Cytosolic and Plastoglobule-targeted Carotenoid Dioxygenases from Crocus sativus Are Both Involved in β-Ionone Release

Abstract

Saffron, the processed stigma of Crocus sativus, is characterized by the presence of several apocarotenoids that contribute to the color, flavor, and aroma of the spice. However, little is known about the synthesis of aroma compounds during the development of the C. sativus stigma. The developing stigma is nearly odorless, but before and at anthesis, the aromatic compound ␤-ionone becomes the principal norisoprenoid volatile in the stigma. In this study, four carotenoid cleavage dioxygenase (CCD) genes, CsCCD1a, CsCCD1b, CsCCD4a, and CsCCD4b, were isolated from C. sativus. Expression analysis showed that CsCCD1a was constitutively expressed, CsCCD1b was unique to the stigma tissue, but only CsCCD4a and -b had expression patterns consistent with the highest levels of ␤-carotene and emission of ␤-ionone derived during the stigma development. The CsCCD4 enzymes were localized in plastids and more specifically were present in the plastoglobules. The enzymatic activities of CsCCD1a, CsCCD1b, and CsCCD4 enzymes were determined by Escherichia coli expression, and subsequent analysis of the volatile products was generated by GC/MS. The four CCDs fell in two phylogenetically divergent dioxygenase classes, but all could cleave ␤-carotene at the 9,10(9 ,10 ) positions to yield ␤-ionone. The data obtained suggest that all four C. sativus CCD enzymes may contribute in different ways to the production of ␤-ionone. In addition, the location and precise timing of ␤-ionone synthesis, together with its known activity as a fragrance and insect attractant, suggest that this volatile may have a role in Crocus pollination.Crocus sativus is a triploid sterile plant characterized by its long red stigmas. These stigmas, when desiccated, constitute the spice known as saffron. Saffron is considered a complex mixture of volatile and non-volatile compounds that contribute to its overall aroma and flavor. The main components of saffron responsible for coloring strength are the derived carotenoids cis-and trans-crocins, picrocrocin, and its degradation product, the odor-active safranal that composes up to 70% of total volatiles (1). In addition to safranal, 150 volatile compounds are estimated to be present in the spice, and ϳ60 constituents have been identified (2). The structures of several of these reveal an isoprenoid-based origin like safranal, and are assumed to be the products of the oxidative cleavage of carotenoids.In recent years, a family of enzymes that cleave carotenoid substrates at different double bond positions have been described in plants. This family, the carotenoid cleavage dioxygenases (CCDs), 2 are specific for the location of the double bond in the molecule they cleave, but many are promiscuous in their carotenoid substrate choice (3, 4). The founding member of the CCD family was Vp14, a 9-cis-epoxycarotenoid dioxygenase (NCED). This and other closely related dioxygenases catalyze the cleavage at the 11,12 double bond of both 9-cis-violaxanthin and 9-cis-neoxanthin to produce xanthoxin (5-7), which is subsequently conv...

Novel carotenoid cleavage dioxygenase catalyzes the first dedicated step in saffron crocin biosynthesis

“…Like the related CCD1 enzymes (21,34), CCD2 lacks a recognizable plastid transit peptide and is localized to the cytoplasm. Note the discoloration of zeaxanthin in CsCCD2-expressing cells.…”

“…full-length form cleaves β-carotene at the 9,10 and/or the 9′,10′ positions, yielding β-ionone (21).…”

Carotenoid cleavage dioxygenases and their apocarotenoid products in plants

“…Both CsZCD and BoLCD fall into the same clade as CCD4 (Bouvier et al 2005). Recent work has shown that CsZCD represents an incomplete CsCCD4 sequence and that full-length CsCCD4 catalyzes the conversion of b-carotene to bionone in E. coli, implying the 9,10(9Ј,10Ј) cleavage activity of CsCCD4 (Rubio et al 2008). These results indicate that the in planta activity of both CsZCD and BoLCD should be reconsidered.…”

Origins of Grape and Wine Aroma. Part 1. Chemical Components and Viticultural Impacts

“…Four subfamilies have been identified-CCD1, CCD4, CCD7, and CCD8-and cleavage may be symmetric or asymmetric depending on the enzyme and carotenoid substrate (Auldridge et al 2006, Walter et al 2010, Young et al 2012). Recently, studies in Crocus sativa, rice, and mycorrhizal roots of Medicago truncatula indicate that CCD4 and CCD7 may be localized in the plastid and the C 13 -and C 27 -apocarotenoids obtained from carotenoid cleavage are exported to the cytosol where further cleavage by CCD1 occurs, yielding C 13 -and C 14 -apocarotenoid products (Floss et al 2008, Rubio et al 2008, Ilg et al 2010). CCD7 and CCD8 are thought to be involved in formation of strigolactone, a plant hormone that inhibits shoot branching (Ruyter-Spira et al 2013).…”