Abstract
Saffron is composed of the dried stigmas of Crocus sativus and is the most expensive spice on Earth. Its red color is due to the apocarotenoid glycosides, crocins, which accumulate in the vacuole and reach up to 10% of the stigma dry weight. We have previously characterized the first dedicated enzyme in crocin biosynthesis, CsCCD2, which cleaves zeaxanthin to yield crocetin dialdehyde. In this work, we identified six putative aldehyde dehydrogenase (ALDH) transcripts expressed in saffron stigmas. When expressed in E. coli, only one of corresponding proteins (CsALDH3I1), was able to convert crocetin dialdehyde into the crocin precursor, crocetin. CsALDH3I1 carries a C-terminal hydrophobic domain, similar to that of a Neurospora membrane-associated apocarotenoid dehydrogenase, YLO-1. We also characterized a UDP-glycosyltransferase enzyme, CsUGT74AD1, able to convert crocetin to crocins 1 and 2'. In vitro assays showed high specificity of CsALDH3I1 for crocetin dialdehyde and long chain apocarotenals, and of CsUGT74AD1 for crocetin. Upon extract fractionation, the CsCCD2, CsALDH3I1 and CsUGT74AD1 enzymes partitioned in the insoluble fraction, suggesting that they are associated to membranes or to large insoluble complexes. Immunogold labeling of saffron stigmas and confocal microscopy of fusions to Green Fluorescent Protein expressed in N. benthamiana leaves revealed that CsCCD2 localizes to plastids, CsALDH3I1 to the endoplasmic reticulum (ER) and CsUGT74AD1 to the cytoplasm, in association with cytoskeletal-like structures. Based on our and on literature data, we propose that the ER and cytoplasm function as
| Original language | English (US) |
|---|---|
| Pages (from-to) | 990-1006 |
| Number of pages | 17 |
| Journal | Plant Physiology |
| Volume | 177 |
| Issue number | 3 |
| DOIs | |
| State | Published - May 29 2018 |
Bibliographical note
KAUST Repository Item: Exported on 2020-10-01Acknowledgements: We thank Elisabetta Bennici (ENEA) for the growth of N. benthamiana plants; Salvatore Chiavarini (ENEA) for suggestions on 2,4-dinitrophenylhydrazine derivatization; Lourdes Gomez-Gomes and Angela Rubio-Moraga (University of Albacete) and Zafferanami (Milano) for provision of C. sativus flowers. Gaetano Perrotta, Paolo Facella, and Fabrizio Carbone (ENEA) for 454 sequencing, and Aparna Balakrishna (KAUST) for technical assistance. Part of the computing resources used for this work have been kindly provided by CRESCO/ENEAGRID High Performance Computing infrastructure and its staff (Ponti et al., 2014). This work was supported by the European Union [From discovery to products: A next generation pipeline for the sustainable generation of high-value plant products, FP7 Contract 613153] and King Abdullah University of Science and Technology (KAUST), and benefited from the networking activities within the European Cooperation in Science and Technology Action CA15136 (EUROCAROTEN).