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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

2022 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, shoot weight from stressed plants (heat-replicate 1, drought-replicate 2 and combined stress-replicate 2) and corresponding controls were collected for all the 165 lines. This research has led to the identification of four lines with tolerance to heat and drought stress. A second replication of the phenotyping for drought in greenhouse for the recombinant inbred line (RIL) population (approximately 200 lines) derived from a cross between stress tolerant Otis and sensitive Golden Promise has been completed (root and shoot biomass). Dry weight of roots and shoots and seed yield data have been collected for these lines. The 200 RILs have been genotyped using the 50K single nucleotide polymorphism chips. Furthermore, in collaboration with an ARS scientist in Aberdeen, Idaho, we have obtained seed yield data for the RIL population grown under irrigated and rainfed conditions in 2020 and 2021 seasons. For Objective 2, whole genome bisulfite sequencing was employed to obtain the global methylation status of the eight germplasm (3 parents, 3 lys3a mutants, 2 lys3 allelic mutants) to address Sub-objective 2.5. All germplasm were sequenced at 9X or higher with the exception of Sloop (5X). Bisulfite conversion was successful as determined by approximately 99% conversion rate. There were no differences in global methylation pattern at CG, CHG, or CHH methylation islands in the tested lines. Developing-endosperm transcriptomes were sequenced to address Sub-objective 2.5, Goal 2.5a. Expression of the predominant beta-amylase (Bmy1) was reduced over 98% in the Bomi lys3a mutant whereas expression was reduced 82 and 19% in Bomi lys3b and lys3c, respectively. Sloop, a malting cultivar, carrying the lys3a locus resulted in a 73% reduction in Bmy1 transcript whereas in Bowman, a non-malting cultivar, the lys3a mutant had a 74 % increase in Bmy1 levels. Bowman lys3a mutant was backcrossed six times and the lys3a locus segregated out demonstrated by the increase in Bmy1 expression and the mutant not having the causal mutation in the barley prolamin binding factor (BPBF) gene. Bmy1 expression data was corroborated by beta-amylase activity, best demonstrated by the Bowman lys3a mutant having the highest Bmy1 gene expression and beta-amylase activity and the Bomi lsy3a mutant having Bmy1 expression reduced 98% and enzyme activity reduced 93%. Addressing Subobjective 2.5, Goal 2.5b, expression levels of HvDME were determined by RNA sequencing in the developing endosperm from 3 parents and 5 lys3 mutants. HvDME is a demethyltransferase originally thought to be the lys3a causal mutation. HvDME expression was determined to be relatively low in all germplasm. In the Bomi background, HvDME transcript levels were 20 % higher in the lys3a mutant, 17% lower in the lys3b mutant, and 40 % lower in the lys3c mutant. The Sloop lsy3a mutant had 15 % lower transcripts than Sloop. The Bowman lys3a mutant had 41% higher HvDME expression than Bowman. The lys3a causal gene was recently determined to be a mutation in the BPBF gene that Bomi lsy3c and Bowman lys3a mutants do not have. BPBF expression patterns in the mutants with the causal genes revealed compensation effects. Bomi lys3a mutant had 684% more BPBF transcript, the Sloop lys3a mutant had 302% more transcript, whereas the Bowman lys3a had 46% more transcript. The Bomi lys3b allelic mutant had no BPBF transcript caused by a large deletion in part of the gene and the Bomi lys3c mutant had 9% lower expression levels compared to Bomi. Another transcription factor could play a role in Bmy1 gene expression given the increase in beta-amylase activity during late grain development and the relatively high Bmy1 expression during grain development. Further addressing Subobjective 2.5, RNAseq analysis revealed a bevy of malting quality genes affected. A common feature of lys3a mutants is low beta-glucan values and RNAseq analysis identified extremely high expression of beta-glucosidase, an enzyme that catalyzes the breakdown of beta-glucans derived from cell walls. Also, lys3a mutants carrying the causal polymorphism have reduced hordein levels and three B1, C, and gamma hordein genes were identified with extreme reduction in transcript levels. Some compensation effects were observed with increases observed in D hordein genes. BPBF is known to bind to the promoter of seed storage proteins but even in the Bomi lys3b germplasm containing zero transcripts of BPBF some hordein expression is observed indicating other transcription factors needed for expression. A total of 305 DEG were similar between the lys3a, lys3b, and lys3c mutants compared to their parent Bomi and 118 DEG were common amongst the three lys3a mutant and 338 common between the two lys3a mutants carrying the causal polymorphism and 184 DEG in all mutants carrying the causal polymorphism.


Review Publications
Walling, J.G., Sallam, A.H., Steffenson, B.J., Henson, C.A., Vinje, M.A., Mahalingam, R. 2022. Quantitative trait loci impacting grain beta-glucan content in wild barley (Hordeum vulgare ssp. spontaneum) reveal genes associated with cell wall modification and carbohydrate metabolism. Crop Science. 62(3):1213-1227.
Matusinec, D., Maule, A., Wiesman, E.C., Atucha, A., Mura, J.D., Zalapa, J.E. 2022. The New Cranberry Wisconsin Research Station: Renovation priorities of a ‘Stevens’ cranberry marsh based on visual mapping, genetic testing, and yield data. International Journal of Fruit Science. 22:1, 121-132.
Mucci, N., Jones, K., Cao, M., Wyatt, M., Foye, S., Kauffman, S., Taufer, M., Takizawa, Y., Chikaraishi, Y., Steffan, S.A., Campagna, S., Goodrich-Blair, H., Richards, G.R. 2022. Chemical ecology of a tripartite symbiosis. American Society for Microbiology. 00312-22.
Dharampal, P., Danforth, B., Steffan, S.A. 2022. Exosymbiotic microbes within fermented pollen provisions are as important for the development of solitary bees as the pollen itself. Journal of Experimental Biology. 12:e8788.
Barcelo, G., Perrig, P., Dharampal, P., Donadio, E., Steffan, S.A., Pauli, J. 2022. More than just meat: Carcass decomposition shapes trophic identities in a terrestrial vertebrate. Proceedings of the Royal Society B. 36:1473–1482.