Bulk carbohydrate grain filling of barley β-glucan mutants studied by 1H HR MAS NMR

Temporal and genotypic differences in bulk carbohydrate acclumulation in three barley genotypes differing in the content of mixed linkage beta-(1 -> 3),(1 -> 4)-D-glucan (beta-glucan) and starch were investigated using proton high-resolution, magic angle spinning, nuclear magnetic resonance (H-1 HR MAS NMR) during grain filling. For the first time, H-1 HR MAS NMR spectra of flour front immature barley seeds are analyzed. Spectral assignments are made using two-dimensional (2D) NMR methods. Both alpha- and beta-glucan biosynthesis were characterized by inspection of the spectra as well as by calibration to the reference methods for starch and P-glucan content. Starch was quantified with very good calibrations to the alpha-(1 -> 4) peak (5.29-5.40 ppm) and the region 3.67-3.83 ppm covering starch glycopyranosidic protons from H5 and H6. In contrast, the spectral inspection of the beta-anomeric region 4.45-4.85 ppm showed unexpected lack of intensity in the high beta-glucan mutant lys5f at seed maturity, resulting in poor calibration to reference beta-glucan content. We hypothesize that the lack of beta-glucan signal in lys5f indicates partial immobilization of the P-glucan that appears to be either genotypic dependent or water/beta-glucan ratio dependent.