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Front. Plant Sci. | doi: 10.3389/fpls.2019.01075

NegFluo, a fast and efficient method to determine starch granule size and morphology in situ in plant chloroplasts.

Camille Vandromme1, Angelina Kasprowicz1,  Adeline Courseaux1, Dave Trinel1, Maud Facon1,  Jean-Luc PUTAUX2,  Christophe D'HULST1,  Fabrice Wattebled1* and  Corentin Spriet1*
  • 1Centre National de la Recherche Scientifique (CNRS), France
  • 2UPR5301 Centre de Recherches sur les Macromolecules Végétales (CERMAV), France

Starch granules that accumulate in the plastids of plants vary in size, shape, phosphate or protein content according to their botanical origin. Depending on their size, the applications in food and non-food industries differ. Being able to master starch granule size for a specific plant, without alteration of other characteristics (phosphate content, protein content, etc.) is challenging. The development of a simple and effective screening method to determine the size and shape of starch granules in a plant population is therefore of prime interest. In this study, we propose a new method, NegFluo, that combines negative confocal autofluorescence imaging in leaf and machine learning-based image analysis. It provides a fast, automated and easy-to-use pipeline for both in situ starch granule imaging and its morphological analysis. NegFluo was applied to Arabidopsis leaves of wild-type and ss4 mutant plants. We validated its accuracy by comparing morphological quantifications using NegFluo and state-of-the-art methods relying either on starch granule purification or on preparation-intensive electron microscopy combined with manual image analysis. NegFluo thus opens the way to high-throughput in situ analysis of starch granules.

Keywords: Starch, Confocal fluorescence imaging, machine learning, Arabidopsis, Starch granule morphology, autofluorescence

Received: 08 Apr 2019; Accepted: 07 Aug 2019.

Copyright: © 2019 Vandromme, Kasprowicz, Courseaux, Trinel, Facon, PUTAUX, D'HULST, Wattebled and Spriet. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

* Correspondence:
Dr. Fabrice Wattebled, Centre National de la Recherche Scientifique (CNRS), Paris, France,
Dr. Corentin Spriet, Centre National de la Recherche Scientifique (CNRS), Paris, France,