Starch structure in developing barley endosperm.

Barley spikes of the cultivars/breeding lines Gustav, Karmosé and SLU 7 were harvested at 9, 12 and 24 days after flowering in order to study starch structure in developing barley endosperm. Kernel dry weight, starch content and amylose content increased during development. Structural analysis was performed on whole starch and included the chain-length distribution of the whole starches and their ß-limit dextrins. Karmosé, possessing the amo1 mutation, had higher amylose content and a lower proportion of long chains (DP ≥38) in the amylopectin component than SLU 7 and Gustav. Structural differences during endosperm development were seen as a decrease in molar proportion of chains of DP 22-37 in whole starch. In ß-limit dextrins, the proportion of Bfp-chains (DP 4-7) increased and the proportion of BSmajor-chains (DP 15-27) decreased during development, suggesting more frequent activity of starch branching enzymes at later stages of maturation, resulting in amylopectin with denser structure.

Thermal properties of barley starch and its relation to starch characteristics.

Amylopectin fine structure and starch gelatinization and retrogradation were studied in 10 different barley cultivars/breeding lines. Clusters and building blocks were isolated from the amylopectin by α-amylase from Bacillus amyloliquefaciens and their structure was characterized. Gelatinization was studied at a starch:water ratio of 1:3, and retrogradation was studied on gelatinized starch at starch:water ratio of 1:2, by differential scanning calorimetry. Three barley cultivars/breeding lines possessed the amo1 mutation, and they all had a lower molar proportion of chains of DP ≥38 and more of large building blocks. The amo1 mutation also resulted in a higher gelatinization temperature and a broader temperature interval during gelatinization. Overall, small clusters with a dense structure resulted in a higher gelatinization temperature while retrogradation was promoted by short chains in the amylopectin and many large building blocks.

Molecular structure of starches from maize mutants deficient in starch synthase III.

Molecular structures of starches from dull1 maize mutants deficient in starch synthase III (SSIII) with a common genetic background (W64A) were characterized and compared with the wild type. Amylose content with altered structure was higher in the nonwaxy mutants (25.4-30.2%) compared to the wild type maize (21.5%) as revealed by gel permeation chromatography. Superlong chains of the amylopectin component were found in all nonwaxy samples. Unit chain length distribution of amylopectins and their φ,ß-limit dextrins (reflecting amylopectin internal structure) from dull1 mutants were also characterized by anion-exchange chromatography after debranching. Deficiency of SSIII led to an increased amount of short chains (DP ≤36 in amylopectin), whereas the content of long chains decreased from 8.4% to between 3.1 and 3.7% in both amylopectin and φ,ß-limit dextrins. Moreover, both the external and internal chain lengths decreased, suggesting a difference in their cluster structures. Whereas the molar ratio of A:B-chains was similar in all samples (1.1-1.2), some ratios of chain categories were affected by the absence of SSIII, notably the ratio of "fingerprint" A-chains to "clustered" A-chains. This study highlighted the relationship between SSIII and the internal molecular structure of maize starch.

On the interconnection of clusters and building blocks in barley amylopectin.

Amylopectin is a highly branched starch component built up of a large number of clustered α-D-glucose chains. A single C-chain possesses the reducing end and carries the rest of the macromolecule. The aim of this study was to investigate the interconnection of clusters and domains (groups of clusters) in barley amylopectin by isolation of the units with α-amylolysis and subsequent labelling of the C-chain in the φ,ß-limit dextrins of these structural units with the fluorescent compound 2-aminopyridine. Because these C-chains were formed by α-amylolysis of B-chains in amylopectin, they were designated bc-chains to be distinguished from C-chains in amylopectin. Four barley samples were selected for the study, of which two had the amo1 genetic background. Longer bc-chains were found in domains suggesting their role in cluster interconnection. The average chain length of bc-chains was longer than the average chain length of B-chains and the size-distribution of the bc-chains was unimodal implying that the bc-chains comprise a unique category of chains. Extensive α-amylolysis of labelled amylopectin and clusters revealed the distribution of branched building blocks situated at the reducing end of these molecules. Any type of size group of building blocks can be situated at the reducing end, because the size-distribution of these blocks was similar to the distribution of all building blocks present in the sample. This suggested certain randomness in the distribution of the types of building blocks within the amylopectin macromolecule.

The building block structure of barley amylopectin.

Amylopectin branchpoints are present in amorphous lamellae of starch granules and organised into densely branched areas, referred to as building blocks. One single amylopectin cluster contains several building blocks. This study investigated the building block structure of domains (groups of clusters) and clusters in four different barley genotypes. Two of the barleys possessed the amo1 mutation, Glacier Ac38 and the double recessive SW 49427 with both wax and amo1 mutations, and were compared with the two waxy type barleys Cinnamon and Cindy. A previous detailed study on these four barley genotypes showed that the amo1 mutation affected the internal structure of amylopectin as manifested in the composition of clusters. In this work the building blocks were isolated from domains and clusters by extensive treatment with liquefying α-amylase of Bacillus subtilis and structurally characterised with enzymatic and chromatographic techniques. The proportion of large building blocks with a high number of chains was increased in the amo1 barleys, and the chain length between the blocks was short, which explained the previous findings of large clusters with more dense structure in the amo1 amylopectins.

The cluster structure of barley amylopectins of different genetic backgrounds.

The unit chains of amylopectin are organized into clusters. In this study, the cluster structure was analysed in detail in four different genotypes of barley, of which two possessed the amo1 genetic background. Amylose content of the barley starches differed from 0 to 32.6%. Isolated amylopectin was hydrolysed with α-amylase from Bacillus subtilis into domains, defined as groups of clusters, which were size-fractionated by methanol. The domain fractions were further treated with α-amylase to release single clusters. Amylopectin, domains and clusters were subsequently treated with phosphorylase and ß-amylase to produce φ,ß-limit dextrins and the detailed internal structures of these different structure levels were investigated. Analysis was performed with gel-permeation and anion-exchange chromatography. Equal amount of A-chains were detected in all barleys, but the distribution of B-chains differed. At least two types of domain structures were identified in all four barley varieties. Large domains were built up by large clusters and small domains by small clusters. In all four barley samples the number of long chains was small suggesting that shorter chains with a degree of polymerization of 25-35 also are involved in the interconnection of clusters. The cluster structure of the amylopectin correlated with the genetic background. The two barley samples with amo1 genetic background possessed a more dense structure. Internal chain lengths in these two barleys were shorter resulting in larger domains built up by larger clusters.