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Red coloration is a widely distributed phenotype among animals, yet the pigmentary and genetic bases for this phenotype have been described in relatively few taxa. Here we show that the Hawaiian endemic anchialine shrimp Halocaridina rubra is red because of the accumulation of astaxanthin. Laboratory colonies of phylogenetically distinct lineages of H. rubra have colony-specific amounts of astaxanthin that are developmentally, and likely genetically, fixed. Carotenoid supplementation and restriction experiments failed to change astaxanthin content from the within-colony baseline levels, suggesting that dietary limitation is not a major factor driving coloration differences. A possible candidate gene product predicted to be responsible for the production of astaxanthin in H. rubra and other crustaceans is closely related to the bifunctional cytochrome P450 family 3 enzyme CrtS found in fungi. However, homologs to the enzyme thought to catalyze ketolation reactions in birds and turtles, CYP2J19, were not found. This work is one of the first steps in linking phenotypic variation in red coloration of H. rubra to genotypic variation. Future work should focus on (1) pinpointing the genes that function in the bioconversion of dietary carotenoids to astaxanthin, (2) examining what genomic variants might drive variation in coloration among discrete lineages, and, Introduction Variation in coloration within animal species is hypothesized to result from selective pressures of the environment (Hasson, 1997; Hadfield and Owens, 2006; Sommaruga, 2010), physiological constraints (Szamado, 2011; Weaver [...]

We report the cloning and characterization of the biosynthetic gene cluster (crtE, crtB, crtI, crtE2, crtYg, crtYh, and crtX) of the [gamma]-cyclic [C.sub.50] carotenoid sarcinaxanthin in Micrococcus luteus NCTC2665. Expression of the complete and partial gene cluster in Escherichia coli hosts revealed that sarcinaxanthin biosynthesis from the precursor molecule farnesyl pyrophosphate (FPP) proceeds via [C.sub.40] lycopene, [C.sub.45] nonaflavuxanthin, [C.sub.50] flavuxanthin, and [C.sub.50] sarcinaxanthin. Glucosylation of sarcinaxanthin was accomplished by the crtX gene product. This is the first report describing the biosynthetic pathway of a [gamma]-cyclic [C.sub.50] carotenoid. Expression of the corresponding genes from the marine M. luteus isolate Otnes7 in a lycopene-producing E. coli host resulted in the production of up to 2.5 mg/g cell dry weight sarcinaxanthin in shake flasks. In an attempt to experimentally understand the specific difference between the biosynthetic pathways of sarcinaxanthin and the structurally related [epsilon]-cyclic decaprenoxanthin, we constructed a hybrid gene cluster with the [gamma]-cyclic [C.sub.50] carotenoid cyclase genes crtYg and crtYh from M. luteus replaced with the analogous [epsilon]-cyclic [C.sub.50] carotenoid cyclase genes crtYe and crtYf from the natural decaprenoxanthin producer Corynebacterium glutamicum. Surprisingly, expression of this hybrid gene cluster in an E. coli host resulted in accumulation of not only decaprenoxanthin, but also sarcinaxanthin and the asymmetric [epsilon]- and [gamma]-cyclic [C.sub.50] carotenoid sarprenoxanthin, described for the first time in this work. Together, these data contributed to new insight into the diverse and multiple functions of bacterial [C.sub.50] carotenoid cyclases as key catalysts for the synthesis of structurally different carotenoids. doi: 10.1128/JB.00724-10

High light can be lethal for photosynthetic organisms. Similar to plants, most cyanobacteria protect themselves from high irradiance by increasing thermal dissipation of excess absorbed energy. The photoactive soluble orange carotenoid protein (OCP) is essential for the triggering of this photoprotective mechanism. Light induces structural changes in the carotenoid and the protein, leading to the formation of a red active form. Through targeted gene interruption we have now identified a protein that mediates the recovery of the full antenna capacity when irradiance decreases. In Synechocystis PCC 6803, this protein, which we called the fluorescence recovery protein (FRP), is encoded by the slr1964 gene. Homologues of this gene are present in all of the OCP-containing strains. The FRP is a 14kDa protein, strongly attached to the membrane, which interacts with the active red form of the OCP. In vitro this interaction greatly accelerates the conversion of the red OCP form to the orange form. We propose that in vivo, FRP plays a key role in removing the red OCP from the phycobilisome and in the conversion of the free red OCP to the orange inactive form. The discovery of FRP and its characterization are essential elements in the understanding of the OCPrelated photoprotective mechanism in cyanobacteria. carotenoid | nonphotochemical quenching | phycobilisome | photoreceptor | Synechocystis doi/ 10.1073/pnas.1002912107

Horizontal gene transfer and selection are major forces driving microbial evolution. However, interactions between them are rarely studied. Phylogenetic analyses of purple bacterial carotenoid biosynthesis genes suggest two lineages: one producing spheroidenone and the other producing spirilloxanthin. Of the latter lineage, Rubrivivax gelatinosus S1 and Hoeflea phototrophica DFL-43 also or instead produce spheroidenone. Evolution of the spheroidenone pathway from that producing spirilloxanthin theoretically requires changes in the substrate specificity of upstream pathway enzymes and acquisition of a terminal ketolase (CrtA). In R. gelatinosus and likely also in H. phototrophica, CrtA was acquired from the Bacteroidetes, in which it functions as a hydroxylase. Estimation of nonsynonymous and synonymous mutations using several pairwise methods indicated positive selection upon both genes, consistent with their functional changes from hydroxylases to ketolases. Relaxed negative selection upon all other carotenoid biosynthetic genes in these organisms was also apparent, likely facilitating changes in their substrate specificities. Furthermore, all genes responsible for terminal carotenoid biosynthetic pathway steps were under reduced negative selection compared to those known to govern biosynthetic pathway specificity. Horizontal transfer of crtA into R. gelatinosus and H. phototrophica has therefore likely been promoted by (i) the apparent selective advantage of spheroidenone production relative to spirilloxanthin production, (ii) reduced negative selection upon other carotenoid biosynthetic genes, facilitating changes in their substrate specificities, and (iii) preexisting low enzyme substrate specificities due to relaxed negative selection. These results highlight the importance and complexity of selection acting upon both a horizontally transferred gene and the biochemical network into which it is integrating. doi: 10.1128/JB.01060-09

To elucidate the biosynthetic pathways of carotenoids, especially myxol 2'-glycosides, in cyanobacteria, Anabaena sp. strain PCC 7120 (also known as Nostoc sp. strain PCC 7120) and Synechocystis sp. strain PCC 6803 deletion mutants lacking selected proposed carotenoid biosynthesis enzymes and GDP-fucose synthase (WcaG), which is required for myxol 2'-fucoside production, were analyzed. The carotenoids in these mutants were identified using high-performance liquid chromatography, field desorption mass spectrometry, and [sup.1]H nuclear magnetic resonance. The wcaG (all4826) deletion mutant of Anabaena sp. strain PCC 7120 produced myxol 2'-rhamnoside and 4-ketomyxol 2'-rhamnoside as polar carotenoids instead of the myxol 2'-fucoside and 4-ketomyxol 2'-fucoside produced by the wild type. Deletion of the corresponding gene in Synechocystis sp. strain PCC 6803 (sll1213; 79% amino acid sequence identity with the Anabaena sp. strain PCC 7120 gene product) produced free myxol instead of the myxol 2'-dimethyl-fucoside produced by the wild type. Free myxol might correspond to the unknown component observed previously in the same mutant (H. E. Mohamed, A. M. L. van de Meene, R. W. Roberson, and W. F. J. Vermaas, J. Bacteriol. 187:6883-6892, 2005). These results indicate that in Anabaena sp. strain PCC 7120, but not in Synechocystis sp. strain PCC 6803, rhamnose can be substituted for fucose in myxol glycoside. The [beta]-carotene hydroxylase orthologue (CrtR, Alr4009) of Anabaena sp. strain PCC 7120 catalyzed the transformation of deoxymyxol and deoxymyxol 2'-fucoside to myxol and myxol 2'-fucoside, respectively, but not the [beta]-carotene-to-zeaxanthin reaction, whereas CrtR from Synechocystis sp. strain PCC 6803 catalyzed both reactions. Thus, the substrate specificities or substrate availabilities of both fucosyltransferase and CrtR were different in these species. The biosynthetic pathways of carotenoids in Anabaena sp. strain PCC 7120 are discussed.

Vitamin A deficiency affects approximately 250 million people in semiarid regions of Africa and Asia, where sorghum (Sorghum bicolor Moench) is a major staple crop. Yellow endosperm sorghums contain carotenoids, some of which can be transformed by humans into vitamin A. Our objective was to study the genetic basis of variation in carotenoid levels in sorghum endosperm by mapping quantitative trait loci (QTL) associated with carotenoid content and endosperm color, as a putative predictor of carotenoid concentration. A recombinant inbred line population developed from a yellow ('KS115') by a white endosperm ('Macia') parental cross was evaluated in two locations in 2005. A genetic map was generated using 112 molecular markers including nine carotenoid candidate genes. Lutein, zeaxanthin, and B-carotene were the major carotenoids identified. Several QTL were detected for each compound as well as for color and total carotenoids. Color was significantly correlated with the levels of all compounds, and color QTL co-localized with carotenoid QTL. For [beta]-carotene (provitamin A), five QTL were localized on chromosomes 1, 2, and 10. One of them, on chromosome 2, was stable across both environments, had positive additive effects (1.179 and 1.379), explained large proportions of the phenotypic variance (11.6% and 15.15%), and was associated with a new phytoene synthase gene (Psy3). This first report of QTL for carotenoid content in sorghum grain provides a starting point for breeding high-provitamin A sorghums.

Vitamin E and carotenoids are fat-soluble micronutrients carried by plasma lipoproteins. Their plasma concentrations are governed by several factors, some of which are genetic, but data on these genetic factors remain scarce. We hypothesized that genes involved in lipid metabolism, i.e. the genes implicated in intestinal uptake, intracellular trafficking, and the lipoprotein distribution of lipids, play a role in the plasma concentrations of these micronutrients. To verify this hypothesis, we assessed whether the plasma status of vitamin E and carotenoids is related to genes involved in lipid metabolism. Fasting plasma vitamin E ([alpha]- and [gamma]-tocopherol) and carotenoid ([alpha]- and [beta]-carotene, lutein, lycopene, [beta]-cryptoxanthin, and zeaxanthin) concentrations were measured in 48 males and 80 females. The following genes were genotyped [single nucleotide polymorphisms (SNP)]: apolipoprotein (apo) A-IV, apo B, apo E, lipoprotein lipase, and scavenger-receptor class B type I (SR-BI). Plasma [alpha]-tocopherol concentrations were different (P< 0.05) in subjects bearing different SNP in apo A-IV, apo E, and SR-BI. Plasma [gamma]-tocopherol concentrations were different (P < 0.05) in subjects bearing different SNP in apo A-IV and SR-BI. [alpha]-Carotene concentrations were different (P < 0.05) in subjects bearing different SNP in SR-BI. [beta]-Carotene concentrations were different (P< 0.05) in subjects bearing different SNP in apo B and SR-BI. Lycopene concentrations were different (P< 0.05) in subjects bearing different SNP in apo A-IV and apo B. [beta]-Cryptoxanthin concentrations were different (P < 0.05) in subjects bearing different SNP in SR-BI. Plasma lutein and zeaxanthin concentrations did not differ in subjects bearing different SNP. Most of the differences remained significant after the plasma micronutrients were adjusted for plasma triglycerides and cholesterol. These results suggest that genes involved in lipid metabolism influence the plasma concentrations of these fat-soluble micronutrients. J. Nutr. 137: 2653-2659, 2007.

The ATP binding cassette G5 (ABCG5) polymorphisms have been postulated to play a role in the response to dietary cholesterol. The objective of this study was to examine the contribution of the ABCG5 polymorphism on the plasma response to consumption of cholesterol and carotenoids from eggs. For this purpose, genotyping was conducted for 40 men and 51 premenopausal women who were randomly assigned to consume an egg (EGG, 640 mg/d additional dietary cholesterol and 600 [micro]g lutein + zeaxanthin) or placebo (SUB, 0 mg/d cholesterol, 0 [micro]g lutein + zeaxanthin) diet for 30 d. The two arms of the dietary intervention were separated by a 3-wk washout period. Plasma concentrations of total cholesterol, LDL cholesterol (LDL-C), and HDL cholesterol were determined. Because eggs are an excellent source of lutein and zeaxanthin, the plasma levels of these carotenoids were also measured in a subset of subjects to determine whether the response to carotenoid intake was similar to that seen for dietary cholesterol and to evaluate the contribution of ABCG5 polymorphism to both responses. Individuals possessing the C/C genotype experienced a greater increase in both LDL-C (P < 0.05) and a trend for lutein (P = 0.08) during the EGG period compared with those individuals with the C/G (heterozygote) or G/G genotypes (homozygotes). These results, although obtained from a small number of subjects, suggest that the ABCG5 polymorphism may play a role in the plasma response to dietary cholesterol and carotenoids. KEY WORDS: * eggs * lutein * dietary cholesterol * ABCGS polymorphism * LDL cholesterol

Research has been conducted on the female preference evolving the condition-dependence of male traits. The genetic divergence in the female preferences for the cartenoid condition between high- and low-cartenoid has been investigated and tested on the cartenoid coloration and the results indicate that this coloration which affects the sexual selection of the male trait varies geographically.

The Erwinia herbicola crtE gene is contained in a gene cluster which is involved in the synthesis of carotenoids. The exact function of crtE within the carotenoid synthesis pathway was determined. The E. herbicola crtE gene was isolated and expressed in Escherichia coli. Geranylgeranyl diphosphate (GGPP) synthase asssays as well as cell extract activities on the GGPP precursors isopentenyl diphosphate and farnesyl phosphate showed that crtE codes for GGPP synthase. This conclusion was confirmed by sequence comparisons with other prokaryotic and eukaryotic GGPP synthases.

Research has been conducted on carotenoid-based plumage coloration in birds. The hypothesis that this coloration is evolutionary process and that it implies the presence of plumage color genetic variance has been tested, and the results are presented.

Both estrogen-receptor (ER) positive MCF-7 and ER-negative Hs578T and MDA-MB-231 human breast cancer cells were treated with carotenoids ([Beta]-carotene, canthaxanthin and lycopene) and retinoids (all-trans-, 9-cis- and 13-cis-retinoic acid and all-trans-retinol). Among carotenoids, [Beta]-carotene significantly reduced the growth of MCF-7 and Hs578T cells, and lycopene inhibited the growth of MCF-7 and MDA-MB-231 cells. Canthaxanthin did not affect the proliferation of any of the three cell lines. AII-trans- and 9-cis-retinoic acid significantly reduced the growth of both MCF-7 and Hs578T cells, whereas 13-cis-retinoic acid and all-trans-retinol had a significant effect only on MCF-7 cells. MCF-7 and Hs578T cells treated with all-trans-retinoic acid (all-t-RA) were further studied for the mechanism behind growth inhibition. Retinoic acid receptors [Alpha] and [Gamma] (RAR[Alpha], [Gamma]) in MCF-7 cells and RAR[Alpha], [Beta] and [Gamma] in Hs578T cells were not induced by all-t-RA treatment at either the protein or mRNA level. Hs578T cells treated with all-t-RA had significantly more cells in the G0/G1 stage of the cell cycle, but the same was not observed for MCF-7 cells. AII-t-RA induced a dose-dependent cell death in MCF-7 cells, which may be a necrotic phenomenon. These results demonstrate that ER status is an important, although not essential factor for breast cancer cell response to carotenoid and retinoid treatments, and the mode of action of all-t-RA in MCF-7 and Hs578T cells is not through the induction of RAR. Other mechanistic pathways that are either followed by or concomitant with growth inhibition are possible. J. Nutr. 131: 1574-1580, 2001. KEY WORDS: * [Beta]-carotene * retinoic acid * breast cancer cells * cell cycle * retinoic acid receptors

Ten structural genes from the Capsicum (pepper) carotenoid biosynthetic pathway have been localized on a (Capsicum annuum x Capsicum chinense)[F.sub.2] genetic map anchored in Lycopersicon (tomato). The positions of these genes were compared with positions of the same genes in tomato when known, and with loci from pepper, potato, and tomato that affect carotenoid levels in different tissues. C2, one of three phenotypically defined loci determining pepper fruit color, cosegregated with phytoene synthase. The capsanthin-capsorubin synthase (Ccs) locus, shown previously to cosegregate with Y, another pepper fruit color locus, mapped to pepper chromosome 6. Other structural genes in pepper corresponded to loci affecting carotenoid production as follows: Ccs in pepper and the B locus for hyperaccumulation of [Beta]-carotene in tomato fruit mapped to homeologous regions; the position of the lycopene [Beta]-cyclase gene in pepper may correspond to the lutescent-2 mutation in tomato; and the lycopene [Epsilon]-cyclase locus in pepper corresponded to the lycopene [Epsilon]-cyclase locus/Del mutation for hyperaccumulation of[Delta] carotene in tomato fruit. Additional associations were seen between the structural genes and previously mapped loci controlling quantitative variation in pepper and tomato fruit color. These results demonstrate that comparative analyses using candidate genes may be used to link specific metabolic phenotypes and loci that affect these phenotypes in related species.

Carotenoid pigments in plants fulfill indispensable functions in photosynthesis. Carotenoids that accumulate as secondary metabolites in chromoplasts provide distinct coloration to flowers and fruits. In this work we investigated the genetic mechanisms that regulate accumulation of carotenoids as secondary metabolites during ripening of tomato fruits. We analyzed two mutations that affect fruit pigmentation in tomato (Lycopersicon esculentum): Beta (B), a single dominant gene that increases [Beta]-carotene in the fruit, and old-gold (og), a recessive mutation that abolishes [Beta]-carotene and increases lycopene. Using a map-based cloning approach we cloned the genes B and og. Molecular analysis revealed that B encodes a novel type of lycopene [Beta]-cyclase, an enzyme that converts lycopene to [Beta]-carotene. The amino acid sequence of B is similar to capsanthin-capsorubin synthase, an enzyme that produces red xanthophylls in fruits of pepper (Capsicum annum). Our results prove that [Beta]-carotene is synthesized de novo during tomato fruit development by the B lycopene cyclase. In wild-type tomatoes B is expressed at Iow levels during the breaker stage of ripening, whereas in the Beta mutant its transcription is dramatically increased. Null mutations in the gene B are responsible for the phenotype in og, indicating that og is an allele of B. These results confirm that developmentally regulated transcription is the major mechanism that governs lycopene accumulation in ripening fruits. The cloned B genes can be used in various genetic manipulations toward altering pigmentation and enhancing nutritional value of plant foods.

The 4,4'-diketo-[Beta]-carotene, canthaxanthin, alters tocopherol status when fed to Balb/c mice, suggesting an involvement of carotenoids in the modulation of oxidative stress in vivo. We investigated further the modifications induced by an oral administration of canthaxanthin on lipid peroxidation, antioxidant enzymes and iron status in liver of Balb/c mice. Female 6-wk-old Balb/c mice were randomly divided into two groups (n = 10/group). The control group (C) received olive oil alone (vehicle) and the canthaxanthin-treated group (Cx) received canthaxanthin at a dose of 14 [micro]g/(g body wt [multiplied by] d). The 15-d canthaxanthin treatment resulted in carotenoid incorporation but did not modify lipid peroxidation as measured by endogenous production of malondialdehyde (MDA). However, glutathione peroxidase activity was 35% lower (P [is less than] 0.01) and catalase (59%, P [is less than] 0.005) and manganese superoxide dismutase (MnSOD) (28%, P [is less than] 0.05) activities were higher in canthaxanthin-treated mice than in controls. Moreover, carotenoid feeding caused a significant (P [is less than] 0.05) overexpression of the MnSOD gene; mRNA levels of the enzyme were greater in treated mice than in controls. Concomitantly, a 27% (P [is less than] 0.05) greater iron concentration was found in liver from canthaxanthin-treated mice compared with controls. These findings support the hypothesis that canthaxanthin alters the protective ability of tissues against oxidative stress in vivo. J. Nutr. 130: 1303-1308, 2000. KEY WORDS: * canthaxanthin * carotenoids * antioxidant enzymes * iron status * mice