Search

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.

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.

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 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.

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.

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.

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.

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.

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.

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.

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: 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.

Background: Lipid transfer proteins (LTPs) are relevant fruit allergens, recently proposed as model plant food allergens. No citrus fruit allergen has been characterized to date. We sought to identify and isolate citrus fruit LTPs and to explore their relevance in orange allergy. Methods: Twenty-seven patients, showing mainly oral allergy syndrome after orange ingestion, as well as positive prick responses and serum-specific IgE levels to orange, were selected. Natural orange and lemon LTPs, as well as a recombinant orange LTP isoform expressed in Pichia pastoris, were isolated by chromatographic methods and characterized by N-terminal amino acid sequencing and matrix-assisted laser desorption/ionizaion mass spectrometry, and DNA sequencing of the corresponding cDNA in the case of the recombinant allergen. Specific IgE determination, immunodetection, ELISA-inhibition assays and in vivo skin prick tests (SPTs) were performed with all three purified allergens and with the major peach LTP allergen, Pru p 3. Results: The natural allergens purified from orange (nCit s 3) and lemon (nCit l 3) showed very similar N-terminal amino acid sequences (18 out of 20 identical residues), typical of LTPs, and molecular masses of 9,610 and 9,618 Da, respectively. The recombinant orange isoform (rCit s 3) expressed in P. pastoris (16 out of 20 residues identical to its natural counterpart in the N-terminal region) presented 92 amino acid residues and 9,463 Da, and 67% sequence identity with rPru p 3. Of the 27 sera analyzed, specific IgE to the purified allergens was found in 54% for nCit l 3, 48% for nCit s 3, 46% for rCit s 3 and 37% for rPru p 3. Positive SPT responses were obtained in 7 out of 26 patients tested for nCit s 3, 3 out of 8 for nCit l 3 and 10 out of 26 for nPru p 3. ELISA-inhibition assays showed an equivalent IgE-binding pattern for the natural and recombinant orange LTPs, and IgE cross-reactivity among the purified orange, lemon and peach LTP allergens. Conclusions: Members of the LTP allergen family are involved in allergy to oranges, displaying positive in vitro and in vivo reactions in 30–50% of the patients studied. Both orange and lemon allergens show cross-reactivity with the major peach allergen Pru p 3.

The water-soluble polysaccharide fraction of the cell wall alkali extract (F1SS) from the mycelial phase of the dimorphic fungus Paracoccidioides brasiliensis is compared with F1SS polysaccharides obtained from the Onygenalean mycelial fungi Ascocalvatia alveolata, Onygena equina and Aphanoascus terreus. These polymers were exclusively composed of mannose and galactose. Data from methylation and NMR analyses reveal that F1SS polysaccharides from the four fungi contain the same residues although in different proportions: [-->2,6)-alpha-D-Manp-(1 -->]; [2)-alpha-D-Manp-(1 -->]; [ -->6)-alpha-D-Manp-(1 -->]; and [alpha-D-Galf-(1 -->]. In P. brasiliensis, the repeating unit of the polysaccharide consists of a backbone of [(1 -->6)-alpha-D-Manp] substituted at the 0-2 position by the disaccharide [alpha-D-Galf-(1 -->6)-alpha-D-Manp-(1 -->], while the remaining 0-2 positions are substituted by single residues of mannose or short chains of (1 -->2)-mannose. The other species had a lower proportion of galactofuranose-containing side chains and higher proportion of mannose-containing side chains. The similarities found among the F1SS polysaccharides from P. brasiliensis and the Onygenalean A. alveolata, A. terreus and O. equina, reveal the close relatedness of all these fungi, show differences with polysaccharides from other fungal genera and agree with the molecular evidence provided in the scientific literature for the placement of P. brasiliensis within the Onygenales.

The alkali‐extractable water‐soluble polysaccharide F1SS isolated from the cell wall of Cubonia bulbifera has been studied by methylation analysis, NMR spectroscopy, and partial hydrolysis, and its structure was established as: being n ≈ 4 and m ≈ 1. †This paper is dedicated to Prof. Gerard Descotes, on the occasion of his 70th birthday.

11 p.-5 fig.-2 tab., Pneumococcal bacteriophage-encoded lysins are modular choline binding proteins that have been shown to act as enzymatic antimicrobial agents (enzybiotics) against streptococcal infections. Here we present the crystal structures of the free and choline bound states of the Cpl-1 lysin, encoded by the pneumococcal phage Cp-1. While the catalytic module displays an irregular (β/α)5β3 barrel, the cell wall-anchoring module is formed by six similar choline binding repeats (ChBrs), arranged into two different structural regions: a left-handed superhelical domain configuring two choline binding sites, and a β sheet domain that contributes in bringing together the whole structure. Crystallographic and site-directed mutagenesis studies allow us to propose a general catalytic mechanism for the whole glycoside hydrolase family 25. Our work provides the first complete structure of a member of the large family of choline binding proteins and reveals that ChBrs are versatile elements able to tune the evolution and specificity of the pneumococcal surface proteins., This work was supported by grants BIO2000-1307 and BIO2002-02887 from Dirección General de Investigación and by grant of Contrato-Programa de Grupos Estratégicos (BMC2000-1002) de la Comunidad Autónoma de Madrid.

Paracoccidioides brasiliensis is a pathogenic dimorphic fungus causing paracoccidioidomycosis, the most widespread systemic mycosis in Latin America. We have studied the structure of the alkali-extracted water-soluble cell wall polysaccharides (F1SS) from both mycelial and yeast phases of this fungus by using chemical analysis and NMR spectroscopic techniques. The F1SS polysaccharide from the mycelial phase consists of a trisaccharidic repeating unit of -->6)-[alpha-Galf -(1-->6)-alpha-Manp-(1-->2)]-alpha-Manp-(1-->. The F1SS polysaccharide of the yeast phase maintains 10% of the structure of the mycelium phase, but the main structure contain a disaccharide repeating unit of -->6)-[-alpha-Manp-(1-->2)]-alpha-Manp-(1-->, alternating with a trisaccharide repeating block of -->6)-[beta-Galf -(1-->6)-alpha-Manp-(1-->2)]-alpha-Manp-(1-->.

The cell wall alkali-extractable water-soluble polysaccharide (F1SS) purified from the three species of Cephalotheca has been characterized. Two different structures have been deduced by chemical and structural analyses. The structure of the polysaccharide F1SS from C. purpurea and C. reniformis is similar, and composed of 3,6-di-O-substituted mannopyranose with terminal residues of rhamnopyranose. The polysaccharide F1SS from C. sulfurea contains 2,6-di-O-substituted mannopyranose and terminal residues of galactofuranose. Our results confirm the relatedness of C. purpurea and C. reniformis with Ophiostomatales and of C. sulfurea with Sordariales.

[EN] Apocarotenoid compounds play diverse communication functions in plants, some of them being as hormones, pigments and volatiles. Apocarotenoids are the result of enzymatic cleavage of carotenoids catalyzed by carotenoid cleavage dioxygenase (CCD). The CCD4 family is the largest family of plant CCDs, only present in flowering plants, suggesting a functional diversification associated to the adaptation for specific physiological capacities unique to them. In saffron, two CCD4 genes have been previously isolated from the stigma tissue and related with the generation of specific volatiles involved in the attraction of pollinators. The aim of this study was to identify additional CCD4 members associated with the generation of other carotenoid-derived volatiles during the development of the stigma. The expression of CsCCD4c appears to be restricted to the stigma tissue in saffron and other Crocus species and was correlated with the generation of megastigma-4,6,8-triene. Further, CsCCD4c was up-regulated by wounding, heat, and osmotic stress, suggesting an involvement of its apocarotenoid products in the adaptation of saffron to environmental stresses. The enzymatic activity of CsCCD4c was determined in vivo in Escherichia coli and subsequently in Nicotiana benthamiana by analyzing carotenoids by HPLC-DAD and the volatile products by GC/MS. beta-Carotene was shown to be the preferred substrate, being cleaved at the 9,10 (9',10') bonds and generating beta-ionone, although beta-cyclocitral resulting from a 7,8 (7',8') cleavage activity was also detected at lower levels. Lutein, neoxanthin and violaxanthin levels in Nicotiana leaves were markedly reduced when CsCCD4c is over expressed, suggesting that CsCCD4c recognizes these carotenoids as substrates., We thank J. Argandona (Instituto Botanico, Universidad de Castilla-La Mancha, Albacete, Spain) for excellent technical support, and K. A. Walsh for language revision. The laboratory is supported by the Spanish Ministerio de Ciencia e Innovacion (BIO2013-44239-R) and participates in the IBERCAROT network (112RT0445). Dr. Ahrazem was funded by FPCYTA through the INCRECYT Programme.

The structure of a cell wall alkali-extractable and water-soluble polysaccharide isolated from two species of Arachniotus has been established by reductive cleavage and NMR spectroscopy. The linear polysaccharide consists of a regular disaccharide-repeating unit with the structure: [ →6 )- β - d - Gal f-(1 →5 )- β - d - Gal f-(1 → ] n →mannan core.

The structure of acidic fungal polysaccharides, isolated from the cell wall of Cylindrocladium and Calonectria species, has been investigated by chemical analysis, methylation and reductive cleavage analyses, and 1D and 2D 1H and 13C NMR spectroscopy. The polysaccharides have an idealized repeating unit: [formula: see text] linked to a small mannan core (< 5%), with n = 3 for Cylindrocladium penicilloides and C. quinqueseptatum and n = 5 for Calonectria theae, C. crotalariae, and C. colhounii.

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., ζ-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 Economía 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 networ

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.

Oxidative cleavage of cis-epoxycarotenoids by 9-cis-epoxycarotenoid dioxygenase (NCED) is the critical step in the regulation of abscisic acid (ABA) synthesis in higher plants. ABA has been associated with dormancy and flower senescence, while also regulating plant adaptive responses to various environmental stresses. An NCED gene, CstNCED, was cloned from Crocus sativus stigmas. The deduced amino acid sequence of the CstNCED protein shared high identity with other monocot NCEDs, and was closely related to the liliopsida enzymes. At the N-terminus of CstNCED a chloroplast transit peptide sequence is located. However, its expression in chloroplast-free tissues suggested localization in other plastid types. The relationship between expression of CstNCED and the endogenous ABA level was investigated in the stigma and corms, where it was developmentally regulated. The senescence of the unpollinated stigma is preceded by an increase in ABA levels and CstNCED expression. In corms, a correlation was observed between CstNCED expression and dormancy. Furthermore, CstNCED expression was correlated with the presence of zeaxanthin in the dormant corms. When detached C. sativus leaves and stigmas were water and salt stressed, increases in CstNCED mRNA were observed. The results provided evidence of the involvement of CstNCED in the regulation of ABA-associated processes such as flower senescence and corm dormancy in monocotyledonous saffron.

Saffron, the desiccated stigmas of Crocus sativus, is highly appreciated by its peculiar colour, flavour and aroma. The main compounds that accumulated throughout stigma development in C. sativus are crocetin, its glucoside derivatives, crocins, and picrocrocin, all of which increased as stigmas reached a fully developed stage. After anthesis, and in the absence of fertilization, the flower enters in a senescence programme, which represents the ultimate stage of floral development and results in wilting of whole flower. The programmed senescence of flowers allows the removal of a metabolically active tissue. We studied the composition of saffron apocarotenoids during the senescence of C. sativus flowers, and observed that changes in crocins were due to their transport from the senescent stigma to the ovaries and the developing corm. Afterwards, deglucosylation of crocins in these tissues results in crocetin accumulation. This mobilization mimics the export to storage cells (resorbed) of different compounds during leaf senescence avoiding loss of nutrients in leaves that would otherwise be cycled back into the soil system through leaf litter decomposition. In C. sativus, the resorbed apocarotenoids are stored within the developing corm, where they are not further detected in the advanced stages of development, suggesting that they are metabolized during the early and active phases of corm development, where the glucose molecules from crocins might contribute to cell initiation and elongation.

Background Flavonol glucosides constitute the second group of secondary metabolites that accumulate in Crocus sativus stigmas. To date there are no reports of functionally characterized flavonoid glucosyltransferases in C. sativus, despite the importance of these compounds as antioxidant agents. Moreover, their bitter taste makes them excellent candidates for consideration as potential organoleptic agents of saffron spice, the dry stigmas of C. sativus. Results Using degenerate primers designed to match the plant secondary product glucosyltransferase (PSPG) box we cloned a full length cDNA encoding CsGT45 from C. sativus stigmas. This protein showed homology with flavonoid glucosyltransferases. In vitro reactions showed that CsGT45 catalyses the transfer of glucose from UDP_glucose to kaempferol and quercetin. Kaempferol is the unique flavonol present in C. sativus stigmas and the levels of its glucosides changed during stigma development, and these changes, are correlated with the expression levels of CsGT45 during these developmental stages. Conclusion Findings presented here suggest that CsGT45 is an active enzyme that plays a role in the formation of flavonoid glucosides in C. sativus.

Background: Orange allergens are virtually unknown, in spite of the large consumption of this fruit. Germin-like proteins, together with vicilins and legumins, form the cupin superfamily of plant proteins, which includes many seed allergens. Methods: Twenty-nine patients with allergy to oranges were studied. A major IgE-binding protein from orange extracts was isolated by means of a two-step cation-exchange chromatographic protocol. The allergen was characterized by N-terminal amino acid sequencing and MALDI analysis, and its reactivity explored by specific IgE determination in individual sera, ELISA inhibition assays and in vivo skin prick tests (SPT). Chemical deglycosylation of the purified allergen was achieved by trifluoromethylsulfonate acid treatment. Results: The 24-kDa purified allergen, designated Cit s 1, was identified as a germin-like glycoprotein, based on its N-terminal amino acid sequence, molecular size and recognition by rabbit anti-complex N-linked glycan antibodies. Specific IgE to Cit s 1 was detected in 62% of 29 individual sera from orange-allergic patients, whereas positive SPT responses to the purified allergen were obtained in only 10% of such patients. Deglycosylation of Cit s 1 resulted in a loss of its IgE-binding capacity. Moreover, the unrelated plant glycoprotein horseradish peroxidase inhibited over 70% the IgE-binding to Cit s 1. Conclusions: Over 60% of patients with allergy to oranges show specific IgE to Cit s 1. However, the purified allergen exerts a low in vivo reactivity. Complex N-linked glycans seem to play a prominent role in the IgE-binding capacity of Cit s 1. This characteristic of Cit s 1, as well as of other orange glycoproteins, could lead to false positives if the diagnosis of allergy to oranges is mainly based on in vitro specific IgE determination.

The structure of acidic fungal polysaccharides isolated from the cell wall of Plectosphaerella cucumerina, Verticillium dahliae, and V. albo-atrum has been investigated by chemical analysis, methylation analysis, and 1D and 2D 1H and 13C NMR spectroscopy. The polysaccharides have an idealized repeating block of the type: [carbohydrates: see text] linked to a small mannan core (

The alkali extractable and water-soluble cell wall polysaccharides F1SS from Aspergillus wentii and Chaetosartorya chrysella have been studied by methylation analysis, 1D- and 2D-NMR, and MALDI-TOF analysis. Their structures are almost identical, corresponding to the following repeating unit: [→ 3)-β-D-Galf-(1 → 5)-β-D-Galf-(1 →] n → mannan core. The structure of this galactofuranose side chain differs from that found in the pathogenic fungus Aspergillus fumigatus, in other Aspergillii and members of Trichocomaceae: [→ 5)-β-D-Galf-(1 →] n → mannan core. The mannan cores have also been investigated, and are constituted by a (1 → 6)-α-mannan backbone, substituted at positions 2 by chains from 1 to 7 residues of (1 → 2) linked α-mannopyranoses. Published in 2004.

The alkali-extracted water-soluble galactomannan F1SS isolated from the cell wall of two species each of Geotrichum, Galactomyces, and Dipodascus have been studied by methylation analysis and NMR spectroscopy, and their structure is established as the following: Download : Download full-size image

The alkali-extractable water-soluble polysaccharide (F1SS) isolated from the cell wall of Apodus deciduus has been studied by methylation analysis and NMR spectroscopy, and its structure established as:where n approximately 130+/-10.

The structure of a polysaccharide isolated from the cell wall of Hypocrea gelatinosa has been investigated by means of chemical analyses and 1D and 2D NMR spectroscopy. The polysacharide has an irregular structure, idealized as follows: Figure options Download full-size image Download as PowerPoint slide

The polysaccharides obtained from the alkali-extractable, water-soluble fraction (F1SS) from the cell wall of Myrothecium verrucaria and Myrothecium atroviride were shown to be composed of beta-(1--6)-galactofuranose fully substituted at O-2 by terminal residues of alpha-glucopyranose and alpha-glucuronic acid. Glucuronic acid was substituted at O-4 by glucopyranose in the Myrothecium species M. inundatum, M. setiramosum, M. prestonii, M. tongaense and M. roridum. The acidic polysaccharides from Phaeostilbella atra (=Myrothecium atrum) and Myrothecium gramineum lacked the backbone of 2,6 di-O-substituted galactofuranose and contained a high amount of O-5-substituted beta-galactofuranose. The structures of the polysaccharides isolated from Myrothecium cinctum and Myrothecium penicilloides were unrelated to each other and to the polysaccharides from the other species analysed. The usefulness of these polysaccharides as a characteristic for delimitation of the genus Myrothecium is discussed.

Alkali-extractable and water-soluble cell-wall polysaccharides were purified from cell walls of some species of Fusarium and Gibberella. Their structures were determined by chemical analysis and NMR. The polysaccharides consisted of a main chain of β-(1 → 6)-linked galactofuranose units almost fully branched at positions O-2 by single residues of glucopyranose or acidic chains containing glucuronic acid and mannose. Individual differences were found, concerning the proportion of neutral and acidic side chains. These polysaccharides showed major differences from those of Microdochium nivale, Plectosphaerella cucumerina, Fusarium ciliatum, F. aquaeductuum and F. cavispermum. Highly specific polyclonal antibodies were raised against this structure, which were used in immunocompetence and immunofluorescence experiments.