Metabolite and target transcript analyses during Crocus sativus stigma development Ángela Rubio Moraga a , José Luis Rambla b , Oussama Ahrazem a , Antonio Granell b , Lourdes Gómez-Gómez a, * a Departamento de Ciencia y Tecnología Agroforestal y Genética, ETSIA, Universidad de Castilla-La Mancha, Campus Universitario s/n, 02071 Albacete, Spain b Instituto de Biología Molecular y Celular de Plantas, Consejo Superior de Investigaciones Científicas, Universidad Politécnica de Valencia, Camino de Vera s/n, 46022 Valencia, Spain article info Article history: Received 19 December 2008 Received in revised form 10 March 2009 Available online 25 May 2009 Keywords: Crocus sativus Iridaceae Saffron Stigma Volatiles Apocarotenoids Crocetin Terpenoids Gene expression abstract Saffron, the desiccated stigmas of Crocus sativus, is highly appreciated for its peculiar colour, flavour and aroma. Several studies have been conducted with the spice, but little is known about the evolution of vol- atile and non-volatile compounds generated during the development of the stigma. In this study, we have followed these compounds, with special attention to those of isoprenoid origin (carotenoids and mono- terpenes), which are responsible for the organoleptic properties of saffron. The main compounds that accumulated throughout stigma development in C. sativus were crocetin, its glucoside derivatives and picrocrocin, all of which increased as stigmas reached a fully developed stage. The volatile composition of C. sativus stigmas changed notably as stigmas developed with each developmental stage being charac- terized by a different volatile combination. In red stigmas, b-cyclocitral, the 7,8 cleavage product of b-car- otene, was highly produced, suggesting the implication of both b-carotene and zeaxanthin in crocetin formation. As stigmas matured, hydroxy-b-ionone and b-ionone were produced while safranal, the most typical aroma compound of the processed spice, was only detected at low levels. However, a safranal- related compound 2,2,2-trimethyl-2-cyclohexene-1,4-dione (4-oxoisophorone) increased rapidly at the anthesis stage and also in senescent stigmas. Monoterpenes were mainly emitted at the time of anthesis and the emission patterns followed the expression patterns of two putative terpene synthases CsTS1 and CsTS2. Fatty acid derivates, which predominated at the earlier developmental stages, were observed at low levels in later stages. Ó 2009 Elsevier Ltd. All rights reserved. 1. Introduction Crocus sativus L. is an autumn-flowering geophyte extensively grown in the Mediterranean basin and Near East since the Late Bronze Age (Negbi, 1999). Saffron, the dried red stigmas of C. sati- vus, has been used as a flavouring and colouring agent since then and is currently considered the world’s most expensive spice. Saf- fron is made up of a complex mixture of volatile and non-volatile compounds that contribute to its overall aroma and flavour (Taran- tilis and Polissiou, 1997). The major components of saffron are the apocarotenoids cis- and trans-crocins, picrocrocin (b-D-glucopy- ranoside of hydroxyl-b-cyclocitral), and its degradation product, the odour-active safranal (Kanakis et al., 2004)(Fig. 1A). However, recent studies reveal a different volatile composition in unpro- cessed stigma tissues (Rubio et al., 2008), suggesting that degrada- tion processes are responsible for the organoleptic properties of saffron from preformed apocarotenoid compounds (Dauria et al., 2006) as has also been observed in other spices (Mookherjee et al., 1990). Apocarotenoids have been extensively studied due to their high concentration in the stigma tissue and because of their organoleptic properties (Pfander and Wittwer, 1975; Tarantil- is et al., 1995). It has been proposed that the biogenesis of the main colour principles, crocins, and the odour active compound, safr- anal, is derived by bio-oxidative cleavage of zeaxanthin (Pfander and Schurtenberger, 1982) by a 7,8(7 0 ,8 0 ) cleavage reaction. How- ever, the ability to synthesize crocetin is not restricted to C. sativus and other related species, but is also present in the flower extract of Buddleja (Liao et al., 1999), in Jacquinia angustifolia (Eugster et al., 1969), in Coleus forskolii (Tandon et al., 1979), in the fruits of Gar- denia (Pfister et al., 1996), and even in the cyanobacterium Micro- cystis which produces crocetin by cleavage of zeaxanthin and b-carotene at 7,8(7 0 ,8 0 ) positions (Jütner and Höflacher, 1985). Studies concerning components in saffron stigma other than those mentioned above are scarce (Tarantilis et al., 1995; Straubin- ger et al., 1997, 1998; Carmona et al., 2007), or the compounds have only been analysed with some detail in petals (Nørbk et al., 2002). The majority of the studies performed with C. sativus concentrate on the spice, and little is known about the synthesis and accumulation of these compounds during stigma development. In the present study, we have used a combination of approaches to study the accumulation of colour and aroma compounds of C. sativus during stigma development, and also to determine whether this accumulation could be paralleled by changes in the 0031-9422/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2009.04.022 * Corresponding author. Tel.: +34 967599200x2854; fax: +34 967599238. E-mail address: marialourdes.gomez@uclm.es (L. Gómez-Gómez). Phytochemistry 70 (2009) 1009–1016 Contents lists available at ScienceDirect Phytochemistry journal homepage: www.elsevier.com/locate/phytochem