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

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 [H, H] (gCOSY and TOCSY and ROESY) and [H-C] 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.This work was supported by the Spanish Ministerio de Economía y Competitividad (BIO2013-44239-R) and participates in the IBERCAROT network (112RT0445). OA was funded by FPCYTCLM through the INCRECYT Programme.Peer Reviewe

Gene-Metabolite Networks of Volatile Metabolism in Airen and Tempranillo Grape Cultivars Revealed a Distinct Mechanism of Aroma Bouquet Production

Volatile compounds are the major determinants of aroma and flavor in both grapes and wine. In this study, we investigated the emission of volatile and non-volatile compounds during berry maturation in two grape varieties (Airén and Tempranillo) throughout 2010 and 2011. HS-SPME coupled to gas chromatography and mass spectrometry was applied for the identification and relative quantitation of these compounds. Principal component analysis was performed to search for variability between the two cultivars and evolution during 10 developmental stages. Results showed that there are distinct differences in volatile compounds between cultivars throughout fruit development. Early stages were characterized in both cultivars by higher levels of some apocarotenoids such as β-cyclocitral or β-ionone, terpenoids (E)-linalool oxide and (Z)-linalool oxide and several furans, while the final stages were characterized by the highest amounts of ethanol, benzenoid phenylacetaldehyde and 2-phenylethanol, branched-amino acid-derived 3-methylbutanol and 2-methylbutanol, and a large number of lipid derivatives. Additionally, we measured the levels of the different classes of volatile precursors by using liquid chromatography coupled to high resolution mass spectrometry. In both varieties, higher levels of carotenoid compounds were detected in the earlier stages, zeaxanthin and α-carotene were only detected in Airén while neoxanthin was found only in Tempranillo; more variable trends were observed in the case of the other volatile precursors. Furthermore, we monitored the expression of homolog genes of a set of transcripts potentially involved in the biosynthesis of these metabolites, such as some glycosyl hydrolases family 1, lipoxygenases, alcohol dehydrogenases hydroperoxide lyases, O-methyltransferases and carotenoid cleavage dioxygenases during the defined developmental stages. Finally, based on Pearson correlation analyses, we explored the metabolite-metabolite fluctuations within VOCs/precursors during the berry development; as well as tentatively linking the formation of some metabolites detected to the expression of some of these genes. Our data showed that the two varieties displayed a very different pattern of relationships regarding the precursor/volatile metabolite-metabolite fluctuations, being the lipid and the carotenoid metabolism the most distinctive between the two varieties. Correlation analysis showed a higher degree of overall correlation in precursor/volatile metabolite-metabolite levels in Airén, confirming the enriched aroma bouquet characteristic of the white varieties.This work was supported by the “Junta de comunidades de Castilla-La Mancha” (JCCM) [PPII10-0062-7718] and benefited from the networking activities within the European Cooperation in Science and Technology Action CA15136 (EUROCAROTEN). GD was supported by short-term fellowships of the Quality Fruit (FA1106) European Cooperation in Science and Technology actions. OA was funded by FPCYTCLM through the INCRECYT Programme.Peer reviewedPeer Reviewe

Fortification and bioaccessibility of saffron apocarotenoids in potato tubers

Carotenoids are C40 isoprenoids with well-established roles in photosynthesis, pollination, photoprotection, and hormone biosynthesis. The enzymatic or ROS-induced cleavage of carotenoids generates a group of compounds named apocarotenoids, with an increasing interest by virtue of their metabolic, physiological, and ecological activities. Both classes are used industrially in a variety of fields as colorants, supplements, and bio-actives. Crocins and picrocrocin, two saffron apocarotenoids, are examples of high-value pigments utilized in the food, feed, and pharmaceutical industries. In this study, a unique construct was achieved, namely O6, which contains CsCCD2L, UGT74AD1, and UGT709G1 genes responsible for the biosynthesis of saffron apocarotenoids driven by a patatin promoter for the generation of potato tubers producing crocins and picrocrocin. Different tuber potatoes accumulated crocins and picrocrocin ranging from 19.41-360 to 105-800 mu g/g DW, respectively, with crocetin, crocin 1 [(crocetin-(beta-D-glucosyl)-ester)] and crocin 2 [(crocetin)-(beta-D-glucosyl)-(beta-D-glucosyl)-ester)] being the main compounds detected. The pattern of carotenoids and apocarotenoids were distinct between wild type and transgenic tubers and were related to changes in the expression of the pathway genes, especially from PSY2, CCD1, and CCD4. In addition, the engineered tubers showed higher antioxidant capacity, up to almost 4-fold more than the wild type, which is a promising sign for the potential health advantages of these lines. In order to better investigate these aspects, different cooking methods were applied, and each process displayed a significant impact on the retention of apocarotenoids. More in detail, the in vitro bioaccessibility of these metabolites was found to be higher in boiled potatoes (97.23%) compared to raw, baked, and fried ones (80.97, 78.96, and 76.18%, respectively). Overall, this work shows that potatoes can be engineered to accumulate saffron apocarotenoids that, when consumed, can potentially offer better health benefits. Moreover, the high bioaccessibility of these compounds revealed that potato is an excellent way to deliver crocins and picrocrocin, while also helping to improve its nutritional value

LA LAGUNA. VALLESECO [Material gráfico]

Copia digital. Madrid : Ministerio de Educación, Cultura y Deporte, 201

Unravelling massive crocins transport and accumulation through proteome and microscopy tools during the development of saffron stigma

Crocins, the glucosides of crocetin, are present at high concentrations in saffron stigmas and accumulate in the vacuole. However, the biogenesis of the saffron chromoplast, the changes during the development of the stigma and the transport of crocins to the vacuole, are processes that remain poorly understood. We studied the process of chromoplast differentiation in saffron throughout stigma development by means of transmission electron microscopy. Our results provided an overview of a massive transport of crocins to the vacuole in the later developmental stages, when electron dense drops of a much greater size than plastoglobules (here defined crocinoplast ) were observed in the chromoplast, connected to the vacuole with a subsequent transfer of these large globules inside the vacuole. A proteome analysis of chromoplasts from saffron stigma allowed the identification of several well-known plastid proteins and new candidates involved in crocetin metabolism. Furthermore, expressions throughout five developmental stages of candidate genes responsible for carotenoid and apocarotenoid biogenesis, crocins transport to the vacuole and starch metabolism were analyzed. Correlation matrices and networks were exploited to identify a series of transcripts highly associated to crocetin (such as 1-Deoxy-D-xylulose 5-phosphate synthase (DXS), 1-Deoxy-D-xylulose 5-phosphate reductoisomerase (DXR), carotenoid isomerase (CRTISO), Crocetin glucosyltransferase 2 (UGT2), etc.) and crocin (e.g., -carotene desaturase (ZDS) and plastid-lipid-associated proteins (PLAP2)) accumulation; in addition, candidate aldehyde dehydrogenase (ADH) genes were highlighted.This work was supported by the Spanish Ministerio de Economia y Competitividad (BIO2013-44239-R), participates in the IBERCAROT network (112RT0445) and benefited from the networking activities within the European Cooperation in Science and Technology Action CA15136 (EUROCAROTEN). The proteomics analysis LC-MS/MS by LTQ Orbitrap Velos was carried out in the Proteomics and Genomics Facility (CIB-CSIC), a member of ProteoRed-ISCIII network. We wish to thank Javier Argandona and Carmen Cifuentes for technical assistance and Kathy Walsh for language revision.Gómez Gómez, L.; Parra Vega, V.; Rivas Sendra, A.; Seguí Simarro, JM.; Molina Romero, RV.; Pallotti Sagripanti, CG.; Rubio Moraga, Á.... (2017). Unravelling massive crocins transport and accumulation through proteome and microscopy tools during the development of saffron stigma. International Journal of Molecular Sciences. 18(1)(76):1-22. https://doi.org/10.3390/ijms18010076S12218(1)7

Characterization of a glucosyltransferase enzyme involved in the formation of kaempferol and quercetin sophorosides in Crocus sativus

UGT707B1 is a new glucosyltransferase isolated from saffron (Crocus sativus) that localizes to the cytoplasm and the nucleus of stigma and tepal cells. UGT707B1 transcripts were detected in the stigma tissue of all the Crocus species analyzed, but expression analysis of UGT707B1 in tepals revealed its absence in certain species. The analysis of the glucosylated flavonoids present in Crocus tepals reveals the presence of two major flavonoid compounds in saffron: kaempferol-3-O-β-D-glucopyranosyl-(1-2)-β-D-glucopyranoside and quercetin-3-O-β-D-glucopyranosyl-(1-2)-β-D-glucopyranoside, both of which were absent from the tepals of those Crocus species that did not express UGT707B1. Transgenic Arabidopsis (Arabidopsis thaliana) plants constitutively expressing UGT707B1 under the control of the cauliflower mosaic virus 35S promoter have been constructed and their phenotype analyzed. The transgenic lines displayed a number of changes that resembled those described previously in lines where flavonoid levels had been altered. The plants showed hyponastic leaves, a reduced number of trichomes, thicker stems, and flowering delay. Levels of flavonoids measured in extracts of the transgenic plants showed changes in the composition of flavonols when compared with wild-type plants. The major differences were observed in the extracts from stems and flowers, with an increase in 3-sophoroside flavonol glucosides. Furthermore, a new compound not detected in ecotype Columbia wild-type plants was detected in all the tissues and identified as kaempferol-3-O-sophoroside-7-O-rhamnoside. These data reveal the involvement of UGT707B1 in the biosynthesis of flavonol-3-O-sophorosides and how significant changes in flavonoid homeostasis can be caused by the overproduction of a flavonoid-conjugating enzyme. © 2012 American Society of Plant Biologists. All Rights Reserved.Peer Reviewe

Multi-species transcriptome analyses for the regulation of crocins biosynthesis in Crocus

Abstract Background Crocins are soluble apocarotenoids that mainly accumulate in the stigma tissue of Crocus sativus and provide the characteristic red color to saffron spice, in addition to being responsible for many of the medicinal properties of saffron. Crocin biosynthesis and accumulation in saffron is developmentally controlled, and the concentration of crocins increases as the stigma develops. Until now, little has been known about the molecular mechanisms governing crocin biosynthesis and accumulation. This study aimed to identify the first set of gene regulatory processes implicated in apocarotenoid biosynthesis and accumulation. Results A large-scale crocin-mediated RNA-seq analysis was performed on saffron and two other Crocus species at two early developmental stages coincident with the initiation of crocin biosynthesis and accumulation. Pairwise comparison of unigene abundance among the samples identified potential regulatory transcription factors (TFs) involved in crocin biosynthesis and accumulation. We found a total of 131 (up- and downregulated) TFs representing a broad range of TF families in the analyzed transcriptomes; by comparison with the transcriptomes from the same developmental stages from other Crocus species, a total of 11 TF were selected as candidate regulators controlling crocin biosynthesis and accumulation. Conclusions Our study generated gene expression profiles of stigmas at two key developmental stages for apocarotenoid accumulation in three different Crocus species. Differential gene expression analyses allowed the identification of transcription factors that provide evidence of environmental and developmental control of the apocarotenoid biosynthetic pathway at the molecular level

The francoists television newscasts. An investigation on the sources

Introduction. A major drawback of the existing studies on the history of television in Spain under Franco is their failure to analyse direct sources. Objectives. The aim of this research is to analyse TVE´s telediarios (television news bulletins) during the Franco era, using new and specific data. Method. Rather than using them purely as examples, this research makes comprehensive use of the programme schedules found in La Vanguardia, Abc and Tele Radio, as well as TVE’s official broadcast reports. Conclusions. Through the use of these sources, this paper intends to demonstrate the availability of a wide range of resources, on the basis of which there is a need for new critical studies on the history of television in Spain to be carried out.Introducción. Los estudios sobre historia de la televisión en España durante el franquismo no recurren a las fuentes directas. Objetivos. El objetivo de esta investigación es analizar desde datos nuevos y concretos, los telediarios de TVE en el periodo franquista. Método. Para el desarrollo de esta investigación se han empleado de manera exhaustiva, no como mero ejemplo, las parrillas de programación recuperadas de La Vanguardia, Abc y Tele Radio, y los partes de emisión de TVE. Conclusiones. Con el manejo de estas fuentes se pretende mostrar la existencia de una amplia gama de recursos de información accesibles, desde las que es preciso abordar nuevos estudios críticos de historia de la televisión en España

Identification and Cloning of Differentially Expressed SOUL and ELIP Genes in Saffron Stigmas Using a Subtractive Hybridization Approach

<div><p>Using a subtractive hybridization approach, differentially expressed genes involved in the light response in saffron stigmas were identified. Twenty-two differentially expressed transcript-derived fragments were cloned and sequenced. Two of them were highly induced by light and had sequence similarity to early inducible proteins (ELIP) and SOUL heme-binding proteins. Using these sequences, we searched for other family members expressed in saffron stigma. <i>ELIP</i> and <i>SOUL</i> are represented by small gene families in saffron, with four and five members, respectively. The expression of these genes was analyzed during the development of the stigma and in light and dark conditions. <i>ELIP</i> transcripts were detected in all the developmental stages showing much higher expression levels in the developed stigmas of saffron and all were up-regulated by light but at different levels. By contrast, only one <i>SOUL</i> gene was up-regulated by light and was highly expressed in the stigma at anthesis. Both the <i>ELIP</i> and <i>SOUL</i> genes induced by light in saffron stigmas might be associated with the structural changes affecting the chromoplast of the stigma, as a result of light exposure, which promotes the development and increases the number of plastoglobules, specialized in the recruitment of specific proteins, which enables them to act in metabolite synthesis and disposal under changing environmental conditions and developmental stages.</p></div