A New Glycosyltransferase Enzyme from Family 91, UGT91P3, Is Responsible for the Final Glucosylation Step of Crocins in Saffron (Crocus sativus L.).

Alberto José López-Jimenez,Gianfranco Diretto,Lourdes Gómez-Gómez,Enrique Niza,Oussama Ahrazem,Sarah Frusciante,Ángela Rubio-Moraga

doi:10.3390/ijms22168815

Abstract

Crocetin is an apocarotenoid formed from the oxidative cleavage of zeaxanthin, by the carotenoid cleavage enzymes CCD2 (in Crocus species) and specific CCD4 enzymes in Buddleja davidii and Gardenia jasminoides. Crocetin accumulates in the stigma of saffron in the form of glucosides and crocins, which contain one to five glucose molecules. Crocetin glycosylation was hypothesized to involve at least two enzymes from superfamily 1 UDP-sugar dependent glycosyltransferases. One of them, UGT74AD1, produces crocins with one and two glucose molecules, which are substrates for a second UGT, which could belong to the UGT79, 91, or 94 families. An in silico search of Crocus transcriptomes revealed six candidate UGT genes from family 91. The transcript profiles of one of them, UGT91P3, matched the metabolite profile of crocin accumulation, and were co-expressed with UGT74AD1. In addition, both UGTs interact in a two-hybrid assay. Recombinant UGT91P3 produced mostly crocins with four and five glucose molecules in vitro, and in a combined transient expression assay with CCD2 and UGT74AD1 enzymes in Nicotiana benthamiana. These results suggest a role of UGT91P3 in the biosynthesis of highly glucosylated crocins in saffron, and that it represents the last missing gene in crocins biosynthesis.

Highlights

Six nucleotide sequences encoding for UDP-glucose-dependent glycosyltransferases (UGTs) that belong to the UGT91 subfamily were identified as possible candidates for crocin glucosylation in the stigmas of saffron (c367693_g2_i1, c411064_g1_i1, c411064_g2_i1, c370708_g1_i2, c370708_g1_i1, c365848_g1_i1) (Supplementary Figure S1), while no sequences were identified with similarities to UGT73, UGT79 and UGT94
Crocins are characteristically non-volatile glucosylated apocarotenoids that confer the red color of saffron stigmas
The glycosylation of secondary metabolites is usually catalyzed by uridine diphosphate-dependent glycosyltransferases (UGTs) that belong to the carbohydrate-active enzyme (CAZY) glycosyltransferase 1 (GT1) family [20]

Summary

Introduction

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Crocins (crocetin esters) are glycosylated apocarotenoid compounds present in a limited group of plants [1]. The presence of these pigments in flowers and fruits suggests their implications in pollinator- and seed-disperser attraction [2]. Crocins accumulate at huge levels in the stigmas of saffron (Crocus sativus L.), and are responsible for the bright red color showed by the saffron spice, one of the most appreciated in the world

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Results

Conclusion