Expression of bacterial starch-binding domains in Arabidopsis increases starch granule size

Starch is a readily renewable resource that is very widely used for food and industrial purposes; however, greater variation in the functional properties of starch would further extend the use of this biodegradable polymer. Genetic engineering may provide a way to produce designer starches that have the desired properties. Starch-binding domains (SBD) from bacterial enzymes that catabolise starches have the ability to bind two helices of starch and thus have the potential to crosslink starch and / or to be used as anchors for other enzymes that can modify starch properties. In a first step towards novel modification of starch we have investigated the effect of expressing SBDs, singly and in tandem, in planta, and targeting them to the chloroplast in the model plant Arabidopsis thaliana (L.) Heynh. Transgenic plants that contained the SBD from the cyclomaltodextrin glucanotransferase (CGTase) of Thermoanaerobacterium thermosulfurigenes in the chloroplast were produced in both the wild type and the starch excess mutant (sex 1-1) backgrounds. Analysis of starch isolated from the chloroplasts of these lines revealed no significant changes in the amylose : amylopectin ratio, the chain-length distribution of debranched amylopectin or the gelatinisation temperature when compared to the parental line. However, significant changes were observed in the starch granule size with the plants expressing the construct having larger granules. The effect was more pronounced in the sex 1-1 background, and expression of two starch-binding domains linked in tandem had an even greater effect. Despite the starch granules being larger in lines expressing the starch-binding domain, no difference was seen in the starch content of the leaves when compared to parental lines. As the presence of the SBDs in the starch granule only altered granule size, and not other granule properties, they may provide an ideal anchor for targeting starch-modifying enzymes to the site of starch synthesis. This will allow the development of novel modifications of starch during synthesis.