The effects of anti-gibberellin treatment, high growth temperature treatment and increase of pullulanase-type debranching enzyme activity on sorghum starch structure and properties have been examined in this study. These treatments can affect the starch biosynthesis process of sorghum plants. In addition, a new starch extraction procedure was established in this study for the accurate structural analysis of sorghum starch.
A complete dissolution is important for an accurate characterization of sorghum starch structure. Current milling and dissolution methods have limitations including incomplete dissolution, molecular degradation, and starch loss. The new extraction/dissolution method introduced in this study involved mild cryo-grinding, protease pre-treatment, dissolution in dimethyl sulfoxide solution containing 0.5% (w/w) LiBr (DMSO/LiBr) at 80 °C, centrifugation, ethanol precipitation, and, finally, re-dissolution in the DMSO/LiBr solution at 80 °C. The methods involving cryo-grinding and dissolution in the DMSO/LiBr solution were found to have negligible effects on the size distribution of starch molecules. The peaks of non-starch components were completely separated from the amylose and amylopectin peaks in the chromatograms without causing starch loss. The larger size of amylopectin as compared with the wet-milled sample and the sample without protease pre-treatment suggested that molecular degradation and aggregation were avoided.
Inhibition of plant growth by Trinexapac-ethyl, TE, a gibberellin-biosynthesis inhibitor, can produce a shorter stemmed plant, requiring less nutrients and water to grow, while maintaining grain yield. Although TE and other plant growth regulators are commonly used in grain crops, their effects on starch biosynthesis in the grains have not been systematically examined. The changes in the structural and functional properties of starch in grains harvested from TE-treated sorghum (Sorghum bicolor (L.) Moench) were examined, and the results compared with those from the untreated controls. TE treatment had little or no effects on the molecular structures of starch, starch granule morphology, and starch and amylose contents, but increased the protein content of the grains significantly. Consistent with the lack of change in the molecular structure, there were no significant effects on the thermal properties of the starch. The pasting properties of TE-treated sorghum flours, however, showed lower peak viscosities, troughs, and final viscosities, which were attributed to their higher protein contents. The TE treatment thus does not have an appreciable effect on the biosynthesis of starch during grain development in sorghum.
The effects of high growth temperature on sorghum starch structures were examined from the grains of three inbred lines (BTx623, I8S525 and Karper669), and the possible mechanism by which the growth of amylopectin molecules is terminated was discussed. Sorghum plants were grown at a high temperature (38/21°C day/night) and a control temperature (32/22°C day/night) from sowing to maturity. The grains sampled from plants grown at a high temperature have significantly lower starch weights per grain (except BTx623) and smaller starch granule sizes than those grown at the control temperature. However the amylose contents are similar. BTx623 and IS8525 samples grown at a high temperature also have higher ratios of long to short amylopectin branches and a lower degree of branching than their control counterparts. These results suggest that the activities of starch biosynthetic enzymes were evidently affected by an elevated growth temperature. However, the weight-average molecular weight and the z-average radius of gyration of sorghum starch molecules were not significantly affected by the growth temperatures, suggesting that the effects of growth temperature on starch yield, starch granule size, and the branching structure of amylopectin molecules do not influence the events which stop the overall growth of amylopectin molecules. This is consistent with the cessation of whole-molecule growth being through increasing hindrance to enzyme access as the size of the starch molecule increases, controlled largely by geometric factors such as the molecular density of the outermost part of an amylopectin molecule.
The effects of increased activity of pullulanase (PUL)-type starch debranching enzyme (DBE) are examined in sorghum grains on multiple levels of starch structure: the size distributions of individual branches and of whole molecules, crystalline structure and granule morphology. A newly described sorghum allele-type with increased PUL-type DBE activity has increased starch content, amylopectin molecular size, amylose fraction, and the proportion of long amylose branches, but shows small or no changes in the size and morphology of starch granules, starch crystalline structure, amylose content, degree of branching, and the chain-length distribution of amylopectin branches. This indicates that PUL-type DBE is an integral part of the biosynthesis of both amylose and amylopectin in sorghum grains. Mechanistic inferences from the structural data, especially amylose fine structure, support a number of biosynthetic hypotheses made in the literature from quite different types of data, especially the central role of this enzyme in removing pre-amylopectin branches which have an inter-branch spacing detrimental to crystallization. In vitro digestibility of the isolated starch is not affected by increased PUL-type DBE activity, whereas that of the flour is increased, indicating that the flour digestibility change is not a direct result of the structural changes in the starch.