The function of carotenoids during chloroplast development. V. Correlation between carotenoid content, ultrastructure and chlorophyll b to chlorophyll a ratio

The ultrastructural effect of carotenoid deficiency in wheat ( Triticum aestivum L. ) was studied after adding the herbicide SAN-9789 to the growth medium. The presence of SAN-9789 (28 mg I^-1) resulted in an almost complete absence of carotenoids. For plants grown in darkness the lack of carotenoids was accompanied by a reduction in partitions between primary thylakoids as well as a change in appearance of the plastoglobuli from small and black (osmiophilic) to large and greyish white (less osmiophilic). When plants were grown in weak red light (16 mW m^-2), the presence of SAN-9789 also resulted in an almost complete absence of grana, a decrease in the ratio of chlorophyll b /chlorophyll a from 0.25 to 0.1, and an almost complete absence of prolamellar bodies. The greatest differences in carotenoid content, in amount of grana, in chlorophyll b /chlorophyll a ratio, and in number of prolamellar bodies, all occurred between 0.28 and 2.8 mg I^-1 of SAN-9789. The change in appearance of plastoglobuli occurred at a lower SAN-9789 concentration, between 0.028 and 0.28 mg I^-1. The ultrastructural responses to the SAN-9789 treatment could be related to the effect on the carotenoid synthesis in different ways. The disappearance of thylakoid partitions was connected to a structural role of carotenoids, while the disappearance of the prolamellar bodies was dependent on the photoprotective role of carotenoids. The change in shape and size of plastoglobuli was not correlated to the presence of carotenoids. However, a connection to the accumulation of the carotenoid precursors is still possible.     

Accumulation of phytoene in plastoglobuli of SAN-9789 (Norflurazon)-treated dark-grown wheat

Grains of wheat ( Triticum aestivum L. cv. Starke II) were treated with SAN-9789 (Norflurazon), and grown in darkness for 6 days. The SAN treatment resulted in an inhibition of the carotenoid synthesis at the level of phytoene. The plastids of SAN-treated plants contained enlarged and non-osmiophilic plastoglobuli, compared to the plastoglobuli of the control. The plastoglobuli were isolated and purified by means of a flotation technique, and their lipid composition was determined. Efforts were made to avoid contamination of epicuticular soluble lipids in the plastoglobuli suspension during isolation. The most suitable method was found to be a mechanical removal of the lipids from the surface of the intact leaves prior to homogenization. Membrane lipids, i.e. galacto-, phospho- and sulpholipids could not be detected in plastoglobuli from either SAN-treated or control plants, indicating that contamination with membranes was negligible. In plastoglobuli of SAN-treated plants, large amounts of phytoene and, to a lesser extent, phytofluene accumulated. The proportion of triacylglycerols to quinones was lower than in the control. The main lipids in control plastoglobuli were triacylglycerols, plastoquinones and α-tocopherol. The possible function of plastoglobuli in etioplasts is discussed.     

Biosynthesis of acyclic and cyclic carotenoids in higher plants and the control by phytochrome

Radish plants ( Raphanus sativus L. cv. Saxa treib) were grown in the presence of three different herbicides interfering with the biosynthesis of cyclic carotenoids. The herbicides caused an accumulation of acyclic biosynthetic intermediates. Plants were then irradiated using four different light programs in order to gain more insight into the first steps of carotenoid biosynthesis and their control by light and phytochrome. Plants grown in the dark in the presence of SAN 6706 or aminotriazole accumulated the acyclic intermediate phytoene, and those treated with J 852, the intermediates phytoene, phytofluene and zeta-carotene. In herbicide-treated plants short time irradiation with red light enhanced the formation of phytoene, phytofluene, zeta-carotene or lycopene, consistent with an effect of phytochrome on the early steps of carotenoid biosynthesis. Biosynthesis of cyclic carotenoids was also enhanced by red light in the untreated controls. In amitrole-treated plants formation of β-carotene, but not that of xanthophylls was stimulated by red light. In many cases neither the red light-induced biosynthesis of cyclic carotenoids nor the formation of acyclic intermediates could be prevented by a subsequent irradiation with far-red light. Similar enhancement as with red light was also obtained after treatment with far-red light only. Presented data may be taken as evidence that the biosynthesis and dehydrogenation of phytoene and the cyclization of lycopene are activated by a low threshold of active phytochrome. This may be further supported by the observation that far-red light itself stimulated carotenoid biosynthesis.     

Events Surrounding the Early Development of Euglena Chloroplasts: 7. Inhibition of Carotenoid Biosynthesis by the Herbicide SAN 9789 (4-Chloro-5-(methylamino)-2-(alpha,alpha,alpha,-trifluoro-m-tolyl)-3-(2H)pyridazinone) and Its Developmental Consequences

The herbicide SAN 9789 (4-chloro-5-(methylamino)-2-(α,α,α-trifluoro-m-tolyl-3- (2H)pyridazinone) blocks carotenoid synthesis in growing and resting cells of Euglena at concentrations of 20 to 100 μg/ml without affecting cell viability. Although the inhibition is immediate and complete, in resting cells no decrease in already synthesized carotenoids is found indicating a lack of turnover. In cells growing in the dark, carotenoids are diluted out as the cells divide. Cells dividing in the light in the presence of SAN 9789, eventually lose viability, presumably because of photooxidations usually prevented by carotenoids. During 72 hours of light-induced plastid development in dark-grown resting cells, none of the usual carotenoids increase while phytoene accumulates, indicating that SAN 9789 blocks carotenoid synthesis at this point. Chlorophyll synthesis and membrane formation are also blocked by the herbicide, but these inhibitions appear to be secondary to the inhibition of carotenoid synthesis. That carotenoid levels are strongly correlated with and may control the synthesis of chlorophyll and the formation of plastid membranes is suggested by the following data. (a) If dark-grown dividing cells are placed in the presence of the herbicide for various periods, rested and exposed to light in the presence of the drug, different amounts of carotenoids remain in the cells and the amount of chlorophyll finally synthesized is proportional to the amount of carotenoids present. (b) Photodestruction of chlorophyll is excluded, since the same amounts of chlorophyll are formed at intensities of 10 to 100 foot-candles of light. (c) Photoconversion of protochlorophyll(ide) to chlorophyll(ide) in dark-grown cells is not blocked by the herbicide. (d) Initial rates of chlorophyll synthesis are the same in treated and nontreated cells. (e) The extent of membrane formation appears to parallel the amount of carotenoids present as judged by electron microscopy.     

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.     

Light sensitivity of plastids and plastid pigments present in the albescent maize mutant

Dark grown albescent corn seedlings are deficient in colored carotenoids but accumulate phytoene, phytofluene and an unidentified substance in the carotenol fraction. They bleach upon exposure to bright light and appear albino. Seedlings grown under low level incandescent light are normal in appearance and contain almost as much colored carotenoid as control seedlings. The existing leaf tissue of seedlings grown under low level light does not bleach upon exposure to bright light. The enhanced carotenoid synthesis and stabilization of plastids is not affected by brief illumination with red light but requires several hours of low level incandescent light.     

Phytoene,phytofluene and ζ-carotene isomers from a Scenedesmus obliquus mutant

A number of mutant strains of the green alga, Scenedesmus obliquus, when grown in the dark, accumulated ζ-carotene as their major carotenoid together with appreciable concentrations of phytoene and phytofluene. The phytoene was almost entirely the 15-cis isomer, and phytofluene was also present mainly as the 15-cis form, whereas the ζ-carotene could be separated into three isomers, predominantly all-trans ζ-carotene accompanied by the 15-cis and an unidentified cis isomer. All the ζ-carotene isomers, when illuminated in the presence of iodine, gave the same equilibrium mixture of stereo-isomers, including a product with unusual spectroscopic and chromatographic properties, which may be a cyclic compound. The pathway of carotenoid biosynthesis in S. obliquus is discussed. On illumination, most of the ζ-carotenic strains were killed, but PGI strain survived, due to the production of cyclic carotenoids with chromophores long enough to protect chlorophyll from photosensitized oxidation.     

Changes in the Pools of Carotenoids and Protochlorophyll(ide) in Etiolated Cucumber (Cucumis Sativus) Cotyledons Treated with Norflurazon and KC 6361

Changes in the pools of carotenoids and protochlorophyll(ide) were investigated in etiolated cucumber cotyledons treated with norflurazon (NF) and an experimental herbicide KC 6361 (KC). Both the NF- and the KC-treated tissues considerably accumulated the colourless carotenes phytoene and phytofluene with a concomitant depletion of the coloured carotenoids lutein and β-carotene in darkness. However, the profiles of changes in chlorophylls (Chls) and carotenoids were different for the two herbicides. The plants were also influenced by the photosynthetic photon flux densities (PPFD's), with a more pronounced decline of Chl under high PPFD than under low PPFD. The ratios of protochlorophyll (PChl)/protochlorophyllide (PChlide) were greatly altered due to a decrease and an increase of PChl in the NF- and the KC-treated etiolated tissues, respectively, whereas the PChlide content was not significantly influenced by the inhibitors. Large increase of PChls in the KC-treated tissues seems to derive from the binding of accumulated geranylgeraniol (GG) equivalents, through carotenogenic inhibition, to PChlide. Therefore, the alterations of PChl and PChlide occurring under disturbed carotenogenesis may suggest an interaction between the biosynthetic pathways of Chls and carotenoids. In addition, the great proportion of PChl GG and PChl dihydro-GG in the KC-treated tissues implies that PChl formation is regulated at the level of hydrogenation. This revised version was published online in June 2006 with corrections to the Cover Date.     

Bioavailability of the isomer mixture of phytoene and phytofluene-rich alga Dunaliella bardawil in rat plasma and tissues

Dunaliella bardawil, a beta-carotene-accumulating alga was treated by the bleaching herbicide norflurazon to select sub-species rich with a mixture of 9-cis and all-trans stereoisomers of phytoene and phytofluene. The present study determines the bioavailability of phytoene and phytofluene with their stereoisomers in rats fed on a diet supplemented with Dunaliella phytoene-rich spray dried powder. Three groups of female weanling rats, eight animals each, were fed AIN diets for two weeks. The control consumed the diet as is. The experimental group was supplemented with 50 g Dunaliella powder to give phytoene/phytofluene at a level of 1 g/kg diet, and the placebo was provided with the oxidized algae free of carotenoids at the same amount. Weight gain and tissues weight of rats fed on the control diet, or on the experimental diets were statistically same. Tissue analyses were carried out by liquid chromatography at the end of two weeks feeding for vitamin A, carotenoids, phytoene and phytofluene and theirs stereoisomers. Liver analyses revealed high hepatic storage of phytoene in the experimental group. Analysis of the other tissues, adrenal, brain, heart, kidney, lung, and spleen detected small amounts of phytoene in the adrenal, kidney and spleen and in the plasma. High-pressure liquid chromatography for stereoisomeric composition was performed to all phytoene-containing tissues. The original algal diet content of 9-cis-to-all-trans ratio of 1:1 was maintained in the plasma and adrenal while in the liver, spleen and kidney the ratio was reduced to 1:3. The preferential accumulation of all-trans phytoene over 9-cis phytoene in the liver, spleen and kidney may be interpreted as indicating stronger antioxidative effect of 9-cis phytoene over the all-trans isomer or alternatively, in vivo streoisomerization of 9-cis phytoene to the all-trans structure.     

Light-dark regulation of carotenoid biosynthesis in pepper (Capsicum annuum) leaves

The carotenoid content in photosynthetic plant tissue reflects a steady state value resulting from permanent biosynthesis and concurrent photo-oxidation. The contributions of both reactions were determined in illuminated pepper leaves. The amount of carotenoids provided by biosynthesis were quantified by the accumulation of the colourless carotenoid phytoene in the presence of the inhibitor norflurazon. When applied, substantial amounts of this rather photo-stable intermediate were formed in the light. However, carotenoid biosynthesis was completely stalled in darkness. This switch off in the absence of light is related to the presence of very low messenger levels of the phytoene synthase gene, psy and the phytoene desaturase gene, pds. Other carotenogenic genes, such as zds, ptox and Icy-b also were shown to be down-regulated to some extent. By comparison of the carotenoid concentration before and after transfer of plants to increasing light intensities and accounting for the contribution of biosynthesis, the rate of photo-oxidation was estimated for pepper leaves. It could be demonstrated that light-independent degradation or conversion of carotenoids e.g. to abscisic acid is a minor process.     

Effect of certain nitrogenous heterocyclic compounds on carotenogenesis in Verticillium agaricinum.

Pyridine, isonicotinoylhydrazide and 1-methylamidazole have been used to investigate carotenoid biosynthesis in V. agaricinum. The results suggest that both torulin (C40) and neurosporaxanthin (C35) are formed from the precursors phytoene and phytofluene. These was no evidence of lycopene accumulation under these conditions. After 4 days' growth in the presence of isocotinolyhydrazine the fungus contained torulin and neurosporaxanthin only, whereas after 7 days, seven other carotenoids appeared as well, some of which were at the early stages of carotenoid biosynthesis. There results cannot be explained on the basis of a system consisting of free enzymes but of an enzyme aggregate already proposed for Phycomyces.     

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.     

Characterization of Pinalate,a novel Citrus sinensis mutant with a fruit-specific alteration that results in yellow pigmentation and decreased ABA content

The characterization of a novel mutant, named Pinalate, derived from the orange (Citrus sinensis L. Osbeck) Navelate, which produces distinctive yellow fruits instead of the typical bright orange colouration, is reported. The carotenoid content and composition, and ABA content in leaf and flavedo tissue (coloured part of the skin) of fruits at different developmental and maturation stages were analysed. No important differences in leaf carotenoid pattern of both phenotypes were found. However, an unusual accumulation of linear carotenes (phytoene, phytofluene and zeta- carotene) was detected in the flavedo of Pinalate. As fruit maturation progressed, the flavedo of mutant fruit accumulated high amounts of these carotenes and the proportion of cyclic and oxygenated carotenoids was substantially lower than in the parental line. Full-coloured fruit of Pinalate contained about 44% phytoene, 21% phytofluene, 25% zeta-carotene, and 10% of xanthophylls, whereas, in Navelate, 98% of total carotenoids were xanthophylls and apocarotenoids. The ABA content in the flavedo of Pinalate mature fruit was 3-6 times lower than in the corresponding tissue of Navelate, while no differences were found in leaves. Other maturation processes were not affected in Pinalate fruit. Taken together, the results indicate that Pinalate is a fruit-specific alteration defective in zeta-carotene desaturase or in zeta-carotene desaturase-associated factors. Possible mechanisms responsible for the Pinalate phenotype are discussed. Because of the abnormal fruit-specific carotenoid complement and ABA deficiency, Pinalate may constitute an excellent system for the study of carotenogenesis in Citrus and the involvement of ABA in fruit maturation and stress responses.     

Regulation of carotenoid biosynthesis during fruit maturation in the red-fleshed orange mutant Cara Cara

Cara Cara is a spontaneous bud mutation of Navel orange (Citrus. sinensis L. Osbeck) characterized by developing fruits with a pulp of bright red coloration due to the presence of lycopene. Peel of mutant fruits is however orange and indistinguishable from its parental. To elucidate the basis of lycopene accumulation in Cara Cara, we analyzed carotenoid profile and expression of three isoprenoid and nine carotenoid genes in flavedo and pulp of Cara Cara and Navel fruits throughout development and maturation. The pulp of the mutant accumulated high amounts of lycopene, but also phytoene and phytofluene, from early developmental stages. The peel of Cara Cara also accumulated phytoene and phytofluene. The expression of isoprenoid genes and of carotenoid biosynthetic genes downstream PDS (phytoene desaturase) was higher in the pulp of Cara Cara than in Navel. Not important differences in the expression of these genes were observed between the peel of both oranges. Moreover, the content of the plant hormone ABA (abscisic acid) was lower in the pulp of Cara Cara, but the expression of two genes involved in its biosynthesis was higher. The results suggest that an altered carotenoid composition may conduct to a positive feedback regulatory mechanism of carotenoid biosynthesis in citrus fruits. Increased levels of isoprenoid precursors in the mutant that could be channeled to carotenoid biosynthesis may be related to the red-fleshed phenotype of Cara Cara.     

Bleaching activity of 4-phenyl-3-(substituted benzylthio)-4H-1,2,4-triazoles

A variety of 4-aryl- and 4-alkyl-3-(substituted benzylthio)-4H-1,2,4-triazoles were prepared and evaluated for their bleaching activity by the lettuce seedling test. Among the series of tested compounds, 4-(3-fluorophenyl)-3-(4-trifluoromethylbenzylthio)-4H-1,2,4-triazole (39) exhibited the highest bleaching activity, causing complete bleaching symptoms at 10 microM. In the dark condition, compound 39 inhibited the formation of such carotenoids as beta-carotene, violaxanthin, neoxanthin and lutein, resulting in the formation of zeta-carotene, phytoene, phytofluene and beta-zeacarotene, which were not detected in the untreated control. Treatment by compound 39 at 50 microM resulted in the amount of accumulated zeta-carotene being seven-fold higher than that of phytoene, phytofluene and beta-zeacarotene. These results suggest that compound 39 might have interfered with desaturation, especially zeta-carotene desaturation, during carotenoid biosynthesis.     

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.     

Correlation between pigment composition and apoproteins of the light-harvesting complex II (LHC II) in wheat (Triticum aestivum)

Seedlings of wheat (Triticum aestivum L. cv. Walde, Weibull) grown in continuous weak red light (16 mW m⁻²) with or without SAN-9789, contained significantly lower amounts of chlorophylls and carotenoids compared to untreated plants grown in a greenhouse. The Chl alb ratios were 3.6 in the greenhouse-grown plants, 5.1 in untreated and ca 16 in SAN-treated plants grown in weak red light, respectively. The main difference in polypeptide composition of thylakoids isolated from red light-grown plants, compared to those grown in the greenhouse, was a lower amount of proteins of the light-harvesting complex (LHC) II. The amount of apo-LHC and LHC were correlated to the xanthophyll to β-carotene ratios in these plants. The absence of grana and the absence of proteins of the light-harvesting complex 11 in SAN-treated plants, support the general dogma that these proteins are involved in the formation of grana. Since the amount of apo-LHC and LHC could be correlated to the presence of carotenoids as well as the chlorophylls, it is concluded that the carotenoids are necessary for the correct assembly and stabilization of the apoproteins of LHC II in the thylakoid membranes.     

Characterization of intergeneric hybrids obtained by protoplast fusion between Phaffia rhodozyma, Cryptoococcus laurentii and Saccharomyces cerevisiae

Protoplast fusion was used to obtain hybrids between Phaffia rhodozymaand Cryptococcus laurentii or Saccharomyces cerevisiae(petite form). Hybrids were obtained which produced the carotenoids b-carotene, phytoene and phytofluene, and were respiratory-competent and prototrophic.     

Effects of the herbicide san 9789 on photomorphogenic responses

The herbicide, 4-chloro-5-(methylamino)-2-(α,α,α-trifluoro-m-tolyl)- 3(2H)-pyridazinone (San 9789), an inhibitor that prevents both carotenoid and chlorophyll accumulation and normal chloroplast development in white light, does not affect the physiological effectiveness of phytochrome in dark-and light-grown plants. Red/far red reversibility of growth inhibition, stimulation of anthocyanin synthesis, and stimulation of phenylalanine ammonia-lyase synthesis are not significantly different in plants grown with and without San 9789. Despite the complete absence of photosynthesis, flowering could be induced in the long day plant Hordeum vulgare L. when sucrose was provided to the leaves. Since the nonphotochemical reactions of phytochrome also are not affected by the herbicide, San 9789 may be used as a tool to study the phytochrome system spectrophotometrically in plants grown for relatively long periods under high intensity white light.