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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-?-ionone ring and that the conversion of zeaxanthin to crocetin dialdehyde proceeds via the C30 intermediate 3-OH-?-apo-8'-carotenal. In contrast, zeaxanthin cleavage dioxygenase (ZCD), an enzyme previously claimed to mediate crocetin formation, did not cleave zeaxanthin or 3-OH-?-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 ?-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.

SUBMITTER: Frusciante S

PROVIDER: S-EPMC4143034 | biostudies-literature | 2014 Aug

REPOSITORIES: biostudies-literature

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

Frusciante Sarah S Diretto Gianfranco G Bruno Mark M Ferrante Paola P Pietrella Marco M Prado-Cabrero Alfonso A Rubio-Moraga Angela A Beyer Peter P Gomez-Gomez Lourdes L Al-Babili Salim S Giuliano Giovanni G

Proceedings of the National Academy of Sciences of the United States of America 20140805 33

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 featu ...[more]

PMID: 25097262

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Project description:The dietary needs of humans for provitamin A carotenoids arise from their inability to synthesize vitamin A de novo. To improve the status of this essential micronutrient, special attention has been given to biofortification of staple foods, such as wheat grains, which are consumed in large quantities but contain low levels of provitamin A carotenoids. However, there remains an unclear contribution of metabolic genes and homoeologs to the turnover of carotenoids in wheat grains. To better understand carotenoid catabolism in tetraploid wheat, Targeting Induced Local Lesions in Genomes (TILLING) mutants of CCD4, encoding a Carotenoid Cleavage Dioxygenase (CCD) that cleaves carotenoids into smaller apocarotenoid molecules, were isolated and characterized. Our analysis showed that ccd4 mutations co-segregated with Poltergeist-like (pll) mutations in the TILLING mutants of A and B subgenomes, hence the ccd-A4 pll-A, ccd-B4 pll-B, and ccd-A4 ccd-B4 pll-A pll-B mutants were analyzed in this study. Carotenoid profiles are comparable in mature grains of the mutant and control plants, indicating that CCD4 homoeologs do not have a major impact on carotenoid accumulation in grains. However, the neoxanthin content was increased in leaves of ccd-A4 ccd-B4 pll-A pll-B relative to the control. In addition, four unidentified carotenoids showed a unique presence in leaves of ccd-A4 ccd-B4 pll-A pll-B plants. These results suggested that CCD4 homoeologs may contribute to the turnover of neoxanthin and the unidentified carotenoids in leaves. Interestingly, abnormal spike, grain, and seminal root phenotypes were also observed for ccd-A4 pll-A, ccd-B4 pll-B, and ccd-A4 ccd-B4 pll-A pll-B plants, suggesting that CCD4 and/or PLL homoeologs could function toward these traits. Overall, this study not only reveals the role of CCD4 in cleavage of carotenoids in leaves and grains, but also uncovers several critical growth traits that are controlled by CCD4, PLL, or the CCD4-PLL interaction.

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

Publications

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

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