Interaction of Chloroplasts with Inhibitors: Location of Carotenoid Synthesis and Inhibition during Chloroplast Development

The inhibitor SAN 6706 [4-chloro-5-(dimethylamino)-2-(α,α,α,-trifluoro- m-tolyl-3(2H)-pyridazinone] has been used to study the synthesis of carotenes and xanthophylls during the conversion of etioplasts to chloroplasts in developing barley (Hordeum vulgare) shoots. SAN 6706 inhibits carotenoid synthesis and causes an accumulation of phytoene, but it is also a potent inhibitor of chloroplast electron transport. When developing barley is treated with SAN 6706, carotenoid synthesis is inhibited but total photosynthesis is unaffected. The ability of SAN 6706 to inhibit carotenoid synthesis becomes progressively less if etiolated shoots are illuminated for increasing lengths of time before treatment. During the greening of treated barley shoots only light-induced β-carotene synthesis is immediately inhibited; xanthophyll synthesis is not affected until after about 8 hours. The hypothesis that SAN 6706 cannot enter the chloroplast but inhibits carotenoid synthesis from the cytoplasm is discussed, and the question as to whether there are not two separate groups of enzymes for the synthesis of carotenes and xanthophylls is considered.     

Bleaching herbicide flurtamone interferes with phytoene desaturase

The mode of action of the furanone herbicide flurtamone and derivatives was investigated with cress seedlings and with the unicellular cyanobacterium Anacystis. Either in the light or in the dark these compounds inhibited the formation of α- and β-carotene and all of the xanthophylls in the seedlings. Instead, phytoene, a precursor of colored carotenoids, was accumulated. In illuminated seedlings photooxidative destruction of chlorophyll was observed. The I₅₀ value of flurtamone inhibition of carotenoid biosynthesis in intact Anacystis cells and the K₁ value for interaction of flurtamone with phytoene desaturase with Anacystis thylakoids were 30 and 18 nanomoles, respectively. Concentrations of flurtamone which strongly inhibited carotenoid synthesis had no direct peroxidative activities and did not inhibit photosynthetic electron transport.     

The Carotenoid Hydrocarbons of Euglena gracilis and Derived Mutants

An examination of the carotene fractions extracted from Euglena gracilis Z and pressure-bleached Euglena mutants PR-1, PR-2, PR-3, and PR-4 revealed phytoene in mutants PR-1, PR-2, and PR-3. Photosynthetic E. gracilis Z cultured at different light intensities showed no detectable phytoene, nor was phytoene found in mutant PR-4. However, dark-cultured E. gracilis Z yielded readily assayable amounts of phytoene. With the exception of PR-4, in which no C₄₀ carotenoids were detected, the following carotenes were identified in all from their mass spectra: phytoene, phytofluene, ζ-carotene, β-zeacarotene, and β-carotene. Of these, phytoene and β-zeacarotene had not previously been unequivocally identified in Euglena.     

Inhibition of carotenoid synthesis as a mechanism of action of amitrole, dichlormate, and pyriclor

Amitrole (3-amino-s-triazole), dichlormate (3,4-dichlorobenzyl methylcarbamate), and pyriclor (2,3,5-trichloro-4-pyridinol) inhibited normal carotenogenesis in etiolated wheat (Triticum aestivum L. var. Coker 65-20) seedlings. Carotenoid precursors accumulated in treated plants. In dichlormate-treated plants, ζ-carotene accumulated, whereas phytofluene, phytoene, and ζ-carotene accumulated in amitrole- and pyriclor-treated plants. None of the herbicides interfered with protochlorophyllide synthesis or its conversion to chlorophyllide when etiolated plants were illuminated. Chlorophyll accumulated in treated plants exposed to light at 60 foot candles, but was unstable and partially destroyed by illumination at 4000 foot candles. These data suggest that the phytotoxicity of amitrole, pyriclor, and dichlormate is due to inhibition of the synthesis of carotenoids and to the consequent photodestruction of chlorophyll and chloroplast disruption.     

Isolation and Characterization of Carotenoid-rich Lipid Globules from Peridinium foliaceum

Carotenoid-rich oil globules were isolated from the cytoplasm of the binucleate dinoflagellate, Peridinium foliaceum. These orange globules were collected from ruptured cells by ultracentrifugation on a sucrose density gradient, and checked for purity by electron microscopy. The osmiophilic globules were assayed for lipid (including pigment) and protein content. The lipid to protein ratio was 1.39:1, with a calculated density of the globules of 1.05 grams per cubic centimeter. The lipids were composed of hydrocarbon, wax ester (phytyl ester), triglyceride, and polar (no phospholipid) fractions. The biochemical composition indicated that the globules function as a reservoir of energy-rich components in the cell. Microspectrophotometric observations were consistent with pigment analyses which demonstrated that the globules were carotenoid-rich. In addition to β-carotene, γ-carotene, and canthaxanthin, the carotenogenic precursors: phytoene, phytofluence, ζ-carotene and β-zeacarotene were isolated from the globules. Corrected fluorescence maxima of phytoene and phytofluene in hexane were recorded at 340 and 490 nanometers, respectively. Carotenes constituted 3.3% of the total oil globule lipid. The possibility of an extraplastidic carotenogenic enzyme system in P. foliaceum is discussed.     

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.     

Ketocarotenoid Production in Soybean Seeds through Metabolic Engineering

The pink or red ketocarotenoids, canthaxanthin and astaxanthin, are used as feed additives in the poultry and aquaculture industries as a source of egg yolk and flesh pigmentation, as farmed animals do not have access to the carotenoid sources of their wild counterparts. Because soybean is already an important component in animal feed, production of these carotenoids in soybean could be a cost-effective means of delivery. In order to characterize the ability of soybean seed to produce carotenoids, soybean cv. Jack was transformed with the crtB gene from Pantoea ananatis, which codes for phytoene synthase, an enzyme which catalyzes the first committed step in the carotenoid pathway. The crtB gene was engineered together in combinations with ketolase genes (crtW from Brevundimonas sp. strain SD212 and bkt1 from Haematococcus pluvialis) to produce ketocarotenoids; all genes were placed under the control of seed-specific promoters. HPLC results showed that canthaxanthin is present in the transgenic seeds at levels up to 52 μg/g dry weight. Transgenic seeds also accumulated other compounds in the carotenoid pathway, such as astaxanthin, lutein, β-carotene, phytoene, α-carotene, lycopene, and β-cryptoxanthin, whereas lutein was the only one of these detected in non-transgenic seeds. The accumulation of astaxanthin, which requires a β-carotene hydroxylase in addition to a β-carotene ketolase, in the transgenic seeds suggests that an endogenous soybean enzyme is able to work in combination with the ketolase transgene. Soybean seeds that accumulate ketocarotenoids could potentially be used in animal feed to reduce or eliminate the need for the costly addition of these compounds.     

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.     

On the Factors Which Determine Massive beta-Carotene Accumulation in the Halotolerant Alga Dunaliella bardawil

Dunaliella bardawil, a β-carotene-accumulating halotolerant alga, has been analyzed for the effect of various growth conditions on its pigment content, and compared with Dunaliella salina, a β-carotene nonaccumulating species. In D. bardawil, increasing light intensity and light period or inhibiting growth by various stress conditions such as nutrient deficiency or high salt concentration caused a decrease in the content of chlorophyll per cell and an increase in the amount of β-carotene per cell. As a result, the β-carotene-to-chlorophyll ratio increased from about 0.4 to 13 grams per gram and the alga changed its visual appearance from green to deep orange. D. salina grown similarly decreased in content of both chlorophyll and β-carotene per cell and the culture turned from green to yellowish. Low chlorophyll-containing cells of D. bardawil or D. salina exhibit very high photosynthetic rates when expressed on a chlorophyll basis (~600 micromoles O₂ evolved per milligram chlorophyll per hour).

Variation of pigment content in D. bardawil by a large variety of environmental agents has been correlated with the integral irradiance received by the algal culture during a division cycle. The higher the integral irradiance per division cycle, the lower the chlorophyll content per cell; the higher the β-carotene content per cell, and therefore the higher the β-carotene-to-chlorophyll ratio. The results are interpreted as indicating a protecting effect of β-carotene against injury by high irradiance under conditions of impairment in chlorophyll content per cell.

     

Stereoisomers of beta-Carotene and Phytoene in the Alga Dunaliella bardawil

Dunaliella bardawil, a halotolerant green alga, was previously shown to accumulate high concentrations of β-carotene when grown outdoors under defined conditions. The β-carotene of algae cultivated under high light intensity in media containing a high salt concentration is composed of approximately 50% all-trans β-carotene and 40% 9-cis β-carotene. We show here that the 9-cis to all-trans ratio is proportional to the integral light intensity to which the algae are exposed during a division cycle. In cells grown under a continuous white light of 2000 microeinsteins per square meter per second, the ratio reached a value of around 1.5, while in cells grown under a light intensity of 50 microeinsteins per square meter per second, the ratio was around 0.2. As previously shown, algae treated with the herbicide norflurazon accumulate phytoene in place of β-carotene. Electron micrographs showed that the phytoene is accumulated in many distinct globules located in the interthylakoid spaces of the chloroplast. Here too, two isomers are present, apparently all-trans and 9-cis phytoene, and their ratio is dependent upon the integral light intensity to which the algae are exposed during a division cycle. In the presence of norflurazon, Dunaliella bardawil grown under a light intensity of 2000 microeinsteins per square meter per second contained about 8% phytoene with a 9-cis to all-trans ratio of about 1.0. This ratio decreased to about 0.1 when the light intensity was reduced to 50 microeinsteins per square meter per second. These data suggest that the isomerization reaction which leads to the production of the 9-cis isomer occurs early in the path of carotene biosynthesis, at or before the formation of all-trans phytoene. The presence of the 9-cis isomer of β-carotene and the dependence of its preponderance on light intensity seem to be a common feature of many plant parts. Thus carrots which are exposed to minimal light contain no 9-cis isomer while sun-exposed leaves, fruits, and flowers contain 20 to 50% of the 9-cis isomer.     

Pigment study of Sphaerobolus stellatus

Five carotenes:B-carotene,-carotene, lycopene, phytoene and phytofluene; as well as two xanthophylls: canthaxanthin and cryptoxanthin have been extracted from mature fruit-bodies ofSphaerobolus stellatus. The most abundant of the pigments isB-carotene. None of the extracted pigments showed appreciable absorption in the region 600–720 nm.     

Increase in linolenic Acid is not a prerequisite for development of freezing tolerance in wheat

Seedlings of winter wheat (Triticum aestivum L. cv. Kharkov) were acclimated at 2 C in the dark in the presence of two inhibitors of linolenic acid synthesis, 4-chloro-5(dimethylamino)-2-phenyl-3(2H)pyridazinone-(BASF 13-338) and 4-chloro-5(dimethylamino)-2-(α,α,α-trifluoro-m-tolyl)- 3(2H)pyridazinone (Sandoz 6706). Although the increase in the proportion of linolenic acid generally observed at low temperature was completely inhibited, the development of freezing tolerance was unaffected. These results demonstrated that an enrichment in linolenic acid is not a prerequisite for low temperature acclimation.     

Carotenoid composition of spinach chloroplast grana and stroma lamellae

Stroma lamellae and grana stacks prepared by French press rupture of spinach (Spinacia oleracea) chloroplasts contain similar amounts of β-carotene on a protein basis. The grana fraction has considerably more xanthophylls than does the stroma fraction. Total carotenoid to chlorophyll ratios are similar for both fractions.     

C-glycosylflavone accumulation in Sandoz 6706-treated barley seedlings is a photocontrolled response

Sandoz 6706 pretreatment of white light grown barley seedlings causes a 60% increase in saponarin (6-C-glucosyl-7-O-glucosylapigenin) but a 300% increase in lutonarin (3′-hydroxysaponarin). Norflurazon has little effect on saponarin levels but is almost as effective as Sandoz 6706 in enhancing lutonarin net synthesis. Barley roots contain saponarin and lutonarin only after herbicide treatment. Mung bean seedlings respond to Sandoz 6706 by accumulating higher levels of rutin and delphinidin 3-glucoside. The results are discussed in relation to the site of action of the herbicides, the High Energy photoresponse, and control of flavonoid 3′-hydroxylation.     

Metabolic Engineering of the Carotenoid Biosynthetic Pathway in the Yeast Xanthophyllomyces dendrorhous (Phaffia rhodozyma)

The crtYB locus was used as an integrative platform for the construction of specific carotenoid biosynthetic mutants in the astaxanthin-producing yeast Xanthophyllomyces dendrorhous. The crtYB gene of X. dendrorhous, encoding a chimeric carotenoid biosynthetic enzyme, could be inactivated by both single and double crossover events, resulting in non-carotenoid-producing transformants. In addition, the crtYB gene, linked to either its homologous or a glyceraldehyde-3-phosphate dehydrogenase promoter, was overexpressed in the wild type and a β-carotene-accumulating mutant of X. dendrorhous. In several transformants containing multiple copies of the crtYB gene, the total carotenoid content was higher than in the control strain. This increase was mainly due to an increase of the β-carotene and echinone content, whereas the total content of astaxanthin was unaffected or even lower. Overexpression of the phytoene synthase-encoding gene (crtI) had a large impact on the ratio between mono- and bicyclic carotenoids. Furthermore, we showed that in metabolic engineered X. dendrorhous strains, the competition between the enzymes phytoene desaturase and lycopene cyclase for lycopene governs the metabolic flux either via β-carotene to astaxanthin or via 3,4-didehydrolycopene to 3-hydroxy-3′-4′-didehydro-β-ψ-caroten-4-one (HDCO). The monocylic carotenoid torulene and HDCO, normally produced as minority carotenoids, were the main carotenoids produced in these strains.     

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.     

An Uncharacterized Apocarotenoid-Derived Signal Generated in ζ-Carotene Desaturase Mutants Regulates Leaf Development and the Expression of Chloroplast and Nuclear Genes in Arabidopsis

In addition to acting as photoprotective compounds, carotenoids also serve as precursors in the biosynthesis of several phytohormones and proposed regulatory signals. Here, we report a signaling process derived from carotenoids that regulates early chloroplast and leaf development. Biosynthesis of the signal depends on ζ-carotene desaturase activity encoded by the ζ-CAROTENE DESATURASE (ZDS)/CHLOROPLAST BIOGENESIS5 (CLB5) gene in Arabidopsis thaliana. Unlike other carotenoid-deficient plants, zds/clb5 mutant alleles display profound alterations in leaf morphology and cellular differentiation as well as altered expression of many plastid- and nucleus-encoded genes. The leaf developmental phenotypes and gene expression alterations of zds/clb5/spc1/pde181 plants are rescued by inhibitors or mutations of phytoene desaturase, demonstrating that phytofluene and/or ζ-carotene are substrates for an unidentified signaling molecule. Our work further demonstrates that this signal is an apocarotenoid whose synthesis requires the activity of the carotenoid cleavage dioxygenase CCD4.     

Accumulation of Trehalose and Sucrose in Cyanobacteria Exposed to Matric Water Stress

The drought-resistant cyanobacteria Phormidium autumnale, strain LPP₄, and a Chroococcidiopsis sp. accumulated trehalose, sucrose, and both trehalose and sucrose, respectively, in response to matric water stress. Accumulated sugar concentrations reached values of up to 6.2 μg of trehalose per μg of chlorophyll in P. autumnale, 6.9 μg of sucrose per μg of chlorophyll in LPP₄, and 4.1 μg of sucrose and 3.2 μg of trehalose per μg of chlorophyll in the Chroococcidiopsis sp. The same sugars were accumulated by these cyanobacteria in similar concentrations under osmotic water stress. Cyanobacteria that did not show drought resistance (Plectonema boryanum and Synechococcus strain PCC 7942) did not accumulate significant amounts of sugars when matric water stress was applied.     

Carotene Hydroxylase Activity Determines the Levels of Both α-Carotene and Total Carotenoids in Orange Carrots

The typically intense carotenoid accumulation in cultivated orange-rooted carrots (Daucus carota) is determined by a high protein abundance of the rate-limiting enzyme for carotenoid biosynthesis, phytoene synthase (PSY), as compared with white-rooted cultivars. However, in contrast to other carotenoid accumulating systems, orange carrots are characterized by unusually high levels of α-carotene in addition to β-carotene. We found similarly increased α-carotene levels in leaves of orange carrots compared with white-rooted cultivars. This has also been observed in the Arabidopsis thaliana lut5 mutant carrying a defective carotene hydroxylase CYP97A3 gene. In fact, overexpression of CYP97A3 in orange carrots restored leaf carotenoid patterns almost to those found in white-rooted cultivars and strongly reduced α-carotene levels in the roots. Unexpectedly, this was accompanied by a 30 to 50% reduction in total root carotenoids and correlated with reduced PSY protein levels while PSY expression was unchanged. This suggests a negative feedback emerging from carotenoid metabolites determining PSY protein levels and, thus, total carotenoid flux. Furthermore, we identified a deficient CYP97A3 allele containing a frame-shift insertion in orange carrots. Association mapping analysis using a large carrot population revealed a significant association of this polymorphism with both α-carotene content and the α-/β-carotene ratio and explained a large proportion of the observed variation in carrots.