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.     

Carotenoids of Heterosigma akashiwo: A chemosystematic contribution

The presence of C₃₇-norcarotenoids (peridinin and probably pyrrhoxanthin, together 87% of total carotenoids) and the carotenoid pattern in general, including dinoxanthin, diatoxanthin and β,β-carotene, but no fucoxanthin, strongly suggest that H. akashiwo is a dinoflagellate and not a chrysophyte.     

Carotenoids of cryptophyceae

The carotenoids of selected Cryptophyceae, Rhodomonas D3 and Cryptomonas ovata, have been examined by methods including HPLC, mass spectrometry ¹H NMR and circular dichroism. $\text{3′R,6′R-}\text{Chirality}$ has been assigned to monadoxanthin from ¹H NMR and CD data; β,?-carotene possessed the common $\text{6′R-}\text{chirality}$. The quantitative distribution pattern of carotenoids in Cryptophyceae established here and previously, totalling five species' is discussed in chemosystematic context. β,?-Carotene (3–8% of total) is the major carotene, accompanied by ?,?-carotene (0.2%), β,β-carotene (0–1%) and lycopene (0-trace). Zeaxanthin (2%) was identified in C. ovate. The diacetylenic alloxanthin is the major carotenoid (70–88%), and the monoacetylenic crocoxanthin (5–15%) and monadoxanthin (0–16%) less abundant. No epoxidic or allenic carotenoids could be detected. The biosynthetic precursor of acetylenic carotenoids in this primitive algal class is discussed. The significance of Cryptophyceae in the marine food chain is commented on, using alloxanthin as an indicator.     

CHLOROPLAST PIGMENT PATTERNS IN DINOFLAGELLATES1

The chlorophylls and carotenoids of 22 species of dinoflagellates were analysed by thin layer chromatography, using 2-dimensional sucrose plates, and 1-dimensional polyethylene plates for chlorophylls c₁ and c₂. Peridinin was the major carotenoid in 19 of the species, while fucoxanthin was the major carotenoid in 3. In the peridinin-containing species, 5 carotenoid fractions, constituting more than 95% of the total carotenoids, were always present. These were peridinin (± neo-peridinin), averaging 64% of the total carotenoid, diadinoxanthin, dinoxanthin, β-carotene and a polar, unidentified pink xanthophyll. Six other carotenoid fractions occurred in minor or trace quantities among the species, but were not identified. Two of these had, a wide distribution; the other 4 were restricted to one or 2 species. The chlorophyll content of the dinoflagellate cultures ranged from 1–141 μg chlorophyll a + c/10⁶ cells, a pattern which was broadly correlated with cell size. In the peridinin-containing species the ratio of chlorophyll a to c on a molar basis was approximately 2 (range 1.60–4.39); in the fucoxanthin-containing species this ratio was approximately 4 (range 2.65–5.73). Both chlorophylls c₁ and c₂ occurred in the fucoxanthin-containing dinoflagellates, and only chlorophyll c₂ (one exception) occurred in the peridinin-containing dinoflagellates. These patterns of chlorophyll c and major carotenoid correspond to patterns previously observed in the Pyrrhophyta and the Chrysophyta, suggesting different phylogenetic origins for the “dinoflagellate” chloroplasts.     

PIGMENTS OF BATHYCOCCUS PRASINOS (PRASINOPHYCEAE): METHODOLOGICAL AND CHEMOSYSTEMATIC IMPLICATIONS1,2

Bathycoccus prasinos Eikrem et Throndsen exhibited a complex carotenoid distribution pattern including the carotenes β,β-carotene (0.8% of total carotenoids) and β, ° Carotene (0.4%) and several xanthophylls. These were prasinoxanthin (49% of total carotenoids), micromonal (16%), neoxanthin (14%), uriolide (7%), violaxanthin (0.8%), 3¹-dehydrouriolide (0.8%), dihydrolutein (0.1%), two partly characterized esterified carotenols (together 10%), and five minor unidentified carotenols (together 2%). The identifications were based on high-performance liquid chromatography (HPLC), thin-layer chromatography (TLC), visible spectroscopy (VIS), and mass spectra (MS) and in part on ¹H nuclear magnetic resonance (NMR), circular dichroism (CD), and chemical derivatization. The carotenoid composition of B. prasinos was related to that of other prasinoxanthin / uriolide / micromonal-producing prasinophytes (Mantoniella squamata, Micromonas pusilla, and Pseudoscourfieldia marina). The relative distribution of chlorophylls (w/w) were chlorophyll a (chl a; 63%), chl b (31%), and an unknown chl c-like chlorophyll (7%) with spectral characteristics similar to magnesium 2,4-divinylphaeoporphyrin a, monomethyl ester, compatible with other prasinophytes. The chemosystematic data and ultrastructural characteristics for the order Mamiellales are discussed. We conclude that HPLC studies alone are insufficient for the identification and characterization of the carotenoids, including the minor carotenoids essential for biosynthetic/chemosystematic considerations.     

Formation of neoxanthin,diadinoxanthin and peridinin from [14C]zeaxanthin by a cell-free system from Amphidinium carterae

A cell-free system prepared from an axenic culture of the alga Amphidinium carterae converted [¹⁴C]zeaxanthin into neoxanthin and then into peridinin (62%) and diadinoxanthin (38%). Peridinin, therefore, is made by the excision of three carbon atoms from a C₄₀ carotenoid and the acetylene group of diadinoxanthin is formed from the allene of neoxanthin, rather than the reverse.     

Hyperdiversity of Genes Encoding Integral Light-Harvesting Proteins in the Dinoflagellate Symbiodinium sp

The superfamily of light-harvesting complex (LHC) proteins is comprised of proteins with diverse functions in light-harvesting and photoprotection. LHC proteins bind chlorophyll (Chl) and carotenoids and include a family of LHCs that bind Chl a and c. Dinophytes (dinoflagellates) are predominantly Chl c binding algal taxa, bind peridinin or fucoxanthin as the primary carotenoid, and can possess a number of LHC subfamilies. Here we report 11 LHC sequences for the chlorophyll a-chlorophyll c ₂-peridinin protein complex (acpPC) subfamily isolated from Symbiodinium sp. C3, an ecologically important peridinin binding dinoflagellate taxa. Phylogenetic analysis of these proteins suggests the acpPC subfamily forms at least three clades within the Chl a/c binding LHC family; Clade 1 clusters with rhodophyte, cryptophyte and peridinin binding dinoflagellate sequences, Clade 2 with peridinin binding dinoflagellate sequences only and Clades 3 with heterokontophytes, fucoxanthin and peridinin binding dinoflagellate sequences.     

Triplet state spectra and dynamics of peridinin analogs having different extents of π-electron conjugation

The Peridinin-Chlorophyll a-Protein (PCP) complex has both an exceptionally efficient light-harvesting ability and a highly effective protective capacity against photodynamic reactions involving singlet oxygen. These functions can be attributed to presence of a substantial amount of the highly-substituted and complex carotenoid, peridinin, in the protein and the facts that the low-lying singlet states of peridinin are higher in energy than those of chlorophyll (Chl) a, but the lowest-lying triplet state of peridinin is below that of Chl a. Thus, singlet energy can be transferred from peridinin to Chl a, but the Chl a triplet state is quenched before it can sensitize the formation of singlet oxygen. The present investigation takes advantage of Chl a as an effective triplet state donor to peridinin and explores the triplet state spectra and dynamics of a systematic series of peridinin analogs having different numbers of conjugated carbon–carbon double bonds. The carotenoids investigated are peridinin, which has a C₃₇ carbon skeleton and eight conjugated carbon–carbon double bonds, and three synthetic analogs: C₃₃-peridinin, having two less double bonds than peridinin, C₃₅-peridinin which has one less double bond than peridinin, and C₃₉-peridinin which has one more double bond than peridinin. In this study, the behavior of the triplet state spectra and kinetics exhibited by these molecules has been investigated in polar and nonpolar solvents and reveals a substantial effect of both π-electron conjugated chain length and solvent environment on the spectral lineshapes. However, only a small dependence of these factors is observed on the kinetics of triplet energy transfer from Chl a and on carotenoid triplet state deactivation to the ground state.     

A nitromethane‐based HPLC system alternative to acetonitrile for carotenoid analysis of fruit and vegetables

Acetonitrile‐based HPLC systems are the most commonly used for carotenoid analysis from different plant tissues. Because of the acetonitrile shortage, an HPLC system for the separation of carotenoids on C₁₈ reversed‐phase columns was developed in which an acetonitrile–alcohol‐based mobile phase was replaced by nitromethane. This solvent comes closest to acetonitrile with respect to its elutrophic property. Our criterion was to obtain similar separation and retention times for a range of differently structured carotenoids. This was achieved by further increase in the lipophilicity with ethylacetate. For all the carotenoids which we tested, we found co‐elution only of β‐cryptoxanthin and lycopene. By addition of 1% of water, separation of this pair of carotenoids was also achieved. The final recommended mobile phase consisted of nitromethane : 2‐propanol : ethyl acetate : water (79 : 10 : 10 : 1, by volume). On Nucleosil C₁₈ columns and related ones like Hypersil C₁₈, we obtained separation of carotenes, hydroxyl, epoxy and keto derivatives, which resembles the excellent separation properties of acetonitrile‐based mobile phases on C₁₈ reversed phase columns. We successfully applied the newly developed HPLC system to the separation of carotenoids from different vegetables and fruit. Copyright © 2010 John Wiley & Sons, Ltd.     

Magnetic field as a trigger of carotenoid production by Phaffia rhodozyma

This study aimed to evaluate the influence of magnetic fields (MF) on inoculum cultivation and carotenoid production by Phaffia rhodozyma. The application of MF in the inoculum culture was evaluated (0 m T – control and 30 m T). Cellular concentration increased by 12.8 % after 24 h-culture with MF application compared to the control assay, and this was the best alternative for the preparation of inoculum. Different intervals of MF application were evaluated over 168 h. The highest volumetric carotenoids concentration was achieved by applying MF throughout cultivation, with values of 1146.39 ± 26.18 μg L⁻¹ and carotenoid productivity of 11.94 ± 1.11 μg L⁻¹ h⁻¹ in 96 h. As a result, carotenoid production increased by 59.4 % and carotenoid productivity by 99.3 %. This study is one of the first to consider MF application in carotenoid production using P. rhodozyma as a viable and low-cost alternative for carotenoid production in a shorter cultivation time.     

A C35 Carotenoid Biosynthetic Pathway

Upon coexpression with Erwinia geranylgeranyldiphosphate (GGDP) synthase in Escherichia coli, C₃₀ carotenoid synthase CrtM from Staphylococcus aureus produces novel carotenoids with the asymmetrical C₃₅ backbone. The products of condensation of farnesyldiphosphate and GDP, C₃₅ structures comprise 40 to 60% of total carotenoid accumulated. Carotene desaturases and carotene cyclases from C₄₀ or C₃₀ pathways accepted and converted the C₃₅ substrate, thus creating a C₃₅ carotenoid biosynthetic pathway in E. coli. Directed evolution to modulate desaturase step number, together with combinatorial expression of the desaturase variants with lycopene cyclases, allowed us to produce at least 10 compounds not previously described. This result highlights the plastic and expansible nature of carotenoid pathways and illustrates how combinatorial biosynthesis coupled with directed evolution can rapidly access diverse chemical structures.     

Effect of the carbon source on the carotenoid profiles of Phaffia rhodozyma strains

Phaffia rhodozyma strains ATCC 24202, ATCC 24203, ATCC 24228, ATCC 24229, ATCC 24261, NRRL Y-10921, NRRL Y-10922 and NRRL Y-17268 were grown on culture media containing glucose, sucrose or xylose as carbon sources. Carotenoids were extracted from biomass and analyzed by HPLC with diode-array detection. The carotenoid profiles depended on both the strain considered and the carbon source employed. Astaxanthin, the main pigment found in P. rhodozyma, accounted for 42–91% of total carotenoids. Other carotenoids such as canthaxanthin, echinenone, 3-hydroxyechinenone, lycopene, 4-hydroxy-3′, 4′-didehydro-β-ψ-carotene and phoenicoxanthin were detected. The highest volumetric carotenoid concentration (3.60 mg L⁻¹) was obtained with strain NRRL Y-17268 growing on xylose. In this case, astaxanthin accounted for 82% of total carotenoids. Received 29 May 1997/ Accepted in revised form 08 August 1997     

In planta cleavage of carotenoids by <Emphasis Type="Italic">Arabidopsis</Emphasis> carotenoid cleavage dioxygenase 4 in transgenic rice plants

A family of carotenoid cleavage dioxygenases (CCDs) produces diverse apocarotenoid compounds via the oxidative cleavage of carotenoids as substrates. Their types are highly dependent on the action of the CCD family to cleave the double bonds at the specific position on the carotenoids. Here, we report in vivo function of the AtCCD4 gene, one of the nine members of the Arabidopsis CCD gene family, in transgenic rice plants. Using two independent single-copy rice lines overexpressing the AtCCD4 transgene, the targeted analysis for carotenoids and apocarotenoids showed the markedly lowered levels of β-carotene (74 %) and lutein (72 %) along with the changed levels of two β-carotene (C₄₀) cleavage products, a two-fold increase of β-ionone (C₁₃) and de novo generation of β-cyclocitral (C₁₀) at lower levels, compared with non-transgenic rice plants. It suggests that β-carotene could be the principal substrate being cleaved at 9–10 (9′–10′) for β-ionone and 7–8 (7′–8′) positions for β-cyclocitral by AtCCD4. This study is in planta report on the generation of apocarotenal volatiles from carotenoid substrates via cleavage by AtCCD4. We further verified that the production of these volatiles was due to the action of exogenous AtCCD4 and not the expression of endogenous rice CCD genes (OsCCD1, 4a, and 4b).     

Carotenoids of Prymnesiophyceae (Haptophyceae)

A quantitative study, including mass spectrometric identification, of the carotenoids isolated from some selected prymnesiophytes harvested in exponential growth phase has been carried out. Isochrysis galbana, Hymenomonas carterae, Prymnesium parvum, Pavlova (Monochrysis) lutheri and a Pavlova sp. all produce β,β-carotene, diatoxanthin, diadinoxanthin and fucoxanthin (major carotenoid) in various proportions, in addition to several minor carotenoids were found characteristic of each alga. 19'-Hexanoyloxyfucoxanthin, previously shown to be the major carotenoid in the prymnesiophyte Emeliana (Coccolithus) huxleyi, was not encountered in the Prymnesiophyceae studied here, and we conclude that this carotenoid should be critically looked for in other members of the Gephyrocapsaceae to which E. huxleyi belongs. We further conclude that the carotenoid complement of the Chrysophyceae (in the narrow sense) should be compared with that of the Prymnesiophyceae.     

Structural Confirmation of a Unique Carotenoid Lactoside,P457, in Symbiodinium sp. Strain nbrc 104787 Isolated from a Sea Anemone and its Distribution in Dinoflagellates and Various Marine Organisms

The molecular structure of the carotenoid lactoside P457, (3S,5R,6R,3′S,5′R,6′S)‐13′‐cis‐5,6‐epoxy‐3′,5′‐dihydroxy‐3‐(β‐d ‐galactosyl‐(1→4)‐β‐d ‐glucosyl)oxy‐6′,7′‐didehydro‐5,6,7,8,5′,6′‐hexahydro‐β,β‐caroten‐20‐al, was confirmed by spectroscopic methods using Symbiodinium sp. strain NBRC 104787 cells isolated from a sea anemone. Among various algae, cyanobacteria, land plants, and marine invertebrates, the distribution of this unique diglycosyl carotenoid was restricted to free‐living peridinin‐containing dinoflagellates and marine invertebrates that harbor peridinin‐containing zooxanthellae. Neoxanthin appeared to be a common precursor for biosynthesis of peridinin and P457, although neoxanthin was not found in peridinin‐containing dinoflagellates. Fucoxanthin‐containing dinoflagellates did not possess peridinin or P457; green dinoflagellates, which contain chlorophyll a and b, did not contain peridinin, fucoxanthin, or P457; and no unicellular algae containing both peridinin and P457, other than peridinin‐containing dinoflagellates, have been observed. Therefore, the biosynthetic pathways for peridinin and P457 may have been coestablished during the evolution of dinoflagellates after the host heterotrophic eukaryotic microorganism formed a symbiotic association with red alga that does not contain peridinin or P457.     

Lipid-dissolved γ-carotene, β-carotene,and lycopene in globular chromoplasts of peach palm (Bactris gasipaes Kunth) fruits

Main conclusion

High levels of β-carotene, lycopene, and the rare γ-carotene occur predominantly lipid-dissolved in the chromoplasts of peach palm fruits. First proof of their absorption from these fruits is reported. The structural diversity, the physical deposition state in planta, and the human bioavailability of carotenoids from the edible fruits of diverse orange and yellow-colored peach palm (Bactris gasipaes Kunth) varieties were investigated. HPLC–PDA–MSⁿ revealed a broad range of carotenes, reaching total carotenoid levels from 0.7 to 13.9 mg/100 g FW. Besides the predominant (all-E)-β-carotene (0.4–5.4 mg/100 g FW), two (Z)-isomers of γ-carotene (0.1–3.9 mg/100 g FW), and one (Z)-lycopene isomer (0.04–0.83 mg/100 g FW) prevailed. Approximately 89–94 % of total carotenoid content pertained to provitamin A carotenoids with retinol activity equivalents ranging from 37 to 609 µg/100 g FW. The physical deposition state of these carotenoids in planta was investigated using light, transmission electron, and scanning electron microscopy. The plastids found in both orange and yellow-colored fruit mesocarps were amylo-chromoplasts of the globular type, containing carotenoids predominantly in a lipid-dissolved form. The hypothesis of lipid-dissolved carotenoids was supported by simple solubility estimations based on carotenoid and lipid contents of the fruit mesocarp. In our study, we report first results on the human bioavailability of γ-carotene, β-carotene, and lycopene from peach palm fruit, particularly proving the post-prandial absorption of the rarely occurring γ-carotene. Since the physical state of carotenoid deposition has been shown to be decisive for carotenoid bioavailability, lipid-dissolved carotenoids in peach palm fruits are expected to be highly bioavailable, however, further studies are required.     

CD36 homolog divergence is responsible for the selectivity of carotenoid species migration to the silk gland of the silkworm Bombyx mori

Dietary carotenoids are absorbed in the intestine and delivered to various tissues by circulating lipoproteins; however, the mechanism underlying selective delivery of different carotenoid species to individual tissues remains elusive. The products of the Yellow cocoon (C) gene and the Flesh (F) gene of the silkworm Bombyx mori determine the selectivity for transport of lutein and β-carotene, respectively, to the silk gland. We previously showed that the C gene encodes Cameo2, a CD36 family member, which is thought to function as a transmembrane lipoprotein receptor. Here, we elucidated the molecular identity of the F gene product by positional cloning, as SCRB15, a paralog of Cameo2 with 26% amino acid identity. In the F mutant, SCRB15 mRNA structure was severely disrupted, due to a 1.4 kb genomic insertion in a coding exon. Transgenic expression of SCRB15 in the middle silk gland using the binary GAL4-UAS expression system enhanced selective β-carotene uptake by the middle silk gland, while transgenic expression of Cameo2 enhanced selective lutein uptake under the same GAL4 driver. Our findings indicate that divergence of genes in the CD36 family determines the selectivity of carotenoid species uptake by silk gland tissue and that CD36-homologous proteins can discriminate among carotenoid species.     

Isolation and identification of 2-hydroxyplectaniaxanthin from Rhodotorula aurantiaca

A new trihydroxyl carotenoid has been isolated from the yeast Rhodotorula aurantiaca (Saito) Lodder C.B.S. 317 and identified as 2-hydroxyplectaniaxanthin (3′,4′-didehydro,1′,2′-dihydro-β, ψ-caroten-2,1′,2′-triol). Its m.p., partition coefficient, _Rf, extinction coefficient, ms and NMR spectra are reported. Since the hydroxyl group at C-2 of the β-ionone ring is unusual, a possible mechanism for the biosynthesis of this carotenoid has been proposed.     

Carotenoid to bacteriochlorophyll energy transfer in the RC–LH1–PufX complex from <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis> containing the extended conjugation keto-carotenoid diketospirilloxanthin

RC–LH1–PufX complexes from a genetically modified strain of Rhodobacter sphaeroides that accumulates carotenoids with very long conjugation were studied by ultrafast transient absorption spectroscopy. The complexes predominantly bind the carotenoid diketospirilloxanthin, constituting about 75% of the total carotenoids, which has 13 conjugated C=C bonds, and the conjugation is further extended to two terminal keto groups. Excitation of diketospirilloxanthin in the RC–LH1–PufX complex demonstrates fully functional energy transfer from diketospirilloxanthin to BChl a in the LH1 antenna. As for other purple bacterial LH complexes having carotenoids with long conjugation, the main energy transfer route is via the S₂–Q_x pathway. However, in contrast to LH2 complexes binding diketospirilloxanthin, in RC–LH1–PufX we observe an additional, minor energy transfer pathway associated with the S₁ state of diketospirilloxanthin. By comparing the spectral properties of the S₁ state of diketospirilloxanthin in solution, in LH2, and in RC–LH1–PufX, we propose that the carotenoid-binding site in RC–LH1–PufX activates the ICT state of diketospirilloxanthin, resulting in the opening of a minor S₁/ICT-mediated energy transfer channel.     

Partial quantification of pigments extracted from the zooxanthellate octocoral <Emphasis Type="Italic">Sinularia flexibilis</Emphasis> at varying irradiances

Chlorophyll-a (chl-a) and carotenoid pigments of the zooxanthellate octocoral Sinularia flexibilis were analyzed using high performance liquid chromatography following exposure to three light intensities for over 30 days. From the coral fragments located at different light intensities, a total carotenoid of >41 μg g⁻¹ dry weight, including peridinin, xanthophylls (likely diadinoxanthin + diatoxanthin), and chl-a as the most abundant pigments, with minor contents of astaxantin and β-carotene were detected. The whole content of chl-a weighed 5 μg g⁻¹ dry weight in all coral colonies. Chl-a and carotenoids contributed 11.2% and 88.2%, respectively, to all pigments detected, and together accounted for 99.4% of the total pigments present. The highest contents of carotenoids and chl-a was observed in the coral grafts placed in an irradiance of 100 μmol quanta m⁻² s⁻¹; they showed lower ratios of total carotenoids: chl-a compared to those exposed to 400 μmol quanta m⁻² s⁻¹ after >30 days of incubation. The ratios of peridinin and xanthophylls with respect to chl-a from the colonies at 400 μmol quanta m⁻² s⁻¹ were approximately double those observed at irradiances of 100 and 200 μmol quanta m⁻² s⁻¹. Partial quantification of pigments in this study showed that the carotenoids of S. flexibilis showed a decrease at irradiances above 100 μmol quanta m⁻² s⁻¹, with the exception of an increase in β-carotene at 200 μmol quanta m⁻² s⁻¹.