Expression of bacterial starch-binding domains in Arabidopsis increases starch granule size
Crispin A. Howitt A B , Sadequr Rahman A and Matthew K. Morell AA CSIRO Plant Industry, GPO Box 1600, Canberra, ACT 2601, Australia.
B Corresponding author. Email: Crispin.Howitt@csiro.au
Functional Plant Biology 33(3) 257-266 https://doi.org/10.1071/FP05277
Submitted: 18 November 2005 Accepted: 17 January 2006 Published: 2 March 2006
Abstract
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.
Keywords: Arabidopsis, granule size, starch-binding domain.
Acknowledgments
We thank Dr Jean Finnegan for help with growth and transformation of Arabidopsis, Dr Bryan Clarke for provision of the wheat cDNA clone, Oscar Larroque for help with the HPLC and CE analysis and Celia Miller for the scanning electron microscopy. This work was carried out as part of the Graingene research program. Graingene is a research consortium between the Australian Wheat Board, the CSIRO and the Grains Research and Development Corporation.
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