Interspecific differences in plasma carotenoid profiles in nestlings of three sympatric vulture species.

Carotenoids are diet-based micronutrients important in health and coloration signaling. Related species with similar diets can differ in the kinds and levels of circulating carotenoids, which suggests specific physiological mechanisms to efficiently utilize these micronutrients, regardless of their availability. We explored whether diet and parental provisioning of unusual sources of carotenoids (fresh vegetal matter and vertebrate feces) can explain the occurrence and concentrations of carotenoids in the cinereous vulture Aegypius monachus, griffon vulture Gyps fulvus, and Egyptian vulture Neophron percnopterus nestlings, even when these pigments appear to not be deposited in their integumentary system. A greater diversity of wild prey in diet could be behind the profile of higher concentrations of carotenoids in the Egyptian vulture, the species with carotenoid-dependent coloration during adulthood, while differences in diet composition between cinereous and griffon vultures do not translate to different carotenoid profiles. The carotenoid profile appears to not be related to the ingestion of unusual matter rich in these compounds, although the infrequent occurrence of lycopene and unidentified γ-carotene-like compounds suggest that these vultures may be exploiting vegetal matter that left no identifiable unconsumed remains in the nest of Egyptian vultures. The consumption of green plant material by griffon vultures does not result in especially high levels of carotenoids when compared to the carotenoids found in cinereous vultures, which do not consume green plant material. Ungulate feces were not provisioned to Egyptian vulture nestlings, despite the fact they contain carotenoids that adults need for appropriate coloration. Overall, this study indicates that diet differences alone appear insufficient to explain contrasting interspecific carotenoid profiles, especially since all types of food consumed are considered to be poor in carotenoids, except vegetable matter. We suggest that nestling Egyptian vultures are comparatively efficient in uptaking carotenoids present in low concentrations in food when these compounds are not deposited in their integument, which suggests allocation to other functions.

Functional Characterization of Four Olive Squalene Synthases with Respect to the Squalene Content of the Virgin Olive Oil.

The release of new olive cultivars with an increased squalene content in their virgin olive oil is considered an important target in olive breeding programs. In this work, the variability of the squalene content in a core collection of 36 olive cultivars was first studied, revealing two olive cultivars, 'Dokkar' and 'Klon-14', with extremely low and high squalene contents in their oils, respectively. Next, four cDNA sequences encoding squalene synthases (SQS) were cloned from olive. Sequence analysis and functional expression in bacteria confirmed that they encode squalene synthases. Transcriptional analysis in distinct olive tissues and cultivars indicated that expression levels of these four SQS genes are spatially and temporally regulated in a cultivar-dependent manner and pointed to OeSQS2 as the gene mainly involved in squalene biosynthesis in olive mesocarp and, therefore, in the olive oil. In addition, the biosynthesis of squalene appears to be transcriptionally regulated in water-stressed olive mesocarp.

Bioavailability and provitamin A activity of neurosporaxanthin in mice.

Various species of ascomycete fungi synthesize the carboxylic carotenoid neurosporaxanthin. The unique chemical structure of this xanthophyll reveals that: (1) Its carboxylic end and shorter length increase the polarity of neurosporaxanthin in comparison to other carotenoids, and (2) it contains an unsubstituted ß-ionone ring, conferring the potential to form vitamin A. Previously, neurosporaxanthin production was optimized in Fusarium fujikuroi, which allowed us to characterize its antioxidant properties in in vitro assays. In this study, we assessed the bioavailability of neurosporaxanthin compared to other provitamin A carotenoids in mice and examined whether it can be cleaved by the two carotenoid-cleaving enzymes: ß-carotene-oxygenase 1 (BCO1) and 2 (BCO2). Using Bco1-/-Bco2-/- mice, we report that neurosporaxanthin displays greater bioavailability than ß-carotene and ß-cryptoxanthin, as evidenced by higher accumulation and decreased fecal elimination. Enzymatic assays with purified BCO1 and BCO2, together with feeding studies in wild-type, Bco1-/-, Bco2-/-, and Bco1-/-Bco2-/- mice, revealed that neurosporaxanthin is a substrate for either carotenoid-cleaving enzyme. Wild-type mice fed neurosporaxanthin displayed comparable amounts of vitamin A to those fed ß-carotene. Together, our study unveils neurosporaxanthin as a highly bioavailable fungal carotenoid with provitamin A activity, highlighting its potential as a novel food additive.

Bread Wheat Biofortification for Grain Carotenoid Content by Inter-Specific Breeding.

Bread wheat has traditionally been selected for whitish derived flours. As a consequence, the current varieties carry carotenogenic alleles associated with low grain carotenoid. In contrast, high grain yellow pigment content (YPC) has been a major target in durum wheat programs since yellow colour is an important aesthetic factor for pasta production. Phytoene synthase 1 (Psy1) genes have an important role in the determination of the carotenoid content in wheat. In this work, we have transferred the genes Psy1-A1 and Psy1-B1 from durum to bread wheat by inter-specific hybridization in order to evaluate the combined effect of these genes for the improvement of grain carotenoid content, as well as the development of carotenoid-enriched bread wheat lines. Inter-specific breeding coupled with a MAS approach based on Psy1-A1 and Psy1-B1 alleles has allowed the development of bread wheat pre-breeding lines with enhanced grain carotenoid content (16-23% mean). These biofortified lines have the potential to become new varieties or to be used as recurrent parents in bread wheat breeding programs.

The squalene route to C30 carotenoid biosynthesis and the origins of carotenoid biosynthetic pathways.

Carotenoids are isoprenoid lipids found across the tree of life with important implications in oxidative stress adaptations, photosynthetic metabolisms, as well as in membrane dynamics. The canonical view is that C40 carotenoids are synthesized from phytoene and C30 carotenoids from diapophytoene. Squalene is mostly associated with the biosynthesis of polycyclic triterpenes, although there have been suggestions that it could also be involved in the biosynthesis of C30 carotenoids. However, demonstration of the existence of this pathway in nature is lacking. Here, we demonstrate that C30 carotenoids are synthesized from squalene in the Planctomycetes bacteria and that this squalene route to C30 carotenoids is the most widespread in prokaryotes. Using the evolutionary history of carotenoid and squalene amino oxidases, we propose an evolutionary scenario to explain the origin and diversification of the different carotenoid and squalene-related pathways. We show that carotenoid biosynthetic pathways have been constantly transferred and neofunctionalized during prokaryotic evolution. One possible origin of the squalene pathway connects it with the one of C40 carotenoid synthesis of Cyanobacteria. The widespread occurrence of the squalene route to C30 carotenoids in Bacteria increases the functional repertoire of squalene, establishing it as a general hub of carotenoids and polycyclic triterpenes synthesis.

Marker-Trait Associations for Total Carotenoid Content and Individual Carotenoids in Durum Wheat Identified by Genome-Wide Association Analysis.

Yellow pigment content is one of the main traits considered for grain quality in durum wheat (Triticum turgidum L.). The yellow color is mostly determined by carotenoid pigments, lutein being the most abundant in wheat endosperm, although zeaxanthin, α-carotene and ß-carotene are present in minor quantities. Due to the importance of carotenoids in human health and grain quality, modifying the carotenoid content and profile has been a classic target. Landraces are then a potential source for the variability needed for wheat breeding. In this work, 158 accessions of the Spanish durum wheat collection were characterized for carotenoid content and profile and genotyped using the DArTSeq platform for association analysis. A total of 28 marker-trait associations were identified and their co-location with previously described QTLs and candidate genes was studied. The results obtained confirm the importance of the widely described QTL in 7B and validate the QTL regions recently identified by haplotype analysis for the semolina pigment. Additionally, copies of the Zds and Psy genes on chromosomes 7B and 5B, respectively, may have a putative role in determining zeaxanthin content. Finally, genes for the methylerythritol 4-phosphate (MEP) and isopentenyl diphosphate (IPPI) carotenoid precursor pathways were revealed as additional sources of untapped variation for carotenoid improvement.

The breeder's tool-box for enhancing the content of esterified carotenoids in wheat: From extraction and profiling of carotenoids to marker-assisted selection of candidate genes.

Carotenoid esterification is a new target for cereal biofortification since esterification increases both accumulation and stability of carotenoids. A xanthophyll acyl transferase is responsible for carotenoid esterification in the endosperm of wheat and related cereals. In this chapter we describe the procedures for transferring the carotenoid esterification attribute into wheat using the wild barley Hordeum chilense as donor of the esterification trait, the outline of the breeding program and the protocols for marker assisted selection and the analysis of carotenoids in grain. Biofortified cereals with increased lutein ester content will help to reduce the risk of developing age-related macular degeneration in human populations with limited access to other dietary sources.

A comprehensive review on carotenoids in foods and feeds: status quo, applications, patents, and research needs.

Carotenoids are isoprenoids widely distributed in foods that have been always part of the diet of humans. Unlike the other so-called food bioactives, some carotenoids can be converted into retinoids exhibiting vitamin A activity, which is essential for humans. Furthermore, they are much more versatile as they are relevant in foods not only as sources of vitamin A, but also as natural pigments, antioxidants, and health-promoting compounds. Lately, they are also attracting interest in the context of nutricosmetics, as they have been shown to provide cosmetic benefits when ingested in appropriate amounts. In this work, resulting from the collaborative work of participants of the COST Action European network to advance carotenoid research and applications in agro-food and health (EUROCAROTEN, www.eurocaroten.eu, https://www.cost.eu/actions/CA15136/#tabs|Name:overview) research on carotenoids in foods and feeds is thoroughly reviewed covering aspects such as analysis, carotenoid food sources, carotenoid databases, effect of processing and storage conditions, new trends in carotenoid extraction, daily intakes, use as human, and feed additives are addressed. Furthermore, classical and recent patents regarding the obtaining and formulation of carotenoids for several purposes are pinpointed and briefly discussed. Lastly, emerging research lines as well as research needs are highlighted.

Distinct Roles of N-Terminal Fatty Acid Acylation of the Salinity-Sensor Protein SOS3.

The Salt-Overly-Sensitive (SOS) pathway controls the net uptake of sodium by roots and the xylematic transfer to shoots in vascular plants. SOS3/CBL4 is a core component of the SOS pathway that senses calcium signaling of salinity stress to activate and recruit the protein kinase SOS2/CIPK24 to the plasma membrane to trigger sodium efflux by the Na/H exchanger SOS1/NHX7. However, despite the well-established function of SOS3 at the plasma membrane, SOS3 displays a nucleo-cytoplasmic distribution whose physiological meaning is not understood. Here, we show that the N-terminal part of SOS3 encodes structural information for dual acylation with myristic and palmitic fatty acids, each of which commands a different location and function of SOS3. N-myristoylation at glycine-2 is essential for plasma membrane association and recruiting SOS2 to activate SOS1, whereas S-acylation at cysteine-3 redirects SOS3 toward the nucleus. Moreover, a poly-lysine track in positions 7-11 that is unique to SOS3 among other Arabidopsis CBLs appears to be essential for the correct positioning of the SOS2-SOS3 complex at the plasma membrane for the activation of SOS1. The nuclear-localized SOS3 protein had limited bearing on the salt tolerance of Arabidopsis. These results are evidence of a novel S-acylation dependent nuclear trafficking mechanism that contrasts with alternative subcellular targeting of other CBLs by S-acylation.

Effect of fertilisation with black table olive wastewater solutions on production and quality of tomatoes cultivated under open field conditions.

This study was aimed at making progress on the valorisation of table olive wastewater that currently represent a big environmental problem for factories. Concentrates from vacuum evaporation of the wastewater generated during processing of black ripe olives treated with KOH were tested as fertilisers of tomato plants in open field assays for three consecutive crops. Fertilisation was performed by drip irrigation every 15 days; the first treatment being 15 days after transplanting, and a total of five fertilisation treatments were carried out. A phytotoxic effect was not observed on plants or fruit in any case but higher yield (fruit/plant and g/plant) was obtained in comparison to irrigation with only tap water. Moreover, the combined use of the olive concentrate with inorganic nitrogen supply (NH4NO3), in order to comply with the nutrient needs of plants, was also tested. The results demonstrated that the olive concentrates could be a good substitute for inorganic potassium (as KNO3) during the cultivation of tomato plants without any negative effect on the tomato quality (pH, °Brix, sugars, organic acids) or content of bioactive substances (phenolic compounds and carotenoids).

Durum Wheat (Triticum durum L.) Landraces Reveal Potential for the Improvement of Grain Carotenoid Esterification in Breeding Programs.

Carotenoids are essential in the human diet for their important functions in health. Besides, they are responsible for the yellow pigments desirable for industrial quality in durum wheat. The remarkable carotenoid content of durum wheat endosperm is mostly due to lutein. However, lutein esters have not been previously detected in durum wheat as in other cereals such as common wheat, tritordeum or Hordeum chilense. Esterification increases carotenoid stability and allows greater retention and accumulation through the food chain. Therefore, carotenoid esterification is revealed as a new key target in breeding. We characterized the carotenoid profile of 156 accessions of the Spanish durum wheat collection, searching for landraces with esterification ability. Interestingly, four accessions produced lutein monoesters and diesters. Also, traces of lutein monoesters were detected in eleven accessions. The identification of the first durum wheat accessions with esterification ability reported herein is a remarkable advance for carotenoid biofortification. Furthermore, variation for the relative content of zeaxanthin, α-carotene and ß-carotene was also observed. This diversity for the ß,ε and ß,ß branches of the carotenogenic pathway also represents a new opportunity for breeding for specific carotenoids in biofortification programs.

Plasma carotenoids and immunity in a despotic avian scavenger.

Carotenoids are pigmented compounds acquired through diet that have important functions as antioxidants and immune modulators. We studied the association between immunity and circulating carotenoids in Andean condors (Vultur gryphus). We evaluated the relationship between α-, ß-, and γ-globulin blood concentrations and different circulating carotenoids in two groups of Andean condors that differ in their mean health status, rehabilitating (suffering different pathologies), and wild individuals (trapped when displaying their physiological behavior). In rehabilitating individuals, α-, ß-, and γ-globulin concentrations were higher than in wild individuals. This shows that rehabilitating individuals were developing an immune response associated with the pathologies that they were suffering at the time of sampling. In addition, circulating carotenoids were lower in rehabilitating than in wild individuals. We found negative correlations between α-, ß-, and γ-globulins and different circulating carotenoids in rehabilitating individuals, but not in wild condors. Xanthophylls were strongly related to α-, ß-, and γ-globulin blood concentrations in rehabilitating, but not in wild condors. Our results suggest that there is a potential relationship between circulating carotenoids and immunity in the Andean condor. Given that this species may display a carotenoid-based pigmentation, our results could suggest that a trade-off between the immune system and external coloration could operate in this species, which may have implications in their access to food resources and mate selection and, thus, in their conservation.

Neurosporaxanthin Overproduction by Fusarium fujikuroi and Evaluation of Its Antioxidant Properties.

Neurosporaxanthin (NX) is a carboxylic carotenoid produced by some filamentous fungi, including species of the genera Neurospora and Fusarium. NX biosynthetic genes and their regulation have been thoroughly investigated in Fusarium fujikuroi, an industrial fungus used for gibberellin production. In this species, carotenoid-overproducing mutants, affected in the regulatory gene carS, exhibit an upregulated expression of the NX pathway. Based on former data on a stimulatory effect of nitrogen starvation on carotenoid biosynthesis, we developed culture conditions with carS mutants allowing the production of deep-pigmented mycelia. With this method, we obtained samples with ca. 8 mg NX/g dry mass, in turn the highest concentration for this carotenoid described so far. NX-rich extracts obtained from these samples were used in parallel with carS-complemented NX-poor extracts obtained under the same conditions, to check the antioxidant properties of this carotenoid in in vitro assays. NX-rich extracts exhibited higher antioxidant capacity than NX-poor extracts, either when considering their quenching activity against [O2(1g)] in organic solvent (singlet oxygen absorption capacity (SOAC) assays) or their scavenging activity against different free radicals in aqueous solution and in liposomes. These results make NX a promising carotenoid as a possible feed or food additive, and encourage further studies on its chemical properties.

Biotechnological production of zeaxanthin by an Antarctic Flavobacterium: Evaluation of culture conditions.

Statistical experimental designs were used to formulate a culture medium for zeaxanthin production by an Antarctic Flavobacterium sp. P8 strain. Eleven nutritional factors were assayed in shaken flasks. The effect of temperature on zeaxanthin and carotenoid production was also studied. Peptone, yeast extract, and sodium chloride were the nutrients that caused the principal impact on the biomass growth. These components were further studied to enhance zeaxanthin and total carotenoid concentrations. Although a high production rate of zeaxanthin and carotenoids was achieved, the aerobic characteristics of the bacterial strain and the oxygen requirements for zeaxanthin biosynthesis incorporate a factor that requires additional consideration. Scaling up the process to a 5 L-bioreactor that increased dissolved oxygen availability resulted in a 4.5-fold increase in the total carotenoid content and an almost 9-fold increase in zeaxanthin, which represented 98% of the total carotenoids produced. The results reveal that Flavobacterium sp. P8 is a promising strain for zeaxanthin production.

Mediation of a GDSL Esterase/Lipase in Carotenoid Esterification in Tritordeum Suggests a Common Mechanism of Carotenoid Esterification in Triticeae Species.

Carotenoids are essential in human diet, so that the development of programs toward carotenoid enhancement has been promoted in several crops. The cereal tritordeum, the amphiploid derived from the cross between Hordeum chilense Roem. et Schulz. and durum wheat has a remarkable carotenoid content in the endosperm. Besides, a high proportion of these carotenoids are esterified with fatty acids. The identification of the gene(s) responsible for xanthophyll esterification would be useful for breeding as esterified carotenoids show an increased ability to accumulate within plant cells and have a higher stability during post-harvest storage. In this work, we analyzed five genes identified as candidates for coding the xanthophyll acyltransferase (XAT) enzyme responsible for lutein esterification in H. chilense genome. All these genes were expressed during grain development in tritordeum, but only HORCH7HG021460 was highly upregulated. Sequence analysis of HORCH7HG021460 revealed a G-to-T transversion, causing a Glycine to Cysteine substitution in the protein of H290 (the only accession not producing quantifiable amounts of lutein esters, hereinafter referred as zero-ester) of H. chilense compared to the esterifying genotypes. An allele-specific marker was designed for the SNP detection in the H. chilense diversity panel. From the 93 accessions, only H290 showed the T allele and the zero-ester phenotype. Furthermore, HORCH7HG021460 is the orthologue of XAT-7D, which encodes a XAT enzyme responsible for carotenoid esterification in wheat. Thus, HORCH7HG021460 (XAT-7Hch) is a strong candidate for lutein esterification in H. chilense and tritordeum, suggesting a common mechanism of carotenoid esterification in Triticeae species. The transference of XAT-7Hch to wheat may be useful for the enhancement of lutein esters in biofortification programs.

A Routine Method for the Extraction and HPLC-DAD Profiling of Major Plant and Food Carotenoids.

Carotenoids are ubiquitously present in Nature, and especially in plants and derived foods. The carotenoid profiling is necessary to understand relevant aspects in relation to their biochemistry and genetics, as well as their important roles on human health and animal ecophysiology. Here we propose a simple methodology for the routine extraction and efficient HPLC separation of the most common plant and food carotenoids from plant and animal origins.

Carotenoids from heterotrophic bacteria isolated from Fildes Peninsula, King George Island, Antarctica.

Carotenoids are isoprenoid pigments used by pharmaceutical, cosmetic, food and feed industry as antioxidants and colorants. Although traditional sources of carotenoids are fruits, vegetables and chemical synthesis, prospecting for alternative sinks of common and/or unusual carotenoids is important for the development of natural carotenoid industry. In this work, 30 pigmented bacterial strains from Fildes Peninsula in King George Island, Antarctica, were isolated and identified by 16S rRNA gene sequencing and classified in three phyla, Bacteroidetes, Firmicutes and Actinobacteria. After cells extraction, ten different carotenoids were identified based on the chromatographic and spectroscopic characteristic obtained by HPLC-PDA and HPLC-PDA-APCI-MS analyses. Strains assigned to Bacteroidetes affiliated to Flavobacterium, Chryseobacterium and Zobellia genera, presented a pigment profile composed of zeaxanthin, ß-cryptoxanthin and ß-carotene. Firmicutes strains of Planococcus genus produced a C50 carotenoid, identified as C.p. 450 glucoside. Actinobacteria isolates were mainly assigned to Arthrobacter genus, and few to Salinibacterium and Cryobacterium genera. Arthrobacter strains produced C50 carotenoids such as decaprenoxanthin and its glucosylated derivatives, as well as some C40 carotenoids such as lycopene which is used as synthesis precursors of the C50 carotenoids. Salinibacterium and Cryobacterium genera produced C.p. 450 free form and its glucosylated derivatives. Although most isolates produce carotenoids similar in diversity and quantity than those already reported in the literature, novel sources for C50 carotenoids results from this work. According to their carotenoid content, all isolates could be promising candidates for carotenoids production.

Exposure of black-necked grebes (Podiceps nigricollis) to metal pollution during the moulting period in the Odiel Marshes, Southwest Spain.

European populations of black-necked grebes (Podiceps nigricollis) congregate every year to moult at the salt ponds of the Odiel Marshes (SW Spain). However, the Odiel Marshes are part of one of the most metal-polluted coastal estuaries in the world, which may pose risks to wildlife. We assessed the exposure of grebes to metal pollution during the critical moulting period in the Odiel Marshes and its potential to cause adverse health effects. Levels of metals in red blood pellet (as a biomarker of exposure), plasma carotenoids, eye redness, and body condition (as biomarkers of effects) were studied. Metal content was also analyzed in the brine shrimp Artemia parthenogenetica, the most important food for grebes in this hypersaline ecosystem during the moulting period. Results showed that, in comparison to toxicity thresholds, grebes had relatively high blood levels of arsenic (As), mercury (Hg) and zinc (Zn). The high loads found in Artemia and the way blood levels vary during the moulting period indicate that shrimp consumption may be the main route of metal exposure for grebes. Plasma carotenoids and body condition showed a positive association with exposure to As, while the relationship of lutein-like carotenoids with Hg accumulation was negative at the beginning of the moulting period to become positive afterwards. Moreover, eye redness was negatively affected by As accumulation. Factors including food resource availability, seasonal fluctuations in physiological status, and interannual variations in the degree of environmental contamination should be considered in monitoring efforts when using moult migrant waterbirds as sentinel species.

HPLC Analysis of Carotenoids in Neurosporaxanthin-Producing Fungi.

The ascomycetous fungi Fusarium fujikuroi and Neurospora crassa are widely used as research models in the study of secondary metabolism and photobiology, respectively. Both fungi exhibit a similar carotenoid pathway, for which all the genes and enzymes have been identified. Under standard laboratory conditions, either F. fujikuroi or N. crassa accumulate a mixture of neurosporaxanthin, a carboxylic apocarotenoid acid, and several of its carotene precursors. We formerly described methods for the identification and quantification of neurosporaxanthin. However, the differences in polarity between this acidic xanthophyll and neutral carotenes make their global analysis cumbersome. Here we propose a simple HPLC methodology for the efficient separation of neurosporaxanthin and earlier pathway intermediates in a single HPLC run. This method should be useful to check the abundance of neurosporaxanthin under different experimental conditions and to evaluate the relative proportions of their different carotene precursors. To assess the validity of the method, we have compared the carotenoid profiles in samples of mycelia of F. fujikuroi and conidia of N. crassa, in both cases obtained from surface cultures of a wild strain of each species.

ß-Cryptoxanthin is more bioavailable in humans from fermented orange juice than from orange juice.

Carotenoids, especially ß-cryptoxanthin, exert multiple biological activities in the organism. Various processing techniques can improve carotenoid bioavailability in relation to the food matrix. The study objective was to compare the bioavailability of carotenoids from orange juice (OJ) with that from a beverage obtained by alcoholic fermentation of orange juice (FOB). Seven volunteers were recruited for a randomized, controlled, and crossover study. Post-intake plasma carotenoid concentrations were measured by HPLC in the subjects at 0-8 h after their consumption of OJ or FOB. ß-Cryptoxanthin and lutein absorption was significantly higher from FOB than from OJ, but no significant difference in zeaxanthin absorption was found. The mean baseline-corrected area under the concentration curve (AUC0-8 h) for ß-cryptoxanthin, lutein and zeaxanthin was 24.6-, 1.3- and 4.65-fold larger, respectively, after FOB versus OJ consumption. This fermented orange beverage could be an abundant source of bioavailable carotenoids, and its regular consumption may exert healthy effects.