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Candidate enzymes for saffron crocin biosynthesis are localized in multiple cellular compartments
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Demurtas, Olivia Costantina, Frusciante, Sarah, Ferrante, Paola, Diretto, Gianfranco, Azad, Noraddin Hosseinpour, Pietrella, Marco, Aprea, Giuseppe, Taddei, Anna Rita, Romano, Elena, Mi, Jianing, Al-Babili, Salim, Frigerio, Lorenzo and Giuliano, Giovanni (2018) Candidate enzymes for saffron crocin biosynthesis are localized in multiple cellular compartments. Plant Physiology, 177 (3). pp. 990-1006. doi:10.1104/pp.17.01815 ISSN 0032-0889.
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Official URL: http://dx.doi.org/10.1104/pp.17.01815
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 "transit centers" for metabolites whose biosynthesis starts in the plastid and are accumulated in the vacuole.
Item Type: | Journal Article | ||||||||
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Divisions: | Faculty of Science, Engineering and Medicine > Science > Life Sciences (2010- ) | ||||||||
Journal or Publication Title: | Plant Physiology | ||||||||
Publisher: | American Society of Plant Biologists | ||||||||
ISSN: | 0032-0889 | ||||||||
Official Date: | 13 July 2018 | ||||||||
Dates: |
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Volume: | 177 | ||||||||
Number: | 3 | ||||||||
Page Range: | pp. 990-1006 | ||||||||
DOI: | 10.1104/pp.17.01815 | ||||||||
Status: | Peer Reviewed | ||||||||
Publication Status: | Published | ||||||||
Access rights to Published version: | Open Access (Creative Commons) | ||||||||
Date of first compliant deposit: | 31 May 2018 | ||||||||
Is Part Of: | European Union [From discovery to products: A next generation pipeline for the sustainable generation of high-value plant products] FP7 Contract 613153 |
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