1) gene expression during the early stages of grain development with high expression levels persisting throughout development. Origins of the high ß-amylase activity trait are perplexing considering GSHO 2096 is not supposed to have grain ß-amylase activity. GSHO 2096 is reported to be derived from a Bowman x Risø 1508 cross followed by recurrent backcrossing to Bowman (BC5). Risø 1508 carries a mutated form of the barley prolamin binding factor, which is responsible for Bmy1 expression during grain development. Thus, the pedigree of GSHO 2096 was explored to determine the potential origins of the high grain ß-amylase trait. Genotyping using the barley 50k iSelect SNP array revealed Bowman and GSHO 2096 were very similar (95.4 %) and provided evidence that both Risø 56 and 1508 are in the pedigree. Risø mutants 56 and 1508 both have perturbed hordein gene expression leading to a discernable pattern using SDS-PAGE. GSHO 2096 and Risø 56 have the same hordein pattern whereas Bowman and Risø 1508 have unique patterns. RNAseq revealed that Hor2 (B-hordein) gene expression was completely downregulated making it unique as the only known line with Bmy1 expression without Hor2 co-expression. Regardless of pedigree, GSHO 2096 remains an extremely valuable high ß-amylase activity line with potential utilization in breeding for malt quality.

The CDPK-related protein kinase HvCRK2 and HvCRK4 interact with HvCML32 to negatively regulate drought tolerance in transgenic Arabidopsis thaliana.

Calcium-dependent protein kinase (CDPK) as one of calcium sensors were play important roles in stress responses. CDPK-related protein kinase (CRK) was identified as subgroup III of CDPK has been characterized in many plants, but the members and functions of CRK genes in hulless barley (Hordeum vulgare L.) has not been clarified. Here, we identified four HvCRK genes and named HvCRK1-4 according to chromosomes localization. Moreover, the physiological function of highly induced genes of HvCRK2 and HvCRK4 were investigated in drought stress tolerance by examining their overexpression transgenic lines functions generated in Arabidopsis thaliana. Under drought stress, both overexpression HvCRK2 and HvCRK4 displayed reduced drought resistance, and accompanied by higher accumulation levels of ROS. Notably, overexpression of HvCRK2 and HvCRK4 reduced sensitivity to exogenous ABA, meanwhile the expression of ABA-responsive genes in transgenic plants were down-regulated compared to the wild type in response to drought stress. Furthermore, the physically interaction of HvCRK2 and HvCRK4 with calmodulin (CaM) and calmodulin-like (CML) proteins were determined in vivo, the further results showed that HvCML32 binds to HvCRK2/4 S_TKC structural domains and negatively regulates drought tolerance. In summary, this study identified HvCRK members and indicated that HvCRK2 and HvCRK4 genes play negative roles in drought tolerance, and provide insight into potential molecular mechanism of HvCRK2 and HvCRK4 in response to drought stress.

A GDSL-motif Esterase/Lipase Affects Wax and Cutin Deposition and Controls Hull-Caryopsis Attachment in Barley.

The cuticle covering aerial organs of land plants is well known to protect against desiccation. Cuticles also play diverse and specialized functions, including organ separation, depending on plant and tissue. Barley shows a distinctive cuticular wax bloom enriched in ß-diketones on leaf sheaths, stem nodes and internodes and inflorescences. Barley also develops a sticky surface on the outer pericarp layer of its grain fruit leading to strongly adhered hulls, 'covered grain', important for embryo protection and seed dispersal. While the transcription factor-encoding gene HvNUDUM (HvNUD) appears essential for adherent hulls, little is understood about how the pericarp cuticle changes during adhesion or whether changes in pericarp cuticles contribute to another phenotype where hulls partially shed, called 'skinning'. To that end, we screened barley lines for hull adhesion defects, focussing on the Eceriferum (= waxless, cer) mutants. Here, we show that the cer-xd allele causes defective wax blooms and compromised hull adhesion, and results from a mutation removing the last 10 amino acids of the GDS(L) [Gly, Asp, Ser, (Leu)]-motif esterase/lipase HvGDSL1. We used severe and moderate HvGDSL1 alleles to show that complete HvGDSL1 function is essential for leaf blade cuticular integrity, wax bloom deposition over inflorescences and leaf sheaths and pericarp cuticular ridge formation. Expression data suggest that HvGDSL1 may regulate hull adhesion independently of HvNUD. We found high conservation of HvGDSL1 among barley germplasm, so variation in HvGDSL1 unlikely leads to grain skinning in cultivated barley. Taken together, we reveal a single locus which controls adaptive cuticular properties across different organs in barley.

A pipeline for identification of causal mutations in barley identifies Xantha-j as the chlorophyll synthase gene.

Thousands of barley (Hordeum vulgare L.) mutants have been isolated over the last century, and many are stored in gene banks across various countries. In the present work, we developed a pipeline to efficiently identify causal mutations in barley. The pipeline is also efficient for mutations located in centromeric regions. Through bulked segregant analyses using whole genome sequencing of pooled F2 seedlings, we mapped 2 mutations and identified a limited number of candidate genes. We applied the pipeline on F2 mapping populations made from xan-j.59 (unknown mutation) and xan-l.82 (previously known). The Xantha-j (xan-j) gene was identified as encoding chlorophyll synthase, which catalyzes the last step in the chlorophyll biosynthetic pathway: the addition of a phytol moiety to the propionate side chain of chlorophyllide. Key amino acid residues in the active site, including the binding sites of the isoprenoid and chlorophyllide substrates, were analyzed in an AlphaFold2-generated structural model of the barley chlorophyll synthase. Three allelic mutants, xan-j.19, xan-j.59, and xan-j.64, were characterized. While xan-j.19 is a 1 base pair deletion and xan-j.59 is a nonsense mutation, xan-j.64 causes an S212F substitution in chlorophyll synthase. Our analyses of xan-j.64 and treatment of growing barley with clomazone, an inhibitor of chloroplastic isoprenoid biosynthesis, suggest that binding of the isoprenoid substrate is a prerequisite for the stable maintenance of chlorophyll synthase in the plastid. We further suggest that chlorophyll synthase is a sensor for coordinating chlorophyll and isoprenoid biosynthesis.

Substrate Specificities of Variants of Barley (1,3)- and (1,3;1,4)-ß-d-Glucanases Resulting from Mutagenesis and Segment Hybridization.

Barley (1,3;1,4)-ß-d-glucanase is believed to have evolved from an ancestral monocotyledon (1,3)-ß-d-glucanase, enabling the hydrolysis of (1,3;1,4)-ß-d-glucans in the cell walls of leaves and germinating grains. In the present study, we investigated the substrate specificities of variants of the barley enzymes (1,3;1,4)-ß-d-glucan endohydrolase [(1,3;1,4)-ß-d-glucanase] isoenzyme EII (HvEII) and (1,3)-ß-d-glucan endohydrolase [(1,3)-ß-d-glucanase] isoenzyme GII (HvGII) obtained by protein segment hybridization and site-directed mutagenesis. Using protein segment hybridization, we obtained three variants of HvEII in which the substrate specificity was that of a (1,3)-ß-d-glucanase and one variant that hydrolyzed both (1,3)-ß-d-glucans and (1,3;1,4)-ß-d-glucans; the wild-type enzyme hydrolyzed only (1,3;1,4)-ß-d-glucans. Using substitutions of specific amino acid residues, we obtained one variant of HvEII that hydrolyzed both substrates. However, neither protein segment hybridization nor substitutions of specific amino acid residues gave variants of HvGII that could hydrolyze (1,3;1,4)-ß-d-glucans; the wild-type enzyme hydrolyzed only (1,3)-ß-d-glucans. Other HvEII and HvGII variants showed changes in specific activity and their ability to degrade the (1,3;1,4)-ß-d-glucans or (1,3)-ß-d-glucans to larger oligosaccharides. We also used molecular dynamics simulations to identify amino-acid residues or structural regions of wild-type HvEII and HvGII that interact with (1,3;1,4)-ß-d-glucans and (1,3)-ß-d-glucans, respectively, and may be responsible for the substrate specificities of the two enzymes.

Hordeum vulgare CYP76M57 catalyzes C2 shortening of tryptophan side chain by C-N bond rearrangement in gramine biosynthesis.

The indole alkaloid gramine, 3-(dimethylaminomethyl)indole, is a defensive specialized metabolite found in some barley cultivars. In its biosynthetic process, the tryptophan (Trp) side chain is shortened by two carbon atoms to produce 3-(aminomethyl)indole (AMI), which is then methylated by N-methyltransferase (HvNMT) to produce gramine. Although side chain shortening is one of the crucial scaffold formation steps of alkaloids originating from aromatic amino acids, the gene and enzyme involved in the Trp-AMI conversion reactions are unknown. In this study, through RNA-seq analysis, 35 transcripts were shown to correlate with gramine production; among them, an uncharacterized cytochrome P450 (CYP) gene, CYP76M57, and HvNMT were identified as candidate genes for gramine production. Transgenic Arabidopsis thaliana and rice overexpressing CYP and HvNMT accumulate AMI, N-methyl-AMI, and gramine. CYP76M57, heterologously expressed in Pichia pastoris, was able to act on Trp to produce AMI. Furthermore, the amino group nitrogen of Trp was retained during the CYP76M57-catalyzed reaction, indicating that the C2 shortening of Trp proceeds with an unprecedented biosynthetic process, the removal of the carboxyl group and Cα and the rearrangement of the nitrogen atom to Cß. In some gramine-non-accumulating barley cultivars, arginine 104 in CYP76M57 is replaced by threonine, which abolished the catalytic activity of CYP76M57 to convert Trp into AMI. These results uncovered the missing committed enzyme of gramine biosynthesis in barley and contribute to the elucidation of the potential functions of CYPs in plants and undiscovered specialized pathways.

Genome-Wide Identification and Transcriptional Expression Profiles of PP2C in the Barley (Hordeum vulgare L.) Pan-Genome.

The gene family protein phosphatase 2C (PP2C) is related to developmental processes and stress responses in plants. Barley (Hordeum vulgare L.) is a popular cereal crop that is primarily utilized for human consumption and nutrition. However, there is little knowledge regarding the PP2C gene family in barley. In this study, a total of 1635 PP2C genes were identified in 20 barley pan-genome accessions. Then, chromosome localization, physical and chemical feature predictions and subcellular localization were systematically analyzed. One wild barley accession (B1K-04-12) and one cultivated barley (Morex) were chosen as representatives to further analyze and compare the differences in HvPP2Cs between wild and cultivated barley. Phylogenetic analysis showed that these HvPP2Cs were divided into 12 subgroups. Additionally, gene structure, conserved domain and motif, gene duplication event detection, interaction networks and gene expression profiles were analyzed in accessions Morex and B1K-04-12. In addition, qRT-PCR experiments in Morex indicated that seven HvMorexPP2C genes were involved in the response to aluminum and low pH stresses. Finally, a series of positively selected homologous genes were identified between wild accession B1K-04-12 and another 14 cultivated materials, indicating that these genes are important during barley domestication. This work provides a global overview of the putative physiological and biological functions of PP2C genes in barley. We provide a broad framework for understanding the domestication- and evolutionary-induced changes in PP2C genes between wild and cultivated barley.

QTL x environment modeling of malting barley preharvest sprouting.

KEY MESSAGE: HvMKK3 alleles are temperature sensitive and are major contributors to environmental stability of preharvest sprouting in barley. Preharvest sprouting (PHS) can severely damage barley (Hordeum vulgare L.) malting quality, but PHS resistance is often negatively correlated with malting quality. Seed dormancy is closely related to PHS. Increased temperature during grain fill can decrease seed dormancy in barley, but genetic components of seed dormancy temperature sensitivity are poorly understood. Six years of PHS data were used to fit quantitative trait locus (QTL) x environment mixed models incorporating marker data from seed dormancy genes HvAlaAT1, HvGA20ox1, and HvMKK3 and weather covariates in spring and winter two-row malting barley. Variation in winter barley PHS was best modeled by average temperature range during grain fill and spring barley PHS by total precipitation during grain fill. Average high temperature during grain fill also accurately modeled PHS for both datasets. A highly non-dormant HvMKK3 allele determined baseline PHS susceptibility and HvAlaAT1 interactions with multiple HvMKK3 alleles conferred environmental sensitivity. Polygenic variation for PHS within haplotype was detected. Residual genotype and QTL by environment interaction variance indicated additional environmental and genetic factors involved in PHS. These models provide insight into genotype and environmental regulation of barley seed dormancy, a method for PHS forecasting, and a tool for breeders to improve PHS resistance.

Corn starch modification during endogenous malt amylases: The impact of synergistic hydrolysis time of α-amylase and ß-amylase and limit dextrinase.

To expand the utility of barley malts and decrease the cost of enzyme-modified starch production, the structural and physicochemical characteristics of corn starch modified with fresh barley malts at different hydrolysis time were investigated. The results indicated that compared to native starch, A chain (DP 6-12) of the enzyme-treated starches increased at hydrolysis time (≤12 h), but it decreased at hydrolysis time (>12 h). Inversely, B chains (DP > 13) decreased at hydrolysis time (≤12 h) and they generally increased at hydrolysis time (>12 h). The relative crystallinity decreased from 25.63% to 21.38% and 1047 cm-1/1022 cm-1 reduced from 1.042 to 0.942 after endogenous malt amylases at hydrolysis time from 0 to 72 h, and the thermal degradation temperatures decreased from 323.19 to 295.94 °C, whereas the gelatinization temperatures slightly increased. The gel strength decreased at hydrolysis time less than 12 h, but it increased at hydrolysis time more than 12 h. The outcomings would provide a theoretical and applicative basis about how endogenous malt amylases with lower price modify starches to obtain desirable starch derivatives and industrial production.

Quality attributes for barley malt: "The backbone of beer".

Malting is the process of preparing barley for brewing through partial germination followed by drying. This process softens the grain cell wall and stimulates the production of diastatic enzymes, which convert starch into malt extract. The suitability of a barley grain for malt production depends upon a large number of quality parameters that are crucial for the identification and release of high-quality malt varieties. Maintaining tight control of these quality attributes is essential to ensure high processing efficiency and final product quality in brewery and malt house. Therefore, we have summarized the basic malting process and various physiological and biochemical quality parameters that are desirable for better malt quality. This study may provide an understanding of the process, problems faced, and opportunities to maltsters and researchers to improve the malt efficiency by altering the malting process or malt varieties.

Tandem Protein Kinases Emerge as New Regulators of Plant Immunity.

Plant-pathogen interactions result in disease development in a susceptible host. Plants actively resist pathogens via a complex immune system comprising both surface-localized receptors that sense the extracellular space as well as intracellular receptors recognizing pathogen effectors. To date, the majority of cloned resistance genes encode intracellular nucleotide-binding leucine-rich repeat receptor proteins. Recent discoveries have revealed tandem kinase proteins (TKPs) as another important family of intracellular proteins involved in plant immune responses. Five TKP genes-barley Rpg1 and wheat WTK1 (Yr15), WTK2 (Sr60), WTK3 (Pm24), and WTK4-protect against devastating fungal diseases. Moreover, a large diversity and numerous putative TKPs exist across the plant kingdom. This review explores our current knowledge of TKPs and serves as a basis for future studies that aim to develop and exploit a deeper understanding of innate plant immunity receptor proteins.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Identification and spatio-temporal expression analysis of barley genes that encode putative modular xylanolytic enzymes.

Arabinoxylans are cell wall polysaccharides whose re-modelling and degradation during plant development are mediated by several classes of xylanolytic enzymes. Here, we present the identification and new annotation of twelve putative (1,4)-ß-xylanase and six ß-xylosidase genes, and their spatio-temporal expression patterns during vegetative and reproductive growth of barley (Hordeum vulgare cv. Navigator). The encoded xylanase proteins are all predicted to contain a conserved carbohydrate-binding module (CBM) and a catalytic glycoside hydrolase (GH) 10 domain. Additional domains in some xylanases define three discrete phylogenetic clades: one clade contains proteins with an additional N-terminal signal sequence, while another clade contains proteins with multiple CBMs. Homology modelling revealed that all fifteen xylanases likely contain a third domain, a ß-sandwich folded from two non-contiguous sequence segments that bracket the catalytic GH domain, which may explain why the full length protein is required for correct folding of the active enzyme. Similarly, predicted xylosidase proteins share a highly conserved domain structure, each with an N-terminal signal peptide, a split GH 3 domain, and a C-terminal fibronectin-like domain. Several genes appear to be ubiquitously expressed during barley growth and development, while four newly annotated xylanase and xylosidase genes are expressed at extremely high levels, which may be of broader interest for industrial applications where cell wall degradation is necessary.

The Effects of Cold Atmospheric Pressure Plasma on Germination Parameters, Enzyme Activities and Induction of DNA Damage in Barley.

Climate change, environmental pollution and pathogen resistance to available chemical agents are part of the problems that the food industry has to face in order to ensure healthy food for people and livestock. One of the promising solutions to these problems is the use of cold atmospheric pressure plasma (CAPP). Plasma is suitable for efficient surface decontamination of seeds and food products, germination enhancement and obtaining higher yields in agricultural production. However, the plasma effects vary due to plasma source, treatment conditions and seed type. In our study, we tried to find the proper conditions for treatment of barley grains by diffuse coplanar surface barrier discharge, in which positive effects of CAPP, such as enhanced germination or decontamination effects, would be maximized and harmful effects, such as oxidation and genotoxic potential, minimized. Besides germination parameters, we evaluated DNA damage and activities of various germination and antioxidant enzymes in barley seedlings. Plasma exposure resulted in changes in germination parameters and enzyme activities. Longer exposures had also genotoxic effects. As such, our findings indicate that appropriate plasma exposure conditions need to be carefully optimized in order to preserve germination, oxidation balance and genome stability, should CAPP be used in agricultural practice.

Structures of two novel crystal forms of Aspergillus oryzae alpha amylase (taka-amylase).

The structures of Aspergillus oryzae α-amylase were determined in a tetragonal crystal, having one molecule as asymmetric unit, and a monoclinic crystal with two molecules as asymmetric unit. Both crystal forms were obtained from trace contaminants of an old commercial lipase preparation. Structures were determined and refined to 1.65 Å and 1.43 Å resolution respectively. The latter crystal has a non-crystallographic (NCS) twofold axis within the asymmetric unit. Glycosylation at Asn197 is evident, and in the tetragonal crystal can be seen to include three, partially disordered sugar residues following the initial N-acetyl glucosamine (NAG). Superposition of the tetragonal crystal model on the α-amylases from Bacillus subtilis (PDB:1BAG), pig pancreas (PDB:3L2L), and barley (PDB:1AMY), show a high degree of coincidence, particularly for the (ß/α)8-barrel domains, and especially within the active site. Using this structural agreement between amylases, we extrapolated the binding model of a six residue, limit dextrin found in pig pancreas α-amylase to the A. oryzae enzyme model, which predicts substrate interacting amino acid residues.

Grain filling in barley relies on developmentally controlled programmed cell death.

Cereal grains contribute substantially to the human diet. The maternal plant provides the carbohydrate and nitrogen sources deposited in the endosperm, but the basis for their spatial allocation during the grain filling process is obscure. Here, vacuolar processing enzymes have been shown to both mediate programmed cell death (PCD) in the maternal tissues of a barley grain and influence the delivery of assimilate to the endosperm. The proposed centrality of PCD has implications for cereal crop improvement.

Overexpression of isochorismate synthase enhances drought tolerance in barley.

Isochorismate synthase (ICS) is a key enzyme for the synthesis of salicylic acid (SA) in plants. SA mediates plant responses to both biotic and abiotic stresses. In previous studies, we found that overexpression of ICS (ICSOE) or suppression of ICS (ICSRNAi) affected the host response to Fusarium graminearum in barley. However, whether the barley ICS gene plays a role in adapting to abiotic stresses remains to be determined. In the present study, expression of the ICS gene was upregulated when treated with 20 % PEG6000, and ICSOE lines were more drought tolerant than wild type (WT) and ICSRNAi. In addition, the abscisic acid (ABA) levels in the ICSOE lines were higher than those in the WT and ICSRNAi lines under drought stress. High ABA levels significantly reduced Gs and E, which may impact water retention under drought stress. Under drought conditions, the activity of antioxidant enzymes was significantly higher in the ICSOE lines, correlating with a lower levels of reactive oxygen species (ROS) and malondialdehyde (MDA). Enhanced antioxidant competence also contributed to drought tolerance in ICSOE lines. These findings help elucidate the abiotic stress resistance of the ICS pathway in barley.

Stability of EPSPS gene copy number in Hordeum glaucum Steud (barley grass) in the presence and absence of glyphosate selection.

BACKGROUND: Gene amplification has been shown to provide resistance to glyphosate in several weed species, including Hordeum glaucum populations in South Australia. The stability of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene copies in resistant populations in the presence or absence of glyphosate selection has not been determined. RESULTS: Applying glyphosate to a cloned plant resulted in an increase in resistance and EPSPS copy number in the progeny of that plant compared to the untreated clone. The LD50 (herbicide concentration required for 50% mortality) increased by 75% to 79% in the progeny of the treated clones compared to the untreated in both populations (YP-17 and YP-16). EPSPS copy number estimates were higher in treated individuals compared to untreated individuals with an average of seven copies compared to six in YP-16 and 11 compared to six in YP-17. There was a positive correlation (R2  = 0.78) between EPSPS copy number and LD50 of all populations. CONCLUSION: EPSPS gene copy number and resistance to glyphosate increased in H. glaucum populations under glyphosate selection, suggesting the number of EPSPS gene copies present is dependent on glyphosate selection. © 2021 Society of Chemical Industry.

Expression of barley oxalate oxidase confers resistance against Sclerotinia sclerotiorum in transgenic Brassica juncea cv Varuna.

Sclerotinia Stem Rot (SSR) caused by the oxalic acid (OA)-secreting necrotrophic fungal pathogen Sclerotinia sclerotiorum, causes significant yields losses in the crop Brassica sps. Oxalate oxidase (OxO) can metabolize OA to CO2 and H2O2. Degradation of OA during the early phase of fungal-host interaction can interfere with the fungal infection and establishment processes. The present study demonstrates the potential of barley oxalate oxidase (BOxO) gene in conferring stable resistance against stem rot in a productive and highly susceptible Brassica juncea cv Varuna under field conditions. Four stable, independent, single-copy transgenic lines (B16, B17, B18, and B53) exhibited a significant reduction in the rate of lesion expansion i.e. 11-26%, 39-47%, and 24-35% reproducibly over the three-generation i.e. T2, T3, and T4 respectively. The enhanced resistance in the transgenic lines correlated with high OxO activity, accumulation of higher levels of H2O2, and robust activation of defense responsive genes upon infection by S. sclerotiorum.

Starch hydrolysis during mashing: A study of the activity and thermal inactivation kinetics of barley malt α-amylase and ß-amylase.

Hydrolysis of starch is key in several industrial processes, including brewing. Here, the activity and inactivation kinetics of amylases throughout barley malt mashing are investigated, as a prerequisite for rational optimisation of this process. Varietal differences were observed in the activity of α- and ß-amylases as a function of temperature for six barley and malt varieties. These differences were not reflected in the resulting wort composition after mashing, using three isothermal phases of 30 min at 45 °C, 62 °C and 72 °C with intermediate heating by 1 °C/min. Thermal inactivation kinetics parameters determined for α- and ß-amylases of an industrially relevant malt variety in a diluted system showed that enzymes were inactivated at lower temperatures than expected. The obtained kinetic parameters could predict α-amylase, but not ß-amylase inactivation in real mashing conditions, suggesting that ß-amylase stability is enhanced during mashing by components present or formed in the mash.