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.     

Interference in Carotenogenesis as a Mechanism of Action of the Pyridazinone Herbicide Sandoz 6706: Accumulation of C-40 Carotenoid Precursors Inhibition of beta-Carotene Synthesis and Enhancement of Phytoene Epoxidation

The herbicide Sandoz 6706 (4-chloro-5-(dimethylamino)-2-α,α,α, (trifluoro-m-tolyl)-3(2H)-pyridazinone), when applied as a preplant soil treatment at a concentration of 0.05 μg/g reduced the content of β-carotene and chlorophylls in 21-day-old wheat seedlings (Triticum aestivum L.) by 55% and 29%, respectively, without affecting the fresh or dry matter of the seedlings. At 0.8 μg/g, the herbicide reduced the content of β-carotene and chlorophyll by as much as 98%, while the fresh weight of the albino seedlings was reduced by only 24%. The effect of the herbicide on chlorophyll b was much stronger than on chlorophyll a. Time course studies of pigment synthesis in Sandoz 6706-treated seedlings showed that chlorophyll, β-carotene, cyclic xanthophylls, phytoene, phytofluene, and ζ-carotene were accumulating during the first 7 days after sowing. Later on, there was a sharp decline in the content of chlorophyll and β-carotene and a gradual reduction in the content of phytofluene, ζ-carotene, and cyclic xanthophylls; the content of phytoene remained essentially unchanged. Coinciding with the drop in content of β-carotene and chlorophyll, there was a remarkable increase in the content of epoxy phytoene. It is suggested that Sandoz 6706 might act as an inhibitor of the cyclization reaction in the biosynthetic pathway of carotenoids and that other effects, such as the bleaching of chlorophyll, are a consequence of this inhibition.     

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.     

Mode of Action of the Massively Accumulated beta-Carotene of Dunaliella bardawil in Protecting the Alga against Damage by Excess Irradiation

When grown under defined conditions Dunaliella bardawil accumulates a high concentration of β-carotene, which is composed primarily of two isomers, all-trans and 9-cis β-carotene. The high β-carotene alga is substantially resistant to photoinhibition of photosynthetic oxygen evolution when compared with low β-carotene D. bardawil or with Dunaliella salina which is incapable of accumulating β-carotene. Protection against photoinhibition in the high β-carotene D. bardawil is very strong when blue light is used as the photoinhibitory agent, intermediate with white light, and nonexistent with red light. These observations suggest that the massively accumulated β-carotene in D. bardawil protects the alga against damage by high irradiation by screening through absorption of the blue region of the spectrum. Irradiation of D. bardawil by high intensity blue light results in the following temporal sequence of events: photoinhibition of oxygen evolution, photodestruction of 9-cis β-carotene, photodestruction of all-trans β-carotene, photodestruction of chlorophyll and cell death.     

Selection of Astaxanthin-Overproducing Mutants of Phaffia rhodozyma with β-Ionone

β-Ionone, an end ring analog of β-carotene, inhibits astaxanthin production in the red yeast Phaffia rhodozyma. Astaxanthin-overproducing mutants of this yeast are easily spotted on β-ionone-containing yeast malt agar plates. β-Ionone appears to block astaxanthin synthesis at the β-carotene level.     

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.     

The Inhibition of Macrophage Foam Cell Formation by 9-Cis β-Carotene Is Driven by BCMO1 Activity

Atherosclerosis is a major cause of morbidity and mortality in developed societies, and begins when activated endothelial cells recruit monocytes and T-cells from the bloodstream into the arterial wall. Macrophages that accumulate cholesterol and other fatty materials are transformed into foam cells. Several epidemiological studies have demonstrated that a diet rich in carotenoids is associated with a reduced risk of heart disease; while previous work in our laboratory has shown that the 9-cis β-carotene rich alga Dunaliella inhibits atherogenesis in mice. The effect of 9-cis β-carotene on macrophage foam cell formation has not yet been investigated. In the present work, we sought to study whether the 9-cis β-carotene isomer, isolated from the alga Dunaliella, can inhibit macrophage foam cell formation upon its conversion to retinoids. The 9-cis β-carotene and Dunaliella lipid extract inhibited foam cell formation in the RAW264.7 cell line, similar to 9-cis retinoic acid. Furthermore, dietary enrichment with the algal powder in mice resulted in carotenoid accumulation in the peritoneal macrophages and in the inhibition of foam cell formation ex-vivo and in-vivo. We also found that the β-carotene cleavage enzyme β-carotene 15,15’-monooxygenase (BCMO1) is expressed and active in macrophages. Finally, 9-cis β-carotene, as well as the Dunaliella extract, activated the nuclear receptor RXR in hepa1-6 cells. These results indicate that dietary carotenoids, such as 9-cis β-carotene, accumulate in macrophages and can be locally cleaved by endogenous BCMO1 to form 9-cis retinoic acid and other retinoids. Subsequently, these retinoids activate the nuclear receptor RXR that, along with additional nuclear receptors, can affect various metabolic pathways, including those involved in foam cell formation and atherosclerosis.     

Effect of gamma-Irradiation on the Biosynthesis of Carotenoids in the Tomato Fruit

Fruits of the lutescent tomato genetic line were exposed to γ-radiation at different stages of maturity to determine the effect of ionizing radiation on carotenoid synthesis in the ripening fruit. Irradiation generally resulted in the inhibition of carotenogenesis. The effect was more pronounced at the higher dosage and in less mature fruit. Lycopene synthesis was inhibited more extensively than β-carotene synthesis. The total carotenoid content was also generally lower in irradiated fruits. It was proposed that the β-carotene in the tomato fruit is formed by a pathway not involving lycopene.     

Evaluation of Structurally Different Carotenoids in Escherichia coli Transformants as Protectants against UV-B Radiation

Escherichia coli cells transformed with several carotenogenic genes to mediate the formation of ζ-carotene, neurosporene, lycopene, β-carotene, and zeaxanthin were exposed to UV-B radiation. Short-term kinetics revealed that endogenous levels of neurosporene and β-carotene protected E. coli against irradiation with UV-B. Zeaxanthin protected against only the photosensitized UV-B treatment. All other carotenoids were ineffective.     

Impact of the microalga Dunaliella salina (Dunal) Teodoresco culture and its β-carotene extract on the development of salt-stressed squash (Cucurbita pepo L. cv. Mabrouka)

Salinity is a major threat to crop production and global food security. Algae and their extracts containing bioactive compounds can enhance the salt tolerance of plants, including the salt-sensitive plants. The current study evaluated the efficacy of Dunaliella salina (Dunal) Teodoresco culture and/or its β-carotene extract in improving the salt tolerance of squash (Cucurbita pepo L. cv. Mabrouka). Amendment of C. pepo with D. salina culture and/or its β-carotene extract was more effective in alleviating the impact of moderate salinity imposed by seawater dilution of 2.5 dS m⁻¹ than either low (0.55 dS m⁻¹) or high (3.5 dS m⁻¹) salinity, with a comparable effect to that of salicylic acid (SA). Plants that received a combination of D. salina culture and its β-carotene extract showed significantly higher growth (total biomass, fruit productivity) and physiological attributes (photosynthetic pigments, nitrogen (N), phosphorus (P), and potassium (K⁺) contents) than those receiving either amendment alone, reaching up to 80–90% of the SA-treated plants at moderate salinity (2.5 dS m⁻¹). The combination could enhance the antioxidant activity of moderately salt-stressed C. pepo via increasing carotenoids and phenolics contents, suggesting that this combination could enhance the adaptation of C. pepo to the moderate salinity. The present study recommends using the blooms of D. salina and its β-carotene that is naturally secreted in situ in natural or synthetic open systems in improving the salt tolerance of C. pepo instead of using the expensive synthetic hormones.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12298-022-01176-6.     

Controls on chlorophyll synthesis in barley

In 7- to 10-day-old leaves of etiolated barley (Hordeum vulgare), all of the enzymes that convert δ-aminolevulinic acid to chlorophyll are nonlimiting during the first 6 to 12 hours of illumination, even in the presence of inhibitors of protein synthesis. The limiting activity for chlorophyll synthesis appears to be a protein (or proteins) related to the synthesis of δ-aminolevulinic acid, presumably δ-aminolevulinic acid synthetase. Protein synthesis in both the cytosol and plastids may be required to produce nonlimiting amounts of δ-aminolevulinic acid. The half-life of a limiting protein controlling the synthesis of δ-aminolevulinic acid appears to be about 1½ hours, when determined with inhibitors of protein synthesis. Acceleration of chlorophyll synthesis by light is not inhibited by inhibitors of nucleic acid synthesis, but is inhibited by inhibitors of protein synthesis. A model for control of chlorophyll synthesis is proposed, based on a light-induced activation at the translational level of the synthesis of proteins forming δ-aminolevulinic acid, as well as the short half-life of these proteins. Evidence is presented confirming the idea that the holochrome on which protochlorophyllide is photoreduced to chlorophyllide functions enzymatically.     

Conversion of carotenoids into vitamins A(1) and A(2) in two species of freshwater fish

1. Examination of two zooplankton species predominating in fish ponds, Daphnia magna and Chironomus larvae, revealed the presence of α- and β-carotene, echinenone, canthaxanthin and 3-hydroxy-4-oxo-β-carotene in Daphnia, and β-carotene and cryptoxanthin ester in Chironomus. No specific provitamins A₂ (containing a 3,4-dehydro-β-ionone ring) were detected. 2. Guppies (Lebistes reticulatus) and platies (Xiphophorus variatus) were found to form vitamin A from β-carotene and from its oxygen-containing derivatives isozeaxanthin, canthaxanthin and astaxanthin. Slight conversion into vitamin A₂ seemed to occur simultaneously. 3,4-Dehydro-3′-hydroxy-β-carotene formed little vitamin A, and the latter was mainly of the A₂ type. Lutein was devoid of provitamin A properties. 3. In addition to vitamin A, β-carotene was detected in fish receiving the 4-oxo- and 4-hydroxy-carotenoids. A reaction scheme for the conversion of carotenoids into retinal and and 3,4-dehydroretinal is presented. 4. It is concluded that natural 4-oxo derivatives of β-carotene may play a significant role as vitamin A precursors for fish.     

β-Carotene Attenuates Angiotensin II-Induced Aortic Aneurysm by Alleviating Macrophage Recruitment in Apoe?/? Mice

Abdominal aortic aneurysm (AAA) is a common chronic degenerative disease characterized by progressive aortic dilation and rupture. The mechanisms underlying the role of α-tocopherol and β-carotene on AAA have not been comprehensively assessed. We investigated if α-tocopherol and β-carotene supplementation could attenuate AAA, and studied the underlying mechanisms utilized by the antioxidants to alleviate AAA. Four-months-old Apoe^−/− mice were used in the induction of aneurysm by infusion of angiotensin II (Ang II), and were orally administered with α-tocopherol and β-carotene enriched diet for 60 days. Significant increase of LDL, cholesterol, triglycerides and circulating inflammatory cells was observed in the Ang II-treated animals, and gene expression studies showed that ICAM-1, VCAM-1, MCP-1, M-CSF, MMP-2, MMP-9 and MMP-12 were upregulated in the aorta of aneurysm-induced mice. Extensive plaques, aneurysm and diffusion of inflammatory cells into the tunica intima were also noticed. The size of aorta was significantly (P = 0.0002) increased (2.24±0.20 mm) in the aneurysm-induced animals as compared to control mice (1.17±0.06 mm). Interestingly, β-carotene dramatically controlled the diffusion of macrophages into the aortic tunica intima, and circulation. It also dissolved the formation of atheromatous plaque. Further, β-carotene significantly decreased the aortic diameter (1.33±0.12 mm) in the aneurysm-induced mice (β-carotene, P = 0.0002). It also downregulated ICAM-1, VCAM-1, MCP-1, M-CSF, MMP-2, MMP-9, MMP-12, PPAR-α and PPAR-γ following treatment. Hence, dietary supplementation of β-carotene may have a protective function against Ang II-induced AAA by ameliorating macrophage recruitment in Apoe^−/− mice.     

Photoinduced Carotenogenesis in Chlorotic Euglena gracilis

Light induces β-carotene synthesis in streptomycin-bleached Euglena gracilis Z. Light-adapted, chemostat-grown cells have up to 10-fold as much β-carotene and 25% more protein than similarly grown, dark-adapted cells. Carotenogenesis does not occur under anaerobic conditions or in the presence of diphenylamine, cyanide, or cycloheximide. The blue portion of the spectrum (360-560 nm) is most active in initiating carotenogenesis. The level of cellular carotenoids is influenced by the type of carbon source and to some degree by pH. Phytofluence and ζ-carotene are present in dark-grown cells but not in cells grown aerobically in white light (360-1120 nm). These pigments, however, were present in cells grown in yellow or green light (above 486 nm) or in cells exposed to white light anaerobically. The carotenoids are localized in two types of structures at the light microscope level. A protoporphyrin was isolated from Euglena, and its role as a possible photoreceptor during carotenogenesis is suggested.     

Genetic Modification of the Soybean to Enhance the β-Carotene Content through Seed-Specific Expression

The carotenoid biosynthetic pathway was genetically manipulated using the recombinant PAC (Phytoene synthase-2A-Carotene desaturase) gene in Korean soybean (Glycine max L. cv. Kwangan). The PAC gene was linked to either the β-conglycinin (β) or CaMV-35S (35S) promoter to generate β-PAC and 35S-PAC constructs, respectively. A total of 37 transgenic lines (19 for β-PAC and 18 for 35S-PAC) were obtained through Agrobacterium-mediated transformation using the modified half-seed method. The multi-copy insertion of the transgene was determined by genomic Southern blot analysis. Four lines for β-PAC were selected by visual inspection to confirm an orange endosperm, which was not found in the seeds of the 35S-PAC lines. The strong expression of PAC gene was detected in the seeds of the β-PAC lines and in the leaves of the 35S-PAC lines by RT-PCR and qRT-PCR analyses, suggesting that these two different promoters function distinctively. HPLC analysis of the seeds and leaves of the T₂ generation plants revealed that the best line among the β-PAC transgenic seeds accumulated 146 µg/g of total carotenoids (approximately 62-fold higher than non-transgenic seeds), of which 112 µg/g (77%) was β-carotene. In contrast, the level and composition of the leaf carotenoids showed little difference between transgenic and non-transgenic soybean plants. We have therefore demonstrated the production of a high β-carotene soybean through the seed-specific overexpression of two carotenoid biosynthetic genes, Capsicum phytoene synthase and Pantoea carotene desaturase. This nutritional enhancement of soybean seeds through the elevation of the provitamin A content to produce biofortified food may have practical health benefits in the future in both humans and livestock.     

CAROTENOID CLEAVAGE DIOXYGENASE4 Is a Negative Regulator of β-Carotene Content in Arabidopsis Seeds

Experimental approaches targeting carotenoid biosynthetic enzymes have successfully increased the seed β-carotene content of crops. However, linkage analysis of seed carotenoids in Arabidopsis thaliana recombinant inbred populations showed that only 21% of quantitative trait loci, including those for β-carotene, encode carotenoid biosynthetic enzymes in their intervals. Thus, numerous loci remain uncharacterized and underutilized in biofortification approaches. Linkage mapping and genome-wide association studies of Arabidopsis seed carotenoids identified CAROTENOID CLEAVAGE DIOXYGENASE4 (CCD4) as a major negative regulator of seed carotenoid content, especially β-carotene. Loss of CCD4 function did not affect carotenoid homeostasis during seed development but greatly reduced carotenoid degradation during seed desiccation, increasing β-carotene content 8.4-fold relative to the wild type. Allelic complementation of a ccd4 null mutant demonstrated that single-nucleotide polymorphisms and insertions and deletions at the locus affect dry seed carotenoid content, due at least partly to differences in CCD4 expression. CCD4 also plays a major role in carotenoid turnover during dark-induced leaf senescence, with β-carotene accumulation again most strongly affected in the ccd4 mutant. These results demonstrate that CCD4 plays a major role in β-carotene degradation in drying seeds and senescing leaves and suggest that CCD4 orthologs would be promising targets for stabilizing and increasing the level of provitamin A carotenoids in seeds of major food crops.     

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.