Effect of CPTA and cycocel on the biosynthesis of carotenoids by Phycomyces blakesleeanus mutants

CPTA and cycocel cause accumulation of lycopene and γ-carotene, simultaneously inhibiting the formation of β-carotene and β-zeacarotene in Phycomyces blakesleeanus mutant strain C115. Phytoene synthesis is enhanced. CPTA is more effective than cycocel. Kinetic studies show that with increasing concentrations of CPTA, lycopene and γ-carotene increase with the concomitant decrease in β-carotene, the total of these three carotenes being almost equal to β-carotene present in the control. When CPTA-treated mycelium is washed free of the chemical and resuspended in phosphate buffer solution containing 2·5% glucose (pH 5·6), β-carotene is formed at the expense of both γ-carotene and lycopene. β-Zeacarotene, which is not present in the mycelium, reappears upon resuspension. These results indicate that CPTA is inhibiting the enzymes causing cyclization both at neurosporene and lycopene levels. Studies on the effect of CPTA on the high lycopene mutant strain C9 reveal that with increasing concentrations of the compound, lycopene increases slightly and both β-carotene and γ-carotene decrease. Phytoene synthesis is stimulated up to a certain level of CPTA and then becomes steady. In the albino mutant strain C5, there is a slight increase in phytoene formation on the addition of CPTA to the medium. No other carotenoid is formed, suggesting that CPTA cannot remove the block caused by genetic mutation and exerts its influence in an already existing biosynthetic pathway.     

In vitro and in vivo biosynthesis of xanthophylls by the cyanobacterium Aphanocapsa

Of the six carotenoids identified in the cyanobacterium Aphanocapsa, β-carotene, zeaxanthin, echinenone and myxoxanthophyll are the major pigments, whilst β-cryptoxanthin and 3-hydroxy-4-keto-β-carotene are present only in trace amounts. With the exception of zeaxanthin, the other xanthophylls could be formed in vitro from [¹⁴C]phytoene in high yields, especially β-cryptoxanthin and 3-hydroxy-4-keto-β-carotene. In a time course experiment of xanthopyll biosynthesis the flow of radioactivity from [¹⁴C]phytoene was followed through the pools of phytofluene, lycopene, and β-carotene. The reaction sequence from phytoene to xanthophylls is sensitive in vitro to both difunone, an inhibitor of carotene desaturation, and CPTA, an inhibitor of cyclization.     

Cyclization of lycopene in the biosynthesis of β-carotene

Mycobacterium marinum produces carotenoids when exposed to light or when antimycin A is added. Although the major pigment synthesized is β-carotene, lycopene is accumulated when the induced bacteria are incubated in the presence of nicotine (5 mM) or 2-(4-chlorophenylthio)-triethylamine hydrochloride (CPTA) (50 μM). Both of these compounds inhibit β-carotene synthesis by blocking the cyclization of lycopene. When nicotine is removed by washing the cells, the accumulated lycopene is cyclized to form β-carotene. The cyclization of lycopene is not an energy-requiring reaction and, furthermore, does not require oxygen or any other electron acceptor. Chloramphenicol addition also does not inhibit the conversion of lycopene to β-carotene indicating that no de novo protein synthesis is involved. Nicotine appears to act by inhibiting the activity of the enzyme required for the cyclization of lycopene.Although the mode of action of CPTA is similar to nicotine, it cannot be removed by washing once the cells have been incubated in its presence, suggesting that the molecule is tightly bound to the enzyme. The possible active molecular sites of nicotine and CPTA are discussed.     

Effect of CPTA and of Nicotine on Chlorophyll and Carotenoid Contents of an Ornamental Gourd

The three coloured parts: green, yellow-green and yellow offruits of an ornamental gourd contained carotenoids and chlorophyllsin decreasing amounts, while the orange parts had only smallamounts of carotenoids. There were 14 individual carotenoids(typically leaf carotenoids) in the green parts with three cis-xanthophylls(normally found in maturing fruits) which were in smaller amountsin the other coloured parts and disappeared completely in theorange parts. When the fruits were stored for 15 days, both carotenoids andchlorophylls (where present) decreased. 2-(4-chlorophenylthio)-triethylaminehydrochloride (CPTA)-treated fruits unusually contained eitherslightly less or slightly more carotenoids than controls, whilechlorophylls were always less. Lycopene and -carotene, absentin controls, accumulated in the treated fruits. These resultssupport the suggestion that CPTA may work as a cyclization reactioninhibitor. Nicotine however, inhibited total chlorophylls but increasedtotal carotenoids without accumulating either -carotene or lycopene.The results cannot be explained on the basis that only the non-ionicform of nicotine is effective as a carotenoid cyclization inhibitor. Ornamental gourd, Cucurbita, carotenoids, lycopene, xanthophylls, chlorophylls, 2-(4-chlorophenylthio)-triethylamine hydrochloride     

Carotenoids of Eriobotrya japonica

The carotenoids of the loquat fruit Eriobotrya japonica Golden Nugget variety, were investigated. They were identified according to their chromatographic, spectrophotometric and chemical properties and compared with standard pigments. For some of the carotenoids, MS were determined. Pulp and peels were investigated separately. The main pattern of the pulp carotenoids was β-carotene (33%), γ-carotene (6%), cryptoxanthin (22%), lutein, violaxanthin and neoxanthin, each about 3–4%. The peel, with a carotenoid content 5 times as high, had a similar pattern, but the ratio between the main pigments differed: β-carotene (50%); γ-carotene (5%); cryptoxanthin (5%); lutein (13%); violaxanthin, neoxanthin, 3–4%. The carotenoids of the loquat (subfamily Maloideae) were very similar to those of the apricot (Prunus armeniaca-subfamily Prunoideae) both of the family of Rosaceae. The intergeneric differences are more pronounced, which is of possible taxonomic significance. The lower concentration of cryptoxanthin and the high concentration of lutein in the peels is noteworthy and of biosynthetic interest.     

Effects of CPTA upon carotenogenesis and lipoidal constituents in Rhodotorula species

The effects of 2-(4-chlorophenyl)-triethylamine (CPTA) upon carotenogenesis in Rhodotorula glutinis, and upon various lipoidal constituents of R. rubra were studied. CPTA was found to cause the accumulation of lycopene and γ-carotene and to inhibit the formation of fatty acids and ergosterol. Upon removal of the inhibitor lycopene was metabolized to the cyclic carotenes and the levels of ergosterol increased. It was proposed that CPTA inhibits the cyclase enzyme of carotenogenesis, inhibits the formation of ergosterol, and causes the build-up of intermediates of these compounds.     

Carotenoid synthesis in Turkish lemons and oranges as influenced by triethylamine derivatives

Two triethylamines, 2-(4-chloro-phenylthio)-triethylamine (CPTA) and 4-chloro[β-(diethylamino)-ethyl]-benzoate (CDEB), were used to investigate C₃₀ carotenoid biosynthesis in Turkish oranges and lemons. It was not possible to determine the pathway, since not all the intermediates from phytoene to the C₃₀ compounds were observed. Lycopene and ζ-carotene, absent in controls, were identified in large amounts in certain CPTA- and CDEB- treated lemons and oranges. Furthermore there were varietal differences, since CDEB-treated Turkish oranges did not produce the increases in α- and β-carotene earlier observed in Californian Citrus fruits.     

Abstracts of papers read at the Winter Meeting, 1962

The effect of a range of inhibitors on the carotenoid biosynthetic pathway of the microalga Haematococcus pluvialis has been studied during normal growth and during the induction of astaxanthin synthesis. Diflufenican and norflurazon had similar effects and resulted in the almost complete inhibition of secondary carotenoid synthesis together with a build up of the acyclic carotenoid precursor, phytoene. In contrast, the inhibitor CPTA blocked cyclisation of lycopene and was seen to act differentially on the β- and ?-cyclases. Both diphenylamine and 1-aminobenzotriazole had the effect of blocking the synthesis of astaxanthin and the other secondary carotenoids by preventing the introduction of oxygen functions. As a direct result treated cells accumulated large levels of β-carotene instead. Selective use of inhibitors of carotenogenesis demonstrated that the accumulated lycopene and β-carotene could act as a precursor for astaxanthin synthesis.     

Biosynthesis of 1,2-dihydrocarotenoids in Rhodopseudomonas viridis: Experiments with inhibitors

The effects of the inhibitors diphenylamine (DPA), 2-(4-chlorophenylthio) triethylammonium chloride (CPTA) and nicotine on the biosynthesis of 1,2-dihydrocarotenoids by Rhodopseudomonas viridis (Rhodospirillaceae) have been investigated. Small amounts of 1,2-dihydro derivatives of phytoene, phytofluene and ξ-carotene and its unsymmetrical isomer, and 1,2,1′,2′,-tetrahydro derivatives of neurosporene and lycopene were isolated from R. viridis grown in the presence of DPA, although there was virtually no quantitative effect on the levels of the normal main carotenoids, neurosporene and lycopene and their 1,2-dihydro derivatives. Nicotine also had little effect on the overall carotenoid composition, but the formation of 1,2-dihydrocarotenoids was inhibited to some extent by CPTA. The 1,2-dihydro end group may thus be introduced by a hydrogenation reaction similar to the more familiar C-1,2 hydration reaction characteristic of carotenoid biosynthesis in other photo synthetic bacteria.     

The effect of CPTA analogs and other nitrogenous compounds on the biosynthesis of carotenoids in Phycomyces blakesleeanus mutants

The inhibitory effect of a series of analogs of CPTA, 2-(4-chlorophenylthio)-triethylamine-HCl, and ammonia derivatives on carotenoid biosynthesis in Phycomyces blakesleeanus mutants was studied. The types of inhibition exhibited allowed no firm conclusions about the biosynthetic route to β-carotene from either β-zeacarotene or lycopene. However, the evidence suggests at present that both pathways are operative. It was found that a slight change in structure of inhibitor resulted in a different type of action. Conclusions based on a single inhibitor could be cited as “evidence” for a certain pathway.     

Carotenoids in fish IV. Salmonidae and Thymallidae from Polish waters

The author investigated the presence of various carotenoids in the Salmonidae and Thymallidae family by means of columnar and thin-layer chromatography. The investigations revealed the presence of the following carotenoids:

Abstract

- in the muscles of Salmo salar: astaxanthin (pure and ester), canthaxanthin, lutein and zeaxanthin.

- in the eggs of Salmo trutta m. trutta: β-carotene, iso- and zeaxanthin, lutein, taraxanthin and astaxanthin.

- in the eggs of Salmo trutta m. fario: β-carotene, canthaxanthin, 4-keto-4-hydroxy-β-carotene, astaxanthin (pure and ester), lutein, taraxanthin and astacene.

- in the eggs of Salmo gairdneri: β-carotene, γ-carotene (?), canthacanthin, isozeaxanthin, lutein and astaxanthin, and in the sperm Salmo gairdneri: β-carotene, γ-carotene (?), 4-keto-4-hydroxy-β-carotene, canthaxanthin, lutein and astaxanthin.

- in the eggs of Salvelinus fontinalis: ester astaxanthin, canthaxanthin, isozeaxanthin, lutein and astacene.

- in the eggs of Hucho hucho: β-carotene, tunaxanthin, lutein, taraxanthin and astaxanthin.

- in the eggs of Coregonus albula: β-carotene, 4-keto-4-hydroxy-β-carotene, ester astaxanthin, zeaxanthin, taraxanthin and astacene.

- in Coregonus lavaretus: a) in eggs: β-carotene, ester astaxanthin, canthaxanthin, iso- and zeaxanthin, lutein, taraxanthin and astacene b) in the sperm: canthaxanthin, 4-hydroxy-4-keto-β-carotene, isozeaxanthin and astaxanthin, and other organs: 4-hydroxy-α-carotene, canthaxanthin, tunaxanthin, monoepoxy lutein, lutein, iso- and zeaxanthin and astaxanthin.

- in the eggs of Coregonus peled: β-carotene, 4-keto-4-hydroxy-β-carotene, lutein, zeaxanthin, taraxanthin and astacene.

- in the eggs of Thymallus thymallus: β-carotene, tunaxanthin, lutein and astaxanthin.

     

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

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

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

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

     

Biosynthesis of carotenoids by Phycomyces blakesleeanus mutants in the presence of nitrogenous heterocyclic compounds

Pyridine, imidazole and some of their derivatives stimulate lycopene and γ-carotene synthesis-simultaneously inhibiting β-carotene formation in Phycomyces blakesleeanus Strain C115. Isonicotinoly-hydrazine has a toxic effect on Strains C9 and C115 and 1-methylimidazole on Strain C115 in the concentrations of 1 g/l. Compounds which cause an accumulation of lycopene and γ-carotene usually cause an increase in phytoene synthesis and the disappearance of β-zeacarotene. The effect of succinimide, 4-hydroxypyridine, and isonicotinoylhydrazine on Strain C9 has also been studied. When β-picoline and 2-methylimidazole treated C115 mycelia were washed and resuspended in phosphate buffer at pH 5·6 β-zeacarotene reappeared and β-carotene increased with the simultaneous decrease in lycopene and γ-carotene. The sum of β-carotene, γ-carotene up to 3days of resuspension was almost equal to the total of these at zero time. These results show that the inhibitory action of these compounds is on the enzymes responsible for cyclization of acyclic carotenes. This inhibition varies with the nature of the substituent on the heterocyclic ring and pyridine derivatives having pK_a values of 6 ± 1 show the greatest degree of inhibition.     

Lipid-dissolved γ-carotene, β-carotene,and lycopene in globular chromoplasts of peach palm (Bactris gasipaes Kunth) fruits

Main conclusion

High levels of β-carotene, lycopene, and the rare γ-carotene occur predominantly lipid-dissolved in the chromoplasts of peach palm fruits. First proof of their absorption from these fruits is reported. The structural diversity, the physical deposition state in planta, and the human bioavailability of carotenoids from the edible fruits of diverse orange and yellow-colored peach palm (Bactris gasipaes Kunth) varieties were investigated. HPLC–PDA–MSⁿ revealed a broad range of carotenes, reaching total carotenoid levels from 0.7 to 13.9 mg/100 g FW. Besides the predominant (all-E)-β-carotene (0.4–5.4 mg/100 g FW), two (Z)-isomers of γ-carotene (0.1–3.9 mg/100 g FW), and one (Z)-lycopene isomer (0.04–0.83 mg/100 g FW) prevailed. Approximately 89–94 % of total carotenoid content pertained to provitamin A carotenoids with retinol activity equivalents ranging from 37 to 609 µg/100 g FW. The physical deposition state of these carotenoids in planta was investigated using light, transmission electron, and scanning electron microscopy. The plastids found in both orange and yellow-colored fruit mesocarps were amylo-chromoplasts of the globular type, containing carotenoids predominantly in a lipid-dissolved form. The hypothesis of lipid-dissolved carotenoids was supported by simple solubility estimations based on carotenoid and lipid contents of the fruit mesocarp. In our study, we report first results on the human bioavailability of γ-carotene, β-carotene, and lycopene from peach palm fruit, particularly proving the post-prandial absorption of the rarely occurring γ-carotene. Since the physical state of carotenoid deposition has been shown to be decisive for carotenoid bioavailability, lipid-dissolved carotenoids in peach palm fruits are expected to be highly bioavailable, however, further studies are required.     

Effect of light intensity on the formation of carotenoids in normal and mutant maize leaves

Carotenoid composition in leaves of normal, lycopenic and ζ-carotenic mutants of Zea mays were investigated. In lycopenic leaves, in addition to lycopene, phytoene, phytofluene, δ- and γ-carotene, trace amounts of α- and β-carotene and antheraxanthin were identified. Low light promoted accumulation of α- and β-carotene; high light brought about an increase in antheraxanthin content. In the leaves of the ζ-carotenic mutant, phytoene, phytofluene and ζ-carotene were synthesized. Illumination of low intensity stimulated carotenoid synthesis to a slight extent. Relative amounts of carotenoid components were essentially the same as in etiolated material, except for a small increase in cis-ζ-carotene. Under high intensity illumination, carotenoids were rapidly destroyed.     

<Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803 CruA (sll0147) encodes lycopene cyclase and requires bound chlorophyll <Emphasis Type="Italic">a</Emphasis> for activity

The genome of the model cyanobacterium, Synechococcus sp. PCC 7002, encodes two paralogs of CruA-type lycopene cyclases, SynPCC7002_A2153 and SynPCC7002_A0043, which are denoted cruA and cruP, respectively. Unlike the wild-type strain, a cruA deletion mutant is light-sensitive, grows slowly, and accumulates lycopene, γ-carotene, and 1-OH-lycopene; however, this strain still produces β-carotene and other carotenoids derived from it. Expression of cruA from Synechocystis sp. PCC 6803 (cruA ₆₈₀₃) in Escherichia coli strains that synthesize either lycopene or γ-carotene did not lead to the synthesis of either γ-carotene or β-carotene, respectively. However, expression of this orthologous cruA ₆₈₀₃ gene (sll0147) in the Synechococcus sp. PCC 7002 cruA deletion mutant produced strains with phenotypic properties identical to the wild type. CruA₆₈₀₃ was purified from Synechococcus sp. PCC 7002 by affinity chromatography, and the purified protein was pale yellow-green due to the presence of bound chlorophyll (Chl) a and β-carotene. Native polyacrylamide gel electrophoresis of the partly purified protein in the presence of lithium dodecylsulfate at 4 °C confirmed that the protein was yellow-green in color. When purified CruA₆₈₀₃ was assayed in vitro with either lycopene or γ-carotene as substrate, β-carotene was synthesized. These data establish that CruA₆₈₀₃ is a lycopene cyclase and that it requires a bound Chl a molecule for activity. Possible binding sites for Chl a and the potential regulatory role of the Chl a in coordination of Chl and carotenoid biosynthesis are discussed.     

Inhibition of lycopene cyclase results in accumulation of chlorophyll precursors

Free porphyrins and their magnesium complexes, including chlorophylls, are potent photo-sensitizers. Plants usually accumulate these compounds bound to proteins together with protective compounds like carotenoids. Besides their protective role, carotenoids can play a structural role in these complexes. To analyze the effect of impaired carotenogenesis on plastid membranes we applied to barley seedlings the bleaching herbicide 2-(4-chlorophenylthio)triethylamine (CPTA) as a specific inhibitor for the cyclization of lycopene. To avoid interference with photo-oxidation, the essential experiments were performed on seedlings grown in darkness. While the amount of total carotenoids decreased, we found accumulation of more δ-carotene than lycopene in darkness clearly showing that CPTA inhibits the lycopene β-cyclase more effectively than the lycopene ε-cyclase. The CPTA treatment resulted in accumulation of non-photoactive protochlorophyllide a; the amount of photoactive protochlorophyllide and NADPH:protochlorophyllide oxidoreductase remained constant. Further, the level of Mg protophorphyrin and its monomethyl ester increased to an extent similar to that obtained by application of 5-aminolevulinic acid (ALA). The perturbation of the ultrastructure of etioplast inner membranes, observed after CPTA-treatment, was not found after ALA-treatment; this excluded the accumulated tetrapyrroles as responsible for the perturbation. By contrast, the down-regulation of Lhcb and RbcS genes found after CPTA-treatment was compatible with the presumed role of Mg protophorphyrin as “plastid signal” for regulation of nuclear gene expression. Possible mechanisms for enhancement of tetrapyrrole accumulation by non-cyclic carotenoids are discussed.     

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

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