Cytosolic and plastoglobule-targeted carotenoid dioxygenases from Crocus sativus are both involved in beta-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 beta-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 beta-carotene and emission of beta-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 beta-carotene at the 9,10(9',10') positions to yield beta-ionone. The data obtained suggest that all four C. sativus CCD enzymes may contribute in different ways to the production of beta-ionone. In addition, the location and precise timing of beta-ionone synthesis, together with its known activity as a fragrance and insect attractant, suggest that this volatile may have a role in Crocus pollination.

FIGURE 1.

The C. sativus dioxygenases belong to two different subfamilies. A, confocal microscopic detection of CsCCD4a-GFP fluorescence in Arabidopsis leaves at two resolution levels. Merge, overlap of chlorophyll and GFP signals. Scale bars = 10 μm. B, unrooted phylogenetic tree of the CCD proteins based on amino acid sequence similarity. Only full-length members of the family are included. The predicted protein sequences were initially clustered using ClustalW. Accession numbers used are: At, A. thaliana, AtCCD1 (At3g63520), AtNCED2 (At4g18350), AtNCED3 (At3g14440), AtCCD4 (At4g19170), AtNCED5 (At1g30100), AtNCED6 (At3g24220), AtCCD7 (At2g44990), AtCCD8 (At4g32810), and AtNCED9 (At1g78390); Hv, Hordeum vulgare HvCCD4 (ak248229.1); Ca, Capsicum annuum, CaCCD1 (Y14164); Cm, Chrysanthemum × morifolium CmCCD4a (AB247158), CmCCD4b (AB247160); Cc, Citrus clementina CcCCD4 (abc26011); Cofa, Coffea Arabica, CofcaCCD1 (DQ157170); Cs, C. sativus, CsCCD (CsCCD1a) (CAC79592), CsCCD1b (EU523661), CsCCD4a (EU523662), CsCCD4b (EU523663), and CsNCED4 (EU527188); Cm, Cucumis melo, CmCCD1 (DQ269467); Ls, Lycopersicum sculentum LeCCD1a (AB120111), LeCCD1b (AAT68188), LeNCED1 (CAB10168); Ml, Malus × domestica MdCCD (Z93765); Mt, Medicago trunculata MtCCD4 (ac144759); NSC, Nostoc sp NSC1 (NP_485149) NSC3 (NP_488935.1); Os, Oryza sativa OsHTD (XP473418), OsCCD4 (AP005825), OsCCD1(ABG22113), OsNCED3 (NM001057300), OsNCED5 (AY838901); Pa, Persea americana, PaNCED1 (AAK00632), PaNCED3 (AAK00623); Ph, Petunia × hybrida PhCCD1 (AY576003); Ps, Pisum sativum, PsNCED2 (AB080192), PsNCED3 (AB080193), PsRMS1 (Q6Q623), PsRMS5 (ABD67496), PsCCD1(BAC10549), PsCCD4 (BAC10552); Sl, Solanum lycopersicom SlCCD4 (ap009393); Ss, Suaeda salsa, SsCCD1(DQ003599); St, Solanum tuberosum, StNCED1 (AJ276244); Vv, Vitis vinifera, VvNCED1 (AY3337613), VvCCD6 (A7NXT2); Zea mays VP14 (U95953.1), ZmCCD1 (ABF85668.1). The arrows indicate the CsCCD enzymes, and asterisks indicate the tested activity. The numbers refer to the double bond recognized and cleaved. The horizontal scale shows the number of differences per 100 residues derived from the ClustalW alignment.

FIGURE 2.

Expression of CsCCD enzymes in carotenoid-producing strains of E. coli. E. coli engineered to accumulate the carotenoids β-carotene and zeaxanthin were transformed with an arabinose-inducible recombinant vector for CsCCD1a, CsCCD1b, CsCCD4a-211, and CsCCD4. A, representative HPLC elution profiles of zeaxanthin levels without (control) and with induction of CsCCD1a, CsCCD1b, CsCCD4a-211, and CsCCD4 expression. Inset, on-line spectrum and structure of zeaxanthin. B, chromatogram at absorbance of 345 nm of the zeaxanthin + CsCCD1a combination with the cleavage product peak at 15 min present in the lower chromatogram. The absorption maximum of the obtained product was consistent with that of C₁₄ dialdehyde reported previously (3). Inset, in the upper part the structure of zeaxanthin is shown and the 9,10 and 9′,10′ double bonds are indicated; in the lower part the on-line spectrum and structure of the obtained product after a 9,10(9′,10′) cleavage. C, representative HPLC elution profiles of β-carotene levels without (control) and with induction of CsCCD1a, CsCCD1b, CsCCD4a-211, and CsCCD4 expression. Inset, on-line spectrum and structure of β-carotene. D, GC/MS analysis showing β-ionone emitted by bacteria following induction of CsCCD1a, CsCCD1b, CsCCD4a-211, and CsCCD4 expression. Inset, in the upper part the structure of β-carotene is shown and the 9,10 and 9′,10′ double bonds are indicated, in the lower part the mass spectrum of the observed of β-ionone product. Controls are representative chromatograms from uninduced cultures.

FIGURE 3.

Expression analysis of CsCCDs by RT-PCR in flower organs. A, expression levels in stigma, petals, and stamens. B, expression levels in style and stigma tissues: ws, white style; ys, yellow style; and rs, red stigma. RT-PCR experiments were repeated three times, and representative results are shown. The RPS18 gene for 18 S RNA was amplified as a control. The PCR products were separated by 1% (w/v) agarose gel electrophoresis and visualized by ethidium bromide staining.

FIGURE 4.

Expression levels of CsCCDs during stigma development and volatile emission. The mRNA levels were determined by RT-PCR amplification using specific oligonucleotides for each CsCCD gene. A, stigma tissue of C. sativus in different developmental stages: yellow (Y), orange (O), red (R), 2 days before anthesis (-2da), anthesis (da), 1 day after anthesis (+1da), and 3 days after anthesis (+3da). B, CsCCD1a, CsCCD1b, CsCCD4a and -b expression in yellow, orange, and red undeveloped stigmas, and in preanthesis, anthesis, and postanthesis developed stigmas as shown in A. Equal amounts of total RNA were used in each reaction. The levels of the constitutively expressed RPS18 coding gene were assayed as controls. C, carotenoid-derived volatile emissions during the stigma development of C. sativus.

FIGURE 5.

Transcript levels of CsCCDs in response to stress conditions. A, transcript levels of CsCCD1a, CsCCD1b, CsCCD4a and -b analyzed by RT-PCR from total RNA extracted from control (0 h) and leaf disks dehydrated for 2, 5, and 10 h. B, RT-PCR analysis on control (Ct), ABA treated, CaCl₂ treated, heat treated, or senescent leaf disks. RPS18 was used as an internal control.