The impact of diurnal photosynthetic and starch branching enzyme activity on the composition and fine structure of barley starch.

Abstract

In this work, the roles of two universal features of starch biosynthesis were investigated to better understand their impact on starch molecular structure. To investigate the impact of diurnal photosynthetic activity on starch fine structure, normal and waxy barley were cultivated in a greenhouse under normal diurnal, or constant light photosynthetic conditions. The impact of starch branching enzymes on starch structure was investigated by studying the lintners of barley starch, which had all known genes coding for starch branching enzymes (SBE I, SBE IIa, SBE IIb) suppressed in the grain resulting in a novel amylose-only starch (AOS). The structure of AOS lintners was compared to lintners from normal barley starch (NBS) and waxy barley starch (WBS). Unexpectedly, NBS and WBS displayed growth rings regardless of lighting regimes. It was observed that the molecular structure and composition of (NBS) was influenced by the diurnal lighting regime, as NBS contained lower quantities of amylose, and a lower ratio of long chain amylose:short chain amylose (determined by gel permeation chromatography) when cultivated under the diurnal lighting regime compared to the constant light regime. While the composition of WBS remained constant, higher relative crystallinity values (determined by X-ray diffraction), and greater crystalline quality (determined by differential scanning calorimetry) were observed when cultivated under the diurnal lighting regime. When considering the fine structure of amylopectin from NBS and WBS, differences in structure were observed when cultivated under the different lighting regimes. The structure of clusters and building blocks of amylopectin were investigated following their isolation by partial and complete hydrolysis with α-amylase from Bacillus amyloliquefaciens. Clusters of amylopectin from NBS and WBS cultivated under diurnal photosynthetic conditions were larger, and contained a greater number of building blocks compared to their counterparts cultivated in constant light conditions. AOS exhibited irregular morphological features and contained multi-lobed granules with a rough surface texture. When viewed by transmission electron microscopy (TEM), acid hydrolyzed components of AOS displayed strong textured aggregates with an organization not previous seen in other specimens, whereas NBS and WBS displayed expected stacks of elongated elements with a width of 5-7 nm, believed to represent crystalline amylopectin side chains viewed longitudinally. High performance anion-exchange chromatography of lintners at equivalent levels of acid hydrolysis (45 wt%) revealed that the average degree of polymerization of the AOS lintner was 21, substantially smaller than that of NBS and WBS (42). While NBS and WBS lintners displayed size distribution and chain length profiles expected of those from barley starch, the AOS lintner displayed a unique size distribution profile wherein a repeat-size of the molecules corresponding to 5-6 glucose residue was observed, which corresponds to the approximate number of residues required per turn of the helical structure of amylose. These data suggests that both diurnal photosynthetic activity, and the suppression of all genes coding for SBEs had significant impacts on the structure of barley starch granules.