Structural and morphological variability of Potato Starch and associated biological processes toward bio-plasTic production – PoStaTic

With this project, structural and morphological variability of starches from a potato cultivar core collection has been estimated. Potato accumulates the biggest starch granules among common plant species. Inter-species variability of size and shape was estimated with the use of a granulomorphometer following starch isolation. Starch composition (amylose and amylopectin contents) and structure (chain length distribution) were determined by enzymatic assay and fluorescence assisted capillary electrophoresis (FACE), respectively. Starch also contains phosphate groups which influence physico chemical properties of the polymers. We established a FACE method in order to measure phosphate contents and their repartition between C3- and C6-position of the glucose residues. Moreover, starch bound proteins were analyzed by mass spectrometry, some of which were characterized by western blot or by in vitro analysis of the corresponding recombinant protein after purification.

We found that, while amylose content varies poorly between samples of the core collection, amylopectin chain length distribution as well as granule size and shape display significant differences between cultivars. Moreover, we established a FACE method allowing to determine starch phosphorylation with a higher throughput and sensitivity. We apply this method to analyze the core collection and observed a great dispersion regarding total phosphate content, the proportion of C6- and C3-phosphoesters. On the other hand, our proteomic study revealed that, in addition to already known starch bound proteins, all classes of starch synthesizing enzymes as well as several non-characterized proteins physically interact with the starch granule.

New starch bound proteins of unknown function were identified with this project. Deciphering their biological role is of great interest for the understanding of starch metabolism and its regulation as well as for breeding new specific traits. Moreover, the phenotypic data produced during this project suggest that starch-bound protein stoichiometry and phosphate distribution impact granule's morphology and structure. Future investigation is necessary for unravelling the function of new target proteins and understanding the relationships between protein stoichiometry and starch metabolism.

Verbeke J., Penverne C., D’Hulst C., Rolando C., Szydlowski N. (2016). Rapid and sensitive quantification of C3- and C6-phosphoesters in starch by fluorescence-assisted capillary electrophoresis. Carbohydrate polymers 152, 784-791.

Szydlowski N. Rapid and sensitive determination of starch phosphoesters by FACE. European Starch Round Table, Villeneuve d’Ascq, France, November 17 -18, 2016.

Szydlowski N. in vivo optimization of potato starch towards agro-based plastic production. Thematic session: public private partnership to speed-up industrialization. Plant based summit, Lille, France, April 2015.

The production of agro-plastics offers three major advantages compared to that of synthetic polymers: i. less greenhouse gas emissions, ii. the use of renewable raw material, iii. a potential biodegradability. Thermoplastic starch is the most widely used bioplastic since this polymer is cheap, abundant and can be used by most microorganisms as a carbon source following its degradation. There are however some constraints due to the structure and physical properties of the polysaccharides which require chemical modification or blending with synthetic polymers to reach the expected characteristics. The associated carbon balance and costs could however be improved by adapting the raw material in vivo, notably the size of the starch granules and their amylose content (one of the two polymers composing starch).
The PoStaTic project forms part of the upstream research programs of the PIA-supported "Institut Français des Matériaux Agrosourcés" (IFMAS) which aims to develop new starch-based plastic materials. The main objective of this project is to seek for potato varieties and cultivars accumulating the most adapted starch for bioplastic production and to study the associated biological processes. The latter comprise the control of starch initiation and plastid division which determine the size of the granules, and the biosynthesis of amylose as well as long chain amylopectin (the other polymer composing starch).
Two approaches will be implemented: i. a blind screening approach for the characteristics of interest within the collection of potato species and cultivars maintained by the INRA Genetic Resources Center BrACySol at Ploudaniel (Brittany, France), ii. a targeted approach for the functional study of selected granule bound proteins. The project involves state of the art methods for starch structural and morphological analyses, crystallography, proteomics, molecular biology and physiology of the potato tuber. On the other hand, pioneering and challenging methods such as synchrotron based X-ray fluorescence microscopy and transient expression of recombinant proteins in potato tubers will be used or established, respectively.
Project achievement, dissemination of the results as well as exploitation of the identified starch resources will be facilitated by the diversity of expertise brought together within IFMAS. The starches will be made available to chemists and physicists forming part of the consortium for testing the plasticisation, filmability and properties of prototypic derived products. In the long term, we expect important environmental, societal and economic benefits in relation with the sustainable development approach developed by IFMAS. Furthermore, this project will allow 3D and kinetic modelling of the storage starch granule biogenesis and contribute to deciphering starch granule initiation. This will represent a major breakthrough in the understanding of starch granule formation in one of the most important cultivated crops.