Red Ketocarotenoids Research Articles

Specific carotenoid pigments in the diet and a bit of oxidative stress in the recipe for producing red carotenoid-based signals.

Colorful ornaments have been the focus of sexual selection studies since the work of Darwin. Yellow to red coloration is often produced by carotenoid pigments. Different hypotheses have been formulated to explain the evolution of these traits as signals of individual quality. Many of these hypotheses involve the existence of a signal production cost. The carotenoids necessary for signaling can only be obtained from food. In this line, carotenoid-based signals could reveal an individual’s capacity to find sufficient dietary pigments. However, the ingested carotenoids are often yellow and became transformed by the organism to produce pigments of more intense color (red ketocarotenoids). Biotransformation should involve oxidation reactions, although the exact mechanism is poorly known. We tested the hypothesis that carotenoid biotransformation could be costly because a certain level of oxidative stress is required to correctly perform the conversion. The carotenoid-based signals could thus reveal the efficiency of the owner in successfully managing this challenge. In a bird with ketocarotenoid-based ornaments (the red-legged partridge; Alectoris rufa), the availability of different carotenoids in the diet (i.e. astaxanthin, zeaxanthin and lutein) and oxidative stress were manipulated. The carotenoid composition was analyzed and quantified in the ornaments, blood, liver and fat. A number of oxidative stress biomarkers were also measured in the same tissues. First, we found that color and pigment levels in the ornaments depended on food levels of those carotenoids used as substrates in biotransformation. Second, we found that birds exposed to mild levels of a free radical generator (diquat) developed redder bills and deposited higher amounts of ketocarotenoids (astaxanthin) in ornaments. Moreover, the same diquat-exposed birds also showed a weaker resistance to hemolysis when their erythrocytes were exposed to free radicals, with females also enduring higher oxidative damage in plasma lipids. Thus, higher color production would be linked to higher oxidative stress, supporting the biotransformation hypothesis. The recent discovery of an avian oxygenase enzyme involved in converting yellow to red carotenoids may support our results. Nonetheless, the effect could also depend on the abundance of specific substrate carotenoids in the diet. Birds fed with proportionally higher levels of zeaxanthin showed the reddest ornaments with the highest astaxanthin concentrations. Moreover, these birds tended to show the strongest diquat-mediated effect. Therefore, in the evolution of carotenoid-based sexual signals, a biotransformation cost derived from maintaining a well-adjusted redox machinery could coexist with a cost linked to carotenoid acquisition and allocation (i.e. a resource allocation trade-off).

Effects of diet on plumage coloration and carotenoid deposition in red and yellow domestic canaries (Serinus canaria)

ABSTRACT Atlantic Canaries (Serinus canaria) are the most commonly kept caged bird with extensive carotenoid-based plumage coloration. Domestic strains of canaries have been bred for a variety of colors and patterns, making them a valuable model for studies of the genetic bases for feather pigmentation. However, no detailed account has been published on feather pigments of the various strains of this species, particularly in relation to dietary pigments available during molt. Moreover, in the twentieth century, aviculturists created a red canary by crossing Atlantic Canaries with Red Siskins (Carduelis cucullata). This “red-factor” canary is reputed to metabolically transform yellow dietary pigments into red ketocarotenoids, but such metabolic capacity has yet to be documented in controlled experiments. We fed molting yellow and red-factor canaries seed diets supplemented with either β-carotene, lutein/zeaxanthin, or β-cryptoxanthin/β-carotene and measured the coloration and carotenoid content of newly gr...

Copper exposure reduces production of red carotenoids in a marine copepod

Sub-lethal exposure to copper has been shown to modulate both mitochondrial function and antioxidant gene expression in zooplankton. To date, however, researchers have not identified a quantifiable phenotypic trait that reliably indicates such physiological responses to copper exposure. Red ketocarotenoids are abundant in marine zooplankton serving both physiological and coloration roles, and their production is sensitive to environmental stress. In this study the expression of mitochondrial gene cytochrome c oxidase I (COI) and antioxidant gene glutathione reductase (GR), and the production of red ketocarotenoid, astaxanthin, was measured in response to sub-lethal copper exposure. We found that mRNA of COI and GR was more abundant in copper-exposed copepods than controls, suggesting there was a physiological response to copper exposure. At the same time, copper-exposed copepods produced less astaxanthin than controls. We suggest that ketocarotenoid content of zooplankton has the potential to be a sensitive bioindicator of marine environmental pollution. Understanding how cellular responses to environmental stressors manifest in the phenotypes of marine animals will greatly increase our capacity to monitor marine ecosystem health.

Genetic Basis for Red Coloration in Birds

The yellow and red feather pigmentation of many bird species [1] plays pivotal roles in social signaling and mate choice [2, 3]. To produce red pigments, birds ingest yellow carotenoids and endogenously convert them into red ketocarotenoids via an oxidation reaction catalyzed by a previously unknown ketolase [4-6]. We investigated the genetic basis for red coloration in birds using whole-genome sequencing of red siskins (Spinus cucullata), common canaries (Serinus canaria), and "red factor" canaries, which are the hybrid product of crossing red siskins with common canaries [7]. We identified two genomic regions introgressed from red siskins into red factor canaries that are required for red coloration. One of these regions contains a gene encoding a cytochrome P450 enzyme, CYP2J19. Transcriptome analysis demonstrates that CYP2J19 is significantly upregulated in the skin and liver of red factor canaries, strongly implicating CYP2J19 as the ketolase that mediates red coloration in birds. Interestingly, a second introgressed region required for red feathers resides within the epidermal differentiation complex, a cluster of genes involved in development of the integument. Lastly, we present evidence that CYP2J19 is involved in ketocarotenoid formation in the retina. The discovery of the carotenoid ketolase has important implications for understanding sensory function and signaling mediated by carotenoid pigmentation.

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.

Carotenoid profile and vitamins in the combs of the red grouse (Lagopus lagopus scoticus): implications for the honesty of a sexual signal

The carotenoid-based ornaments displayed by many birds often play key roles in social and sexual signalling, revealing information about individual quality. However, the proximate regulation of the honesty of sexual traits remains controversial. Understanding the mechanisms of coloured trait production and maintenance requires an accurate description of their chemical composition and of the physiological pathways involved in pigment production and deposition in the ornaments. Carotenoid-based colouration has been extensively studied in birds, but such information is often lacking for coloured integuments other than feathers, such as fleshy carotenoid-based ornaments. Here we report the carotenoid composition of the combs of the red grouse (Lagopus lagopus scoticus), a sexual trait that honestly reveals individual quality. In the present study, we also investigated blood carotenoid content, as well as associations between carotenoids, retinol and tocopherol (the active forms of vitamin A and E, respectively) within the ornament. We found that comb pigmentation was primarily the result of two red ketocarotenoids (astaxanthin and papilioerythrinone), which are synthesised from their dietary precursors (zeaxanthin and lutein) directly at the comb integument. These red ketocarotenoids are largely deposited esterified with fatty acids. Astaxanthin concentration in the comb was found to negatively correlate with retinol levels but positively correlate with tocopherol levels. Considering evidence from this and other studied species, we suggest that carotenoid esterification is a characteristic of coloured fleshy integuments, probably affecting pigment stability and colouration in living tissues, with subsequent effects on their signalling role and maintenance costs. We found little evidence that the honesty of this signal would result from a direct connection with vitamin A metabolism, as recently proposed. Rather, honest signalling via comb colouration appears more related to potential allocation trade-offs of some specific dietary precursors or to the capacity of individuals to manage the redox reactions interfering with carotenoid metabolism.

Molecular mechanisms of the coordination between astaxanthin and fatty acid biosynthesis in Haematococcus pluvialis (Chlorophyceae).

Astaxanthin, a red ketocarotenoid with strong antioxidant activity and high commercial value, possesses important physiological functions in astaxanthin-producing microalgae. The green microalga Haematococcus pluvialis accumulates up to 4% fatty acid-esterified astaxanthin (by dry weight), and is used as a model species for exploring astaxanthin biosynthesis in unicellular photosynthetic organisms. Although coordination of astaxanthin and fatty acid biosynthesis in a stoichiometric fashion was observed in H.pluvialis, the interaction mechanism is unclear. Here we dissected the molecular mechanism underlying coordination between the two pathways in H.pluvialis. Our results eliminated possible coordination of this inter-dependence at the transcriptional level, and showed that this interaction was feedback-coordinated at the metabolite level. In vivo and in vitro experiments indicated that astaxanthin esterification drove the formation and accumulation of astaxanthin. We further showed that both free astaxanthin biosynthesis and esterification occurred in the endoplasmic reticulum, and that certain diacylglycerol acyltransferases may be the candidate enzymes catalyzing astaxanthin esterification. A model of astaxanthin biosynthesis in H.pluvialis was subsequently proposed. These findings provide further insights into astaxanthin biosynthesis in H.pluvialis.

From genetic improvement to commercial-scale mass culture of a Chilean strain of the green microalga Haematococcus pluvialis with enhanced productivity of the red ketocarotenoid astaxanthin

Astaxanthin is a red ketocarotenoid, widely used as a natural red colourant in marine fish aquaculture and poultry and, recently, as an antioxidant supplement for humans and animals. The green microalga Haematococcus pluvialis is one of the richest natural sources of this pigment. However, its slow growth rate and complex life cycle make mass culture difficult for commercial purposes. The aims of this research were (i) to standardize and apply a genetic improvement programme to a Chilean strain of H. pluvialis in order to improve its carotenogenic capacity and (ii) to evaluate the performance of a selected mutant strain in commercial-sized (125 000 L) open ponds in the north of Chile. Haematococcus pluvialis strain 114 was mutated by ethyl methanesulfonate. The level of mutagen dose (exposure time and concentration) was one that induced at least 90 % mortality. Surviving colonies were screened for resistance to the carotenoid biosynthesis inhibitor diphenylamine (25 µM). Resistant mutants were grown in a 30-mL volume for 30 days, after which the total carotenoid content was determined by spectrophotometry. Tens of mutants with improved carotenogenic capacity compared with the wild-type strain were isolated by the application of these standardized protocols. Some mutants exhibited curious morphological features such as spontaneous release of astaxanthin and loss of flagella. One of the mutants was grown outdoors in commercial-sized open ponds of 125 000 L in the north of Chile. Grown under similar conditions, the mutant strain accumulated 30 % more astaxanthin than the wild-type strain on a per dry weight basis and 72 % more on a per culture volume basis. We show that random mutagenesis/selection is an effective strategy for genetically improving strains of H. pluvialis and that improved carotenogenic capacity is maintained when the volume of the cultures is scaled up to a commercial size.

Carotenoid Distribution in Living Cells of Haematococcus pluvialis (Chlorophyceae)

Haematococcus pluvialis is a freshwater unicellular green microalga belonging to the class Chlorophyceae and is of commercial interest for its ability to accumulate massive amounts of the red ketocarotenoid astaxanthin (3,3′-dihydroxy-β,β-carotene-4,4′-dione). Using confocal Raman microscopy and multivariate analysis, we demonstrate the ability to spectrally resolve resonance–enhanced Raman signatures associated with astaxanthin and β-carotene along with chlorophyll fluorescence. By mathematically isolating these spectral signatures, in turn, it is possible to locate these species independent of each other in living cells of H. pluvialis in various stages of the life cycle. Chlorophyll emission was found only in the chloroplast whereas astaxanthin was identified within globular and punctate regions of the cytoplasmic space. Moreover, we found evidence for β-carotene to be co-located with both the chloroplast and astaxanthin in the cytosol. These observations imply that β-carotene is a precursor for astaxanthin and the synthesis of astaxanthin occurs outside the chloroplast. Our work demonstrates the broad utility of confocal Raman microscopy to resolve spectral signatures of highly similar chromophores in living cells.

Elucidation of the Pathway to Astaxanthin in the Flowers of Adonis aestivalis

A few species in the genus Adonis are the only land plants known to produce the valuable red ketocarotenoid astaxanthin in abundance. Here, we ascertain the pathway that leads from the β-rings of β-carotene, a carotenoid ubiquitous in plants, to the 3-hydroxy-4-keto-β-rings of astaxanthin (3,3'-dihydroxy-β,β-carotene-4,4'-dione) in the blood-red flowers of Adonis aestivalis, an ornamental and medicinal plant commonly known as summer pheasant's eye. Two gene products were found to catalyze three distinct reactions, with the first and third reactions of the pathway catalyzed by the same enzyme. The pathway commences with the activation of the number 4 carbon of a β-ring in a reaction catalyzed by a carotenoid β-ring 4-dehydrogenase (CBFD), continues with the further dehydrogenation of this carbon to yield a carbonyl in a reaction catalyzed by a carotenoid 4-hydroxy-β-ring 4-dehydrogenase, and concludes with the addition of an hydroxyl group at the number 3 carbon in a reaction catalyzed by the erstwhile CBFD enzyme. The A. aestivalis pathway is both portable and robust, functioning efficiently in a simple bacterial host. Our elucidation of the pathway to astaxanthin in A. aestivalis provides enabling technology for development of a biological production process and reveals the evolutionary origin of this unusual plant pathway, one unrelated to and distinctly different from those used by bacteria, green algae, and fungi to synthesize astaxanthin.

Evidence for the involvement of surface carbohydrates in the recognition of Haematococcus pluvialis by the parasitic blastoclad Paraphysoderma sedebokerensis

The unicellular green alga Haematococcus pluvialis (Chlorophyta, Volvocales) is currently the best commercial source of the natural red ketocarotenoid astaxanthin. Paraphysoderma sedebokerensis (Blastocladiomycota), a parasitic blastoclad that is specific for this microalga, was recently isolated and identified in our laboratory. In this study, we investigated the recognition process between the parasite and H. pluvialis. Obligatory requirements for recognition were identified as an ion concentration in the medium of 20 mM, the presence of calcium ions, and neutral to basic conditions; these requirements imply that a protein is involved in the process. In a search for potential lectin–sugar interactions as a major event in the recognition process, we screened for exposed glycosidic moieties on the cell wall of the alga and on the parasite zoospore surface. Competition experiments with the appropriate lectins and monosugars identified Ricinus communis agglutinin (RCA 120) as the lectin that recognizes Gal- N-acetyl- d-glucosamine, an oligosaccharide located on the host. We propose that an RCA 120-like lectin–sugar interaction mediates the highly specific interaction between the blastocladian parasite and its algal host.

Atypical Teratoid/Rhabdoid Tumors in Adult Patients: CT and MR Imaging Features

Primary AT/RT is a rare highly malignant tumor of the CNS, usually occurring in children younger than 5 years of age. The objective of this study was to characterize the CT and MR imaging findings in a series of 5 adult patients with pathologically proved AT/RT. All 5 AT/RTs were supratentorial. In 2 patients who underwent nonenhanced CT, the tumors appeared isoattenuated, and 1 of the 2 tumors contained calcifications. Solid portions of the tumors on MR imaging were isointense on T1-weighted, T2-weighted, and FLAIR images, and 1 case showed restricted diffusion on DWI. The tumors also demonstrated a bandlike rim of strong enhancement surrounding a central cystic area on contrast-enhanced T1-weighted imaging. One tumor was associated with destruction of the calvaria. Although AT/RTs can have nonspecific findings, the tumors in our series were large and isointense on T1-weighted, T2-weighted, and FLAIR images with central necrosis and prominent rim enhancement.

Carotenoid Accumulation in the Tissues of Zebra Finches: Predictors of Integumentary Pigmentation and Implications for Carotenoid Allocation Strategies

Carotenoid pigments produce the bright yellow to red ornamental colors of many animals, especially birds, and must ultimately be derived from the diet. However, they are also valuable for many physiological functions (e.g., antioxidants, immunostimulants, photoprotection, visual tuning, yolk nourishment to embryos), and as a result they are present in numerous internal body tissues (e.g., liver, adipose tissue, retina) whose carotenoid types and amounts are rarely studied in the context of color acquisition. Because male and female animals typically place different priorities on fitness-enhancing activities (e.g., gametic investment in females, sexual attraction in males), carotenoid allocation may track such investment patterns in the two sexes, and we can test for such sex-specific priorities of carotenoids by assessing body-tissue distributions of these pigments. We used high-performance liquid chromatography to identify and quantify carotenoid pigments from the plasma, liver, adipose tissue, and retina as well as the beak and legs of male and female zebra finches (Taeniopygia guttata), a species in which males display sexually attractive, red, carotenoid-based beak coloration and females also display some (albeit a less rich orange) beak color. To our knowledge, this is the first study of the predictors of carotenoid-based leg coloration-another potentially important visual signal-in this species. The same suite of dietary (e.g., lutein, zeaxanthin, beta-cryptoxanthin) and metabolically derived (e.g., dehydrolutein, anhydrolutein) yellow and orange carotenoids was present in plasma, liver, and adipose tissue of both sexes. Retina contained two different metabolites (astaxanthin and galloxanthin) that serve specific functions in association with unique photoreceptor types in the eye. Beaks were enriched with four red ketocarotenoid derivatives in both sexes (alpha-doradexanthin, adonirubin, astaxanthin, and canthaxanthin), while the carotenoid profile of legs was similar to that of plasma/liver/adipose tissue but with the additional presence of canthaxanthin. Sex differences in beak coloration were attributable to different concentrations of all four red ketocarotenoids. Males also had more colorful legs than did females, and this color difference was due to the increased presence of canthaxanthin in males. Males had higher carotenoid concentrations in plasma and retina than did females, but females had higher carotenoid concentrations in liver and adipose tissue than did males. These patterns are consistent with the apparently different life-history strategies employed for carotenoids by adult males and females, with females prioritizing future access to carotenoids (in tissue stores for egg production) and males prioritizing current access (in circulation, for maintaining bright color and/or health).

CAROTENOIDS IN THE SEMINAL FLUID OF WILD BIRDS: INTERSPECIFIC VARIATION IN FAIRY-WRENS

Male secondary sexual characters can provide females with information regarding the fertilizing capacity of a male's sperm. In some fishes and birds, intense nuptial coloration is correlated with male fertilizing capacity, but no mechanistic link between coloration and sperm quality has been established. One plausible mechanism is that carotenoid pigments, which color skin and feathers in many animals, are present in seminal fluid and serve as antioxidant protectors of sperm. We used high-performance liquid chromatography (HPLC) to analyze sperm samples from four species of Australian fairy-wren (Malurus) and detected low concentrations (<1 μg ml−1) of carotenoids in some samples. Xanthophyll carotenoids (including lutein and zeaxanthin), which are typically dietary in origin, were present in the seminal fluid of Superb (M. cyaneus) and Splendid (M. splendens) Fairy-Wrens. In contrast, red ketocarotenoids (including astaxanthin and canthaxanthin), which are likely metabolically derived from dietary precursors, were present in the seminal fluid of Red-backed Fairy-Wrens (M. melanocephalus). This work is the first to report carotenoids in avian seminal fluid and suggests that, although carotenoids are at low levels and thus may have limited antioxidant activity, there may be biological variability in avian semen carotenoids on which selection could act.

Development of an Adonis aestivalis expressed sequence tag population as a resource for genes of the carotenoid pathway

Expressed sequence tag (EST) libraries are a powerful tool for gene discovery in plants with genomes that are not fully sequenced. Adonis aestivalis (Ranunculaceae) is unusual among higher plants in that it accumulates large amounts of the valuable red ketocarotenoid astaxanthin in the flower petals, in addition to other carotenoids, and should therefore be a rich source of transcripts involved in carotenoid biosynthesis. To create a resource for gene discovery and increase our understanding of ketocarotenoid biosynthesis in plants, we sequenced 4189 cloned cDNAs from the floral tissues of A. aestivalis. The EST collection was annotated using sequence homology searches comparing the A. aestivalis ESTs with reference databases. ESTs involved in carotenoid biosynthesis were identified based on sequence homology to published sequences and by functional prediction using InterProScan. This study identified expressed transcripts for most of the known genes involved in the carotenoid pathway. Using an Escherichia coli functional expression system, two candidate transcripts from the A. aestivalis cDNA library were functionally characterized and identified as a novel beta-carotene 3-hydroxylase (CrtH2) and a beta-carotene monooxygenase. The expanded use of this EST collection combined with the E. coli expression system as presented here will greatly facilitate the cloning and characterization of candidate genes involved in carotenoid biosynthesis in plants.

On the relative efficiency of two‐ vs. one‐stage production of astaxanthin by the green alga Haematococcus pluvialis

Haematococcus pluvialis under stress conditions overproduces the valuable red ketocarotenoid astaxanthin. Two proposed strategies for commercial production are under current analysis. One separates in time the production of biomass (optimal growth, green stage) and pigment (permanent stress, red stage), while the other uses an approach based on continuous culture under limiting stress at steady state. The productivities, efficiencies and yields for the pigment accumulation in each case have been compared and analyzed in terms of the algal basic physiology. The two-stage system indoors yields a richer astaxanthin product (4% of dry biomass) with a final astaxanthin productivity of 11.5 mg L(-1) day(-1), is more readily upscalable and amenable to outdoors production. Furthermore, each stage can be optimized for green biomass growth and red pigment accumulation by adjusting independently the respective ratio of effective irradiance to cell density. We conclude that the two-stage system performs better (by a factor of 2.5-5) than the one-stage system, and the former is best fit in an efficient mass production setup.

Optical properties of rhodoxanthin accumulated in Aloe arborescens Mill. leaves under high-light stress with special reference to its photoprotective function

In Aloe arborescens Mill. leaves, strong sunlight or its combination with drought induces the accumulation of the red keto-carotenoid, rhodoxanthin. Simultaneously, the transformation of chloroplasts into chromoplasts accompanied by degradation of thylakoid membranes and formation of plastoglobuli, large in size and number, takes place. Depending on stress conditions the build up of rhodoxantin occurred along with the loss of chlorophyll or on the background of relatively high content of the pigment in the leaves. Microspectrophotometrical measurements showed the presence of chlorophyll-free plastids and retention of carotenoids during leaf adaptation to strong sunlight. The plastid spectra contained absorption bands of common for higher plants carotenoids together with those of rhodoxantin, with absorption maxima situated in the blue (440-480 nm) and the green ranges of the spectrum, respectively. The studies of whole-leaf optical properties revealed a broad band of rhodoxanthin absorption in the blue-green range peaking near 540-550 nm. Within this spectral band the accumulation of rhodoxanthin occurring, probably, in plastoglobuli considerably increased light absorption by stressed Aloe leaves. A possible photoprotective function of rhodoxanthin and other carotenoids as an internal light trap analogous to that accomplished by anthocyanins in other plant species is discussed.

A study in scarlet: enzymes of ketocarotenoid biosynthesis in the flowers of Adonis aestivalis

The red ketocarotenoid astaxanthin (3,3'-dihydroxy-4,4'-diketo-beta,beta-carotene) is widely used as an additive in feed for the pigmentation of fish and crustaceans and is frequently included in human nutritional supplements as well. There is considerable interest in developing a plant-based biological production process for this valuable carotenoid. Adonis aestivalis (Ranunculaceae) is unusual among plants in synthesizing and accumulating large amounts of astaxanthin and other ketocarotenoids. The formation of astaxanthin requires only the addition of a carbonyl at the number 4 carbon of each beta-ring of zeaxanthin (3,3'-dihydroxy-beta,beta-carotene), a carotenoid typically present in the green tissues of higher plants. We screened an A. aestivalis flower library to identify cDNAs that might encode the enzyme that catalyzes the addition of the carbonyls. Two closely related cDNAs selected in this screen were found to specify polypeptides similar in sequence to plant beta-carotene 3-hydroxylases, enzymes that convert beta-carotene (beta,beta-carotene) into zeaxanthin. The Adonis enzymes, however, exhibited neither 4-ketolase nor 3-hydroxylase activity when presented with beta-carotene as the substrate in Escherichia coli. Instead, the products of the Adonis cDNAs were found to modify beta-rings in two distinctly different ways: desaturation at the 3,4 position and hydroxylation of the number 4 carbon. The 4-hydroxylated carotenoids formed in E. coli were slowly metabolized to yield compounds with ketocarotenoid-like absorption spectra. It is proposed that a 3,4-desaturation subsequent to 4-hydroxylation of the beta-ring leads to the formation of a 4-keto-beta-ring via an indirect and unexpected route: a keto-enol tautomerization.

Carotenoid composition and its chemotaxonomic significance in leaves of ten species of the genus Ceratozamia (Cycads)

The qualitative composition and localization of carotenoids in leaflets of ten species of the genus Ceratozamia (Cycads) was investigated, and the distributions of 16 of these carotenoids, which were isolated and differently located in the chloroplast, are discussed. Eight classic carotenoids are located in the thylakoidal membranes of the chloroplasts of all ten of the species examined. In contrast, eight red keto-carotenoids are unusually located in several plastoglobules present in the stroma of nine species Ceratozamia mexicana Brongn. excluded. The characteristic red-brown transitory coloration shown by the newly formed leaflets during the first stages of development is due to this latter keto mixture. It is constituted of three very rare keto-carotenoids, which were, in our case, identified for the first time from the photosynthetic tissues (semi-beta-carotenone, triphasiaxanthin, and beta-carotenone). It is also constituted of four others, which are completely novel (ceratoxanthin, ceratozamiaxanthin, kuesteriaxanthin, and ceratoxanthone). Some hypotheses about their presence and function are presented.

Red‐winged blackbirds Agelaius phoeniceus use carotenoid and melanin pigments to color their epaulets

Over the past three decades, the red‐winged blackbird Agelaius phoeniceus has served as a model species for studies of sexual selection and the evolution of ornamental traits. Particular attention has been paid to the role of the colorful red‐and‐yellow epaulets that are striking in males but reduced in females and juveniles. It has been assumed that carotenoid pigments bestow the brilliant red and yellow colors on epaulet feathers, but this has never been tested biochemically. Here, we use high‐performance liquid chromatography (HPLC) to describe the pigments present in these colorful feathers. Two red ketocarotenoids (astaxanthin and canthaxanthin) are responsible for the bright red hue of epaulets. Two yellow dietary precursors pigments (lutein and zeaxanthin) are also present in moderately high concentrations in red feathers. After extracting carotenoids, however, red feathers remained deep brown in color. HPLC tests show that melanin pigments (primarily eumelanin) are also found in the red‐pigmented barbules of epaulet feathers, at an approximately equal concentration to carotenoids. This appears to be an uncommon feature of carotenoid‐based ornamental plumage in birds, as was shown by comparable analyses of melanin in the yellow feathers of male American goldfinches Carduelis tristis and the red feathers of northern cardinals Cardinalis cardinalis, in which we detected virtually no melanins. Furthermore, the yellow bordering feathers of male epaulets are devoid of carotenoids (except when tinged with a carotenoid‐derived pink coloration on occasion) and instead are comprised of a high concentration of primarily phaeomelanin pigments. The dual pigment composition of red epaulet feathers and the melanin‐only basis for yellow coloration may have important implications for the honesty‐reinforcing mechanisms underlying ornamental epaulets in red‐winged blackbirds, and shed light on the difficulties researchers have had to date in characterizing the signaling function of this trait. As in several other birds, the melanic nature of feathers may explain why epaulets are used largely to settle aggressive contests rather than to attract mates.