Location: Cereal Crops Research2011 Annual Report
1a. Objectives (from AD-416)
Objective 1: To assess allelic variation in Agl1, the gene encoding a-glucosidase, in barley (Hordeum vulgare ssp. vulgare) germplasm adapted to North America and in selected Hordeum vulgare ssp. spontaneaum accessions currently being used to introgress disease resistance genes into North American barley germplasm. Objective 2: To determine the potential contributions of isoamylase to the production of fermentable sugars during mashing and, if isoamylase contributes to fermentable sugar production, assess allelic variation in selected barley populations. Objective 3: To determine metabolic profiles of worts or 20ºC water extracts to determine which metabolites distinguish the best malting barleys from those of lesser malting quality.
1b. Approach (from AD-416)
Metabolic profiling of malts will be conducted using standard methods of derivatization, gas chromatography and mass spectral detection/ identification of sample components followed by multivariate analysis of data to identify metabolites that best define the trait "malting quality". Metabolite identification will be done using several mass spectra databases including the 7th edition of Wiley, Essential Oils/Flavor and Fragrance and my lab's personal database. Examination of enzyme contributions to fermentable sugar production will be by use of an immunological probe and by characterizing the recombinant enzyme in a heterologous expression system. Assessment of allelic variation of carbohydrases will be via PCR amplification of expressed mRNA followed by sequence analysis and exploration of functional consequences of documented polymorphisms.
3. Progress Report
Assessment of gene expression, transcript abundance, and protein abundance of a second gene encoding a starch degrading carbohydrase in barley seeds during development was completed in tissues of two malting quality genotypes and two wild accessions of barley. Studies of the promoter of this gene and of another gene in barley that encodes an enzyme that is functionally identical to identify the precise region of the promoters necessary for controlling expression in the endosperm and other tissues were initiated. Studies of whole metabolome concentrations in malts of varying quality were completed and the application of the technique to measurement and prediction of the concentrations of whole suites of metabolites of industrial importance in mashing were initiated.
1. Differential expression of two carbohydrase genes. The existence of a second gene encoding an enzyme that is critical to the industrial conversion of starch to fermentable sugars has long been known, but the functional expression of the gene and the presence of its product in mature barley and malts was not known. ARS researchers at Madison, Wisconsin, demonstrated that this gene is expressed during kernel development, its gene product is present in developing, mature and germinated (both green and kilned malts) seeds, and determined the abundance of both the transcript and the protein. This work clearly demonstrated the amount this gene product contributes to a malting barley's ability to convert starch to high value fermentable sugars. The impact is that we have demonstrated the relative value of different carbohydrase genes which guides breeders selecting for malting quality to the genes contributing the most to the malting process.
2. Metabolite measurements as indicators of malt quality. Rapid and inexpensive methods of measuring fermentable sugars that can be used to monitor the extent of their production during mashing or their consumption during fermentation in real time as the mashing or fermentation occurs are limited. ARS researchers at Madison, Wisconsin, and collaborators demonstrated that a collective measurement of all metabolites in malts predicts the levels of fermentable sugars produced in mashing. The impact of this research is that a new, inexpensive tool is available which allows mashing time to be optimized.
Vinje, M.A., Willis, D.K., Henson, C.A., Duke, S.H. 2011. Differential expression of two ß-amylase genes (Bmy1 and Bmy2) in developing and mature barley grain. Planta. 233(5):1001-1010.