Structural characterization of highly glucosylated crocins and regulation of their biosynthesis during flower development in Crocus

Abstract

Crocin biosynthesis in Crocus has been proposed to proceed through a zeaxanthin cleavage pathway catalyzed by carotenoid cleavage dioxygenase 2 (CCD2), and followed by glucosylation reactions catalyzed by CsGT2 (UGT74AD1). In Crocus ancyrensis flowers, crocins with eight (crocin-1), seven (crocin-2), and six glucose (crocin-3) moieties accumulated both in stigma and tepals. We have characterized the structure of these highly glucosylated crocins and follow up their accumulation by high-resolution liquid chromatography coupled with diode array detector along the development of both tissues, and coupled to the isolation and analysis of the expression of eighteen genes (PSY-I, PSY-II, PDS-(I-V), ISO-ZDS, ZDS, CtrISO, LYC-I and II, BCH, CaCCD2, UGT74AD2-5) related with the apocarotenoid metabolism in C. ancyrensis tepals and stigmas. Structure elucidation of crocin-1 and crocin-2 was done by the combined use of 1D and 2D [1H, 1H] (gCOSY and TOCSY and ROESY) and [1H-13C] NMR experiments, revealing that for crocin-1 was all-trans-crocetin O-[β-D- Glucopyranosyl)-(1→4)-(β-D-glucopyranosyl)-(1→2)]-O-[β-D-glucopyranosyl-(1→6)]-β-D-glucopyranosyl diester, while crocin-2 showed an identical structure except for the absence of one glucose residue in one end of the molecule. Crocins accumulation was not synchronically regulated in stigma and tepals, although in both cases crocins accumulation parallels tissue development, decreasing at anthesis. The expression of the carotenogenic genes PSY, ZDS-V, BCH, and LCY-II was correlated with crocins accumulation. In addition, CaCCD2 and only one of the four glucosyltransferase encoding genes, UGT74AD2, were highly expressed, and the expression was correlated with high levels of crocins accumulation in stigma and tepals.

Physicochemical Characterization of Crocus serotinus Stigmas Indicates Their Potential as a Source of the Bioactive Apocarotenoid Crocetin

“…On the other hand, all the spectra contained several weak bands at 922, 895, 875–873, and 776–772 cm −1 , which are typical of glucopyranose derivatives (e.g., gentiobiose, sophorose, maltotriose, or tetraose) with both α‐ and β‐configurations, as well as the characteristic absorption at 826–833 cm −1 due to α‐glucose units that could be structural parts of the existing CRTSEs (e.g., peaks 2 and 3 in Figure ). These findings coincide with our HPLC‐DAD results but also with previously reported data about the variability in the type and degree of glycosylation in CRTSEs of wild crocuses …”

In‐depth analysis of crocetin ester glycosides from dried/processed stigmas of Crocus sativus L. by HPLC‐ESI‐MSn (n = 2, 3)

“…the aglycon moiety of crocins, seems to be conserved in its chemical structure, the oligosaccharide moieties of these molecules are highly variable from mono‐ to bis‐ester derivatives with mono‐ester up to trisaccharide substituents in a very complex pattern of substitution and a total number of up to five monosaccharides per crocin derivative. Several efforts to analyse crocins from tepals or dried stigmas of various Crocus species by high‐performance liquid chromatography with diode array detection (HPLC‐DAD) alone or in combination with mass spectrometry (MS) have been performed up to now . We have recently characterised the major crocin component of saffron, bis‐gentiobiosyl‐crocetin, as [M + Na] + adduct ion by different tandem mass spectrometric techniques including low‐energy collision‐induced dissociation (LE‐CID) with three‐dimensional (3D)‐quadrupole ion trap MS and with, high resolution quadrupole‐reflectron time‐of‐flight mass spectrometry (Q/RTOF‐MS) as well as a high‐energy collision‐induced dissociation (HE‐CID) with tandem time‐of‐flight mass spectrometry (TOF/RTOF‐MS) .…”

“…Several efforts to analyse crocins from tepals or dried stigmas of various Crocus species by high-performance liquid chromatography with diode array detection (HPLC-DAD) alone or in combination with mass spectrometry (MS) have been performed up to now. 8,[21][22][23][24][25][26][27] We have recently characterised the major crocin component of saffron, bis-gentiobiosyl-crocetin, as [M + Na] + adduct ion by different tandem mass spectrometric techniques including low-energy collision-induced dissociation (LE-CID) with three-dimensional (3D)quadrupole ion trap MS and with, high resolution quadrupole-reflectron time-of-flight mass spectrometry (Q/RTOF-MS) as well as a highenergy collision-induced dissociation (HE-CID) with tandem time-offlight mass spectrometry (TOF/RTOF-MS). 28 This study followed a detailed fragmentation analysis of variously substituted crocin species by LE-CID 3D quadrupole ion trap tandem mass spectrometry (MS 2 ).…”

Intron retention and rhythmic diel pattern regulation of carotenoid cleavage dioxygenase 2 during crocetin biosynthesis in saffron

“…Our results showed that in fact darkness promotes higher accumulation of crocins and CsCCD2 expression, while continuous light repressed CsCCD2 expression. During the development of the stigma CsCCD2 expression drops in the stigma of buds and flowers that have already emerged to the light (Frusciante et al 2014 ; Rubio et al 2008 ), the same is observed for crocins, which content has been increased as the stigma develops in the underground (Moraga et al 2009 ), showing that flower development in saffron is coordinated with apocarotenoid biosynthesis in the stigma tissue, as observed for other Crocus species (Ahrazem et al 2015a ), suggesting a highly interconnected regulatory network that coordinates flower development and apocarotenoid biosynthesis. Therefore, the control of nuclear gene expression in response to the developmental state of plastids should be critical for the synthesis and accumulation of crocins in the chromoplast by the activity of CsCCD2 .…”