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The current status of controversy regarding the use of certain preservatives in cosmetic products makes it necessary to seek new ecological alternatives that are free of adverse effects on users. In our study, two different natural terpenes Carvacrol and Eugenol were encapsulated in chitosan nanoparticles in different ratios of Chitosan:terpene. The nanoparticles were characterized by DLS and TEM showing a maximum particle size of 100 nm. The chemical structure, thermal properties, and release profile of terpenes were evaluated showing a successful protection of terpene in Chitosan matrix. Two different release profile were observed showing a faster release profile in the case of Eugenol. Antimicrobial properties of nanoparticles were evaluated against typical microbial contaminants found in cosmetic products, showing higher antimicrobial properties with chitosan encapsulation of terpenes. Furthermore, natural moisturizing cream inoculated with beforementioned microorganisms was formulated with Carvacrol-chitosan nanoparticles and Eugenol-chitosan nanoparticles to evaluate the preservative efficiency, indicating a highest preservative efficiency with the use of Eugenol-chitosan nanoparticles.

[EN] 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., This work was supported by grants BIO2016-77000-R and PIB2020-114761RB-I00 from Ministerio de Ciencia e Innovacion (MCIN), SBPLY/17/180501/000234 and SBPLY/21/180501/000012 from the Junta de Comunidades de Castilla-La Mancha (co-financed European Union FEDER funds) to LG and OA, and SBPLY/17/180501/000229 and SBPLY/21/180501/000064 from the Junta de Comunidades de Castilla-La Mancha (co-financed European Union FEDER funds) to RL and Margarita Salas fellowship from Ministerio de Universidades and Universidad de Castilla-La Mancha to AM-B. JR was supported by the Spanish Ministry of Science and Innovation through a "Juan de la Cierva-Incorporacion" grant (IJC2020-045612-I).

In this study, chitosan-coated biogenic silver nanoparticles (AgNP-CH) were obtained through green chemistry by recycling wheat crop leaf residues. The nanoparticles were characterized by UV-VIS spectroscopy, and total reflectance-Fourier transform infrared spectroscopy confirmed the nanoparticle formation, and the incorporation of chitosan surrounding silver nanoparticles. The size and morphology of nanoparticles were evaluated by microscopy techniques, showing a size range of 2-10 nm, with spherical shape and narrow distribution. The antifungal assay indicated a higher antimicrobial activity showing values of minimum inhibitory concentrations of 41.7 μg/mL against Fusarium oxysporum, and 208.37 μg/mL for Aspergillus niger, A. versicolor and A. brasiliensis. Finally, non-phytotoxic effects were observed in germination assays at early plant stage of development, and an increase in chlorophyll levels were observed at the doses tested with AgNP-CH. Thus, the use of AgNP-CH could be a potential alternative for the prevention of fungal infections in cereals in the early stages of wheat crop development.

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

Crocetin biosynthesis in Buddleja davidii flowers proceeds through a zeaxanthin cleavage pathway catalyzed by two carotenoid cleavage dioxygenases (BdCCD4.1 and BdCCD4.3), followed by oxidation and glucosylation reactions that lead to the production of crocins. We isolated and analyzed the expression of 12 genes from the carotenoid pathway in B. davidii flowers and identified four candidate genes involved in the biosynthesis of crocins (BdALDH, BdUGT74BC1, BdUGT74BC2, and BdUGT94AA3). In addition, we characterized the profile of crocins and their carotenoid precursors, following their accumulation during flower development. Overall, seven different crocins, crocetin, and picrocrocin were identified in this study. The accumulation of these apocarotenoids parallels tissue development, reaching the highest concentration when the flower is fully open. Notably, the pathway was regulated mainly at the transcript level, with expression patterns of a large group of carotenoid precursor and apocarotenoid genes (BdPSY2, BdPDS2, BdZDS, BdLCY2, BdBCH, BdALDH, and BdUGT Genes) mimicking the accumulation of crocins. Finally, we used comparative correlation network analysis to study how the synthesis of these valuable apocarotenoids diverges among B. davidii, Gardenia jasminoides, and Crocus sativus, highlighting distinctive differences which could be the basis of the differential accumulation of crocins in the three species.

Crocins are high-value soluble pigments that are used as colorants and supplements, their presence in nature is extremely limited and, consequently, the high cost of these metabolites hinders their use by other sectors, such as the pharmaceutical and cosmetic industries. The carotenoid cleavage dioxygenase 2L (

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).

[EN] Crocins and picrocrocin are high-value hydrophilic pigments produced in saffron and used commercially in the food and pharmaceutical industries. These apocarotenoids are derived from the oxidative cleavage of zeaxanthin by specific carotenoid cleavage dioxygenases. The pathway for crocins and picrocrocin biosynthesis was introduced into tomato using fruit specific and constitutive promoters and resulted in 14.48 mg/g of crocins and 2.92 mg/g of picrocrocin in the tomato DW, without compromising plant growth. The strategy involved expression of CsCCD2L to produce crocetin dialdehyde and 2,6,6-trimethyl-4-hydroxy-1-carboxaldehyde-1-cyclohexene, and of glycosyltransferases UGT709G1 and CsUGT2 for picrocrocin and crocins production, respectively. Metabolic analyses of the engineered fruits revealed picrocrocin and crocetin-(beta-D-gentiobiosyl)-(beta-D-glucosyl)-ester, as the predominant crocin molecule, as well as safranal, at the expense of the usual tomato carotenoids. The results showed the highest crocins content ever obtained by metabolic engineering in heterologous systems. In addition, the engineered tomatoes showed higher antioxidant capacity and were able to protect against neurological disorders in a Caenorhabditis elegans model of Alzheimer's disease. Therefore, these new developed tomatoes could be exploited as a new platform to produce economically competitive saffron apocarotenoids with health-promoting properties., This work was supported by grants BIO2016-77000-R from the Spanish Ministerio de Ciencia, Innovacion y Uni-versidades and SBPLY/17/180501/000234 from the Junta de Comunidades de Castilla-La Mancha (co-financed European Union FEDER funds) and HARNESSTOM, contract number 101000716 Innovation Action ECH2020-SFS-2020-1. GD and LGG are participants of the European COST action CA15136 (EUROCAROTEN). GD and AG are participants of the European COST action CA18210 (ROXY). LGG is a participant of the CARNET network (BIO2015-71703-REDT and BIO2017-90877-RED). JLR was supported by the Spanish Ministry of Economy and Competitiveness through a "Juan de la CiervaFormacion" grant (FJCI-2016-28601). The UCLM and the IBMCP researchers constitute the Associated Unit of CAROTENOID BIOTECHNOLOGY.

Colored apocarotenoids accumulate at high concentrations in few plant species, where display a role in attraction of pollinators and seed dispersers. Among these apocarotenoids, crocins accumulate at high concentrations in the stigma of saffron and are responsible for the organoleptic and medicinal properties of this spice. Phytoene synthase and Orange protein are key for carotenoid biosynthesis and accumulation. We previously isolated four phytoene synthase genes from saffron with differential roles in carotenoid and apocarotenoid biosynthesis. However, the implications of Orange genes in the regulation of apocarotenoid accumulation are unknown. Here, we have identified two Orange genes from saffron, with different expression patterns. CsOr-a was mainly expressed in vegetative tissues and was induced by light and repressed by heat stress. Both CsOr-a and CsOr-b were expressed in stigmas but showed a different profile during the development of this tissue. The interactions of CsOr-a and CsOr-b were tested with all the four phytoene synthase proteins from saffron and with CsCCD2. None interactions were detected with CCD2 neither with the phytoene synthase 2, involved in apocarotenoid biosynthesis in saffron. The obtained results provide evidence of different mechanisms regulating the phytoene synthase enzymes in saffron by Orange for carotenoid and apocarotenoid accumulation in saffron.

Saffron, a spice derived from the dried red stigmas of Crocus sativus, is one of the oldest natural food additives. The flowers have long red stigmas, which store significant quantities of the glycosylated apocarotenoids crocins and picrocrocin. The apocarotenoid biosynthetic pathway in saffron starts with the oxidative cleavage of zeaxanthin, from which crocins and picrocrocin are derived. In the processed stigmas, picrocrocin is converted to safranal, giving saffron its typical aroma. By a targeted search for differentially expressed uridine diphosphate glycosyltransferases (UGTs) in Crocus transcriptomes, a novel apocarotenoid glucosyltransferase (UGT709G1) from saffron was identified. Biochemical analyses revealed that UGT709G1 showed a high catalytic efficiency toward 2,6,6-trimethyl-4-hydroxy-1-carboxaldehyde-1-cyclohexene (HTCC), making it suited for the biosynthesis of picrocrocin, the precursor of safranal. The role of UGT709G1 in picrocrocin/safranal biosynthesis was supported by the absence or presence of gene expression in a screening for HTCC and picrocrocin production in different Crocus species and by a combined transient expression assay with CsCCD2L in Nicotiana benthamiana leaves. The identification of UGT709G1 completes one of the most highly valued specialized metabolic biosynthetic pathways in plants and provides novel perspectives on the industrial production of picrocrocin to be used as a flavor additive or as a pharmacological constituent.

The main goal of this work was to relate the germinative ecology and genetic variability of Chaerolhyllum aureum L., an Apiaceae plant with dormant, underdeveloped embryos at the time of seed dispersal. We compared the seed germination physiology traits between a relictic geographically isolated Mediterranean population (Iberian System; central Spain) and a population located in the main, colder Atlantic Alpine core (Pyrenees; northern Spain). We analyzed both populations? genetic identity in parallel on the basis of ISSR?PCR analyses. Stratification, gibberellin treatment, and seed storage tests showed that C. aureum seeds have deep complex morphophysiological dormancy. The Iberian System population seeds required shorter cold stratification than the Pyrenees population seeds (16 vs. 20 weeks) to germinate. The genetic analyses suggested closer similarity between Iberian System individuals and those from the Pyrenees requiring shorter cold periods to germinate. Our findings denote a consistent parallelism between germination physiology and genetic identity. The records from these germination and genetic analyses suggested selective pressure due to the climate differences between the two geographical regions. Thus, the individuals in the Iberian System population would have been selected from the original pool to a shorter cold-stratification requiring ecotype.

In this study, garlic essential oil (GEO) has been encapsulated in chitosan nanoparticles (NPCH) with sodium tripolyphosphate (TPP). Fourier transform infrared (FT-IR) spectroscopy, UV–vis spectrophotometry, thermogravimetric analysis (TGA) and X-ray diffraction (XRD) techniques were applied to characterize GEO-NPCH. The obtained nanoparticles exhibited a regular distribution and spherical shape with size range of 200–400 nm as revealed by scanning electron microscopy (SEM). The maximum encapsulation efficiency (EE) and loading capacity (LC) of GEO-loaded chitosan nanoparticles were about 32.8% and 19.8% respectively. Nanoparticle formulations of GEO were found to have antifungal activity against Aspergillus versicolor, A. niger and Fusarium oxysporum. In addition, they showed growth promoting effects by increasing emergence, shoot and root fresh weight on wheat, oat and barley.

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.

Biogenic-Silver nanoparticles emerge as new nanosilver platfmorm that allow to obtain silver nanoparticles via “green chemistry”. In our study we obtained biogenic-Silver nanoparticles from Iris tuberosa leaves extract. Nanoparticles were characterized by UV-vis spectroscopy, dinnamical light scattering technique and Transmission electron microscope, showing spheric and irregular nanoparticles with 5 to 50 nm in diameter. Antioxidant and antimicrobial properties were evaluated against typical microbial contaminants found in cosmetic products, showing high antioxidant and antimicrobial properties. We formulated a natural moisturizing cream with biogenic-silver nanoparticles to evaluate the preservative efficiency trought challenge test, confirming that our nanoparticles are a promising alternative to use as cosmetic preservative.

The current status of controversy regarding the use of certain preservatives in cosmetic products makes it necessary to seek new ecological alternatives that are free of adverse effects on users. In our study, the natural terpene thymoquinone was encapsulated in chitosan nanoparticles. The nanoparticles were characterized by DLS and TEM, showing a particle size of 20 nm. The chemical structure, thermal properties, and release profile of thymoquinone were evaluated and showed a successful stabilization and sustained release of terpenes. The antimicrobial properties of the nanoparticles were evaluated against typical microbial contaminants found in cosmetic products, showing high antimicrobial properties. Furthermore, natural moisturizing cream inoculated with the aforementioned microorganisms was formulated with thymoquinone-chitosan nanoparticles to evaluate the preservative efficiency, indicating its promising use as a preservative in cosmetics.

[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).

Carotenoid cleavage products, apocarotenoids, are biologically active compounds exerting important functions as chromophore, hormones, signaling molecules, volatiles, and pigments. Apocarotenoids are generally synthesized by the carotenoid cleavage dioxygenases (CCDs) that comprise a ubiquitous family of enzymes. The activity of plant CCDs was unraveled more than 20 years ago, with the characterization of the maize VP14, the first identified CCD. The protocol developed to determine the activity of this enzyme in vitro is still being used, with minor modifications. In addition, in vivo procedures have been developed during these years, mainly based on the exploitation of Escherichia coli cells engineered to produce specific carotenoid substrates. Further, technological developments have led to significant improvements, contributing to a more efficient detection of the reaction products. This chapter provides an updated set of detailed protocols suitable for the in vitro and in vivo characterization of the activities of CCDs, starting from well-established methods.

Carotenoid cleavage products, apocarotenoids, are biologically active compounds exerting important functions as chromophore, hormones, signaling molecules, volatiles, and pigments. Apocarotenoids are generally synthesized by the carotenoid cleavage dioxygenases (CCDs) that comprise a ubiquitous family of enzymes. The activity of plant CCDs was unraveled more than 20 years ago, with the characterization of the maize VP14, the first identified CCD. The protocol developed to determine the activity of this enzyme in vitro is still being used, with minor modifications. In addition, in vivo procedures have been developed during these years, mainly based on the exploitation of Escherichia coli cells engineered to produce specific carotenoid substrates. Further, technological developments have led to significant improvements, contributing to a more efficient detection of the reaction products. This chapter provides an updated set of detailed protocols suitable for the in vitro and in vivo characterization of the activities of CCDs, starting from well-established methods.

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. Electronic supplementary material The online version of this article (10.1186/s12864-019-5666-5) contains supplementary material, which is available to authorized users.

The carotenoid cleavage dioxygenase 2, a new member of the CCD family, catalyzes the conversion of zeaxanthin into crocetin-dialdehyde in Crocus. CCD2 is expressed in flowers, being responsible for the yellow, orange and red colorations displayed by tepals and stigma. Three CsCCD2 genes were identified in Crocus sativus, the longest contains ten exons and the shorter is a truncated copy with no introns and which lacks one exon sequence. Analysis of RNA-seq datasets of three developmental stages of saffron stigma allowed the determination of alternative splicing in CsCCD2, being intron retention (IR) the prevalent form of alternative splicing in CsCCD2. Further, high IR was observed in tissues that do not accumulate crocetin. The analysis of one CsCCD2 promoter showed cis-regulatory motifs involved in the response to light, temperature, and circadian regulation. The light and circadian regulation are common elements shared with the previously characterized CsLycB2a promoter, and these shared common cis-acting elements may represent binding sites for transcription factors responsible for co-regulation of these genes during the development of the stigma in saffron. A daily coordinated rhythmic regulation for CsCCD2 and CsLycB2a was observed, with higher levels of mRNA occurring at low temperatures during darkness, confirming the results obtained in the in silico promoter analysis. In addition, to the light and temperature dependent regulation of CsCCD2 expression, the apocarotenoid β-cyclocitral up-regulated CsCCD2 expression and could acts as a mediator of chromoplast-to-nucleus signalling, coordinating the expression of CsCCD2 with the developmental state of the chromoplast in the developing stigma. Electronic supplementary material The online version of this article (doi:10.1007/s11103-016-0473-8) contains supplementary material, which is available to authorized users.

Crocus sativus stigmas are the main source of crocins, which are glucosylated apocarotenoids derived from zeaxanthin cleavage that give saffron its red color. Phytoene synthase (PSY) mediates the first committed step in carotenoid biosynthesis in plants. Four PSY genes encoding functional enzymes were isolated from saffron. All the proteins were localized in plastids, but the expression patterns of each gene, CsPSY1a, CsPSY1b, CsPSY2, and CsPSY3, in different saffron tissues and during the development of the stigma showed different tissue specialization. The CsPSY2 transcript was primarily detected in the stigmas where it activates and stimulates the accumulation of crocins, while its expression was very low in other tissues. In contrast, CsPSY1a and CsPSY1b were mainly expressed in the leaves, but only CsPSY1b showed stresslight regulation. Interestingly, CsPSY1b showed differential expression of two alternative splice variants, which differ in the intron retention at their 50 UTRs, resulting in a reduction in their expression levels. In addition, the CsPSY1a and CsPSY1b transcripts, together with the CsPSY3 transcript, were induced in roots under different stress conditions. The CsPSY3 expression was high in the root tip, and its expression was associated with mycorrhizal colonization and strigolactone production. CsPSY3 formed a separate branch to the stress-specific Poaceae homologs but was closely related to the dicot PSY3 enzymes.

With nearly 140 α-glycosidases in 14 different families, plants are well equipped with enzymes that can break the α-glucosidic bonds in a large diversity of molecules. Here, we introduce activity-based protein profiling (ABPP) of α-glycosidases in plants using α-configured cyclophellitol aziridine probes carrying various fluorophores or biotin. In Arabidopsis (Arabidopsis thaliana), these probes label members of the GH31 family of glycosyl hydrolases, including endoplasmic reticulum-resident α-glucosidase-II Radial Swelling3/Priority for Sweet Life5 (RSW3/PSL5) and Golgi-resident α-mannosidase-II Hybrid Glycosylation1 (HGL1), both of which trim N-glycans on glycoproteins. We detected the active state of extracellular α-glycosidases such as α-xylosidase XYL1, which acts on xyloglucans in the cell wall to promote cell expansion, and α-glucosidase AGLU1, which acts in starch hydrolysis and can suppress fungal invasion. Labeling of α-glycosidases generates pH-dependent signals that can be suppressed by α-glycosidase inhibitors in a broad range of plant species. To demonstrate its use on a nonmodel plant species, we applied ABPP on saffron crocus (Crocus sativus), a cash crop for the production of saffron spice. Using a combination of biotinylated glycosidase probes, we identified and quantified 67 active glycosidases in saffron crocus stigma, of which 10 are differentially active. We also uncovered massive changes in hydrolase activities in the corms upon infection with Fusarium oxysporum using multiplex fluorescence labeling in combination with probes for serine hydrolases and cysteine proteases. These experiments demonstrate the ease with which active α-glycosidases and other hydrolases can be analyzed through ABPP in model and nonmodel plants.

In saffron, the cleavage of zeaxanthin by means of CCD2 generates crocetin dialdehyde, which is then converted by an unknown aldehyde dehydrogenase to crocetin. A proteome from saffron stigma was released recently and, based on the expression pattern and correlation analyses, five aldehyde dehydrogenases (ALDHs) were suggested as possible candidates to generate crocetin from crocetin dialdehydes. We selected four of the suggested ALDHs and analyzed their expression in different tissues, determined their activity over crocetin dialdehyde, and performed structure modeling and docking calculation to find their specificity. All the ALDHs were able to convert crocetin dialdehyde to crocetin, but two of them were stigma tissue-specific. Structure modeling and docking analyses revealed that, in all cases, there was a high coverage of residues in the models. All of them showed a very close conformation, indicated by the low root-mean-square deviation (RMSD) values of backbone atoms, which indicate a high similarity among them. However, low affinity between the enzymes and the crocetin dialdehyde were observed. Phylogenetic analysis and binding affinities calculations, including some ALDHs from Gardenia jasmonoides, Crocus sieberi, and Buddleja species that accumulate crocetin and Bixa orellana synthetizing the apocarotenoid bixin selected on their expression pattern matching with the accumulation of either crocins or bixin, pointed out that family 2 C4 members might be involved in the conversion of crocetin dialdehyde to crocetin with high specificity.

The apocarotenoid crocetin and its glycosylated derivatives, crocins, confer the red colour to saffron. Crocetin biosynthesis in saffron is catalysed by the carotenoid cleavage dioxygenase CCD2 (AIG94929). No homologues have been identified in other plant species due to the very limited presence of crocetin and its derivatives in the plant kingdom. Spring Crocus species with yellow flowers accumulate crocins in the stigma and tepals. Four carotenoid CCDs, namely CaCCD1, CaCCD2 and CaCCD4a/b and CaCCD4c were first cloned and characterized. CaCCD2 was localized in plastids, and a longer CCD2 version, CsCCD2L, was also localized in this compartment. The activity of CaCCD2 was assessed in Escherichia coli and in a stable rice gene function characterization system, demonstrating the production of crocetin in both systems. The expression of all isolated CCDs was evaluated in stigma and tepals at three key developmental stages in relation with apocarotenoid accumulation. CaCCD2 expression parallels crocin accumulation, but C14 apocarotenoids most likely are associated to the CaCCD1 activity in Crocus ancyrensis flowers. The specific CCD2 localization and its membrane interaction will contribute to the development of a better understanding of the mechanism of crocetin biosynthesis and regulation in the chromoplast.

Crocins, the red soluble apocarotenoids of saffron, accumulate in the flowers of Crocus species in a developmental and tissue-specific manner. In Crocus sieberi, crocins accumulate in stigmas but also in a distinct yellow tepal sector, which we demonstrate contains chromoplast converted from amyloplasts. Secondary metabolites were analysed by LC-DAD-HRMS, revealing the progressive accumulation of crocetin and crocins in the yellow sector, which were also localized in situ by Raman microspectroscopy. To understand the underlying mechanisms of crocin biosynthesis, we sequenced the C. sieberi tepal transcriptome of two differentially pigmented sectors (yellow and white) at two developmental stages (6 and 8) by Illumina sequencing. A total of 154 million high-quality reads were generated and assembled into 248,099 transcripts. Differentially expressed gene analysis resulted in the identification of several potential candidate genes involved in crocin metabolism and regulation. The results provide a first profile of the molecular events related to the dynamics of crocetin and crocin accumulation during tepal development, and present new information concerning apocarotenoid biosynthesis regulators and their accumulation in Crocus. Further, reveals genes that were previously unknown to affect crocin formation, which could be used to improve crocin accumulation in Crocus plants and the commercial quality of saffron spice.

This study set out to determine the distribution of sulfur compounds and saponin metabolites in different parts of garlic cloves. Three fractions from purple and white garlic ecotypes were obtained: the tunic (SS), internal (IS) and external (ES) parts of the clove. Liquid Chromatography coupled to High Resolution Mass spectrometry (LC-HRMS), together with bioinformatics including Principal Component Analysis (PCA), Hierarchical Clustering (HCL) and correlation network analyses were carried out. Results showed that the distribution of these metabolites in the different parts of garlic bulbs was different for the purple and the white ecotypes, with the main difference being a slightly higher number of sulfur compounds in purple garlic. The SS fraction in purple garlic had a higher content of sulfur metabolites, while the ES in white garlic was more enriched by these compounds. The correlation network indicated that diallyl disulfide was the most relevant metabolite with regards to sulfur compound metabolism in garlic. The total number of saponins was almost 40-fold higher in purple garlic than in the white variety, with ES having the highest content. Interestingly, five saponins including desgalactotigonin-rhamnose, proto-desgalactotigonin, proto-desgalactotigonin-rhamnose, voghieroside D1, sativoside B1-rhamnose and sativoside R1 were exclusive to the purple variety. Data obtained from saponin analyses revealed a very different network between white and purple garlic, thus suggesting a very robust and tight coregulation of saponin metabolism in garlic. Findings in this study point to the possibility of using tunics from purple garlic in the food and medical industries, since it contains many functional compounds which can be exploited as ingredients.

a b s t r a c t Crocus sativus is a triploid sterile plant and autumnal flowering geophyte with corms. As a subterranean organ, the corm is susceptible to diseases caused by fungi, bacteria, nematodes and viruses. Fusarium corm rot incited by F. oxysporum is the most destructive disease in saffron, having caused severe yield losses in saffron producer's countries. Infected plants die off early, resulting in reduction of corm yield, quality and flower and stigma production. Little is known about the formae speciales attacking saffron and less about the relationship among the fungus and the possible hosts. In this study we investigate the formae speciales which colonize saffron and their relationship with some members of iridaceae such as C. vernus (ornamental crocus), gladiolus and narcissus using pathogenicity tests; we determine whether different pathogenic isolates of F. oxysporum can be distinguished by ISSR, and we analyze the genetic relationships and variability among some isolates of these pathogens. We found two formae speciales iridiacearum and croci which attack different iridaceous crops and Crocus sp., respectively and we suggested the creation of a new formae speciales saffrani which shows only pathogenicity on saffron corms. From ISSR analysis, the unweighted paired group method with arithmetic averages cluster analysis (UPGMA) was used to discriminate the F. oxysporum isolates, we were able to differentiate among f. sp. from saffron corms and other formae speciales of F. oxysporum but not among formae speciales iridiacearum, croci and saffrani. Furthermore, we searched for fum1, tri5, tri7 and tri13 genes using PCR assays, however, all the isolates from saffron corms were negative.

Glycosylation plays a major role in the chemical diversity of flavonoids. The wide diversity of the family-1 glycosyltransferase (UGT) impairs the determination of the biochemical function solely from its primary sequence. Here we combined differential expression and target metabolomic analysis in various Crocus species to identify a gene that is key in determining the flavonoid composition of Crocus species that belong to the Crocus series. UGT703B1 recognizes isorhamnetin and kaempferol as substrates in vitro. In addition, UGT703B1 expression was found to be highly correlated with the presence of kaempferol 7-O-biglucoside-3-O-β-glucoside and isorhamnetin-3,7-O-diglucoside. These flavonols were present in C. sativus and C. cartwrightianus albus, both from series Crocus but absent in Crocus species from the other series analyzed. Further, the presence of both flavonols was associated with the expression of UGT703B1, and this expression was correlated with the presence of the UGT703B1 coding gene, with the exception of C. cancellatus, whose genomic sequence was present but contained a shorter intronic sequence and promoter alterations, suggesting the presence of regulatory sequences in the deleted part of that intron and promoter important for UGT703B1 expression. Overall, the data obtained supports the involvement of UGT703B1 in the formation of specific kaempferol and isorhamnetin glucosides, while demonstrating that the integration of metabolomic and differential expression analysis is a versatile tool for understanding a multigene family of UGTs in Crocus.

Ethnopharmacological relevance The Helianthemum genus contains approximately one hundred taxa. Some of them are important medicinal plants used in several countries for many different purposes. However, studies addressing the phytochemistry of many of these species or their biological activities are currently nonexistent. Aim of the study To perform a comparative analysis of the qualitative composition of secondary metabolites and biological activities of the leaves of the most commonly distributed and used Spanish Helianthemum taxa in order to find a relationship between chemotype, biological activity and uses. Materials and methods water and 80% methanolic extracts derived from 11 different Helianthemum taxa were analyzed for their phytochemical composition using standard methods. Furthermore, HPLC analysis was performed for the estimation of gallic acid, ellagic acid, tannins and flavonols as marker compounds. The antioxidant activity was measured by employing the scavenging activity on DPPH (2,2-diphenyl-1-picrylhydrazyl) and ABTS (2,2′-azinobis-3-ethylbenzothiazoline-6-sulfonate) radicals. The 80% methanolic extracts were evaluated for antibacterial (Gram-positive Staphylococcus aureus , Enterococcus faecalis and Listeria monocytogenes and Gram-negative Escherichia coli and Salmonella enterica ) activity using the micro dilution technique. Results The total polyphenolic content and antioxidant capabilities differed significantly among the Helianthemum leaf extracts. A strong correlation between total phenolic content, antioxidant properties and antimicrobial activity was found. The polyphenolic profile was specific for each taxon. Whereas Helianthemum alypoides , Helianthemum cinereum subsp. rotundifolium , Helianthemum hirtum , Helianthemum asperum , and Helianthemum marifolium subsp. marifolium were characterized by the presence of gallic acid, egallic derivatives and ellagitannins; the polyphenolic profile of Helianthemum apenninum subsp. c avanillesianum , Helianthemum syriacum and Helianthemum polygonoides was mostly based on flavonoids. Helianthemum cinereum , Helianthemum alypoides and Helianthemum marifolium consistently presented the strongest radical scavenging activity (in water extracts EC 50 ranges from 29.88 to 38.1 against DPPH and from 8.11 to 20.8 against ABTS, and in 80% MeOH extracts from 25.3 to 31.8 against DPPH and from 6.15 to 8.6 against ABTS), total phenol content (>117 mg GAE/l) and antimicrobial activities. Conclusion The Helianthemum taxa used in folk medicine did not cluster in a unique section, being equally distributed in two out of the four sections analysed. There was not a clear relationship between the chemotype, based on the polyphenolic composition of the taxa, and their taxonomical classification. However, the composition of the methanolic and water extracts from the leaves of plants belonging to the Helianthemum genus was strongly related to their medicinal uses.

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. ELIP and SOUL 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. ELIP 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 SOUL gene was up-regulated by light and was highly expressed in the stigma at anthesis. Both the ELIP and SOUL 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.

Anthillys rupestris Coss. is a rare, endangered and endemic plant from the south of the Iberian Peninsula. The phylogenetic relationships of A. rupestris and its genetic variation were studied to assess the distribution of genetic variability within and among six subpopulations from two remnants locations. We obtained sequences of internal transcribed spacer ITS2 of three Anthyllis species and ISSRs markers to estimate gene diversity and the genetic structure of the subpopulations. A. rupestris was closely related to two other endemic species to southeastern Spain. The ISSR analyses revealed high level of genetic diversity [61.59% average polymorphism; and H T = 0.269] in subpopulations and a moderate level of genetic differentiation [( G ST ) = 0.267] among subpopulations. Furthermore, analysis of molecular variance (AMOVA) revealed that 18% of the total genetic diversity resided among the two analysed areas, 12% among subpopulations and 70% variation was found within subpopulations. Principal Coordinates Analysis (PCA) partially grouped together the genotypes according to their geographical origin. Three out of the four subpopulations from one location were genetically related to each other, and the remaining subpopulation, which was fenced, was isolated from the obtained clusters. The data are interpreted as a result of recent fragmentation and isolation. Threats to this species are mainly anthropogenic. Because A. rupestris currently retains a high genetic variation among individuals within subpopulations, a metapopulation perspective preserving and expanding the habitat at each site, in order to promote connectivity would be an additional strategy to ensure conservation before populations become too small to persist naturally.

Eight Pinus nigra Arn. populations from Southern Spain and Northern Morocco were examined using inter-simple sequence repeat markers to characterize the genetic variability amongst populations. Pair-wise population genetic distance ranged from 0.031 to 0.283, with a mean of 0.150 between populations. The highest inter-population average distance was between PaCU from Cuenca and YeCA from Cazorla, while the lowest distance was between TaMO from Morocco and MA Sierra Mágina populations. Analysis of molecular variance (AMOVA) and Nei’s genetic diversity analyses revealed higher genetic variation within the same population than among different populations. Genetic differentiation (Gst) was 0.233. Cuenca showed the highest Nei’s genetic diversity followed by the Moroccan region, Sierra Mágina, and Cazorla region. However, clustering of populations was not in accordance with their geographical locations. Principal component analysis showed the presence of two major groups—Group 1 contained all populations from Cuenca while Group 2 contained populations from Cazorla, Sierra Mágina and Morocco—while Bayesian analysis revealed the presence of three clusters. The low genetic diversity observed in PaCU and YeCA is probably a consequence of inappropriate management since no estimation of genetic variability was performed before the silvicultural treatments. Data indicates that the inter-simple sequence repeat (ISSR) method is sufficiently informative and powerful to assess genetic variability among populations of P. nigra.

Plants have developed many mechanisms to protect themselves against most potential microbial pathogens and diseases. Among these mechanisms, pathogenesis-related proteins are produced as part of the active defence to prevent attack. In this study, a full-length cDNA encoding the CsPR10 protein was identified in fresh saffron stigmas (Crocus sativus). The deduced amino acid sequence from the nucleotide sequence of the coding region showed homology with PR10 proteins. The clone expressed as a protein in fusion with a GST tag produced a 47-kDa protein in E. coli. CsPR10 had ribonuclease activity, with features common to class II-type ribonucleases; its specific activity was quantified as 68.8 U·mg(-1) protein, thus falling within the range of most PR10 proteins exhibiting RNase activity. Antifungal activity of CsPR10 was assayed against Verticillium dahliae, Penicillium sp. and Fusarium oxysporum. CsPR10 inhibited only F. oxysporum growth, and antifungal potency was reflected in a IC(50) of 8.3 μm. Expression analysis showed the presence of high transcript levels in anther and tepal tissues, low levels in stigmas and roots, and no signal detected in leaves. This protein seems to be involved in the active defence response through activation of the jasmonic acid pathway.

The Crocus genus, which comprehends approximately 80 species, is known mainly for the triploid sterile plant C. sativus, whose dried stigmas provide us with the spice called saffron. Among the species belonging to the C. sativus L. aggregate, C. cartwrightianus cv. albus shares morphological and cytological characters with both C. sativus and C. cartwrightianus, although its positions seem to be uncertain. Due to the complexity of the genus, few studies have revised the classification proposed by Mathew in 1982. In the present study, intersimple sequence repeat (ISSR) and HPLC–ESI-MS analyses have been used in an attempt to clarify the taxonomical position of cv. albus. No differences were found between C. sativus and C. cartwrightianus cv. albus using 12 ISSR primers. Results on apocarotenoid contents from HPLC analysis showed some quantitative differences. According to our findings, C. cartwrightianus cv. albus is more related to C. sativus than to C. cartwrightianus and may be an albinic saffron. These data agree with other authors who reached the same conclusion using different techniques. The results obtained in this work may be used in improving programme of saffron spice.

Flowering and flower formation is a major developmental switch in plants where different genes are expressed in a coordinate manner for reproductive success. Among these genes MADS domain transcription factors and FLOWER LOCUS T like genes play important roles in various developmental processes in flowering plants, from transition to flowering to the specification of floral organ identity. For uncovering and understanding the molecular mechanisms controlling flowering, flower development and flower formation in cultivated Crocus sativus L., we present here the isolation and characterization of a series of flowering genes. Furthermore, based on the isolated genomic sequences of these genes, we have developed and used three SSR molecular markers located at the 5'-UTR of isolated MADS box genes in order to analyze 11 Crocus accessions from different countries.

Crocus sativus is a triploid sterile plant characterized by its long red stigmas, which produce and store significant quantities of carotenoid derivatives formed from the oxidative cleavage of beta-carotene and zeaxanthin. The present study reports on the genomic structures of two lycopene-beta-cyclase genes, CstLcyB1 and CstLcyB2a, and on their transcription patterns in different C. sativus tissues. Phylogenetic analysis showed that both proteins are located in different groups: CstLcyB2a encodes chromoplast-specific lycopene cyclases, with an expression analysis showing strongly in flower stigmas where it activates and boosts beta-carotene accumulation. The CstLcyB1 transcript, however, was present in leaves, tepals, and stigmas at lower levels. In vivo assays in transgenic Arabidopsis demonstrated lycopene beta-cyclase activity of CstLcyB2a. CstLcyB2a is a CstLcyB1 paralogue derived through a gene duplication event, while promoter analysis showed that both genes have diverged in their regulatory sequences after duplication. Furthermore, it was found that the CstLcyB2a gene was absent from Crocus kotschyanus and, although present in C. goulimyi and C. cancellatus, the absence of transcripts suggests that transcriptional regulation of CstLcyB2a is responsible for the low apocarotenoid content in these species.

Saffron, the desiccated stigmas of Crocus sativus, is highly appreciated for its peculiar colour, flavour and aroma. Several studies have been conducted with the spice, but little is known about the evolution of volatile and non-volatile compounds generated during the development of the stigma. In this study, we have followed these compounds, with special attention to those of isoprenoid origin (carotenoids and monoterpenes), which are responsible for the organoleptic properties of saffron. The main compounds that accumulated throughout stigma development in C. sativus were crocetin, its glucoside derivatives and picrocrocin, all of which increased as stigmas reached a fully developed stage. The volatile composition of C. sativus stigmas changed notably as stigmas developed with each developmental stage being characterized by a different volatile combination. In red stigmas, beta-cyclocitral, the 7,8 cleavage product of beta-carotene, was highly produced, suggesting the implication of both beta-carotene and zeaxanthin in crocetin formation. As stigmas matured, hydroxy-beta-ionone and beta-ionone were produced while safranal, the most typical aroma compound of the processed spice, was only detected at low levels. However, a safranal-related compound 2,2,2-trimethyl-2-cyclohexene-1,4-dione (4-oxoisophorone) increased rapidly at the anthesis stage and also in senescent stigmas. Monoterpenes were mainly emitted at the time of anthesis and the emission patterns followed the expression patterns of two putative terpene synthases CsTS1 and CsTS2. Fatty acid derivates, which predominated at the earlier developmental stages, were observed at low levels in later stages.

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.

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., This work was supported by the Ibero-American network for the study of carotenoids as food ingredients (IBERCAROT, CYTED 112RT0445), the Spanish Government, Ministry of Science and Technology (projects AGL2015-70218, BIO2013-44239-R, AGL2012-34573, BIO2012-39716, and BIO2009-11131), the Andalusian Government (projects P07-CVI-02813 and CTS-6638), and the Generalitat Valenciana (PROMETEOII/2014/027). We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI)

The alkali-extractable water-soluble polysaccharides F1SS isolated from the cell wall of four species of Discula destructiva have been studied by methylation analysis and NMR spectroscopy, and their idealized structures established as [structure: see text] where n approximately 2 for strains CBS 109771 and CBS 133.91, n approximately 1 for CBS 132.91, and it has an intermediate value in strain CBS 130.91. The mannan core was obtained by mild hydrolysis of the F1SS polysaccharide and its structure consisted of a skeleton of alpha-(1--6)-mannopyranan, with around one out of eleven residues substituted at C-2 by short chains (one to six units) of 2-substituted mannopyranoses.

Background: Peach allergy has two different patterns: central Europe with oral allergy syndrome (OAS) related to a primary sensitization to birch pollen Bet v 1 and profilins and southern Europe with mostly systemic symptoms, in many cases due to sensitization to lipid-transfer proteins. Methods: Thirty peach-allergic patients with positive skin and food challenge tests and 29 control subjects were included. Skin prick tests (SPT) with inhalant allergens, commercial peach and apple extracts and native Pru p 3 were performed. In vitro specific immunoglobulin (Ig) E to grass pollen, birch pollen, peach, apple, rBet v 1, rBet v 2 and rPhl p 12 was determined by CAP, and rBet v 1, rMal d 1, rMal d 4, rMal d 3 and rPru p 3 using the ADVIA-Centaur platform. Basophil activation test (BAT) with commercial peach extract, commercial apple extract, nPru p 3, rMal d 3, rMal d 1 and rMal d 4 was also performed. Results: Pru p 3 was the major allergen in the patient group from northern Spain. Sensitization to this allergen was found in 100% of the patients with systemic symptoms or contact urticaria. Only 60% of OAS patients were sensitized to Pru p 3, being all of them sensitized to profilins and 60% of them to allergens of the Bet v 1 family. Specific IgE determination and BAT using recombinant allergens (rPru p 3) show specificity and sensitivity values close to 100%. Conclusions: Most peach-allergic patients coming from the north of Spain present systemic symptoms after ingestion of peach, Pru p 3 being the main allergen. Patients with OAS present profilin-Bet v 1-related sensitization. Thus, in the north of Spain our patients show a mixed central-south Europe pattern with LTP-profilin-Bet v 1 sensitization depending on the symptoms presented. The use of natural and recombinant plant allergens, allows establishing the sensitization patterns to the different allergens studied.

We report on a patient with anaphylaxis from mandarin. Temporal relationship between consumption of the fruit, the presence of positive specific IgE, the positive skin test and the basophil activation test for mandarin strongly supported the diagnosis of an IgE-mediated allergy from mandarin. The lipid transfer protein allergen from mandarin fruit was isolated and characterized. Specific IgE levels and IgE immunodetection data indicated the patient’s sensitization to orange (Cit s 3) and mandarin (Cit r 3) lipid transfer protein allergens, as well as to germin-like (Cit s 1) allergen. These results were fully confirmed by skin prick test and basophil activation test (BAT) for lipid transfer proteins, and a BAT for Cit s 1. This case report has several particularities. First, in Central and Northern Europe, it is not widely appreciated that citrus fruits, particularly mandarin, can elicit anaphylaxis. Second, this case report re-emphasizes sensitization from lipid transfer proteins to predispose for severe allergic reactions. Finally, it provides an opportunity to summarize the applications of flow cytometry-assisted analysis and quantification of in vitro activated basophils in the diagnostic approach of anaphylaxis from food.

The management of maritime pine in fire-prone habitats is a challenging task and fine-scale population genetic analyses are necessary to check if different fire recurrences affect genetic variability. The objective of this study was to assess the effect of fire recurrence on maritime pine genetic diversity using inter-simple sequence repeat markers (ISSR). Three maritime pine ( Pinus pinaster Ait.) populations from Northern Portugal were chosen to characterize the genetic variability among populations. In relation to fire recurrence, Seiros population was affected by fire both in 1990 and 2005 whereas Vila Seca-2 population was affected by fire just in 2005. The Vila Seca-1 population has been never affected by fire. Our results showed the highest Nei's genetic diversity (He = 0.320), Shannon information index (I = 0.474) and polymorphic loci (PPL = 87.79%) among samples from twice burned populations (Seiros site). Thus, fire regime plays an important role affecting genetic diversity in the short-term, although not generating maritime pine genetic erosion.

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.

The IgE of sera from patients with a history of allergy to oranges (Citrus sinensis) bind a number of proteins in orange extract, including Cit s 1, a germin-like protein. In the present study, we have analysed its immunological cross-reactivity and its molecular nature. Sera from many of the patients examined recognise a range of glycoproteins and neoglycoconjugates containing β1,2-xylose and core α1,3-fucose on their N-glycans. These reagents also inhibited the interaction of Cit s 1 with patients’ sera, thus underlining the critical role of glycosylation in the recognition of this protein by patients’ IgE and extending previous data showing that deglycosylated Cit s 1 does not possess IgE epitopes. In parallel, we examined the peptide sequence and glycan structure of Cit s 1 using mass spectrometric techniques. Indeed, we achieved complete sequence coverage of the mature protein as compared to the translation of an expressed sequence tag cDNA clone and demonstrated that the single N-glycosylation site of this protein carries oligosaccharides with xylose and fucose residues. Due to the presumed requirement for multivalency for in vivo allergenicity, our molecular data showing that Cit s 1 is monovalent as regards glycosylation and that the single N-glycan is the target of the IgE response to this protein, therefore, explain the immunological cross-reactive properties of Cit s 1 as well as its equivocal nature as a clinically-relevant allergen.

Background Food IgE-mediated allergy to members of the Brassicaceae family has been increasingly reported. Objective To characterize cabbage— Brassica oleracea var capitata —allergy and its major allergens. Methods A prospective study was performed, recruiting 17 patients allergic to cabbage, and control subjects. Skin prick tests and double-blind placebo-controlled food challenges were performed. A major allergen was isolated from cabbage by RP-HPLC and characterized by N-terminal amino acid sequencing and matrix-assisted laser desorption/ionization mass spectrometry analysis. Specific IgE determinations, IgE immunoblots, and CAP-inhibition assays were also performed. Results Skin prick test and specific IgE were positive to cabbage in all patients. Five of them referred anaphylactic reactions when eating cabbage, and in another 5 patients, cabbage allergy was further confirmed by double-blind placebo-controlled food challenge. Most of them showed associated sensitizations to mugwort pollen, mustard, and peach. A 9-kd cabbage IgE-binding protein, Bra o 3, was identified as a lipid transfer protein (LTP) with 50% of identity to peach LTP Pru p 3. Skin prick test with Bra o 3 showed positive results in 12 of 14 cases (86%). On CAP inhibition assays, Bra o 3 managed to inhibit significantly the IgE binding to cabbage, mugwort pollen, and peach. Both Bra o 3 and Pru p 3 were recognized by IgE from the patients' sera. Conclusion Bra o 3, a cabbage LTP, is a major allergen in this food, cross-reacting with mugwort pollen and with other plant foods, such as peach. Clinical implications Cabbage IgE-mediated allergy is a potentially severe condition that can present with other plant food and pollen allergies.

Background: Orange is among the most widely consumed fruits, and among the plant food sources causing allergic reactions according to popular perception. However, its relevant allergenic components are virtually unknown. Profilin is a well-defined minor plant panallergen, showing prevalences around 30% in fruits and vegetables. Methods: Twenty-three orange-allergic patients were studied. Natural orange profilin, named Cit s 2, was purified by affinity chromatography and characterized by N-terminal amino acid sequencing, matrix-assisted laser desorption/ionization mass spectrometry analysis and isolation of its coding cDNA. Reactivity to Cit s 2 was analyzed in vivo by skin prick tests (SPT) and in vitro by IgE immunodetection, specific IgE determination in individual sera and enzyme-linked immunosorbent assay-inhibition assays. Results: The N-terminal amino acid sequence and molecular mass of natural Cit s 2, both fully in agreement with the complete amino acid sequence deduced from its coding cDNA, demonstrated its profilin nature. An unexpectedly high reactivity to Cit s 2 was found in vivo (78% of positive SPT responses) and in vitro (87% of sera from orange allergic patients had specific IgE to Cit s 2). The purified allergen inhibited around 50% of the IgE binding to an orange pulp extract. Conclusion: Orange profilin Cit s 2, unlike other plant food profilins, is a major and highly prevalent allergen.