ORIGINAL ARTICLE Overexpression of the rice carotenoid cleavage dioxygenase 1 gene in Golden Rice endosperm suggests apocarotenoids as substrates in planta Andrea Ilg • Qiuju Yu • Patrick Schaub • Peter Beyer • Salim Al-Babili Received: 22 March 2010 / Accepted: 28 May 2010 / Published online: 13 June 2010 Ó Springer-Verlag 2010 Abstract Carotenoids are converted by carotenoid cleavage dioxygenases that catalyze oxidative cleavage reactions leading to apocarotenoids. However, apocarote- noids can also be further truncated by some members of this enzyme family. The plant carotenoid cleavage dioxy- genase 1 (CCD1) subfamily is known to degrade both carotenoids and apocarotenoids in vitro, leading to differ- ent volatile compounds. In this study, we investigated the impact of the rice CCD1 (OsCCD1) on the pigmentation of Golden Rice 2 (GR2), a genetically modified rice variety accumulating carotenoids in the endosperm. For this pur- pose, the corresponding cDNA was introduced into the rice genome under the control of an endosperm-specific pro- moter in sense and anti-sense orientations. Despite high expression levels of OsCCD1 in sense plants, pigment analysis revealed carotenoid levels and patterns compara- ble to those of GR2, pleading against carotenoids as sub- strates in rice endosperm. In support, similar carotenoid contents were determined in anti-sense plants. To check whether OsCCD1 overexpressed in GR2 endosperm is active, in vitro assays were performed with apocarotenoid substrates. HPLC analysis confirmed the cleavage activity of introduced OsCCD1. Our data indicate that apocarote- noids rather than carotenoids are the substrates of OsCCD1 in planta. Keywords Apocarotenoids Á Carotenoids Á Carotenoid cleavage dioxygenase Á Golden Rice Á Endosperm Abbreviations CCD1 Carotenoid cleavage dioxygenase 1 GR Golden Rice Gt1 Glutelin 1 promoter Introduction Carotenoids are lipophilic pigments synthesized by all photosynthetic organisms as well as by some fungi and heterotrophic bacteria. In plants, carotenoids exert a vital role in protecting the photosynthetic apparatus from pho- tooxidation and represent essential constituents of the light harvesting and reaction center complexes. In addition, carotenoids confer their color to many flowers and fruits, contributing substantially to plant–animal communication (Cunningham and Gantt 1998; Hirschberg 2001; Fraser and Bramley 2004; DellaPenna and Pogson 2006). Besides these functions, carotenoids serve as precursors of physi- ologically important compounds termed apocarotenoids synthesized through oxidative cleavage (Moise et al. 2005; Bouvier et al. 2005; Auldridge et al. 2006a). Representative examples of apocarotenoids are the ubiquitous chromo- phore retinal, the chordate morphogen retinoic acid and the phytohormone abscisic acid (ABA). A further example is represented by the signalling molecules strigolactones responsible for attracting both symbiotic arbuscular mycorrhizal fungi and parasitic plants (Akiyama 2007; Bouwmeester et al. 2007). Strigolactones were also Electronic supplementary material The online version of this article (doi:10.1007/s00425-010-1205-y) contains supplementary material, which is available to authorized users. A. Ilg Á Q. Yu Á P. Schaub Á P. Beyer Á S. Al-Babili (&) Faculty of Biology, Institute of Biology II, Albert-Ludwigs University of Freiburg, Schaenzlestr. 1, 79104 Freiburg, Germany e-mail: salim.albabili@biologie.uni-freiburg.de 123 Planta (2010) 232:691–699 DOI 10.1007/s00425-010-1205-y