The individual variation in carotenoid content of the lichen species Dirinaria applanata (Fée) Awasthi

Column and thin‐layer chromatography revealed the presence of the following carotenoids in thalli of Dirinaria applanata from 13 different sites: α‐carotene, β‐carotene, β‐cryptoxanthin, lutein, 3′‐epilutein, zeaxanthin, antheraxanthin, canthaxanthin, astaxanthin, violaxanthin, mutatoxanthin, neoxanthin, capsochrome, fucoxanthinol, paracentrone and apo‐6′‐lycopenal. In the thalli of all 13 specimens of Dirinaria applanata β‐carotene, lutein, astaxanthin and violaxanthin were found as constant carotenoids. The total content of carotenoids ranged from 21.0 (from Mexico) to 54.9 μg g⁻¹ dry weight (from Antilles).     

Provitamin A accumulation in cassava (Manihot esculenta) roots driven by a single nucleotide polymorphism in a phytoene synthase gene

Cassava (Manihot esculenta) is an important staple crop, especially in the arid tropics. Because roots of commercial cassava cultivars contain a limited amount of provitamin A carotenoids, both conventional breeding and genetic modification are being applied to increase their production and accumulation to fight vitamin A deficiency disorders. We show here that an allelic polymorphism in one of the two expressed phytoene synthase (PSY) genes is capable of enhancing the flux of carbon through carotenogenesis, thus leading to the accumulation of colored provitamin A carotenoids in storage roots. A single nucleotide polymorphism present only in yellow-rooted cultivars cosegregates with colored roots in a breeding pedigree. The resulting amino acid exchange in a highly conserved region of PSY provides increased catalytic activity in vitro and is able to increase carotenoid production in recombinant yeast and Escherichia coli cells. Consequently, cassava plants overexpressing a PSY transgene produce yellow-fleshed, high-carotenoid roots. This newly characterized PSY allele provides means to improve cassava provitamin A content in cassava roots through both breeding and genetic modification.     

Carotenoid Crystal Formation in Arabidopsis and Carrot Roots Caused by Increased Phytoene Synthase Protein Levels

Background

As the first pathway-specific enzyme in carotenoid biosynthesis, phytoene synthase (PSY) is a prime regulatory target. This includes a number of biotechnological approaches that have successfully increased the carotenoid content in agronomically relevant non-green plant tissues through tissue-specific PSY overexpression. We investigated the differential effects of constitutive AtPSY overexpression in green and non-green cells of transgenic Arabidopsis lines. This revealed striking similarities to the situation found in orange carrot roots with respect to carotenoid amounts and sequestration mechanism.

Methology/Principal Findings

In Arabidopsis seedlings, carotenoid content remained unaffected by increased AtPSY levels although the protein was almost quantitatively imported into plastids, as shown by western blot analyses. In contrast, non-photosynthetic calli and roots overexpressing AtPSY accumulated carotenoids 10 and 100-fold above the corresponding wild-type tissues and contained 1800 and 500 µg carotenoids per g dry weight, respectively. This increase coincided with a change of the pattern of accumulated carotenoids, as xanthophylls decreased relative to β-carotene and carotene intermediates accumulated. As shown by polarization microscopy, carotenoids were found deposited in crystals, similar to crystalline-type chromoplasts of non-green tissues present in several other taxa. In fact, orange-colored carrots showed a similar situation with increased PSY protein as well as carotenoid levels and accumulation patterns whereas wild white-rooted carrots were similar to Arabidopsis wild type roots in this respect. Initiation of carotenoid crystal formation by increased PSY protein amounts was further confirmed by overexpressing crtB, a bacterial PSY gene, in white carrots, resulting in increased carotenoid amounts deposited in crystals.

Conclusions

The sequestration of carotenoids into crystals can be driven by the functional overexpression of one biosynthetic enzyme in non-green plastids not requiring a chromoplast developmental program as this does not exist in Arabidopsis. Thus, PSY expression plays a major, rate-limiting role in the transition from white to orange-colored carrots.     

An Ultrastructural and Carotenoid Analysis of the Red Ventrum of the Japanese Newt,Cynops pyrrhogaster

The ventral skin of the wild Japanese newt Cynops pyrrhogaster is creamy at metamorphosis, but turns red when mature. The color of the ventral skin of laboratory (lab)‐reared newts stays yellow throughout their life. However, the mechanism for the red coloration of this animal still remains unknown. In this study, we have performed ultrastructural and carotenoid analyses of the red ventrum of wild and lab‐reared Japanese newts. Using electron microscopy, we observed a number of xanthophores having ring carotenoid vesicles (rcv) and homogenous carotenoid granules (hcg) in the ventral red skin of the wild newt. In the skin, β‐carotene and five other kinds of carotenoids were detected by thin‐layer chromatography (TLC). In the ventral yellow skin of lab‐reared newts, however, only β‐carotene and three other kinds of carotenoids were found. The total amount of carotenoids in the red skin of the wild adult newt was six times more than that of the yellow skin of the lab‐reared newt. Moreover, rcv were more abundant in xanthophores in red skin, but hcg were more abundant in yellow skin. These results, taken together, suggest that the presence of carotenoids in rcv in xanthophores is one of the critical factors for producing the red ventral coloration of the Japanese newt C. pyrrhogaster.     

Characterization of cyanobacterial β‐carotene ketolase and hydroxylase genes in Escherichia coli,and their application for astaxanthin biosynthesis

Carotenoid biosynthesis is highly conserved and well characterized up to the synthesis of β‐carotene. Conversely, the synthesis of astaxanthin from β‐carotene is less well characterized. Regardless, astaxanthin is a highly sought natural product, due to its various industrial applications and elevated antioxidant capacity. In this article, 12 β‐carotene ketolase and 4 β‐carotene hydroxylase genes, isolated from 5 cyanobacterial species, are investigated for their function, and potential for microbial astaxanthin synthesis. Further, this in vivo comparison identifies and applies the most promising genetic elements within a dual expression vector, which is maintained in Escherichia coli. Here, combined overexpression of individual β‐carotene ketolase and β‐carotene hydroxylase genes, within a β‐carotene accumulating host, enables a 23.5‐fold improvement in total carotenoid yield (1.99 mg g^?1), over the parental strain, with >90% astaxanthin. Biotechnol. Bioeng. 2009;103: 944–955. © 2009 Wiley Periodicals, Inc.     

Bottlenecks in carotenoid biosynthesis and accumulation in rice endosperm are influenced by the precursor–product balance

The profile of secondary metabolites in plants reflects the balance of biosynthesis, degradation and storage, including the availability of precursors and products that affect the metabolic equilibrium. We investigated the impact of the precursor–product balance on the carotenoid pathway in the endosperm of intact rice plants because this tissue does not normally accumulate carotenoids, allowing us to control each component of the pathway. We generated transgenic plants expressing the maize phytoene synthase gene (ZmPSY1) and the bacterial phytoene desaturase gene (PaCRTI), which are sufficient to produce β‐carotene in the presence of endogenous lycopene β‐cyclase. We combined this mini‐pathway with the Arabidopsis thaliana genes AtDXS (encoding 1‐deoxy‐D‐xylulose 5‐phosphate synthase, which supplies metabolic precursors) or AtOR (the ORANGE gene, which promotes the formation of a metabolic sink). Analysis of the resulting transgenic plants suggested that the supply of isoprenoid precursors from the MEP pathway is one of the key factors limiting carotenoid accumulation in the endosperm and that the overexpression of AtOR increased the accumulation of carotenoids in part by up‐regulating a series of endogenous carotenogenic genes. The identification of metabolic bottlenecks in the pathway will help to refine strategies for the creation of engineered plants with specific carotenoid profiles.     

Synergistic metabolism in hybrid corn indicates bottlenecks in the carotenoid pathway and leads to the accumulation of extraordinary levels of the nutritionally important carotenoid zeaxanthin

Lutein and zeaxanthin cannot be synthesized de novo in humans, and although lutein is abundant in fruit and vegetables, good dietary sources of zeaxanthin are scarce. Certain corn varieties provide adequate amounts because the ratio of endosperm β : ε lycopene cyclase activity favours the β‐carotene/zeaxanthin branch of the carotenoid pathway. We previously described a transgenic corn line expressing the early enzymes in the pathway (including lycopene β‐cyclase) and therefore accumulating extraordinary levels of β‐carotene. Here, we demonstrate that introgressing the transgenic mini‐pathway into wild‐type yellow endosperm varieties gives rise to hybrids in which the β : ε ratio is altered additively. Where the β : ε ratio in the genetic background is high, introgression of the mini‐pathway allows zeaxanthin production at an unprecedented 56 μg/g dry weight. This result shows that metabolic synergy between endogenous and heterologous pathways can be used to enhance the levels of nutritionally important metabolites.     

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.     

Non‐endogenous ketocarotenoid accumulation in engineered Synechocystis sp. PCC 6803

The cyanobacterium Synechocystis sp. PCC 6803 is a model species commonly employed for biotechnological applications. It is naturally able to accumulate zeaxanthin (Zea) and echinenone (Ech), but not astaxanthin (Asx), which is the highest value carotenoid produced by microalgae, with a wide range of applications in pharmaceutical, cosmetics, food and feed industries. With the aim of finding an alternative and sustainable biological source for the production of Asx and other valuable hydroxylated and ketolated intermediates, the carotenoid biosynthetic pathway of Synechocystis sp. PCC 6803 has been engineered by introducing the 4,4′ β‐carotene oxygenase (CrtW) and 3,3′ β‐carotene hydroxylase (CrtZ) genes from Brevundimonas sp. SD‐212 under the control of a temperature‐inducible promoter. The expression of exogenous CrtZ led to an increased accumulation of Zea at the expense of Ech, while the expression of exogenous CrtW promoted the production of non‐endogenous canthaxanthin and an increase in the Ech content with a concomitant strong reduction of β‐carotene (β‐car). When both Brevundimonas sp. SD‐212 genes were coexpressed, significant amounts of non‐endogenous Asx were obtained accompanied by a strong decrease in β‐car content. Asx accumulation was higher (approximately 50% of total carotenoids) when CrtZ was cloned upstream of CrtW, but still significant (approximately 30%) when the position of genes was inverted. Therefore, the engineered strains constitute a useful tool for investigating the ketocarotenoid biosynthetic pathway in cyanobacteria and an excellent starting point for further optimisation and industrial exploitation of these organisms for the production of added‐value compounds.     

Major QTL for carrot color are positionally associated with carotenoid biosynthetic genes and interact epistatically in a domesticated × wild carrot cross

We performed QTL analyses for pigment content on a carotenoid biosynthesis function map based on progeny of a wild white carrot (QAL) which accumulates no pigments × domesticated orange carrot (B493), one of the richest sources of carotenoid pigments—mainly provitamin A α- and β- carotenes. Two major interacting loci, Y and Y ₂ on linkage groups 2 and 5, respectively, control much variation for carotenoid accumulation in carrot roots. They are associated with carotenoid biosynthetic genes zeaxanthin epoxidase and carotene hydroxylase and carotenoid dioxygenase gene family members as positional candidate genes. Dominant Y allele inhibits carotenoid accumulation. When Y is homozygous recessive, carotenoids that accumulate are either only xanthophylls in Y ₂ __ plants, or both carotenes and xanthophylls, in y ₂ y ₂ plants. These two genes played a major role in carrot domestication and account for the significant role that modern carrot plays in vitamin A nutrition.     

Dissecting the mechanism of Solanum lycopersicum and Solanum chilense flower colour formation

Flowers are the defining feature of angiosperms, and function as indispensable organs for sexual reproduction. Flower colour typically plays an important role in attracting pollinators, and can show considerable variation, even between closely related species. For example, domesticated tomato (S. lycopersicum) has orange/yellow flowers, while the wild relative S. chilense (accession LA2405) has bright yellow flowers. In this study, the mechanism of flower colour formation in these two species was compared by evaluating the accumulation of carotenoids, assessing the expression genes related to carotenoid biosynthetic pathways and observing chromoplast ultrastructure. In S. chilense petals, genes associated with the lutein branch of the carotenoid biosynthetic pathway, phytoene desaturase (PDS), ζ‐carotene desaturase (ZDS), lycopene β‐cyclase (LCY‐B), β‐ring hydroxylase (CRTR‐B) and ε‐ring hydroxylase (CRTR‐E), were highly expressed, and this was correlated with high levels of lutein accumulation. In contrast, PDS, ZDS and CYC‐B from the neoxanthin biosynthetic branch were highly expressed in S. lycopersicum anthers, leading to increased β‐carotene accumulation and hence an orange/yellow colour. Changes in the size, amount and electron density of plastoglobules in chromoplasts provided further evidence of carotenoid accumulation and flower colour formation. Taken together, these results reveal the biochemical basis of differences in carotenoid pigment accumulation and colour between petals and anthers in tomato.     

Single‐molecule real‐time sequencing reveals diverse allelic variations in carotenoid biosynthetic genes in pepper (Capsicum spp.)

The diverse colours of mature pepper (Capsicum spp.) fruit result from the accumulation of different carotenoids. The carotenoid biosynthetic pathway has been well elucidated in Solanaceous plants, and analysis of candidate genes involved in this process has revealed variations in carotenoid biosynthetic genes in Capsicum spp. However, the allelic variations revealed by previous studies could not fully explain the variation in fruit colour in Capsicum spp. due to technical difficulties in detecting allelic variation in multiple candidate genes in numerous samples. In this study, we uncovered allelic variations in six carotenoid biosynthetic genes, including phytoene synthase (PSY1, PSY2), lycopene β‐cyclase, β‐carotene hydroxylase, zeaxanthin epoxidase and capsanthin‐capsorubin synthase (CCS) genes, in 94 pepper accessions by single‐molecule real‐time (SMRT) sequencing. To investigate the relationship between allelic variations in the candidate genes and differences in fruit colour, we performed ultra‐performance liquid chromatography analysis using 43 accessions representing each allelic variation. Different combinations of dysfunctional mutations in PSY1 and CCS could explain variation in the compositions and levels of carotenoids in the accessions examined in this study. Our results demonstrate that SMRT sequencing technology can be used to rapidly identify allelic variation in target genes in various germplasms. The newly identified allelic variants will be useful for pepper breeding and for further analysis of carotenoid biosynthesis pathways.     

Carotenoids,vitamin A,and vitamin E concentrations during egg development in panther chameleons (Furcifer pardalis)

Insects are known to be poor sources of preformed vitamin A, leading to the speculation that insectivorous species, including reptiles, may be able to convert carotenoid precursors to meet dietary requirements for this nutrient. This study was conducted to indirectly evaluate carotenoid and vitamin A metabolism in the panther chameleon (Furcifer pardalis). Eggs were obtained from females in Madagascar that were yolked either early or later in the breeding season, and carotenoid (α‐ and β‐carotene, cryptoxanthin, lutein/zeaxanthin, and lycopene), vitamin A, and vitamin E concentrations were measured in egg contents in early, middle, or late embryonic development. An overall trend of decreased nutrient concentration as eggs matured (from egg period 1 (yolks) to egg period 3 (embryos)) was seen within both clutch groups. The season of clutch deposition was a significant influence on egg weight, α‐carotene, and lutein/zeaxanthin concentrations, but on no other nutrients. Chameleon yolks contained considerably higher levels of carotenoids than levels previously reported from two viviparous lizard species, and β‐carotene concentrations were of the same magnitude as reported in grazing tortoises. β‐Carotene and β‐cryptoxanthin were the predominant carotenoids in yolk and embryos, comprising about 95% of total carotenoids detected. Measurable concentrations of retinol at all stages of egg development in the chameleons suggests effective conversion from carotenoid precursors, with concentrations similar to those measured in other lizard eggs. Information from eggs obtained in native habitats may provide baseline data on nutrient interactions to improve and optimize captive dietary management; preliminary data suggest that micronutrient environments may vary over the protracted breeding season, with possible implications for embryo health and survival. Zoo Biol 21:295–303, 2002. © 2002 Wiley‐Liss, Inc.     

Carotenoid and fatty acid metabolism in light‐stressed Dunaliella salina

β‐Carotene is overproduced in the alga Dunaliella salina in response to high light intensities. We have studied the effects of a sudden light increase on carotenoid and fatty acid metabolism using a flat panel photobioreactor that was run in turbidostat mode to ensure a constant light regime throughout the experiments. Upon the shift to an increased light intensity, β‐carotene production commenced immediately. The first 4 h after induction were marked by constant intracellular levels of β‐carotene (2.2 g LCV^?1), which resulted from identical increases in the production rates of cell volume and β‐carotene. Following this initial phase, β‐carotene productivity continued to increase while the cell volume productivity dropped. As a result, the intracellular β‐carotene concentration increased reaching a maximum of 17 g LCV^?1 after 2 days of light stress. Approximately 1 day before that, the maximum β‐carotene productivity of 30 pg cell^?1 day^?1 (equivalent to 37 mg LRV^?1 day^?1) was obtained, which was about one order of magnitude larger than the average productivity reported for a commercial β‐carotene production facility, indicating a vast potential for improvement. Furthermore, by studying the light‐induced changes in both β‐carotene and fatty acid metabolism, it appeared that carotenoid overproduction was associated with oil globule formation and a decrease in the degree of fatty acid unsaturation. Our results indicate that cellular β‐carotene accumulation in D. salina correlates with accumulation of specific fatty acid species (C16:0 and C18:1) rather than with total fatty acid content. Biotechnol. Bioeng. 2010;106: 638–648. © 2010 Wiley Periodicals, Inc.