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

Saffron is a monomorphic species as revealed by RAPD, ISSR and microsatellite analyses

<p>Abstract</p> <p>Background</p> <p>Saffron (<it>Crocus sativus</it>) is considered the world's most expensive spice. Used mainly as a colorant for foodstuffs, it is highly appreciated for its aromatic and flavouring properties. Since no molecular markers for this species have been found in the literature, the objective of this study was to determine whether phenotypical differences found in <it>C. sativus </it>were supported by molecular analyses.</p> <p>Findings</p> <p>Thirty primers from Operon Technologies were used in random amplified polymorphic DNA (RAPD) analysis, forty eight primers were screened using intersimple sequence repeats (ISSR) method and fifteen primers derived from a microsatellites library flanking sequences with repeat motifs were assayed in forty three isolates of <it>C. sativus </it>from eleven different countries and a <it>C. kotschyanus </it>isolate was used as outgroup. No polymorphic bands were detected in any of the accessions combining the different approaches used in this study.</p> <p>Conclusion</p> <p>According to our findings, all accessions appear identical clones, not only because morphological characters but also at a molecular level. These data strongly suggested that <it>C. sativus </it>is a monomorphic species. Thus, genome sequencing is needed to find molecular markers for saffron.</p

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

[EN] 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 (Airen 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 beta-cyclocitral or beta-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 alpha-carotene were only detected in Airen 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 Airen, confirming the enriched aroma bouquet characteristic of the white varieties.We thank J. Argandona (Institute Botanico, Universidad de Castilla-La Mancha, Albacete, Spain) for excellent technical support, and K.A. Walsh for language revision. 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.Rambla Nebot, JL.; Trapero-Mozos, A.; Diretto, G.; Rubio-Moraga, A.; Granell Richart, A.; Gomez-Gomez, L.; Ahrazem, O. (2016). Gene-Metabolite Networks of Volatile Metabolism in Airen and Tempranillo Grape Cultivars Revealed a Distinct Mechanism of Aroma Bouquet Production. Frontiers in Plant Science. 7(1619):1-23. https://doi.org/10.3389/fpls.2016.01619S1237161

The Biosynthesis of Non-Endogenous Apocarotenoids in Transgenic Nicotiana glauca

Crocins are high-value compounds with industrial and food applications. Saffron is currently the main source of these soluble pigments, but its high market price hinders its use by sectors, such as pharmaceutics. Enzymes involved in the production of these compounds have been identified in saffron, Buddleja, and gardenia. In this study, the enzyme from Buddleja, BdCCD4.1, was constitutively expressed in Nicotiana glauca, a tobacco species with carotenoid-pigmented petals. The transgenic lines produced significant levels of crocins in their leaves and petals. However, the accumulation of crocins was, in general, higher in the leaves than in the petals, reaching almost 302 µg/g DW. The production of crocins was associated with decreased levels of endogenous carotenoids, mainly β-carotene. The stability of crocins in leaf and petal tissues was evaluated after three years of storage, showing an average reduction of 58.06 ± 2.20% in the petals, and 78.37 ± 5.08% in the leaves. This study illustrates the use of BdCCD4.1 as an effective tool for crocin production in N. glauca and how the tissue has an important impact on the stability of produced high-value metabolites during storage.This work was supported by grants BIO2016-77000-R from the Spanish Ministerio de Ciencia; Innovación y Universidades and SBPLY/17/180501/000234 from the Junta de Comunidades de Castilla-La Mancha (co-financed European Union FEDER funds); the National Natural Science Foundation of China (31870278); and the Spanish Ministry of Economy and Competitiveness (MINECO), Spain (RTI2018–097613-B-I00; PGC2018–097655-B-I00). C.Z. and L.G.G. are participants of the European COST action CA15136 (EUROCAROTEN) and Programa Estatal de Investigación Científica y Técnica de excelencia, Spain (BIO2015–71703-REDT and BIO2017–90877-REDT)

Efficient production of saffron crocins and picrocrocin in Nicotiana benthamiana using a virus-driven system

[EN] Crocins and picrocrocin are glycosylated apocarotenoids responsible, respectively, for the color and the unique taste of the saffron spice, known as red gold due to its high price. Several studies have also shown the health-promoting properties of these compounds. However, their high costs hamper the wide use of these metabolites in the pharmaceutical sector. We have developed a virus-driven system to produce remarkable amounts of crocins and picrocrocin in adult Nicotiana benthamiana plants in only two weeks. The system consists of viral clones derived from tobacco etch potyvirus that express specific carotenoid cleavage dioxygenase (CCD) enzymes from Crocus sativus and Buddleja davidii. Metabolic analyses of infected tissues demonstrated that the sole virus driven expression of C. sativus CsCCD2L or B. davidii BdCCD4.1 resulted in the production of crocins, picrocrocin and safranal. Using the recombinant virus that expressed CsCCD2L, accumulations of 0.2% of crocins and 0.8% of picrocrocin in leaf dry weight were reached in only two weeks. In an attempt to improve apocarotenoid content in N. benthamiana, co-expression of CsCCD2L with other carotenogenic enzymes, such as Pantoea ananatis phytoene synthase (PaCrtB) and saffron beta-carotene hydroxylase 2 (BCH2), was performed using the same viral system. This combinatorial approach led to an additional crocin increase up to 0.35% in leaves in which CsCCD2L and PaCrtB were co-expressed. Considering that saffron apocarotenoids are costly harvested from flower stigma once a year, and that Buddleja spp. flowers accumulate lower amounts, this system may be an attractive alternative for the sustainable production of these appreciated metabolites.We thank K. Schreiber and C. Mares (IBMCP, CSIC-UPV, Valencia, Spain) for technical assistance during plant transformation. We thank M. Gasc.on and M.D. G.omez-Jim.enez (IBMCP, CSIC-UPV, Valencia, Spain) for helpful assistance with LSCM analyses. We thank D. Dubbala (IBMCP, CSIC-UPV, Valencia, Spain) for English revision. This work was supported by grants BIO2016-77000-R and BIO2017-83184-R from the Spanish Ministerio de Ciencia e Innovacion (co-financed European Union ERDF), and SBPLY/17/180501/000234 from Junta de Comunidades de Castilla-La Mancha. M.M. was the recipient of a predoctoral fellowship from the Spanish Ministerio de Educacion, Cultura y Deporte (FPU16/05294). G.D. and L.G.G. are participants of the European COST action CA15136 (EUROCAROTEN). L.G.G. is a participant of the CARNET network (BIO2015-71703-REDT and BIO2017-90877-RED).Martí, M.; Diretto, G.; Aragones, V.; Frusciante, S.; Ahrazem, O.; Gómez-Gómez, L.; Daròs, J. (2020). Efficient production of saffron crocins and picrocrocin in Nicotiana benthamiana using a virus-driven system. 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Carotenoid Cleavage Oxygenases from Microbes and Photosynthetic Organisms: Features and Functions

Apocarotenoids are carotenoid-derived compounds widespread in all major taxonomic groups, where they play important roles in different physiological processes. In addition, apocarotenoids include compounds with high economic value in food and cosmetics industries. Apocarotenoid biosynthesis starts with the action of carotenoid cleavage dioxygenases (CCDs), a family of non-heme iron enzymes that catalyze the oxidative cleavage of carbon–carbon double bonds in carotenoid backbones through a similar molecular mechanism, generating aldehyde or ketone groups in the cleaving ends. From the identification of the first CCD enzyme in plants, an increasing number of CCDs have been identified in many other species, including microorganisms, proving to be a ubiquitously distributed and evolutionarily conserved enzymatic family. This review focuses on CCDs from plants, algae, fungi, and bacteria, describing recent progress in their functions and regulatory mechanisms in relation to the different roles played by the apocarotenoids in these organisms.Ministerio de Ciencia y Tecnología AGL2015-70218, BIO2013-44239-R, AGL2012-34573, BIO2012-39716 y BIO2009-11131Junta de Andalucía P07-CVI-02813 y CTS-6638Generalitat Valenciana PROMETEOII/2014/02

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

Ex-situ genetic conservation of Pinus nigra salzmannii endangered populations from the Spanish Central Range: abstract

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