Genotype-Specific Spatial Distribution of Starch Molecules in the Starch Granule:  A Combined CLSM and SEM Approach

Starch granule types from a variety of botanical sources were selected to represent differences in crystalline polymorph, amylose and phosphate content, and amylopectin chain length distribution. Equimolar labeling of starch molecules with the fluorophore 8-amino-1,3,6-pyrenetrisulfonic acid (APTS) was used to construct a detailed map of the distribution of amylose and amylopectin within the granule by confocal laser scanning microscopy (CLSM) analysis. Medium- and high-resolution scanning electron microscopy (SEM) were used to provide detailed images of granule surface structures. By using a combined surface and internal imaging approach, interpretations of a number of previous structural observations is presented. In particular, internal images of high amylose maize and potato suggest that multiple initiations of new granules are responsible for the compound or elongated structures observed in these starches. CLSM optical sections of rice granules revealed an apparent altered distribution of amylose in relation to the proposed growth ring structure, hinting at a novel mechanism of starch molecule deposition. Well-described granule features, such as equatorial grooves, channels, cracks, and growth rings were documented and related to both the internal and external observations. A new method for probing the phosphate distribution in native granules was developed using a phosphate-binding fluorescent dye and CLSM.