Abstract
Mixed-linkage (1,3; 1,4)-β-D-glucan, MLG, also known as β-glucan, is a dietary fiber accumulated in cereal crops such as oats and barley. Unlike starch and sucrose that can result in blood sugar spikes in humans after meals, MLG can only be degraded partially and slowly and does not lead to blood sugar spikes. Supplementation of MLG in human diets is a very effective way of reducing blood sugar and cholesterol levels and is highly recommended for patients with heart and diabetic conditions. Therefore, high MLG content in grains has recently become a major goal of breeding programs for food barley cultivars. In this study, a barley mutant, CM1, that produces about two-fold MLG content of those found in most barley cultivars, was isolated and characterized. Genetic mapping placed the chemically induced mutation locus on a chromosome 7H location where the AGPS1 gene is present. Genomic DNA and cDNA sequencing results indicated that a guanine (G) to adenine (A) substitution was present at the splicing donor site of intron 4 of the AGPS1 gene, leading to the synthesis of the alternative mRNA with the presence of a premature stop codon that would lead to the synthesis of a C-terminus truncated small subunit of ADP-glucose pyrophosphorylase (AGPase) enzyme. Quantitative real-time PCR (qRT-PCR) analysis revealed that the AGPS1 transcript level was significantly decreased in the mutant as compared to the wild-type plants, suggesting that the alternative spliced mutant mRNA was less stable than its wild-type variant. Analysis of AGPase enzyme activity in leaves and developing grain indicated that the AGPase enzyme activity was significantly reduced in the CM1 mutant. Results of LC-MS/MS (liquid chromatography with tandem mass spectrometry) analysis revealed that there were higher levels of glucose, fructose, sucrose, UDP-glucose, glucose-1 phosphate (Glc-1P), and glucose-6 phosphate (Glc-6P), but a lower level of ADP-glucose, in the developing seed extracts of CM1 mutant as compared to the wild type plants. The fact that the high MLG phenotype in CM1 mutant was associated with the reduced starch content implies that the AGPS1 gene could be an ideal target for genetic manipulation aimed at changing the partitioning of carbon metabolites from starch biosynthesis to MLG accumulation in grain crops. The results of this work have provided insight knowledge of polysaccharide metabolism and a useful tool for breeders in the development of high MLG food barley cultivars with improved grain nutrition quality. This mutant is currently being used as valuable germplasm for the development of food barley cultivars with high MLG in the grain.