Hyperphosphorylation of cereal starch Massimiliano Carciofi a , Shahnoor S. Shaik b , Susanne L. Jensen b, c , Andreas Blennow b , Jan T. Svensson b , Éva Vincze a , Kim H. Hebelstrup a, * a Department of Biotechnology and Genetics, Aarhus University, Forsøgsvej 1, 4200 Slagelse, Denmark b VKR Research Center Pro-Active Plants, Department of Plant Biology and Biotechnology, University of Copenhagen, Thorvaldsensvej 40,1871 Frederiksberg C, Denmark c KMC, Herningvej 60, 7330 Brande, Denmark article info Article history: Received 5 October 2010 Received in revised form 29 April 2011 Accepted 8 June 2011 Keywords: Starch Phosphorylation Glucan Water Dikinase (GWD) Transgenic barley abstract Plant starch is naturally phosphorylated at a fraction of the C6 and the C3 hydroxyl groups during its biosynthesis in plastids. Starch phosphate esters are important in starch metabolism and they also generate specific industrial functionality. Cereal grains starch contains little starch bound phosphate compared with potato tuber starch and in order to investigate the effect of increased endosperm starch phosphate, the potato starch phosphorylating enzyme glucan water dikinase (StGWD) was overex- pressed specifically in the developing barley endosperm. StGWD overexpressors showed wild-type phenotype. Transgenic cereal grains synthesized starch with higher starch bound phosphate content (7.5 (0.67) nmol/mg) compared to control lines (0.8 (0.05) nmol/mg) with starch granules showing altered morphology and lower melting enthalpy. Our data indicate specific action of GWD during starch biosynthesis and demonstrates the possibility for in planta production of highly phosphorylated cereal starch. Ó 2011 Elsevier Ltd. All rights reserved. 1. Introduction Starch is the primary storage reserve carbohydrate in many plants and is an important polysaccharide in food, in animal feed and in industry (Damager et al., 2010). Phosphorylation is the only known covalent modification occurring naturally in starch and takes place in virtually all plant species to a different extent (Blennow et al., 2000, 2002; Blennow and Engelsen, 2010)(Table 1). For unknown reasons, cereal endosperm starch contains low concentrations of starch bound phosphate esters (z1 nmol/mg) while tuber starch like potato tuber starch has considerably higher phosphate content (8e33 nmol/mg) (Fernbach, 1904; Baunsgaard et al., 2005). The phosphates are linked through phospho- monoester bonds to the C-3 (w30%) or C-6 (w70%) positions of the glucose units of the amylopectin chains (Baysmidt et al., 1994; Hizukuri et al., 1970). Starch phosphorylation takes place in plastids intimately integrated with the other reactions of starch biosyn- thesis, and it has been demonstrated to be important for degra- dation of plastidial starch in leaves (Edner et al., 2007). Starch phosphorylation is catalyzed by glucan, water dikinases (GWD) and phosphoglucan, water dikinase (PWD) also known as GWD3 (Baunsgaard et al., 2005; Edner et al., 2007; Kotting et al., 2005; Ritte et al., 2006). GWD transfers a phosphate of ATP specifically to the C-6 position glucose residue whereas PWD specifically phosphorylates pre-phosphorylated starch at the C-3 position (Baunsgaard et al., 2005; Kotting et al., 2005; Ritte et al., 2006). Potato glucan water dikinase (StGWD) is a 155 kDa (mature) protein with five domains of 37, 24, 21, 36 and 38kDa (Mikkelsen et al., 2004; Mikkelsen and Blennow, 2005; Ritte et al., 2002). The 38 kDa region contains a plastid-targeting transit peptide and one of the two glucan-binding sites, while the other one is located on the following 24 kDa domain (Mikkelsen and Blennow, 2005; Yu et al., 2001). The nucleotide binding site and the catalytic histi- dine are located respectively in the 38 and 36 kDa domains (Mikkelsen and Blennow, 2005; Yu et al., 2001). The catalytic mechanism follows a dikinase type reaction in which the starch glucose units are esterified with the b-phosphate from ATP (Ritte et al., 2002; Blennow et al., 2002). Histidine (992) at the catalytic site forms a phosphorylated intermediate (-His-P) with the b-phosphate before transferring it to the starch (Mikkelsen et al., 2004). Homologs of StGWD have been identified in many other plants and organs like tubers (sweet potatoes, yams), endosperms of cereals (barley, maize), fruits (banana) and leaves (Arabidopsis) (Mikkelsen et al., 2005; Ritte et al., 2000). This demonstrates the Abbreviations: GWD, glucan water dikinase; Glc-6P, glucose-6-phosphate; qPCR, quantitative real-time PCR; PWD, phosphoglucan water dikinase. * Corresponding author. Tel.: þ45 8999 3675. E-mail address: kim.hebelstrup@agrsci.dk (K.H. Hebelstrup). Contents lists available at ScienceDirect Journal of Cereal Science journal homepage: www.elsevier.com/locate/jcs 0733-5210/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.jcs.2011.06.013 Journal of Cereal Science xxx (2011) 1e8 Please cite this article in press as: Carciofi, M., et al., Hyperphosphorylation of cereal starch, Journal of Cereal Science (2011), doi:10.1016/ j.jcs.2011.06.013