Carotenoids in fish. XXVI. Pungitius pungitius (L.) and Gasterosteus aculeatus L. (Gasterosteidae)

The author investigated the presence of various carotenoids in the different parts of the body of Pungitius pungitius (L.) and Gasterosteus aculeatus L. by means of columnar and thin-layer chromatography. The investigations revealed the presence of the following carotenoids:

in Pungitius pungitius. α-carotene, β-carotene, β-cryptoxanthin, mutatochrome, zeaxanthin and astaxanthin;

in Gasterosteus aculeatus: β-carotene, β-cryptoxanthin, β-carotene epoxide, neothxanthin, canthaxanthin, mutatochrome, lutein, phoenicoxanthin, zeaxanthin, taraxanthin, tunaxanthin, astaxanthin, astaxanthin ester and α-doradexanthin. The total carotenoid content ranged from 2.229 to 138.504 µg/g wet weight.

     

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.     

Antioxidant activity of palm oil carotenes in peroxyl radical-mediatedperoxidation of phosphatidyl choline liposomes

Abstract

The antioxidant efficacy of α-carotene and comparison with β-carotene in multilamellar liposomes prepared from egg yolk phosphatidyl choline (EYPC) exposed to the lipid soluble 2,2′-azobis (2,4-dimethyl valeronitrile) (AMVN) was investigated. Lipid peroxidation was measured as thiobarbituric acid reacting substances (TBARS)at 532 nm or as hydroperoxide formation at 234 nm after separation of phosphatidyl choline hydroperoxide (PCOOH) by high-pressure liquid chromatography (HPLC). Lutein and zeaxanthin, the hydroxyl derivatives of α- and β-carotenes, and the chain breaking antioxidant α-tocopherol were also included in the study.AMVN being a lipid soluble, non polar azo initiator penetrates into the hydrophobic interior of the phospholipid bilayer, forming peroxyl radicals which peroxidate the phospholipid leading to PCOOH accumulation. All the carotenoids tested at 1 mol% relative to EYPC significantly suppressed the formation of PCOOH compared to control samples.In this system, α-carotene retarded PCOOH formation better than β-carotene. Similarly, lutein was a better antioxidant than is zeaxanthin. But lutein and zeaxanthin were more effective antioxidants than α- and β-carotenes, respectively. After 1 h of incubation of the carotenoid with AMVN, α-, β-carotene, lutein and zeaxanthin limited PCOOH formation by 77%, 68%, 85%and 82%, respectively, while α-tocopherol elicited 90%reduction.AMVN incubated with EYPC for 2 h induced the formation of TBARS compared to control (P <0.001). α-Carotene significantly suppressed the TBARS formation by 78% whilst β-carotene, lutein, zeaxanthin and α-tocopherol elicited 60%, 91%and 80% reductions, respectively. Increasing the concentration of the carotenoid >1 mol% to EYPC did not significantly increase protection of the membrane against free radical attack.Our findings suggest that α-carotene is a better antioxidant than is β-carotene in phosphatidyl choline vesicles. It may, therefore, be useful in limiting free radical mediated peroxidative damage against membrane phospholipids _{in vivo}.     

Investigations of carotenoids in some animals of the Adriatic Sea V. Echinodermata

The author investigated the carotenoids in the Echinodermata from Adriatic sea by means of columnar and thin-layer chromatography. The following carotenoids were identified:

- in Coscinasterias tenuispina: β-carotene, isocryptoxanthin lutein, lutein-5, 6-epoxide, 4-hydroxy-4-keto-β-carotene, zeaxanthin, astaxanthin and asterinacid.

- in Marthasterias glacialis: β-carotene, echinenone, cryptoxanthin, lutein, lutein 5, 6-epoxide, 4-hydroxy-4-keto-β-carotene, zeaxanthin, astaxanthin ester, astaxanthin and 3, 4-didehydro-α-carotene.

- in Paracentrotus lividus: β-carotene, echinenone, cryptothin, isocryptoxanthin, lutein, lutein-5, 6-epoxide, 4-hydroxy-4-keto-β-carotene, zeaxanthin, astaxanthin, astaxanthin ester and asterinacid.

- in Sphaerechinus granularis: ,β-carotene, echinenone, cryptoxanthin, lutein, lutein-5, 6-epoxide, astaxanthin and guaraxanthin.

     

Flower color alteration in <Emphasis Type="Italic">Lotus japonicus</Emphasis> by modification of the carotenoid biosynthetic pathway

To establish a model system for alteration of flower color by carotenoid pigments, we modified the carotenoid biosynthesis pathway of Lotus japonicus using overexpression of the crtW gene isolated from marine bacteria Agrobacterium aurantiacum and encoding β-carotene ketolase (4,4′-β-oxygenase) for the production of pink to red color ketocarotenoids. The crtW gene with the transit peptide sequence of the pea Rubisco small subunit under the regulation of the CaMV35S promoter was introduced to L. japonicus. In most of the resulting transgenic plants, the color of flower petals changed from original light yellow to deep yellow or orange while otherwise exhibiting normal phenotype. HPLC and TLC analyses revealed that leaves and flower petals of these plants accumulated novel carotenoids, believed to be ketocarotenoids consisting of including astaxanthin, adonixanthin, canthaxanthin and echinenone. Results indicated that modification of the carotenoid biosynthesis pathway is a means of altering flower color in ornamental crops.     

Over-expression of Arabidopsis thaliana carotenoid hydroxylases individually and in combination with a β-carotene ketolase provides insight into in vivo functions

Carotenoids represent a group of widely distributed pigments derived from the general isoprenoid biosynthetic pathway that possess diverse functions in plant primary and secondary metabolism. Modification of α- and β-carotene backbones depends in part on ring hydroxylation. Two ferredoxin-dependent non-heme di-iron monooxygenases (AtB1 and AtB2) that mainly catalyze in vivo β-carotene hydroxylations of β,β-carotenoids, and two heme-containing cytochrome P450 (CYP) monooxygenases (CYP97A3 and CYP97C1) that preferentially hydroxylate the ε-ring of α-carotene or the β-ring of β,ε-carotenoids, have been characterized in Arabidopsis by analysis of loss-of-function mutant phenotypes. We further investigated functional roles of both hydroxylase classes in modification of the β- and ε-rings of α-carotene and β-carotene through over-expression of AtB1, CYP97A3, CYP97C1, and the hydroxylase candidate CYP97B3. Since carotenoid hydroxylation is required for generation of ketocarotenoids by the bkt1(CrtO) β-carotene ketolase, all hydroxylase constructs were also introduced into an Arabidopsis line expressing the Haematococcus pluvalis bkt1 β-carotene ketolase. Analysis of foliar carotenoid profiles in lines overexpressing the individual hydroxylases indicate a role for CYP97B3 in carotenoid biosynthesis, confirm and extend previous findings of hydroxylase activities based on knock-out mutants, and suggest functions of the multifunctional enzymes in carotenoid biosynthesis. Hydroxylase over-expression in combination with bkt1 did not result in ketocarotenoid accumulation, but instead unexpected patterns of α-carotene derivatives, accompanied by a reduction of α-carotene, were observed. These data suggest possible interactions between the β-carotene ketolase bkt1 and the hydroxylases that impact partitioning of carbon flux into different carotenoid branch pathways.     

Enhancement of carotenoid biosynthesis in the green microalga Dunaliella salina with light-emitting diodes and adaptive laboratory evolution

There is a particularly high interest to derive carotenoids such as β-carotene and lutein from higher plants and algae for the global market. It is well known that β-carotene can be overproduced in the green microalga Dunaliella salina in response to stressful light conditions. However, little is known about the effects of light quality on carotenoid metabolism, e.g., narrow spectrum red light. In this study, we present UPLC-UV-MS data from D. salina consistent with the pathway proposed for carotenoid metabolism in the green microalga Chlamydomonas reinhardtii. We have studied the effect of red light-emitting diode (LED) lighting on growth rate and biomass yield and identified the optimal photon flux for D. salina growth. We found that the major carotenoids changed in parallel to the chlorophyll b content and that red light photon stress alone at high level was not capable of upregulating carotenoid accumulation presumably due to serious photodamage. We have found that combining red LED (75 %) with blue LED (25 %) allowed growth at a higher total photon flux. Additional blue light instead of red light led to increased β-carotene and lutein accumulation, and the application of long-term iterative stress (adaptive laboratory evolution) yielded strains of D. salina with increased accumulation of carotenoids under combined blue and red light.     

Elicitors,SA and MJ enhance carotenoids and tocopherol biosynthesis and expression of antioxidant related genes in Moringa oleifera Lam. leaves

Moringa oleifera Lam. leaves are rich source of carotenoids (provitamin A) and α-tocopherol (vitamin E), and there is a scope for their further enhancement, through elicitor mediation, thereby a great potential for addressing these vitamins deficiency. In the present study, we report the efficacy of foliar administration of biotic elicitors, carboxy-methyl chitosan and chitosan, and signaling molecules, methyl jasmonate (MJ) and salicylic acid (SA) for enhancement of major carotenoids and α-tocopherol. Highest α-tocopherol content of 49.7 mg/100 g FW was recorded upon foliar application of 0.1 mM SA after 24 h of treatment, which represented a 187.5 % increase in comparison to the untreated control. Similarly, a maximum of 52.6 mg/100 g FW lutein, and 21.8 mg/100 g FW β-carotene content were observed in leaves after 24 h of treatment with MJ, which represented a 54.0 and 20.3 % increase in comparison to the untreated control, respectively. Among the major genes of carotenoid biosynthetic pathway, the expression of lycopene β-cyclase (LCY-β) was maximum influenced after treatment with elicitors and signaling molecules, compared to phytoene synthase and phytoene desaturase, suggesting the LCY-β-mediated enhancement in the production of β-carotene in elicitor treated M. oleifera leaves. Enhanced production of α-tocopherol under respective elicitor treatment was further supported by 2.0–2.7 fold up-regulation of γ-tocopherol methyl transferase, compared to untreated control. This is the first report on elicitor-mediated enhanced production of tocopherol and carotenoids in foliage of economically important food plant.     

Antioxidant activity of carotenoids: An electron-spin resonance study on β-carotene and lutein interaction with free radicals generated in a chemical system

β-Carotene is thought to be a chain-breaking antioxidant, even though we have no information about the mechanism of its antioxidant activity. Using electron-spin resonance (ESR) spectroscopy coupled to the spin-trapping technique, we have studied the effect of β-carotene and lutein on the radical adducts of the spin-trap PBN (N-t -butyl-α-phenylnitrone) generated by the metal-ion breakdown of different tert -butyl hydroperoxide (t BOOH) concentrations in methylene chloride. The peroxyl radical, along with an oxidation product of PBN (the PBNOx), trapped at room temperature from the breakdown of high concentration of t BOOH (1 M), were quenched by β-carotene or lutein, in competition with the spin-trapping agent. However, carotenoids were not able to quench the alkoxyl and methyl radicals generated in the reaction carried out in the presence of low t BOOH concentration (1 mM). The reaction between carotenoids and the peroxyl radical was also carried out in the absence of the spin trap, at 77 K: Under these different experimental conditions, we did not detect any radical species deriving from carotenoids. In the same system, a further evidence of the peroxyl radical quenching by β-carotene and lutein was obtained. The antioxidant activity of vitamin E was also tested, for comparison with the carotenoids. In the presence of α-tocopherol, peroxyl and alkoxyl radicals were quenched, and the tocopheroxyl radical was detected. Our data provide the first direct evidence that carotenoids quench peroxyl radicals. Under our experimental conditions, we did not detect any carotenoid radical species that could derive from the interaction with the peroxyl radical. The radical-trapping activity of β-carotene and lutein demonstrated in this chemical reaction contributes to our understanding carotenoid antioxidant action in biological systems. © 1998 John Wiley & Sons, Inc. J Biochem Toxicol 12: 299–304, 1998     

Genetic architecture controlling variation in grain carotenoid composition and concentrations in two maize populations

Key message

Genetic control of maize grain carotenoid profiles is coordinated through several loci distributed throughout three secondary metabolic pathways, most of which exhibit additive, and more importantly, pleiotropic effects.

Abstract

The genetic basis for the variation in maize grain carotenoid concentrations was investigated in two F_2:3 populations, DEexp × CI7 and A619 × SC55, derived from high total carotenoid and high β-carotene inbred lines. A comparison of grain carotenoid concentrations from population DEexp × CI7 grown in different environments revealed significantly higher concentrations and greater trait variation in samples harvested from a subtropical environment relative to those from a temperate environment. Genotype by environment interactions was significant for most carotenoid traits. Using phenotypic data in additive, environment-specific genetic models, quantitative trait loci (QTL) were identified for absolute and derived carotenoid traits in each population, including those specific to the isomerization of β-carotene. A multivariate approach for these correlated traits was taken, using carotenoid trait principal components (PCs) that jointly accounted for 97 % or more of trait variation. Component loadings for carotenoid PCs were interpreted in the context of known substrate-product relationships within the carotenoid pathway. Importantly, QTL for univariate and multivariate traits were found to cluster in close proximity to map locations of loci involved in methyl-erythritol, isoprenoid and carotenoid metabolism. Several of these genes, including lycopene epsilon cyclase, carotenoid cleavage dioxygenase1 and beta-carotene hydroxylase, were mapped in the segregating populations. These loci exhibited pleiotropic effects on α-branch carotenoids, total carotenoid profile and β-branch carotenoids, respectively. Our results confirm that several QTL are involved in the modification of carotenoid profiles, and suggest genetic targets that could be used for the improvement of total carotenoid and β-carotene in future breeding populations.     

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.

     

Effects of salinity increase on carotenoid accumulation in the green alga Dunaliella salina

The effect of sudden salinity increases on the kinetics of growth and carotenogenesis was studied in three geographically diverse isolates of Dunaliella saliva. A sudden increase in salinity results in a lag phase in growth and the length of this lag phase is dependent on the final salinity and the magnitude of the salinity change (no lag at 10–15% w/v NaCl, 4-day lag at 30% NaCl). There is also a lag before an increase in the total carotenoid content can be measured following the salinity up-shock, and the length of the lag depends largely on the initial salinity and the magnitude of the salinity up-shock, whereas the rate of carotenogenesis and the final carotenoid content reached depend on the final salinity. The increase in total carotenoid content is mainly due to β-carotene. Following the salinity up-shock (especially from 10% to 20% NaCl) the proportion of lutein as a percentage of total carotenoids decreases, whereas zeaxanthin increases. This suggests that the pathway synthesising lutein is more sensitive to salt or osmotic stress and is inhibited at higher salinities, thus leading to β-carotene formation. The proportion of α-carotene does not change.     

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).     

Strong and weak plasma response to dietary carotenoids identified by cluster analysis and linked to beta-carotene 15,15'-monooxygenase 1 single nucleotide polymorphisms

The mechanisms as well the genetics underlying the bioavailability and metabolism of carotenoids in humans remain unclear. To begin to address these questions, we used cluster analysis to examine individual temporal responses of plasma carotenoids from a controlled-diet study of subjects who consumed carotenoid-rich beverages. Treatments, given daily for 3 weeks, were watermelon juice at two levels (20-mg lycopene, 2.5-mg β-carotene, n=23 and 40-mg lycopene, 5-mg β-carotene, n=12) and tomato juice (18-mg lycopene, 0.6-mg β-carotene, n=10). Cluster analysis revealed distinct groups of subjects differing in the temporal response of plasma carotenoids and provided the basis for classifying subjects as strong responders or weak responders for β-carotene, lycopene, phytoene and phytofluene. Individuals who were strong or weak responders for one carotenoid were not necessarily strong or weak responders for another carotenoid. Furthermore, individual responsiveness was associated with genetic variants of the carotenoid metabolizing enzyme β-carotene 15,15'-monooxygenase 1. These results support the concept that individuals absorb or metabolize carotenoids differently across time and suggest that bioavailability of carotenoids may involve specific genetic variants of β-carotene 15,15'-monooxygenase 1.     

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.     

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.     

Salt stress (NaCl) affects plant growth and branch pathways of carotenoid and flavonoid biosyntheses in <Emphasis Type="Italic">Solanum nigrum</Emphasis>

In this study, we set out to investigate the effect of sodium chloride (NaCl) on carotenoid and flavonoid production by the black nightshade (Solanum nigrum L.). The study was carried out under green chamber conditions using seedlings subjected to 0, 50, 100 and 150 mM NaCl for 3 weeks. The negative effect of NaCl on dry biomass production of roots and leaves were accompanied by a significant restriction in K⁺, Ca²⁺ and Mg²⁺ ion uptake and by an increase in Na⁺ ion concentrations, the effects of which were most pronounced at the highest NaCl level. Salt stress also induced oxidative stress, according to the amplified levels of thiobarbituric acid reactive substances and relative ion leakage ratio. Expression of some related carotenoid (phytoene synthase 2 and β-lycopene cyclase) and flavonoids genes (phenylalanine ammonialyase, chalcone synthase and flavonol synthase) were induced by NaCl, followed enhanced production of β-carotene, lutein, and quercetin 3-β-d-glucoside. At the highest NaCl level (150 mM NaCl), quercetin 3-β-d-glucoside synthesis came at the expense of reduced β-carotene and lutein, while salt stress treatment affected leaf antioxidant activities to a great extent relative to the control. Our data suggest that the potential antioxidant properties of carotenoids and flavonoids and their related key genes may be efficiently involved in the restriction of salt-induced oxidative damages.