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Novel Carotenoid Cleavage Dioxygenase Catalyzes the First Dedicated Step in Saffron Crocin Biosynthesis

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Novel Carotenoid Cleavage Dioxygenase Catalyzes the First Dedicated Step in Saffron Crocin Biosynthesis Novel carotenoid cleavage dioxygenase catalyzes the first dedicated step in saffron crocin biosynthesis Sarah Frusciantea,b, Gianfranco Direttoa, Mark Brunoc, Paola Ferrantea, Marco Pietrellaa, Alfonso Prado-Cabrerod, Angela Rubio-Moragae, Peter Beyerc, Lourdes Gomez-Gomeze, Salim Al-Babilic,d, and Giovanni Giulianoa,1 aItalian National Agency for New Technologies, Energy, and Sustainable Development, Casaccia Research Centre, 00123 Rome, Italy; bSapienza, University of Rome, 00185 Rome, Italy; cFaculty of Biology, University of Freiburg, D-79104 Freiburg, Germany; dCenter for Desert Agriculture, Division of Biological and Environmental Science and Engineering, King Abdullah University of Science and Technology, Thuwal 23955-6900, Saudi Arabia; and eInstituto Botánico, Facultad de Farmacia, Universidad de Castilla–La Mancha, 02071 Albacete, Spain Edited by Rodney B. Croteau, Washington State University, Pullman, WA, and approved July 3, 2014 (received for review March 16, 2014) Crocus sativus stigmas are the source of the saffron spice and responsible for the synthesis of crocins have been characterized accumulate the apocarotenoids crocetin, crocins, picrocrocin, and in saffron and in Gardenia (5, 6). safranal, responsible for its color, taste, and aroma. Through deep Plant CCDs can be classified in five subfamilies according to transcriptome sequencing, we identified a novel dioxygenase, ca- the cleavage position and/or their substrate preference: CCD1, rotenoid cleavage dioxygenase 2 (CCD2), expressed early during CCD4, CCD7, CCD8, and nine-cis-epoxy-carotenoid dioxygen- stigma development and closely related to, but distinct from, the ases (NCEDs) (7–9). NCEDs solely cleave the 11,12 double CCD1 dioxygenase family. CCD2 is the only identified member of bond of 9-cis-epoxycarotenoids to produce the ABA precursor a novel CCD clade, presents the structural features of a bona fide xanthoxin. CCD7 and CCD8 act sequentially in the strigolactone CCD, and is able to cleave zeaxanthin, the presumed precursor of pathway, leading to strigolactone precursor carlactone (10). saffron apocarotenoids, both in Escherichia coli and in maize en- Enzymes of the CCD1 family cleave a wide spectrum of dif- dosperm. The cleavage products, identified through high-resolu- ferent carotenoids at several different positions (9,10; 9,10,9′,10′; tion mass spectrometry and comigration with authentic standards, 5,6,5′,6′; or 7,8,7′,8′) (11, 12). CCD4 enzymes cleave carotenoids are crocetin dialdehyde and crocetin, respectively. In vitro assays at the 9′,10′ or the 7′,8′ positions and determine the level of show that CCD2 cleaves sequentially the 7,8 and 7′,8′ double pigmentation in plant tissues, including Chrysanthemum petals bonds adjacent to a 3-OH-β-ionone ring and that the conversion (13), peach flesh (14), potato tubers (15), Citrus peel (16, 17), of zeaxanthin to crocetin dialdehyde proceeds via the C30 interme- and Arabidopsis seeds (18). diate 3-OH-β-apo-8′-carotenal. In contrast, zeaxanthin cleavage Structurally, all CCDs are characterized by a rigid, seven- + dioxygenase (ZCD), an enzyme previously claimed to mediate cro- bladed β-propeller structure, at the axis of which a Fe2 atom is cetin formation, did not cleave zeaxanthin or 3-OH-β-apo-8′-caro- located (19). The propeller is covered by a less-conserved dome + tenal in the test systems used. Sequence comparison and structure formed by a series of loops. The reaction is catalyzed by the Fe2 prediction suggest that ZCD is an N-truncated CCD4 form, lacking atom via the introduction of oxygen (20). one blade of the β-propeller structure conserved in all CCDs. These To date, conflicting data have been reported about the identity results constitute strong evidence that CCD2 catalyzes the first of the enzyme catalyzing the cleavage reaction in saffron. A dedicated step in crocin biosynthesis. Similar to CCD1, CCD2 has zeaxanthin cleavage dioxygenase (ZCD) was reported to cleave a cytoplasmic localization, suggesting that it may cleave carote- zeaxanthin symmetrically at the 7,8/7′,8′ positions, yielding the noids localized in the chromoplast outer envelope. crocin precursor crocetin dialdehyde (4). However, later work has suggested that ZCD is a truncated form of a plastoglobule- β-citraurin | symmetric carotenoid cleavage localized CCD4 enzyme, devoid of cleavage activity, and that the he plant Crocus sativus L. (Iridaceae) is a perennial, sterile, Significance T vegetatively propagated triploid widely cultivated in a tem- perate belt extending from Spain to Kashmir (1). Albeit its site of Saffron is a triploid, sterile species whose red stigmas consti- domestication is uncertain, the earliest archaeological evidence tute the most expensive spice on Earth. The color, the taste, of its cultivation is provided by Minoan frescoes dated 1,700– and the aroma of the spice are owed to the crocus-specific 1,500 B.C. Its dried red stigmas (Fig. 1A) constitute the saffron apocarotenoid accumulation of crocetin/crocins, picrocrocin, spice, which is commonly considered the most expensive spice on and safranal. Through deep transcriptome analysis, we identi- Earth, with retail prices ranging between 2,000 and 7,000 V/kg. fied a novel carotenoid cleavage dioxygenase (CCD) whose expression profile parallels the production of crocetin. Using in These high prices are due to the labor associated with its har- bacterio, in vitro, and in planta functional assays, we demon- vesting: because one stigma of saffron weighs about 2 mg, 1 kg of strate that CCD2 is the dioxygenase catalyzing the first dedi- dry saffron requires the manual harvest of stigmas from around cated step in saffron crocetin biosynthesis starting from the 110,000–170,000 flowers (www.europeansaffron.eu) (1). carotenoid zeaxanthin. Saffron stigmas accumulate large amounts (up to 8% on dry weight) of the apocarotenoids crocetin (and its glycosylated Author contributions: S.A.-B. and G.G. designed research; S.F., G.D., and P.F. performed forms, crocins), responsible for the red pigmentation of the research; P.B., L.G.-G., M.B., M.P., A.P.-C., and A.R.-M. contributed new reagents/analytic tools; S.F. and G.D. analyzed data; and G.G. wrote the paper. stigmas; picrocrocin, responsible for their bitter flavor; and The authors declare no conflict of interest. safranal, responsible for the pungent aroma of saffron (Fig. 1A) This article is a PNAS Direct Submission. (2). The proposed biosynthetic pathway (3, 4) starts through the Freely available online through the PNAS open access option. symmetric cleavage of zeaxanthin at the 7,8/7′,8′ positions by Data deposition: The sequence of CCD2 reported in this paper has been deposited in the a nonheme iron carotenoid cleavage dioxygenase (CCD) (Fig. GenBank database (accession no. KJ541749). β 1B). The two cleavage products, 3-OH- -cyclocitral and crocetin 1To whom correspondence should be addressed. Email: [email protected]. dialdehyde, are dehydrogenated and glycosylated to yield pic- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. rocrocin and crocins, respectively. Putative glucosyl transferases 1073/pnas.1404629111/-/DCSupplemental. 12246–12251 | PNAS | August 19, 2014 | vol. 111 | no. 33 www.pnas.org/cgi/doi/10.1073/pnas.1404629111 Downloaded by guest on September 25, 2021 A using Newbler, and the contigs were searched for similarity to known CCD enzymes using BLAST. The search resulted in seven CCDs, including CCD1, CCD7, three allelic forms of CCD4,and a novel transcript, which we called CCD2 due to its evolutionary relation with CCD1 (see below). The identified CCDs differ in their temporal pattern of expression during stigma development (Table S1). In particular, CCD2 expression peaks early, at the Ostage(Fig.2A) coincident with crocetin and crocin accu- mulation (22), whereas ZCD and CCD4 are expressed late Zeaxanthin during stigma development. B 7 A phylogenetic analysis of CCD protein sequences from sev- eral plants was inferred using the neighbor-joining method using 8 Synechocystis apocarotenoid cleavage oxygenase (ACO) as an CCD outgroup (Fig. 2B). The results suggested that Crocus CCD2 is 3-OH- a member of a clade closely related to, but distinct from, an- 7 giosperm CCD1 enzymes. A lettuce enzyme labeled as CCD2 (23) clustered with CCD1 enzymes, whereas an enzyme known to 8 cut zeaxanthin at the 7,8 position, Citrus CCD4b1 (16), clustered CCD with CCD4 enzymes (Fig. 2B). ZCD appeared to be a member of 3-OH- -Cyclocitral Crocetin dialdehyde the CCD4 family (Fig. 2B), truncated at the N terminus (Fig. S1). Because the ZCD cDNA was originally isolated by rapid am- plification of cDNA ends (RACE) (4, 24) that can lead to the ′ ALDH cloning of truncated transcripts, we carried out a 5 -RACE UGT analysis of CCD4 transcripts. Next to a 950-base full-length Picrocrocin Crocetin transcript, whose length is compatible with a full-length CCD4 protein, a series of abundant 5′-truncated transcripts are de- tectable, the longest of which is compatible with the length of the UGTs ZCD protein, which is encoded starting from an internal ATG ? codon (Fig. S2A). It is therefore likely that the original ZCD Crocin clone (4) corresponds to a truncated CCD4 transcript. This Safranal cannot be either CCD4a or CCD4b (25), which are only 98% identical to ZCD at the nucleotide level. To further address this point, we cloned the 400- to 350-bp RACE products shown in Fig. S2A, containing the internal ATG codon, and sequenced Fig. 1. The saffron apocarotenoid pathway. Crocus sativus flower at an- multiple clones. The sequence of eight of the clones corresponds thesis. The yellow arrowheads point at the three stigmas (A). Proposed to CCD4a, of one to CCD4b, of five to ZCD (4), and of four saffron apocarotenoid biosynthesis pathway (B). Zeaxanthin is cleaved at the to a yet-unidentified CCD4. All of them contain the internal ′ ′ 7,8 and 7 ,8 positions by a CCD activity. The C20 cleavage product, crocetin ATG codon. dialdehyde, is converted to crocetin by an aldehyde dehydrogenase, and We modeled the CCD2, ZCD, and CCD4a structures using then to crocins by at least two UDPG-glucosyltransferases.
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