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     

Effects of two Triethylamines on the Carotenoids of Strelitzia reginae and of Hemerocallis flava

A method for the successful uptake of two triethylamines, 2-(4-chlorophenylthio)-triethylaminehydrochloride (CPTA) and 2-(4-ethylphenyloxo)-triethylamine(EPOT), by flowers of Hemerocallis flava (yellow day-lily) andby sepals of Strelitzia reginae is described. CPTA is more effectivethan EPOT in causing lycopene accumulation which appears tobe through a different mechanism in the two plants studied.Whereas inhibition of cyclase(s) may account for lycopene accumulationin H. flava, this is not so in S. reginae. The permeabilityof the cell wall to the triethylamines may be one of the reasonsfor the differences observed. Strelitzia reginae, Hemerocallis flava, day-lily, triethylamine, lycopene, carotenoids     

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.     

Light-dependent switch from formation of poly-cis carotenes to all-trans carotenoids in the Scenedesmus mutant C-6D

The Scenedesmus obliquus mutant C-6D forms untypical poly-cis carotenes and lacks cyclic carotenoids when grown heterotrophically in the dark. After transfer to light, a very fast formation of usual all-trans carotenoids is observed leading to a phenotype very similar to the wild-type. Carotene analysis and quantitative kinetics of carotenoid changes after illumination in the presence of inhibitors which inhibit the three enzymes involved in carotene conversion showed isomerization of poly-cis carotenes, especially pro--carotene, to all-trans isomers. This result and consequently photoisomerization of pro--carotene in solution demonstrated that photoisomerization is the light-dependent mechanism leading to all-trans carotenoids in illuminated cultures. Now the different phenotypes of Scenedesmus C-6D can be explained by a single mutation of phytoene desaturase when photoisomerization of poly-cis carotenes is considered.Non-common abbreviations CPTA 2-(4-chlorophenylthio)-triethylamine HCL     

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.     

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.     

Lycopene induction in Cucurbita moschata cotyledons by CPTA

2-(4-Chlorophenylthio) triethylamine hydrochloride (CPTA) inducedthe rapid accumulation of lycopene in Cucurbita moschata cotyledons.Concomitant with this increase, was a decrease in chlorophyllconcentration in green seedlings, a cessation of further plumuleand cotyledonary growth and an increase in cotyledon respiration.Light was required for maximum lycopene accumulation. (Received August 4, 1973; )     

Photoisomerization of lycopene during carotenogenesis in mutant C-6D of Scenedesmus obliquus

Mutant C-6D of the unicellular green alga Scenedesmus obliquus has lost the ability to form cyclic carotenoids during heterotrophic growth in the dark. In the dark it accumulates acyclic intermediates, i.e. lycopene, neurosporene and ζ-carotene. The lycopene and two neurosporene forms were identified to be cis-isomers. Upon transfer to light, intermediates decrease and a normal set of carotenoids is synthesized. Inhibition of the cyclization reaction by nicotine reveals a lightinduced isomerization of cis-lycopene to trans-lycopene. Since the spectral characteristics of these two isomers differ drastically the isomerization can be followed in vivo by measuring a light-induced absorbance change. This absorbance change has its maximum at 520 nm and shows fast kinetics under high light intensities reaching a saturation level after about 2 min. Fluence-response curves for this absorbance change were performed for different wavelengths of actinic light. From the linear parts of these curves an action spectrum was caculated showing maxima at 670, 630 and 440 nm originating from chlorophyll and a maximum at shorter wavelengths (400–510 nm) which is interpreted to derive from ζ-carotene. A model for the light regulation of carotenogenesis in mutant C-6D is presented and the relation to the so-called 520-change observed in many plants is discussed.     

Regulation of lycopene formation in cell suspension culture of VFNT tomato (Lycopersicon esculentum) by CPTA, growth regulators, sucrose, and temperature

The onium compound 2–(4-chlorophenylthio)-triethylamine(CPTA) was used to increase lycopene formation to levels approximatingthose in field–or glasshousegrown fruit, and then growthregulators, sucrose and temperature were used to regulate lycopeneaccumulation. It was found that the native auxin indole–3–acetic acid (IAA) was substantially more effective than 2,4-dichlorophenoxyaceticacid (2,4–D) in promoting lycopene formation, sucroseinhibited lycopene formation (cell basis), and temperature produceda pattern similar to that observed in the field with a temperatureoptimum between 18 and 26 C. Suggestions for further improvementsin technique are included. Key words: Suspension culture, tomato, lycopene     

Pigment synthesis in Cucurbita moschata cotyledons as influenced by CPTA and several inhibitors

2-(4-Chlorophenylthio) triethylamine hydrochloride (CPTA) inducedthe accumulation of a number of carotenes in pumpkin (Cucurbitamoschata) cotyledons, a tissue which does not normally accumulatethese pigments. Lycopene accumulated concomitantly with a decreasein -carotene and ß-carotene. CPTA appeared to inhibitcyclases involved in the synthesis of -carotene and ß-caroteneand to stimulate enzymes involved in lycopene synthesis. Cycloheximidereduced this CPTA induced enzyme synthesis while diphenylaminereduced CPTA induced carotene accumulation. Actinomycin D alonereduced accumulation of carotenes, but it did not affect CPTAinduced carotene accumulation. Rather lutein, violoxanthin andneoxanthin decreased and -carotene and ß-caroteneaccumulated. ¹Present address: Morioka Branch, Vegetable and Ornamental CropsResearch Station, Ministry of Agriculture and Forestry, Shimokuriyagawa,Morioka, Japan. (Received August 12, 1974; )     

Distribution of carotenoids in sub-cellular and sub-plastidic fractions of radish seedlings (Raphanus sativus) grown in the presence of bleaching herbicides

The intracellular and intraplastidic distribution of carotenoids has been investigated in radish seedlings grown in the presence of the herbicides amitrole and SAN 6706. Both herbicides caused bleaching and the plants became deficient in chlorophylls and the usual chloroplast cyclic carotenoids, but accumulated the acyclic carotenoid biosynthetic intermediates 15-cis-phytoene and all-trans-lycopene. In both the untreated and herbicide-treated plants all carotenoids, including phytoene and lycopene, were contained in the plastid. In all cases the normal cyclic carotenoids were located virtually exclusively in the thylakoid or prothylakoid fraction. In amitrole-treated plants, lycopene also was contained only in the thylakoid fraction, whereas phytoene, in these and in SAN 6706-treated plants, was detected in both the thylakoid fraction and an envelope preparation. Possible implications for the biosynthesis of the carotenoids are discussed.     

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.     

Biosynthesis of Carotenoids in Brevibacterium sp. KY-4313

The biosynthesis of 4-keto and 4,4′-diketo carotenoids in Brevibacterium sp. KY-4313 was studied. Echinenone and canthaxanthin were isolated from the cultures grown on a medium containing several n-alkanes. When glutathione was added to the bacterial cultures, the formation of canthaxanthin was inhibited while β-carotene and its hydroxy derivatives accumulated. It is suggested that these 4-hydroxy compounds, isocryptoxanthin, isozeaxanthin, and 4-hydroxy-4′-keto-β-carotene, are intermediates in the biosynthesis of canthaxanthin. In the presence of 2-(4-chlorophenylthio)-triethylamine hydrochloride or nicotine, lycopene and neurosporene accumulated. The β-carotene level decreased slightly but β-zeacarotene remained unchanged. β-carotene and its derivatives were resynthesized upon removal of the inhibitors. It was concluded that cyclization can take place at either the neurosporene or lycopene level in Brevibacterium sp. KY-4313.     

Effects of amines on the carotenogenesis in Blakeslea trispora

Six amines profoundly affected carotenogenesis in Blakeslea trispora. When cultures were treated with the amines, namely 4-[β-(diethylamino)-ethoxy]-benzaldehyde, 4-[β-(diethylamino)-ethoxy]-acetophenone hydrochloride, 4-β-(diethylamino)-ethoxy]-benzophenone hydrochloride, triethylamine hydrochloride, α-diethylaminopropiophenone hydrochloride and tributylamine hydrochloride, an increase in the lycopene accumulation was observed. The modes of action of these amines appear to be similar to that of 2-(4-chlorophenylthio)triethylamine hydrochloride (CPTA); however, they difrer in relative effectiveness.     

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.     

Phytochrome control of its own accumulation and leaf expansion in tentoxin- and norflurazon-treated mung bean seedlings

Primary leaves of 4-day-old, dark-grown mung bean [ Vigna radiata (L.) Wilczek cv. Berken] seedlings were exposed to 24 h of white light (200 μmol m⁻² s⁻¹) which was terminated by a 15 min, phytochrome-saturating red or far-red light exposure. Phytochrome content (in vivo and in vitro) and leaf area were monitored during the subsequent dark period. Red light treatments resulted in lower phytochrome content and greater leaf expansion than did far-red treatments. Phytochrome accumulation and leaf expansion were less in norflurazon- (no carotenoids and very low Chl) than in tentoxin- (very low Chl) treated leaves. After 3 days of darkness, leaf expansion was about 25% greater and phytochrome content was about 50% less in red- than in far-red-treated leaves of all treatments. These effects generally took longer to develop in norflurazon- than in tentoxin-treated tissues. Norflurazon-treated tissues exposed to long white light periods apparently do not as accurately reflect phytochrome-controlled photomorphogenic events of green tissues as do tentoxin-treated tissues of mung bean seedlings.     

The enzymatic conversion of cis-(14C)phytofluene, trans-(14C)phytofluene, and trans-zeta-(14C)carotene to poly-cis-acyclic carotenes by a cell-free preparation of tangerine tomato fruit plastids

The conversion of cis-[¹⁴C]phytofluene to trans-[¹⁴C]phytofluene and the conversion of the latter to trans-ζ-[¹⁴C]carotene by a soluble enzyme system obtained from plastids of tangerine tomato fruits is reported. Each of these compounds is also converted to cis-ζ-carotene, proneurosporene, prolycopene, neurosporene, lycopene, and γ- and β-carotenes. [¹⁴C]Prolycopene was also incubated with the above enzyme system. No conversion of this compound to trans-lycopene or cyclic carotenes was observed. Proof for the formation of the above carotenes from each of the substrates mentioned above was obtained by cochromatography with authentic samples on an alumina column. A close correspondence between radioactivity and light absorbance of each carotene was observed. Further proof for the formation of acyclic and cyclic carotenes from the above radioactive substrates was obtained by gas-liquid chromatography of the hydrogenated products. Coincidence between mass and radioactivity was observed in each case.     

The Effect of Light on Carotenoids of Etiolated Mung Bean Seedlings

Etiolated mung bean seedlings have been shown to contain thefollowing carotenoids: phytofluene, ß-carotene, ß-zeacarotene,5,6-monoepoxy-ß-carotene, 5,6:5', 6'-diepoxy-ß-carotene,violaxanthin, lutein, 5,6-monoepoxylutein, flavoxanthin, andauroxanthin whereas in the light -carotene and neoxanthin werealso identified. In the dark, total carotenoids after 8 dayswere 71.4 µg/g compared to 926.5 µg/g dry weightin light. In the dark, whereas most of the other individualcarotenoids were decreasing between 6 and 8 days, auroxanthinwas increasing. Further, flavoxanthin (5,8-monoepoxylutein)and auroxanthin (5,8:5', 8'-diepoxyzeaxanthin) were decreasingand disappeared in the light. Total xanthophylls increased much more than total caroteneson illuminating etiolated seedlings; lutein increased much morethan ß-carotene. This is in agreement with Goodwinand Phagpolngarm's (1960) results and in contrast to those ofother workers who suggested that ß-carotene was rapidlysynthesized whereas xanthophyll levels altered only slightlyunder these conditions. However, a critical look at these resultsshowed that a considerable increase in carotenes was more thanmatched by the increase in xanthophylls.     

Identification of carotenoids in Erwinia herbicola and in a transformed Escherichia coli strain

The yellow pigments of Erwinia herbicola Eho 10 and of a transformed Escherichia coli LE392 pPL376 have been identified as carotenoids. HPLC separation, spectra and in some cases mass spectroscopy demonstrated the presence of phytoene (15-cis isomer), beta-carotene (all-trans, 9-cis and 15-cis), beta-cryptoxanthin ( = 3-hydroxy beta-carotene), zeaxanthin (3,3'-dihydroxy beta-carotene) and corresponding carotene glycosides. In addition, lycopene and gamma-carotene accumulated in the presence of the inhibitor 2-(4-chlorophenylthio)-triethylamine.HCl. Carotenoid content in the transformed E. coli was two-fold higher than in E. herbicola. The pattern of the carotenoids was similar in the two organisms. Inactivation of the katF gene in E. coli resulted in an 85% lowering of carotenoid formation, as did the addition of 0.5% glucose to the medium. Suppression of carotenoid formation by inactivation of the katF gene lowered, but did not abolish, the protection offered by carotenoids against inactivation by alpha-terthienyl plus near-ultraviolet light (320-400 nm).     

红闪光对绿豆幼苗生长发育的影响

红闪光对绿豆幼苗的生长发育有明显影响。实验发现,与普通的日光生长条件相比,经红闪光(加普通绿光和蓝光)处理的绿豆幼苗的茎生长比较快,而叶片生长比较缓慢。同时,叶片叶绿素含量较少,叶重也较轻,示红闪光使绿豆幼苗的干物质积累受阻。这种差别随红闪光处理时间的延长越来越明显。此外,红闪光还大大增强了 1叶的UBE强度。这些结果表明,红闪光处理不利于绿豆幼苗的生长发育。