Novel imaging technique looks inside starch granules

11th February 2011

Starch is the major storage compound in plants, and a major component of cereal grains and so is an important part of our food.  For this reason scientists are keen to understand how it is made and stored by the plant, and how changes to the plants genes could affect the composition and properties of the starch in the grain.  IFR scientists have developed a method of visualising and measuring the properties of the starch in a developing maize kernel, revealing previously unidentified properties.  This could have a practical application in screening varieties for novel starch properties, or help in the selection of novel starches for industrial applications.

Starch is made up of a mix of two long sugar chains (polysaccharides), amylose and amylopectin.  Amylose is a long chain molecule, whilst amylopectin is much more branched.  The two different molecules combine to form starch granules within the cell, with amylopectin located in ordered, crystallised regions in the granule and amylose located in more unstructured regions.

Funded by the Biotechnology and Biological Sciences Research Council (BBSRC), IFR scientists Klaus Wellner, Mary Parker and Vic Morris together with Dominique Georget at the University of East Anglia have developed a novel high-resolution method of characterising the properties of starch granule within seeds, and have used it to examine starch in a high-amylose maize mutant lacking an enzyme that adds branches to amylopectin molecules.  Examining starch granules in the intact seeds means that the effects of isolating starch do not change the properties of the granules and the patterns of heterogeneity within the seed are maintained.

Raman maps comparing the starch branching in the inner endosperm of Wild type (a) and branching mutant (b) maize kernels. Blue areas depict a normal ratio of linear amylose and branched amylopectin. Yellow/orange areas have more linear molecules. The maps are overlaid on the visible images of the sections to locate the granule boundaries. Scale bars are 10 μm.

The technique, Raman microscopy, uses the way molecules absorb and re-emit light to give information about the chemical and physical structure of the molecules.  This allows the structure of individual starch granules to be measured and compared with other granules in the seed.  The versatility of using Raman microscopy allows a number of different chemical and physical parameters to be mapped within the sample.

The researchers showed that the starch granules in normal maize are all very similar to each other, but the mutant starch granules showed large differences both within and between granules in the same cell, and between different cells in the seed.  The technique shows that marked differences can occur between starch properties in neighbouring cells.  The degree of heterogeneity was found to be much greater than had previously been reported for studies of isolated granules extracted from seeds.

The spatial difference in the properties within seeds provides information on the structure of starch over time and during growth.  Analysis of the data suggested that the mutation in the branching enzyme leads to defects in the way the granules self-assemble that accumulate to produce the observed heterogeneity, rather than simply producing new homogeneous granular structures with different relative amounts of amylose and amylopectin.

The technique can be developed to provide more quantitative data, and the researchers believe that it could also be used to produce 3D image data sets for seeds.  Using Raman microscopy in this way could have a practical application in screening varieties for novel starch properties, potentially aiding in the selection of novel starches for specific industrial applications.

These imaging methods can also be used to study many other biological tissues. There is interest in using chemical imaging for determination of inflammatory status and cancer detection in intestinal tissue. Initial infrared mapping studies together with IFR scientist Anastasia Sobolewski have shown that it is possible to map the distribution of protein and polysaccharide in gut sections. Raman imaging could be applied to obtain high resolution images and to identify the changes in chemical composition for individual cells in normal and diseased tissue.

Reference: In situ Raman microscopy of starch granule structures in wild type and ae mutant maize kernels.  Wellner et al, Starch/Stärke 63(3):128-138 doi: 10.1002/star.201000107