A CD36-related Transmembrane Protein Is Coordinated with an Intracellular Lipid-binding Protein in Selective Carotenoid Transport for Cocoon Coloration

The transport pathway of specific dietary carotenoids from the midgut lumen to the silk gland in the silkworm, Bombyx mori, is a model system for selective carotenoid transport because several genetic mutants with defects in parts of this pathway have been identified that manifest altered cocoon pigmentation. In the wild-type silkworm, which has both genes, Yellow blood (Y) and Yellow cocoon (C), lutein is transferred selectively from the hemolymph lipoprotein to the silk gland cells where it is accumulated into the cocoon. The Y gene encodes an intracellular carotenoid-binding protein (CBP) containing a lipid-binding domain known as the steroidogenic acute regulatory protein-related lipid transfer domain. Positional cloning and transgenic rescue experiments revealed that the C gene encodes Cameo2, a transmembrane protein gene belonging to the CD36 family genes, some of which, such as the mammalian SR-BI and the fruit fly ninaD, are reported as lipoprotein receptors or implicated in carotenoid transport for visual system. In C mutant larvae, Cameo2 expression was strongly repressed in the silk gland in a specific manner, resulting in colorless silk glands and white cocoons. The developmental profile of Cameo2 expression, CBP expression, and lutein pigmentation in the silk gland of the yellow cocoon strain were correlated. We hypothesize that selective delivery of lutein to specific tissue requires the combination of two components: 1) CBP as a carotenoid transporter in cytosol and 2) Cameo2 as a transmembrane receptor on the surface of the cells.     

Analysis of a silkworm F1 hybrid with yellow cocoon generated by crossing two white-cocoon strains: Further evidences for the roles of Cameo2 and CBP in formation of yellow cocoon

In this report, we examined the gene expression related to carotenoid transport for a silkworm F₁ hybrid with yellow cocoon generated by crossing two white-cocoon strains, Qiubai and 12-260. Our results showed that, in Qiubai, Cameo2, a transmembrane protein gene belonging to the CD36 family genes, was expressed normally in the silk gland, but no intact carotenoid-binding protein (CBP) mRNA (only the truncated CBP mRNA) was detected in the midgut. In 12-260, we detected the intact CBP mRNA expression in the midgut, but no Cameo2 expression in the silk gland. Regarding the F₁ hybrid from crossing Qiubai and 12-260, both Cameo2 and intact CBP mRNA expressed normally in the silk gland and midgut. HPLC detection confirmed that in the F₁ hybrid the carotenoids could be absorbed from dietary mulberry leaves through the midgut and transferred to silk gland via the hemolymph, which eventually colored cocoons into yellow. We also identified four CBP mRNA isoforms expressed in the midgut of the F₁ hybrid, subsequently named as variants 5–8. Our results provide further evidences for the roles of Cameo2 and CBP in the formation of yellow cocoon of silkworm.     

Combined Effect of Cameo2 and CBP on the Cellular Uptake of Lutein in the Silkworm,Bombyx mori

Formation of yellow-red color cocoons in the silkworm, Bombyx mori, occurs as the result of the selective delivery of carotenoids from the midgut to the silk gland via the hemolymph. This process of pigment transport is thought to be mediated by specific cellular carotenoids carrier proteins. Previous studies indicated that two proteins, Cameo2 and CBP, are associated with the selective transport of lutein from the midgut into the silk gland in Bombyx mori. However, the exact roles of Cameo2 and CBP during the uptake and transport of carotenoids are still unknown. In this study, we investigated the respective contributions of these two proteins to lutein and β-carotene transport in Bombyx mori as well as commercial cell-line. We found that tissues, expressed both Cameo2 and CBP, accumulate lutein. Cells, co-expressed Cameo2 and CBP, absorb 2 fold more lutein (P<0.01) than any other transfected cells, and the rate of cellular uptake of lutein was concentration-dependent and reached saturation. From immunofluorescence staining, confocal microscopy observation and western blot analysis, Cameo2 was localized at the membrane and CBP was expressed in the cytosol. What’s more, bimolecular fluorescence complementation analysis showed that these two proteins directly interacted at cellular level. Therefore, Cameo2 and CBP are necessarily expressed in midguts and silk glands for lutein uptake in Bombyx mori. Cameo2 and CBP, as the membrane protein and the cytosol protein, respectively, have the combined effect to facilitate the cellular uptake of lutein.     

CAROTENOID COMPOSITIONS OF CLADOPHORA BALLS (AEGAGROPILA LINNAEI) AND SOME MEMBERS OF THE CLADOPHORALES (ULVOPHYCEAE,CHLOROPHYTA): THEIR TAXONOMIC AND EVOLUTIONARY IMPLICATION1

Photosynthetic pigments were analyzed by HPLC for 27 samples of the Cladophorales (Ulvophyceae, Chlorophyta). The carotenoid compositions of the examined algae were classified into three types based on the final compound of biosynthesis of the α‐carotene branch: lutein type, characterized by containing lutein as a major carotenoid and lacking loroxanthin and siphonaxanthin; loroxanthin type, characterized by containing loroxanthin and lacking siphonaxanthin; and siphonaxanthin type, characterized by containing siphonaxanthin. We constructed molecular phylogenetic tree of the species examined in the present study using 18S rRNA gene sequences and mapped the carotenoid types of the species onto the tree. The molecular phylogenetic analysis divided the Cladophorales into two major clades, clade 1 and Aegagropila‐clade (clade 2), and divided clade 1 into subclade 1‐1 and subclade 1‐2. All the examined species positioned in the Aegagropila‐clade and those of the subclade 1‐1 belonged to the loroxanthin type, whereas both lutein type and siphonaxanthin type appeared only in the subclade 1‐2. The clades delineated by molecular phylogenetic analysis were congruent with distribution of the carotenoid types, indicating that the carotenoid types are of taxonomic significance in the Cladophorales. Considering the distribution pattern of these carotenoid types and minimum state changes in the Cladophorales, we concluded that the loroxanthin type was the primitive (plesiomorphic) state and the siphonaxanthin type and lutein type appeared in the subclade 1‐2 as advanced (apomorphic) state within this order and suggested that the cladophoralean siphonaxanthin type would have been secondarily acquired.     

Effects of light intensity and quality on the growth rate and photosynthetic pigment content of Spirulina platensis

The quantitative and qualitative effects of light on carotenoid production by Spirulina were studied. Maximum total carotenoid production was measured in cells grown under white light at an irradiance of 432 μmol photon m^?2 s^?1, the onset of light saturation for this organism as determined by growth rates. A true maximum may exist at irradiances above 1500 μmol photon m^?2 s^?1 under white light. Individual carotenoids responded differently to light conditions. Under white light, β-carotene and echinenone were most abundant at the lowest and highest irradiance levels tested. Myxoxanthophyll and lutein/zeaxanthin did not change over the same irradiance range. Under red and blue light, we found decreased values of myxoxanthophyll, while β-carotene increased and lutein/zeaxanthin and echinenone showed little change. In general, maximum carotenoid production requires optimization of the culture conditions that favor growth.     

Carotenoids of the dinophyceae

The carotenoids of the photosynthetic dinofiagellates Amphidinium carterae (two strains), Glenodinium sp.,Gymnodinium splendens, G. nelsoni and Gyrodinium dorsum have been investigated, quantitatively and qualitatively. Peridinin is the principal carotenoid in all species; also present are β-carotene, diadinoxanthin, dinoxanthin, pyrrhoxanthin, astaxanthin, peridininol, diatoxanthin and pyrrhoxanthinol. New structures have been assigned to dinoxanthin and pyrrhoxanthin while peridininol and pyrrhoxanthinol are new carotenoids not previously reported. A carotenoid glycoside, P-457, found in four species, is a hexoside. Dinoxanthin is the only, plausible biosynthetic precursor of peridinin that could be detected.     

Absorption and Ocular Deposition of Dietary Lutein in Marine Mammals

Cataracts and ocular disease are common lesions of marine mammals in zoological collections. Lutein, an oxygenated carotenoid, may have therapeutic or prophylactic effects on ocular disorder. Therefore, this study examined the ability of marine mammals to absorb dietary lutein. Two preliminary trials examined lutein in two forms (beadlet or ester) in a small sample size of marine mammals representing pinnipeds and cetaceans. Lutein was fed daily in tablets providing 0.89–3.6 mg lutein/kg body weight^0.75 per day for 15 days to 2 years. A third study was conducted using lutein beadlet fed at 3.6 mg lutein/kg body weight^0.75 per day for 15–21 days. Blood was analyzed for lutein pre‐ and postsupplementation. In the preliminary trials, lutein beadlet was observed to result in greater blood lutein levels than lutein esters, and cetaceans had more noticeable responses than pinnipeds. In Study 3, serum lutein and zeaxanthin increased postsupplementation in beluga whales (P < 0.05), and serum lutein tended to increase postsupplementation in dolphins (P < 0.10), but little change was seen in serum lutein in pinnipeds or manatee. Opportunistic retinal samples demonstrated some detectable lutein in the retina of a dolphin and several harp seals. The lutein levels in dolphins after supplementation are similar to those reported in free‐ranging animals. Ocular lutein in harp seals demonstrates that ocular deposition occurs despite low circulating lutein levels. Zoo Biol. 32:316–323, 2013. © 2012 Wiley Periodicals, Inc.     

A new carotenoid glucoside ester from Chondromyces apiculatus

The carotenoid composition of the myxobacterium Chondromyces apiculatus is reported. A new acyclic carotenoid glucoside ester was isolated and its structure determined as 1′-glucosyloxy-3′,4′-didehydro-1′,2′-dihydro-ψ,ψ-carotene monoester.     

Carotenoids in fish II. Carotenoids and vitamin A in some fishes from the coastal region of the Black Sea

The presence of various carotenoids and vitamin A in seven species of fish from the coastal region of the Black Sea was investigated by means of columnar and thinlayer chromatography. The investigations revealed the presence of the following carotenoids: Mugil auratus: ß-carotene, canthaxanthin, lutein, zeaxanthin, astaxanthin ester and astacene. Diplodus annularis: ß-carotene, canthaxanthin, tunaxanthin, lutein, zeaxanthin and astacene. Diplodus sargus: ß-carotene, tunaxanthin, lutein, taraxanthin, zeaxanthin and astaxanthin. Crenilabrus tinca: tunaxanthin, canthaxanthin, lutein, astaxanthin and astacene. Blennius sphinx: ß-carotene, χ-carotene (?), lutein, tunaxanthin, taraxanthin and astaxanthin. Blennius sanguinolentus: ß-carotene, tunaxanthin and astaxanthin (ester and free). Gobius melanostomus: ß-carotene and astacene. Some fractions were not identified. Vitamin A was found in all species investigated.     

Ketocarotenoid Production in Soybean Seeds through Metabolic Engineering

The pink or red ketocarotenoids, canthaxanthin and astaxanthin, are used as feed additives in the poultry and aquaculture industries as a source of egg yolk and flesh pigmentation, as farmed animals do not have access to the carotenoid sources of their wild counterparts. Because soybean is already an important component in animal feed, production of these carotenoids in soybean could be a cost-effective means of delivery. In order to characterize the ability of soybean seed to produce carotenoids, soybean cv. Jack was transformed with the crtB gene from Pantoea ananatis, which codes for phytoene synthase, an enzyme which catalyzes the first committed step in the carotenoid pathway. The crtB gene was engineered together in combinations with ketolase genes (crtW from Brevundimonas sp. strain SD212 and bkt1 from Haematococcus pluvialis) to produce ketocarotenoids; all genes were placed under the control of seed-specific promoters. HPLC results showed that canthaxanthin is present in the transgenic seeds at levels up to 52 μg/g dry weight. Transgenic seeds also accumulated other compounds in the carotenoid pathway, such as astaxanthin, lutein, β-carotene, phytoene, α-carotene, lycopene, and β-cryptoxanthin, whereas lutein was the only one of these detected in non-transgenic seeds. The accumulation of astaxanthin, which requires a β-carotene hydroxylase in addition to a β-carotene ketolase, in the transgenic seeds suggests that an endogenous soybean enzyme is able to work in combination with the ketolase transgene. Soybean seeds that accumulate ketocarotenoids could potentially be used in animal feed to reduce or eliminate the need for the costly addition of these compounds.     

Variations in the carotenoid content of Chlamydomonas reinhardii throughout the cell cycle

Synchronous cultures of Chlamydomonas reinhardii have been examined for the total amounts of carotenoid and chlorophyll present throughout a 12 hrs light–4 hrs dark life cycle. Variations in the carotenoid distribution at different points within the cell cycle have been found. During the greater part of the light period all major carotenoids increased at a proportionally similar rate. However, the increases in lutein and violaxanthin preceded those in β-carotene and neoxanthin by some 2 hrs and that in loroxanthin, an algal xanthophyll, by about 3 hrs. A marked drop in total carotenoid accumulation, corresponding to similar temporary falling away in the accumulation of β-carotene, lutein and violaxanthin occurred at 9 hrs. The correspondence of this with the established drop in RNA accumulation and the break-up of the nucleolus was pointed out. Considerable redistribution among the carotenoids occurred during the dark period, notably the amount of β-carotene increased relative to the total xanthophylls. The full significance of these results can not be estimated in the absence of comparative data on related organisms.     

Hormonal effects on carotenoid uptake by the silk gland in the silkworm, Bombyx mori

The carotenoid uptake by the silk gland of the silkworm (Bombyx mori), which occurs only during the middle to late period of the last (fifth) instar in the natural condition, was studied in relation to the hormonal controls. During certain stages of the fourth and last instars, the corpus allatum hormone (JH) was found to inhibit the activation of the absorbing function of the silk gland. The absorbing activity was inactivated, if the activated silk gland was implanted into larva at the late stage of the fourth instar in the presence of the moulting hormone (MH). As more ponasterone-A (ecdysone-analogue) was injected into decapitated larvae, the pigmentation of the silk gland was increased; but injection of a high titre inhibited its activity. It seems that, through serial transplantations, the silk gland inactivated experimentally at the late stage of the fourth instar is reactivated in the presence of MH during the middle to late period of the last instar. The results indicate that MH and JH at each stage control the activity of the carotenoid uptake.     

Heterologous biosynthesis of lutein in S. cerevisiae enabled by temporospatial pathway control

The market-expanding lutein is currently mainly supplied by plant extraction, with microbial fermentation using engineered cell factory emerging as a promising substitution. During construction of lutein-producing yeast, α-carotene formation through asymmetric ε- and β-cyclization of lycopene was found as the main limiting step, attributed to intra-pathway competition of the cyclases for lycopene, forming β-carotene instead. To solve this problem, temperature-responsive expression of β-cyclase was coupled to constitutive expression of ε-cyclase for flux redirection to α-carotene by allowing ε-cyclization to occur first. Meanwhile, the ε-cyclase was engineered and re-localized to the plasma membrane for further flux reinforcement towards α-carotene. Finally, pathway extension with proper combination of carotenoid hydroxylases enabled lutein (438 μg/g dry cells) biosynthesis in S. cerevisiae. The success of heterologous lutein biosynthesis in yeast suggested temporospatial pathway control as a potential strategy in solving intra-pathway competitions, and may also be applicable for promoting the biosynthesis of other natural products.     

Characterization of the Role of β-Carotene 9,10-Dioxygenase in Macular Pigment Metabolism

A family of enzymes collectively referred to as carotenoid cleavage oxygenases is responsible for oxidative conversion of carotenoids into apocarotenoids, including retinoids (vitamin A and its derivatives). A member of this family, the β-carotene 9,10-dioxygenase (BCO2), converts xanthophylls to rosafluene and ionones. Animals deficient in BCO2 highlight the critical role of the enzyme in carotenoid clearance as accumulation of these compounds occur in tissues. Inactivation of the enzyme by a four-amino acid-long insertion has recently been proposed to underlie xanthophyll concentration in the macula of the primate retina. Here, we focused on comparing the properties of primate and murine BCO2s. We demonstrate that the enzymes display a conserved structural fold and subcellular localization. Low temperature expression and detergent choice significantly affected binding and turnover rates of the recombinant enzymes with various xanthophyll substrates, including the unique macula pigment meso-zeaxanthin. Mice with genetically disrupted carotenoid cleavage oxygenases displayed adipose tissue rather than eye-specific accumulation of supplemented carotenoids. Studies in a human hepatic cell line revealed that BCO2 is expressed as an oxidative stress-induced gene. Our studies provide evidence that the enzymatic function of BCO2 is conserved in primates and link regulation of BCO2 gene expression with oxidative stress that can be caused by excessive carotenoid supplementation.     

Effects of simulated drought stress on carotenoid contents and expression of related genes in carrot taproots

Carotenoids are liposoluble pigments found in plant chromoplasts that are responsible for the yellow, orange, and red colors of carrot taproots. Drought is one of the main stress factors affecting carrot growth. Carotenoids play important roles in drought resistance in higher plants. In the present work, the carotenoid contents in three different-colored carrot cultivars, ‘Kurodagosun’ (orange), ‘Benhongjinshi’ (red), and ‘Qitouhuang’ (yellow), were determined by ultra-high-performance liquid chromatography (UPLC) after 15% polyethylene glycol (PEG) 6000 treatment. Real-time fluorescence quantitative PCR (RT-qPCR) was then used to determine the expression levels of carotenoid synthesis- and degradation-related genes. Increases in β-carotene content in ‘Qitouhuang’ taproots under drought stress were found to be related to the expression levels of DcPSY2 and DcLCYB. Increases in lutein and decreases in α-carotene content in ‘Qitouhuang’ and ‘Kurodagosun’ under PEG treatment may be related to the expression levels of DcCYP97A3, DcCHXE, and DcCHXB1. The expression levels of DcNCED1 and DcNCED2 in the three cultivars significantly increased, thus suggesting that NCED genes could respond to drought stress. Analysis of the growth status and carotenoid contents of carrots under PEG treatment indicated that the orange cultivar ‘Kurodagosun’ has better adaptability to drought stress than the other cultivars and that β-carotene and lutein may be involved in the stress resistance process of carrot.

     

PHOTOINHIBITION IN RED ALGAL SPECIES WITH DIFFERENT CAROTENOID PROFILES1

Members of the Rhodophyta present different carotenoid profiles. In a majority of the species, lutein constitutes >50% of the total carotenoid content, while in other species, it is replaced by zeaxanthin or antheraxanthin. Given that carotenoids have specific roles in photoprotection, different carotenoid profiles of red algae species could be related to their capacity to cope with photoinhibitory stress. Therefore, in the present work, the sensitivity to light stress of red algal species with different carotenoid profiles was investigated. Photoinhibition of photosynthesis induced by high‐light stress and the subsequent recovery in dim‐light conditions was measured using maximal PSII quantum efficiency (F_v/F_m). The degree of decrease and recovery of F_v/F_m and their respective kinetics were related to the carotenoid profile of the species. Although no relationship between sensitivity to high‐light stress and the carotenoid profile was observed, there were clear carotenoid profile‐related differences in the decrease and recovery kinetics. In species with zeaxanthin or antheraxanthin as the major carotenoid, F_v/F_m reduction and recovery was principally associated with slowly activated and relaxed processes. In contrast, in species with lutein as the major carotenoid, rapidly activated processes appear to play a major role in the down‐regulation of photosynthesis during light‐stress conditions. In these species, the repair of D1 is also important during light‐stress conditions. This finding could imply differential expression of mechanisms involved in photoprotection in red algae that seems to be related to the carotenoid profile of the species.