The accumulation of three major carotenoid derivatives-crocetin glycosides picrocrocin and safranal-is

The accumulation of three major carotenoid derivatives-crocetin glycosides picrocrocin and safranal-is in large part responsible for the color bitter taste and aroma of saffron which is obtained from the dried styles of Crocus. water-soluble derivatives into the central vacuole. INTRODUCTION Carotenoids are isoprenoid pigments that have key biological functions in organisms of all major taxa. The oxidation of the rigid backbone of carotenoids by specific dioxygenases leads to the formation of diverse bioactive derivatives such as vitamin A (von Lintig and Vogt 2000 Wyss et al. 2000 the plant hormone abscisic acid (Schwartz et al. 1997 and several aroma compounds (Enzell 1985 Buttery et al. 1988 Sefton et al. 1989 Winterhalter and Rouseff 2002 and apocarotenoid pigments (Winterhalter and Rouseff 2002 In plants this metabolic process generally is stimulated during flowering (Eugster and M?rki-Fischer 1991 fruit ripening (Gross and Eckhardt 1981 Lutz and AURKB Winterhalter 1992 Maoka et al. 2001 Fleischmann et al. 2002 industrial curing of tobacco (Wahlberg et al. 1977 and tea fermentation (Kawakami and Kobayashi 2002 Crocus a cultivated sterile plant offers a convenient model with which to further our understanding of this carotenoid catabolic pathway. Upon flowering in autumn Crocus displays red style branches (Figures 1A and 1B) which once dried out constitute the spice saffron (Mathew 1983 Saffron may be the priciest spice known and in addition is a very important herbal medication (Gainer and Brumgard 1982 Sampathu et al. 1984 Holloway and Gainer 1988 The build up of three main carotenoid derivatives-crocetin PHA-665752 glycosides picrocrocin and safranal-is in huge part in charge of the initial color bitter flavor and aroma of saffron (Shape 1C) (Winterhalter and Rouseff 2002 Stereochemical configurations (Buchecker and Eugster 1973 and an extremely decreased carotenoid level in saffron claim that these supplementary metabolites are shaped by a unique sequence which involves the cleavage of zeaxanthin (Pfander and Schurtenberger 1982 accompanied by oxidative adjustments and glycosylations (Shape 1C). Although these substances generate much curiosity enzymes that start the reaction series never have been identified. Shape 1. Pathway and Build up of Carotenoid-Derived Metabolites in Crocus. The limited event of crocetin and related apocarotenoids in character (Eugster et al. 1969 Tandon et al. 1979 Pfister et al. 1996 Liao et al. 1999 argues against PHA-665752 their synthesis PHA-665752 via an enzymatic cooxidation system concerning ubiquitous lipoxygenases (Wu et al. 1999 or xanthine oxidases (Bosser and Belin 1994 Two types of carotenoid cleavage dioxygenases have already been identified in vegetation. The foremost is the maize Vp14 (Schwartz et al. 1997 which catalyzes the conversion of epoxy-xanthophylls to the abscisic acid precursor xanthoxin. The second is the Arabidopsis broad-substrate-specificity carotenoid dioxygenase (AtCCD1) which cleaves the 9 10 and 9′ 10 double carbon-carbon bonds of carotenoid chromophores (Schwartz et al. 2001 However none of these enzymes cleaves the carotenoid chromophore at the 7 8 and 7′ 8 positions (Figure 1C). These two positions have the highest electron density in the carotenoid chromophore (El-Tinay and Chichester 1970 and represent potential targets of Crocus dioxygenase which leads to the formation of secondary saffron metabolites. Here we report the discovery and the functional characterization of two Crocus carotenoid cleavage dioxygenases CsCCD (carotenoid cleavage dioxygenase) and CsZCD (zeaxanthin cleavage dioxygenase). We demonstrate that CsCCD is a member of the broad-substrate-specificity 9 10 10 cleavage dioxygenase involved in the synthesis of several carotenoid-derived metabolites (Schwartz et al. PHA-665752 2001 Winterhalter and Rouseff 2002 whereas CsZCD specifically catalyzes the synthesis of crocetin dialdehyde and hydroxy-β-cyclocitral from zeaxanthin. Thus CsZCD is the Crocus 7 8 8 cleavage dioxygenase that initiates the synthesis of saffron pigment and aroma (Figure 1C). Further immunological and gene expression PHA-665752 analyses revealed that unlike CsCCD CsZCD is expressed specifically in the chromoplasts of style cells during the active period of zeaxanthin cleavage. We also investigated the ultrastructural changes in the style during the synthesis of secondary saffron metabolites. Our results have led to the hypothesis that in Crocus styles the carotenoid-derived PHA-665752 metabolites are sequestered in the vacuole consistent with their water-soluble nature. RESULTS Carotenoid Analysis of Crocus Style Branches We analyzed the carotenoid content in red Crocus style branches to evaluate the.