Skip to main content
ARS Home » Midwest Area » Madison, Wisconsin » Cereal Crops Research » Research » Research Project #434646

Research Project: Biochemical Pathways and Molecular Networks Involved in Seed Development, Germination and Stress Resilience in Barley and Oat

Location: Cereal Crops Research

2019 Annual Report

Objective 1: Identify and characterize germplasm for barley malt production in suboptimal environmental conditions. Sub-objective 1.1: Barley will be assessed for resilience to combined heat and drought stress. Sub-objective 1.2: Assess the impact of abiotic stress on malting quality. Sub-objective 1.3: SNP genotyping of barley lines using Illumina chips and Genome Wide Association Study (GWAS). Objective 2: Identify molecular networks associated with malting, and functionally characterize known and putative genes with the potential to improve malt quality. Sub-objective 2.1a: Determine the transcriptome and the miRNAs involved in regulating the transcriptome in malting barley. Sub-objective 2.2: Analyze proteome changes during various stages of barley malting. Sub-objective 2.3: Integrate transcriptional, post-transcriptional, and proteomic changes during various stages of malting. Sub-objective 2.4: Functionally characterize the putative malting quality genes Bmy2 and DPE1. Sub-Objective 2.5: Characterize the molecular mechanisms of barley lys3a and determine how its function regulates malting quality genes. Objective 3: Determine biochemical or physiological roles of metabolites in barley and oat. Sub-objective 3.1: Identify abiotic stress-induced seed solutes in malting barley. Sub-objective 3.2: Determine if stress-induced seed solutes function as osmoprotectant molecules to hydrolytic enzymes during mashing.

Objective 1. Accessions from the barley mini-core collection, the Vavilov collection, and selected pre-prohibition and modern elite malting barley cultivars will be grown under optimal and abiotic stress conditions. Evaluation of selected tolerant lines will be for a variety of physical traits including biomass and seed yield, physiological traits such as photosynthesis, transpiration, respiration, stomatal conductance and a variety of malting quality traits including standard metrics of quality plus mashing performance. SNP genotyping of the mini-core collection will aid in GWAS for identification of malting quality and abiotic stress associated QTLs. Objective 2. Changes in the transcriptome, miRNAome and the proteome during malting of selected lines will be evaluated. Omics data from these multiple high throughput platforms will be integrated to develop a systems model of the genetic and biochemical pathways involved in the barley malting process. Genetic confirmation of key genes and proteins associated with malting quality and/or abiotic stress tolerance will be conducted via transformation, CRISPER/Cas or via TILLING populations. Barley lys3.a mutants will be evaluated during grain development to determine the mechanism of action on malting quality genes and to identify the causal gene. Select malting quality genes will be evaluated in modern elite malting cultivars during malting. Objective 3. Stress induced metabolites present in malts and rendered soluble during mashing will be chromatographically separated, then detected and identified by mass spectrometry. Resource spectral databases used for identification will include NIST, Flavor and Fragrances and our in-house authentic compound database. Metabolites identified that are commercially available will be used in relevant concentrations to determine if they affect the activity and thermostability of key enzymes involved in the production of fermentable sugars during high temperature mashing.

Progress Report
Studies to address Objective 1 included subjecting the barley mini-core collection to combined heat and drought stress during the heading stage. Seed yield, root and shoot weight from stressed plants and corresponding controls were collected for all the 165 lines. This research will enable the identification of germplasm for barley malt production in suboptimal environmental conditions. Additionally, DNA from the 165 lines of the barley mini-core collection was extracted and were used for genotyping with the 50,000 single nucleotide polymorphism arrays at Fargo, North Dakota. This will enable the identification of genomic regions associated with heat and drought stress tolerance which can then be used for marker-assisted introgression of stress tolerance from newly identified germplasm. Studies to address Objective 2 included malting an elite U.S. malting barley variety, Conrad, conducting small scale malting and collecting samples at daily time points. Malting samples were prepared by pooling five individual samples grown in four different environments over two crop years. Pooled samples from Conrad were malted in triplicate. Total and small RNA (microRNA) have been isolated from malted samples at all time points to create sequencing libraries. Libraries have been sequenced for both the total RNA and small RNA project and data generated. Data are currently being analyzed by a collaborating scientist from a public university. Once these data have been analyzed an assessment of the molecular networks associated with malting will be possible. Studies to address Objective 2 (2.2) include the analysis of the proteome during five different malting stages, which identified 37 proteins that belonged to the RNA binding protein superfamily. This prompted a genome-wide survey of ‘RNA binding’ proteins in barley. A manuscript detailing the in silico analyses of barley RNA binding proteins is in progress. Further addressing Objective 2 (2.4), this project has discovered two novel genes that are turned on during the malting process. These genes and their respective gene products (enzymes) have great potential to alter the sugar profile in a beneficial way during the mashing process. Beta-amylase 2 expression increased 21-fold in Conrad and peaked at day 3 of malting, whereas, Legacy had an increase of 41-fold with the peak identified at day 4 of malting. Beta-amylase 2 protein levels also increased throughout malting. Both Conrad and Legacy had beta-amylase activity significantly increased throughout malting corroborating the gene expression data. Differences in expression levels between the two malting varieties were observed with Conrad having more beta-amylase 2 transcript than Legacy between 0 and 3 days of malting but Legacy had more beta-amylase 2 transcript than Conrad at 4 and 5 days of malting. Additionally, disproportionating enzyme 1 was found to be regulated during grain development and during malting. Disproportionating enzyme 1 increased about 5-fold during seed development peaking at 17 days of grain development. During the malting procedure, disproportionating enzyme expression increased during malting between 3 and 4-fold depending on the barley variety and Conrad was found to have significantly more disproportionating enzyme 1 transcript than Legacy at all malting time points. Enzyme data was to be collected during this fiscal year, but the furlough prevented this from happening and should be finished soon. Comparing the gene expression of disproportionating enzyme 1 and beta-amylase 2 between two elite barley malting varieties identified significant differences in expression. To further test the expression differences in US malting varieties, twelve American Malting Barley Association recommended varieties were selected that represent spring two-row and six-row and winter two-row and six-row. Five individual samples from each of the twelve varieties were selected based upon their growing environment and equally pooled together. Malting was performed in triplicate and sampled daily. Data from this experiment will help identify varietal differences in gene expression and addresses Objective 2, Sub-objective 2.4. In order to address Sub-objective 2.5 and determine if the beta-amylase 1 gene is regulated by a mutation in the lys3a locus, three different parental backgrounds carrying the mutated lys3a locus were grown in a greenhouse and sampled during grain development. Additionally, novel mutants carrying a potentially different mutation in the lys3 locus were also sampled. A total of 316 developing grain samples have been collected with analysis to take place later. Studies addressing Objective 3 were conducted on stress induced metabolites in barley malts identified in recently published literature and on metabolites widely demonstrated to be induced in stressed plants of many different species. These molecules were evaluated for their ability to provide thermoprotection to the two malt enzymes that produce the majority of the fermentable sugars generated during mashing. One of the metabolites, a small molecule, widely demonstrated to be stress induced in many species had no effect on the thermostability of either alpha- or beta-amylases from barley malts. However, another well-established stress induced molecule with approximately four times more mass did increase the thermostability of both amylases. An even larger molecule, also well-established to impart thermoprotection of many enzymes had no effect on either alpha- or beta-amylases from barley malts.


Review Publications
Mahalingam, R., Bregitzer, P.P. 2019. Impact on physiology and malting quality of barley exposed to heat, drought and their combination during different growth stages under controlled environment. Physiologia Plantarum. 165(2):277-289.
Vinje, M.A., Walling, J.G., Henson, C.A., Duke, S.H. 2019. Comparative gene expression analysis of the beta-amylase and hordein gene families in the developing barley grain. Gene. 693:127-136.
Duke, S.H., Henson, C.A., Vinje, M.A., Walling, J.G., Bockelman, H.E. 2019. Comparisons of modern United States and Canadian malting barley cultivars with those from pre-Prohibition: V. Bmy1 intron III alleles and grain beta-amylase activity and thermostability. Journal of American Society of Brewing Chemists. 77(1):62-68.
Henson, C.A., Duke, S.H., Bockelman, H.E. 2018. Comparisons of modern United States and Canadian malting barley cultivars with those from pre-Prohibition: IV. Malting quality assessments using standard and nonstandard measures. Journal of American Society of Brewing Chemists. 76(3):156-168.