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

Abstract

Crocus sativus stigmas are the source of the saffron spice and accumulate the apocarotenoids crocetin, crocins, picrocrocin, and safranal, responsible for its color, taste, and aroma. Through deep transcriptome sequencing, we identified a novel dioxygenase, carotenoid cleavage dioxygenase 2 (CCD2), expressed early during stigma development and closely related to, but distinct from, the CCD1 dioxygenase family. CCD2 is the only identified member of a novel CCD clade, presents the structural features of a bona fide CCD, and is able to cleave zeaxanthin, the presumed precursor of saffron apocarotenoids, both in Escherichia coli and in maize endosperm. The cleavage products, identified through high-resolution mass spectrometry and comigration with authentic standards, are crocetin dialdehyde and crocetin, respectively. In vitro assays show that CCD2 cleaves sequentially the 7,8 and 7',8' double bonds adjacent to a 3-OH-β-ionone ring and that the conversion of zeaxanthin to crocetin dialdehyde proceeds via the C30 intermediate 3-OH-β-apo-8'-carotenal. In contrast, zeaxanthin cleavage dioxygenase (ZCD), an enzyme previously claimed to mediate crocetin formation, did not cleave zeaxanthin or 3-OH-β-apo-8'-carotenal in the test systems used. Sequence comparison and structure prediction suggest that ZCD is an N-truncated CCD4 form, lacking one blade of the β-propeller structure conserved in all CCDs. These results constitute strong evidence that CCD2 catalyzes the first dedicated step in crocin biosynthesis. Similar to CCD1, CCD2 has a cytoplasmic localization, suggesting that it may cleave carotenoids localized in the chromoplast outer envelope.

Keywords: symmetric carotenoid cleavage; β-citraurin.

Fig. 1.

The saffron apocarotenoid pathway. Crocus sativus flower at anthesis. The yellow arrowheads point at the three stigmas (A). Proposed saffron apocarotenoid biosynthesis pathway (B). Zeaxanthin is cleaved at the 7,8 and 7′,8′ positions by a CCD activity. The C₂₀ cleavage product, crocetin dialdehyde, is converted to crocetin by an aldehyde dehydrogenase, and then to crocins by at least two UDPG-glucosyltransferases. The C₁₀ product, 3-OH-β-cyclocitral, is converted to picrocrocin by an UDPG-glucosyltransferase, and then to safranal.

Fig. 2.

Expression and structural characteristics of carotenoid cleavage dioxygenases from saffron stigma. Transcript levels of saffron CCDs in different stigma developmental stages, based on 454 RNA-Seq data; −2dA, 2 d preanthesis; 0dA, day of anthesis; +2dA, 2 d postanthesis (A). Data expressed as reads per kilobase per million (RPKM). The graph above the heat map indicates the kinetics of accumulation of the different apocarotenoids. Phylogenetic relationships of CCDs from saffron (Cs), Arabidopsis (At), rice (Os), tomato (Sl), lettuce (Ls), clementine (Cc), and Synechocystis (Sy) inferred using the neighbor-joining method; CsCCD1, CAC79592.1; CsCCD4a, ACD62476.1; CsCCD4b, ACD62477.1; CsZCD, CAD33262.1; AtCCD1, AT3G63520; AtCCD4, AT4G19170; OsCCD1, Os12g0640600; OsCCD4A, Os02g0704000; OsCCD4B, Os12g0435200; SlCCD1a, Solyc01g087250.2; SlCCD4a, Solyc08g075480.2; LsCCD2, BAE72095.1; CcCCD4b1, Ciclev10028113m; SyACO, P74334 (B). Topology diagrams of Synechocystis apocarotenoid cleavage oxygenase (ACO) and Crocus sativus CCD2, ZCD, CCD4a (C). Secondary structural elements consisting of α-helices and β-sheets are colored in pink and yellow, respectively. The seven blades are labeled from I to VII for ACO and is the same for the other topology diagrams. The ferrous catalytic iron is colored in green. All structural elements located outside the seven blades form part of the dome. The gray shaded structural elements in ZCD are lacking; please note the alternative N terminus. Most of the dome is lacking in this protein, together with most of blade VII. CCD4a topology diagram is showed for comparison.

Fig. 3.

CCD2 expressed in E. coli cleaves zeaxanthin to yield crocetin dialdehyde. E. coli cells accumulating lycopene, β-carotene, or zeaxanthin were transformed with the empty pThio vector (C−), or the same vector expressing CCD2 or ZCD, induced for 16 h at 20 °C with arabinose and pelleted (A). Note the discoloration of zeaxanthin in CsCCD2-expressing cells. LC-HRMS analysis of zeaxanthin cleavage products (B). Zeaxanthin-accumulating E. coli cells expressing CsCCD2 were induced for 16 h at 20 °C with arabinose, extracted with acetone, and the extracts were run on a LC-HRMS system alongside authentic standards. The accurate masses of zeaxanthin, 3-OH-β-apo-8′-carotenal, and crocetin dialdehyde were extracted. Only crocetin dialdehyde is detectable and has an accurate mass and a chromatographic mobility identical to that of the authentic standard.

Fig. 4.

Cleavage of maize kernel carotenoids by transiently expressed CCD2. Pigmentation of maize kernels after 48 h of agroinfiltration with pBI-GUS, pBI-CCD2, and pBI-ZCD (A). LC-HRMS of hydrophobic kernel extracts (B). The CCD2 extracts show accumulation of crocetin, but not crocetin dialdehyde or 3-OH-β-apo-8′-carotenal.

Fig. 5.

In vitro cleavage assay. In vitro cleavage of zeaxanthin by E. coli extracts (A). Decoloration is diagnostic of cleavage. Cleavage products are identified by HPLC–photodiode array detection (HPLC-PDA) and LC-HRMS (Fig. S5A). Substrates that are not cleaved by CCD2 in the in vitro assay (B). Substrates that are cleaved by CCD2 in the in vitro assay and position of the cleavage, as deduced by HPLC-PDA or Orbitrap LC-HRMS analysis (C) (Figs. S5 and S6). The percentage cleavage of the different substrates in an overnight assay is shown in Table S3.