Tracking sulfur and phosphorus within single starch granules using synchrotron X-ray microfluorescence mapping Alain Buléon a, ⁎, Marine Cotte b,c , Jean-Luc Putaux d,1 , Christophe d'Hulst e , Jean Susini b a INRA-UR 1268 BIA, Rue de la Géraudière, BP 71627, F-44316 Nantes cedex 3, France b ESRF (European Synchrotron Radiation Facility), 6 rue Jules Horowitz, F-38000 Grenoble, France c LAMS (Laboratoire d'Archéologie Moléculaire et Structurale) UMR-8220, 3 rue Galilée, 94200 Ivry-sur-Seine, France d CERMAV-CNRS, BP 53, F-38041 Grenoble Cedex 9, France e UGSF, UMR 8576 CNRS, Université Lille 1, Bât. C9, F-59655 Villeneuve d'Ascq Cedex, France abstract article info Article history: Received 17 April 2013 Received in revised form 24 July 2013 Accepted 29 August 2013 Available online 7 September 2013 Keywords: Micro X-ray fluorescence Synchrotron Starch Sulfur Phosphorus Granule bound starch synthase Background: Native starch accumulates as granules containing two glucose polymers: amylose and amylopectin. Phosphate (0.2–0.5%) and proteins (0.1–0.7%) are also present in some starches. Phosphate groups play a major role in starch metabolism while granule-bound starch synthase 1 (GBSS1) which represents up to 95% of the pro- teins bound to the granule is responsible for amylose biosynthesis. Methods: Synchrotron micro-X-ray fluorescence (μXRF) was used for the first time for high-resolution mapping of GBSS1 and phosphate groups based on the XRF signal of sulfur (S) and phosphorus (P), respectively. Wild-type starches were studied as well as their related mutants lacking GBSS1 or starch-phosphorylating enzyme. Results: Wild-type potato and maize starch exhibited high level of phosphorylation and high content of sulfur respectively when compared to mutant potato starch lacking glucan water dikinase (GWD) and mutant maize starch lacking GBSS1. Phosphate groups are mostly present at the periphery of wild-type potato starch granules, and spread all over the granule in the amylose-free mutant. P and S XRF were also measured within single small starch granules from Arabidopsis or Chlamydomonas not exceeding 3–5 μm in diameter. Conclusions: Imaging GBSS1 (by S mapping) in potato starch sections showed that the antisense technique suppresses the expression of GBSS1 during biosynthesis. P mapping confirmed that amylose is mostly present in the center of the granule, which had been suggested before. General significance: μXRF is a potentially powerful technique to analyze the minor constituents of starch and understand starch structure/properties or biosynthesis by the use of selected genetic backgrounds. © 2013 Elsevier B.V. All rights reserved. 1. Introduction Native starch, the major energy reserve of a large variety of higher plants, accumulates as a complex granular structure containing two dis- tinct α-linked glucose polymers: quasi-linear amylose and moderately branched amylopectin. Amylopectin, the major fraction of starch, is thought to be the basis of the semicrystalline architecture of the granule while amylose is generally considered as an amorphous polymer [1,2]. The relative position of amylose and amylopectin within the granule is not known with certainty yet. Nevertheless, the synthesis of amylose occurs within the amylopectin matrix [3,4] and some reports suggest that amylose is preferably located at the hilum of the granule [5,6]. The size and shape of starch granules strongly depend on their origin with a size typically varying from 1 μm for amaranth to 50–60 μm for potato. Besides lipids, essentially contained in cereal starches, phosphate (0.2–0.5%) and proteins (0.1–0.7%) can be present in some starches depending on their origin [1,7]. Among the numerous enzymes involved in starch biosynthesis, granule-bound starch synthase 1 (GBSS1) is one of the various elongating enzymes (glycosyltransferases of the GT5 family of the CAZy classification; www.cazy.org) only re- sponsible for amylose biosynthesis. GBSS1 naturally represents up to 95% of the proteins non-covalently bound to the granule where it is active. Depending on growth conditions, tissue origin, or genetic back- ground, the GBSS1 content may reach 0.5% of the mass of the granule being by far the most represented protein within the starch granule. GBSS1-deficient starches lack not only the corresponding protein but also more or less completely the amylose fraction such as in waxy maize, [8] amf potato [9] or lam pea [10], for example. Starch is also naturally phosphorylated on some of its glucose resi- dues at C3 and C6 positions. It has been shown that most phosphate groups are bound to amylopectin but not to amylose [11,12]. Phosphate groups are present not only in amorphous regions but also in crystalline domains. Phosphorylation of starch is required to trigger polysaccharide catabolism by degrading enzymes [13]. Specialized enzymes are re- sponsible for starch phosphorylation: glucan water dikinase (GWD) Biochimica et Biophysica Acta 1840 (2014) 113–119 ⁎ Corresponding author. Tel.: +33 2 40 67 50 47; fax: +33 2 40 67 50 43. E-mail address: alain.buleon@nantes.inra.fr (A. Buléon). 1 Affiliated with Université Joseph Fourier, member of Institut de Chimie Moléculaire de Grenoble and Institut Carnot PolyNat. 0304-4165/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbagen.2013.08.029 Contents lists available at ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbagen