The role of mutants in the search for the photoreceptor for phototropism in higher plants

Early attempts to identify the chromophore of the photoreceptor for phototropism are reviewed. Carotenoids and flavins were the principal candidates, but studies with grass coleoptiles devoid of carotenoids suggest that at least in these organs carotenoids are most unlikely to play that role. The status of characterization of a gene for a putative photoreceptor protein is also reviewed. As the action spectrum for phototropism resembles the absorption spectrum of a flavoprotein, flavoproteins are attractive candidates at present, especially since the CRY1 photoreceptor in Arabidopsis thaliana that mediates blue light-dependent hypocotyl growth suppression has flavin adenine dinucleotide as one of its two chromophores. As the second chromophore appears to be pterin, pterins should not be ruled out as candidate chromophores for the photoreceptor for phototropism.     

Experimental Modification of Pigment Content and Phototropic Sensitivity in Excised Avena Coleoptiles

Avena seedlings were imbibed and germinated in the presence of inhibitors of carotenoid biosynthesis. After excision and defoliation, the coleoptiles were cultured in the presence of these basally supplied inhibitors and their growth, phototropic behavior and pigment content were subsequently measured. Total carotenoids could be reduced to ca. 20 percent of the control value without marked influence on the dose-response curve for the first positive curvature. Chromatographic analysis of extracted carotenoids on alumina columns revealed that the inhibitors produced both qualitative and quantitative changes, reducing one fraction and virtually eliminating two others. The total riboflavin content of the coleoptiles was almost completely unaffected by these treatments. The data are applied to an analysis of the nature of the photoreceptor in phototropism of the Avena coleoptile.     

Phototropism and gravitropism in transgenic lines of Arabidopsis altered in the phytochrome pathway

Phytochromes are a family of photoreceptor molecules, absorbing primarily in red and far-red, that are important in many aspects of plant development. These studies investigated the role of phytochromes in phototropism and gravitropism of seedlings of Arabidopsis thaliana. We used two transgenic lines, one which lacked phytochromes specifically in the roots (M0062/UASBVR) and the other lacked phytochromes in the shoots (CAB3::pBVR). These transgenic plants are deficient in the phytochrome chromophore in specific tissues due the expression of biliverdin IXa reductase (BVR), which binds to precursors of the chromophore. Experiments were performed in both light and dark conditions to determine whether roots directly perceive light signals or if the signal is perceived in the shoot and then transmitted to the root during tropistic curvature. Kinetics of tropisms and growth were assayed by standard methods or with a computer-based feedback system. We found that the perception of red light occurs directly in the root during phototropism in this organ and that signaling also may occur from root to shoot in gravitropism.     

Growth Retardant-Induced Changes in Phototropic Reaction of Vigna radiata Seedlings

The effect of growth retardants on phototropism has been studied in mung bean (Vigna radiata) seedlings. Ancymidol, tetcyclacis, and paclobutrazol inhibited phototropism while AMO 1618 and CCC were ineffective. The fluence-response relationships for phototropism of etiolated seedlings were similar to those previously described for monocots and other dicots. Ancymidol caused a shift in the maximum phototropic response to higher fluence of light. It is suggested that ancymidol may affect phototropism through an effect on the photoreceptor system.     

Role of carotenoids in the phototropic response of corn seedlings

The herbicide, 4 chloro-5-(methylamino)-2-(α,α,α,-trifluoro-m-tolyl)-3 (2H)-pyridazinone (SAN 9789), which blocks the synthesis in higher plants of colored carotenoids but not of flavins, was used to examine the involvement of carotenoids in corn seedling phototropism. It was concluded that “bulk” carotenoids are not the photoreceptor pigment based on the results that increasing concentrations of SAN 9789 (up to 100 micromolar) did not alter the phototropic sensitivity to 380 nanometers light (using geotropism as a control) and did not increase the threshold intensities of fluence response curves for both 380 and 450 nanometers light even though carotenoid content was reduced to 1 to 2% of normal. SAN 9789 treatment, however, did reduce seedling sensitivity toward 450 nanometers light indicating that carotenoids are involved in phototropism. Carotenoids, which are located mainly in the primary leaves, may act in phototropism as an internal screen, enhancing the light intensity gradient across the seedling and thus contributing to the ability of the seedling to perceive light direction. These results indicate that the action spectra for phototropic responses can be significantly affected by the absorbance of screening pigments in vivo thus altering its shape from the in vitro absorption spectrum of the photoreceptor pigment.     

Introduction of new carotenoids into the bacterial photosynthetic apparatus by combining the carotenoid biosynthetic pathways of Erwinia herbicola and Rhodobacter sphaeroides.

Carotenoids have two major functions in bacterial photosynthesis, photoprotection and accessory light harvesting. The genes encoding many carotenoid biosynthetic pathways have now been mapped and cloned in several different species, and the availability of cloned genes which encode the biosynthesis of carotenoids not found in the photosynthetic genus Rhodobacter opens up the possibility of introducing a wider range of foreign carotenoids into the bacterial photosynthetic apparatus than would normally be available by producing mutants of the native biosynthetic pathway. For example, the crt genes from Erwinia herbicola, a gram-negative nonphotosynthetic bacterium which produces carotenoids in the sequence of phytoene, lycopene, beta-carotene, beta-cryptoxanthin, zeaxanthin, and zeaxanthin glucosides, are clustered within a 12.8-kb region and have been mapped and partially sequenced. In this paper, part of the E. herbicola crt cluster has been excised and expressed in various crt strains of Rhodobacter sphaeroides. This has produced light-harvesting complexes with a novel carotenoid composition, in which the foreign carotenoids such as beta-carotene function successfully in light harvesting. The outcome of the combination of the crt genes in R. sphaeroides with those from E. herbicola has, in some cases, resulted in an interesting rerouting of the expected biosynthetic sequence, which has also provided insights into how the various enzymes of the carotenoid biosynthetic pathway might interact. Clearly this approach has considerable potential for studies on the control and organization of carotenoid biosynthesis, as well as providing novel pigment-protein complexes for functional studies.     

The effects of dietary carotenoid intake on carotenoid accumulation in the retina of a wild bird, the house finch (Carpodacus mexicanus)

Carotenoid pigments accumulate in the retinas of many animals, including humans, where they play an important role in visual health and performance. Recently, birds have emerged as a model system for studying the mechanisms and functions of carotenoid accumulation in the retina. However, these studies have been limited to a small number of domesticated species, and the effects of dietary carotenoid access on retinal carotenoid accumulation have not been investigated in any wild animal species. The purpose of our studies was to examine how variation in dietary carotenoid types and levels affect retinal accumulation in house finches (Carpodacus mexicanus), a common and colorful North American songbird. We carried out three 8-week studies with wild-caught captive birds: (1) we tracked the rate of retinal carotenoid depletion, compared to other body tissues, on a very low-carotenoid diet, (2) we supplemented birds with two common dietary carotenoids (lutein + zeaxanthin) and measured the effect on retinal accumulation, and (3) we separately supplemented birds with high levels of zeaxanthin - an important dietary precursor for retinal carotenoids - or astaxanthin - a dominant retinal carotenoid not commonly found in the diet (i.e. a metabolic derivative). We found that carotenoids depleted slowly from the retina compared to other tissues, with a significant (∼50%) decline observed only after 8 weeks on a very low-carotenoid diet. Supplementation with lutein + zeaxanthin or zeaxanthin alone significantly increased only retinal galloxanthin and ε-carotene levels, while other carotenoid types in the retina remained unaffected. Concentrations of retinal astaxanthin were unaffected by direct dietary supplementation with astaxanthin. These results suggest highly specific mechanisms of retinal carotenoid metabolism and accumulation, as well as differential rates of turnover among retinal carotenoid types, all of which have important implications for visual health maintenance and interventions.     

Egg yolk carotenoids in relation to habitat and reproductive investment in the great tit Parus major

Maternal allocation of antioxidants to egg yolk has been shown to affect early embryonic development and nestling survival. In environments with high levels of anthropogenic pollution, antioxidants (such as carotenoids) are important to protect the body from elevated oxidative stress. Thus, female allocation of antioxidants to yolk may be traded off against self-maintenance. Here we investigate maternal reproductive investment with respect to yolk carotenoid content and composition in relation to subsequent female condition and carotenoid status in urban and rural great tits Parus major. We found no differences between the urban and rural populations in total yolk carotenoids, egg mass, clutch size, hatching success, or female carotenoid status. Interestingly, however, rural eggs contained more zeaxanthin, a more potent antioxidant than lutein, which suggests that rural embryos have better antioxidant protection than urban embryos. Whether rural females actively transfer more zeaxanthin to the yolk or whether it passively reflects differences in dietary access or uptake needs to be further investigated. This highlights the importance of carotenoid identity and composition in future studies of carotenoid physiology, ecology, and signaling.     

Oxidation of carotenoids by heat and tobacco smoke

The stability to autoxidation of the polar carotenoids, lutein and zeaxanthin, was compared to that of the less polar carotenoids, beta-carotene and lycopene at physiologically or pathophysiologically relevant concentrations of 2 and 6 microM, after exposure to heat or cigarette smoke. Three methodological approaches were used: 1) Carotenoids dissolved in solvents with different polarities were incubated at 37 and 80 degrees C for different times. 2) Human plasma samples were subjected to the same temperature conditions. 3) Methanolic carotenoid solutions and plasma were also exposed to whole tobacco smoke from 1-5 unfiltered cigarettes. The concentrations of individual carotenoids in different solvents were determined spectrophotometrically. Carotenoids from plasma were extracted and analyzed using high performance liquid chromatography. Carotenoids were generally more stable at 37 than at 80 degrees C. In methanol and dichloromethane the thermal degradation of beta-carotene and lycopene was faster than that of lutein and zeaxanthin. However, in tetrahydrofuran beta-carotene and zeaxanthin degraded faster than lycopene and lutein. Plasma carotenoid levels at 37 degrees C did not change, but decreased at 80 degrees C. The decrease of beta-carotene and lycopene levels was higher than those for lutein and zeaxanthin. Also in the tobacco smoke experiments the highest autoxidation rates were found for beta-carotene and lycopene at 2 microM, but at 6 microM lutein and zeaxanthin depleted to the same extent as beta-carotene. These data support our previous studies suggesting that oxidative stress degrade beta-carotene and lycopene faster than lutein and zeaxanthin. The only exception was the thermal degradation of carotenoids solubilized in tetrahydrofuran, which favors faster breakdown of beta-carotene and zeaxanthin.     

Acclimation of chlorophyll a and carotenoid levels to different irradiances in four freshwater cyanobacteria

This study investigated carotenoid and chlorophyll a (Chl-a) contents under two different growth irradiances in four freshwater cyanobacterial strains. We found an increased weight ratio of zeaxanthin to Chl-a after exposure to high irradiances over several days. Two out of four strains showed higher zeaxanthin amounts on a biomass basis as well. It appears that cyanobacteria enhance their carotenoid pool in response to high light conditions, as increased production of other carotenoids with photoprotective abilities has also been observed under high irradiance levels. Cyanobacteria do not possess the violaxanthin cycle, which enables a rapid reversible conversion from violaxanthin into zeaxanthin and functioning as a quencher of excessive energy, and elevated zeaxanthin concentrations could therefore be seen as an adaptive strategy against excess light energy. Some differences in the acclimation pattern were revealed between different cyanobacteria. Anabaena torulosa contained higher amounts of every carotenoid, while Nostoc sp. mainly increased zeaxanthin, and myxoxanthophyll. Anabaenopsis elenkinii produced exceptionally high amounts of myxoxanthophyll and beta-carotene under higher irradiances. Anabaena cylindrica generally showed less variation of carotenoids under different irradiances.     

Effect of aVirido-Xantha mutation in barley on the content of individual plastid pigments

Summary A virido-xantha (vx) barley mutant with reduced chlorophyll content when grown at 15°C was investigated to determine quantitative and qualitative changes in the plastid pigments. Both chlorophyll and the capacity to produce protochlorophyllide from exogeneously supplied δ-aminolevulinic acid are reduced in the mutant. The total carotenoid content is also reduced in the mutant, but individual carotenoids are not reduced coordinately. Antheraxanthin actually accumulates in the mutant, and zeaxanthin was found in significant amounts in mutant seedlings but could not be detected in normal barley seedlings. Included in the methods is a procedure for the preliminary identification of zeaxanthin employing spectrophotometric analysis of fractions eluted from a sucrose column. The presence of recessive suppressors of thevx mutant results in increased contents of the chlorophyll pigments accompanied by a partial reversal of the changes in carotenoid indicated above. The changes in chlorophyll and carotenoid contents are also partially reversed when mutant seedlings are grown at 21°C. Research supported under contract AT(11-1)-332 with the United States Atomic Energy Commission.     

Phototropins and Their LOV Domains：Versatile Plant Blue-Light Receptors

The phototropins phot1 and phot2 are plant blue-light receptors that mediate phototropism, chloroplast movements, stomatal opening, leaf expansion, the rapid Inhibition of hypocotyl growth in etiolated seedlings, and possibly solar tracking by leaves in those species in which It occurs. The phototroplns are plasma membrane-associated hydrophilic proteins with two chromophore domains （designated LOV1 and LOV2 for their resemblance to domains In other signaling proteins that detect light, oxygen, or voltage） in their Nterminal half and a classic serine/threonlne kinase domain in their C-terminal half. Both chromophore domains bind flavin mononucleotide （FMN） and both undergo light-activated formation of a covalent bond between a nearby cystelne and the C（4a） carbon of the FMN to form the signaling state. LOV2-cystelnyl adduct formation leads to the release downstream of a tightly bound amphlpathlc α-helix, a step required for activation of the klnase function. This cysteinyl adduct then slowly decays over a matter of seconds or minutes to return the photoreceptor chromophore modules to their ground state. Functional LOV2 is required for light-activated phosphorylation and for various blue-light responses mediated by the phototroplns. The function of LOV1 is still unknown, although It may serve to modulate the signal generated by LOV2. The LOV domain Is an ancient chromophore module found In a wide range of otherwise unrelated proteins In fungi and prokaryotes, the latter Including cyanobacterla, eubacterla, and archaea. Further general reviews on the phototropins are those by Celaya and Liscum （2005） and Christie and Briggs （2005）.     

ASTER-B regulates mitochondrial carotenoid transport and homeostasis

The scavenger receptor class B type 1 (SR-B1) facilitates uptake of cholesterol and carotenoids into the plasma membrane (PM) of mammalian cells. Downstream of SR-B1, ASTER-B protein mediates the nonvesicular transport of cholesterol to mitochondria for steroidogenesis. Mitochondria also are the place for the processing of carotenoids into diapocarotenoids by β-carotene oxygenase-2. However, the role of these lipid transport proteins in carotenoid metabolism has not yet been established. Herein, we showed that the recombinant StART-like lipid-binding domain of ASTER-A and B preferentially binds oxygenated carotenoids such as zeaxanthin. We established a novel carotenoid uptake assay and demonstrated that ASTER-B expressing A549 cells transport zeaxanthin to mitochondria. In contrast, the pure hydrocarbon β-carotene is not transported to the organelles, consistent with its metabolic processing to vitamin A in the cytosol by β-carotene oxygenase-1. Depletion of the PM from cholesterol by methyl-β-cyclodextrin treatment enhanced zeaxanthin but not β-carotene transport to mitochondria. Loss-of-function assays by siRNA in A549 cells and the absence of zeaxanthin accumulation in mitochondria of ARPE19 cells confirmed the pivotal role of ASTER-B in this process. Together, our study in human cell lines established ASTER-B protein as key player in nonvesicular transport of zeaxanthin to mitochondria and elucidated the molecular basis of compartmentalization of the metabolism of nonprovitamin A and provitamin A carotenoids in mammalian cells.     

Lycopene and beta-carotene decompose more rapidly than lutein and zeaxanthin upon exposure to various pro-oxidants in vitro

Major carotenoids of human plasma and tissues were exposed to radical-initiated autoxidation conditions. The consumption of lutein and zeaxanthin, the only carotenoids in the retina, and lycopene and beta-carotene, the most effective quenchers of singlet oxygen in plasma, were compared. Under all conditions of free radical-initiated autoxidation of carotenoids which were investigated, the breakdown of lycopene and beta-carotene was much faster than that of lutein and zeaxanthin. Under the influence of UV light in presence of Rose Bengal, by far the highest breakdown rate was found for beta-carotene, followed by lycopene. Bleaching of carotenoid mixtures mediated by NaOCl, addition of azo-bis-isobutyronitril (AIBN), and the photoirradiation of carotenoid mixtures by natural sunlight lead to the following sequence of breakdown rates: lycopene > beta-carotene > zeaxanthin > lutein. The slow degradation of the xanthophylls zeaxanthin and lutein may be suggested to explain the majority of zeaxanthin and lutein in the retina of man and other species. In correspondence to that, the rapid degradation of beta-carotene and lycopene under the influence of natural sunlight and UV light is postulated to be the reason for the almost lack of those two carotenoids in the human retina. Nevertheless, a final proof of that theory is lacking.     

The effect of an acute phase response on tissue carotenoid levels of growing chickens (Gallus gallus domesticus)

Plasma, liver and skin carotenoids decrease following infectious disease challenges. Since these challenges often involve substantial host pathology and chronic immune responses, the mechanism underlying altered carotenoid deposition is unclear. Therefore, changes in tissue carotenoid levels were examined during an acute phase response induced by lipopolysaccharide (LPS) or interleukin-1 (IL-1). In two experiments, chicks were hatched from carotenoid-deplete eggs (n=28, n=64, respectively) and fed 0, 8 or 38 mg carotenoids (lutein+canthaxanthin)/kg diet. For chicks fed 38 mg carotenoids, but not those fed 0 or 8 mg, LPS generally reduced plasma lutein, canthaxanthin and total carotenoids (P<0.05), and liver lutein, zeaxanthin, canthaxanthin and total carotenoids (P<0.05). Additionally, LPS reduced thymic total carotenoids (P=0.05) and increased thymocyte lutein (P=0.07), zeaxanthin (P=0.07) and total carotenoids (P=0.07). Finally, LPS increased bursal canthaxanthin (P<0.01), but had no effect on shank carotenoids (P>0.5). In chicks hatched from carotenoid-replete eggs (n=36) and fed dietary lutein (38 mg/kg diet), LPS reduced plasma and liver zeaxanthin and liver total carotenoids (P<0.05); IL-1 reduced plasma and liver lutein, zeaxanthin and total carotenoids (P<0.05). Therefore, an acute phase response plays a role in reduced tissue carotenoids during infectious disease.     

Decrease of serum carotenoids in Crohn's disease.

Crohn's disease (CD) is frequently complicated by various nutritional disturbances. Although it is important to correct these disturbances, the nutritional status of CD patients has been poorly documented, especially concerning vitamin status. The aims of this study were (a) to measure the serum concentrations of vitamin A and six other carotenoids (lutein, zeaxanthin, alpha-, beta-carotene, alpha-, beta-cryptoxanthin) in patients with CD and to compare them with those in healthy controls and (b) to follow the changes of serum carotenoid levels in CD patients during treatment. Twenty-eight patients with CD and 23 healthy persons were included in this study. The results of twelve patients were followed up through one year. The patients were free of any nutritional treatment. The serum concentrations of carotenoids were measured with high-pressure liquid chromatography (HPLC). The serum concentrations of five carotenoids were significantly lower in the patients than in the controls (vitamin A, zeaxanthin: P < 0.001; alpha-, beta-carotene: P < 0.01; lutein: P < 0.05). The carotenoid status of the followed patients advanced to the normal range, but this increase was not significant. These findings suggest that there is a deficiency of vitamin A and its provitamins in Crohn' s disease prior to treatment. However, because we did not evaluate the vitamin intake in this study, we could not conclude which of the factors--poor intake, increased requirement, or malabsorption--was more important in decreasing of carotenoid levels.     

Carotenoids from further prasinophytes

The qualitative and quantitative carotenoid composition of seven prasinophytes (eight clones) have been examined by chromatographic (TLC and HPLC) and spectroscopic methods (VIS, CD and mass spectra).

The prasinophytes studied fall into two pigment types: (A) those producing common green algal carotenoids (β,β-carotene, β,ε-carotene, lutein, zeaxanthin and the epoxides violaxanthin and neoxanthin) and (B) prasinophytes synthesising carotenoids peculiar to this algal class (prasinoxanthin, anhydroprasinoxanthin, uriolide, anhydrouriolide, micromonal, anhydromicromonal, micromonol, anhydromicromonol and dihydrolutein), where prasinoxanthin is a major carotenoid.

Mantoniella squamata (clone 2) was grown under both low and high light intensity, revealing differences in carotenoid composition. Lutein together with lesser amounts of zeaxanthin and its epoxides were only detected at high light intensity.

Three previously unidentified carotenoids were identified as prasinoxanthin (xanthophyll K), micromonal and dihydrolutein.     

Selective absorption of carotenoids in the common green iguana (Iguana iguana)

In the animal kingdom, species-specific differences with regard to the absorption of intact carotenoids are observed. The causes of these differences are not entirely understood. To investigate the absorption of selected carotenoids, 20 juvenile green iguanas (Iguana iguana) were fed a carotenoid deficient basal diet for 56 days. Thereafter, the iguanas were assigned to receive a basal diet supplemented with different carotenoids (80 mg/kg diet) such as beta-carotene, canthaxanthin and apo-8'-carotenoic acid ethyl ester for 28 days. Changes in plasma carotinoid concentrations associated with the individual diets were used as indicators of carotenoid absorption. In both the experimental and control groups, only the oxygenated carotenoids (xanthophylls), lutein, zeaxanthin and canthaxanthin, were found in the plasma. Canthaxanthin and apo-8'-carotenoic acid ethyl ester were readily absorbed and recovered from the plasma. However, the supplementation of beta-carotene caused no increase in plasma beta-carotene concentration. Additionally, beta-carotene, canthaxanthin or apo-8'-carotenoic acid ethyl ester did not affect the concentrations of retinol and alpha-tocopherol in plasma. In conclusion, the study demonstrates that iguanas appear to be selective accumulators of polar xanthophylls. The iguana might, therefore, be a valuable model to investigate the selectiveness of carotenoid absorption as well as the function of xanthophylls in animals.     

Carotenogenesis diversification in phylogenetic lineages of Rhodophyta

Carotenoid composition is very diverse in Rhodophyta. In this study, we investigated whether this variation is related to the phylogeny of this group. Rhodophyta consists of seven classes, and they can be divided into two groups on the basis of their morphology. The unicellular group (Cyanidiophyceae, Porphyridiophyceae, Rhodellophyceae, and Stylonematophyceae) contained only β‐carotene and zeaxanthin, “ZEA‐type carotenoids.” In contrast, within the macrophytic group (Bangiophyceae, Compsopogonophyceae, and Florideophyceae), Compsopogonophyceae contained antheraxanthin in addition to ZEA‐type carotenoids, “ANT‐type carotenoids,” whereas Bangiophyceae contained α‐carotene and lutein along with ZEA‐type carotenoids, “LUT‐type carotenoids.” Florideophyceae is divided into five subclasses. Ahnfeltiophycidae, Hildenbrandiophycidae, and Nemaliophycidae contained LUT‐type carotenoids. In Corallinophycidae, Hapalidiales and Lithophylloideae in Corallinales contained LUT‐type carotenoids, whereas Corallinoideae in Corallinales contained ANT‐type carotenoids. In Rhodymeniophycidae, most orders contained LUT‐type carotenoids; however, only Gracilariales contained ANT‐type carotenoids. There is a clear relationship between carotenoid composition and phylogenetics in Rhodophyta. Furthermore, we searched open genome databases of several red algae for references to the synthetic enzymes of the carotenoid types detected in this study. β‐Carotene and zeaxanthin might be synthesized from lycopene, as in land plants. Antheraxanthin might require zeaxanthin epoxydase, whereas α‐carotene and lutein might require two additional enzymes, as in land plants. Furthermore, Glaucophyta contained ZEA‐type carotenoids, and Cryptophyta contained β‐carotene, α‐carotene, and alloxanthin, whose acetylenic group might be synthesized from zeaxanthin by an unknown enzyme. Therefore, we conclude that the presence or absence of the four enzymes is related to diversification of carotenoid composition in these three phyla.     

Differential accumulation and pigmenting ability of dietary carotenoids in colorful finches

Many animals develop bright red, orange, or yellow carotenoid pigmentation that they use to attract mates. Colorful carotenoid pigments are acquired from the diet and are either directly incorporated as integumentary colorants or metabolized into other forms before deposition. Because animals often obtain several different carotenoids from plant and animal food sources, it is possible that these pigments are accumulated at different levels in the body and may play unique roles in shaping the ultimate color expression of individuals. We studied patterns of carotenoid accumulation and integumentary pigmentation in two colorful finch species--the American goldfinch (Carduelis tristis) and the zebra finch (Taeniopygia guttata). Both species acquire two main hydroxycarotenoids, lutein and zeaxanthin, from their seed diet but transform these into a series of metabolites that are used as colorful pigments in the plumage (goldfinches only) and beak (both species). We conducted a series of carotenoid-supplementation experiments to investigate the relative extent to which lutein and zeaxanthin are accumulated in blood and increase carotenoid coloration in feathers and bare parts. First, we supplemented the diets of both species with either lutein or zeaxanthin and measured plasma pigment status, feather carotenoid concentration (goldfinches only), and integumentary color. Zeaxanthin-supplemented males grew more colorful feathers and beaks than lutein-supplemented males, and in goldfinches incorporated a different ratio of carotenoids in feathers (favoring the accumulation of canary xanthophyll B). We also fed goldfinches different concentrations of a standard lutein-zeaxanthin mix and found that at physiologically normal and high concentrations, birds circulated proportionally more zeaxanthin over lutein than occurred in the diet. Collectively, these results demonstrate that zeaxanthin is preferentially accumulated in the body and serves as a more potent substrate for pigmentation than lutein in these finches.