beta-carotene and norflurazone

Interactions between β-carotene (β-C) and Chl a turnover were investigated in relation to photoinhibition and D1 protein turnover in mature leaves of Arabidopsis (Arabidopsis thaliana) by ¹⁴CO₂ pulse-chase labeling. Following a 2 h treatment of leaves with water, lincomycin (Linco; an inhibitor of chloroplast protein synthesis) or norflurazon (NF; an inhibitor of carotenoid biosynthesis at phytoene desaturation) in the dark, ¹⁴CO₂ was applied to the leaves for 30 min under control light (CL; 130 μmol photons m⁻² s⁻¹) conditions, followed by exposure to either CL or high light (HL; 1,100 μmol photons m⁻² s⁻¹) in ambient CO₂ for up to 6 h. Under both light conditions, ¹⁴C incorporation was strongly decreased for Chl a and moderately suppressed for β-C in Linco-treated leaves, showing a marked decline of PSII efficiency (F(v)/F(m)) and β-C content compared with water-treated leaves. Partial inhibition of carotenoid biosynthesis by NF caused no or only a minor decrease in F(v)/F(m) and Chl a turnover under both conditions, while the β-C content significantly declined and high ¹⁴C labeling was found for phytoene, the substrate of phytoene desaturase. Together, the results suggest coordinated turnover of Chl a and D1, but somewhat different regulation for β-C turnover, in Arabidopsis leaves. Inhibition of carotenoid biosynthesis by NF may initially enhance metabolic flux in the pathway upstream of phytoene, presumably compensating for short supply of β-C. Our observations are also in line with the notion that HL-induced accumulation of xanthophylls may involve a precursor pool which is distinct from that for β-C turnover.

Topics: Arabidopsis; beta Carotene; Carbon Dioxide; Carotenoids; Chlorophyll; Chlorophyll A; Isotope Labeling; Light; Lincomycin; Photosystem II Protein Complex; Plant Leaves; Pyridazines

The cauliflower (Brassica oleracea L. var. botrytis) Or gene is a rare carotenoid gene mutation that confers a high level of beta-carotene accumulation in various tissues of the plant, turning them orange. To investigate the biochemical basis of Or-induced carotenogenesis, we examined the carotenoid biosynthesis by evaluating phytoene accumulation in the presence of norflurazon, an effective inhibitor of phytoene desaturase. Calli were generated from young seedlings of wild type and Or mutant plants. While the calli derived from wild type seedlings showed a pale green color, the calli derived from Or seedlings exhibited intense orange color, showing the Or mutant phenotype. Concomitantly, the Or calli accumulated significantly more carotenoids than the wild type controls. Upon treatment with norflurazon, both the wild type and Or calli synthesized significant amounts of phytoene. The phytoene accumulated at comparable levels and no major differences in carotenogenic gene expression were observed between the wild type and Or calli. These results suggest that Or-induced beta-carotene accumulation does not result from an increased capacity of carotenoid biosynthesis.

Topics: beta Carotene; Brassica; Carotenoids; Gene Expression; Herbicides; Plant Structures; Pyridazines; Reverse Transcriptase Polymerase Chain Reaction; Tissue Culture Techniques

Haematococcus pluvialis was mutated by UV or ethyl methanesulphonate. Mutants resistant to nicotine, diphenylamine, fluridone or norflurazon were then selected. Several nicotine-resistant mutants showed increased (1.9% to 2.5% vs. 1.2% w/w) astaxanthin production. Mutants maintained high astaxanthin production over 4 months of repeated culture.

Topics: beta Carotene; Biotechnology; Chlorophyta; Diphenylamine; Ethyl Methanesulfonate; Mutagenesis; Mutation; Nicotine; Pyridazines; Pyridones; Ultraviolet Rays; Xanthophylls

The carotenoid biosynthetic pathway in algae and plants takes place within plastids. In these organelles, carotenoids occur either in a free form or bound to proteins. Under stress, the unicellular green alga Haematococcus pluvialis accumulates secondary carotenoids, mainly astaxanthin esters, in cytoplasmic lipid vesicles up to 4% of its dry mass. It is therefore one of the favored organisms for the biotechnological production of these antioxidative compounds. We have studied the cellular localization and regulation of the enzyme beta-carotene oxygenase in H. pluvialis that catalyzes the introduction of keto functions at position C-4 of the beta-ionone ring of beta-carotene and zeaxanthin. Using immunogold labeling of ultrathin sections and Western blot analysis of cell fractions, we discovered that under inductive conditions, beta-carotene oxygenase was localized both in the chloroplast and in the cytoplasmic lipid vesicles, which are (according to their lipid composition) derived from cytoplasmic membranes. However, beta-carotene oxygenase activity was confined to the lipid vesicle compartment. Because an early carotenogenic enzyme in the pathway, phytoene desaturase, was found only in the chloroplast (Grünewald, K., Eckert, M., Hirschberg, J., and Hagen, C. (2000) Plant Physiol. 122, 1261-1268), a transport of intermediates from the site of early biosynthetic steps in the chloroplast to the site of oxygenation and accumulation in cytoplasmic lipid vesicles is proposed.

Topics: Antioxidants; beta Carotene; Cell Compartmentation; Chlorophyta; Cytoplasm; Diphenylamine; Industrial Microbiology; Oxygenases; Plastids; Pyridazines; Xanthophylls

Recently, we reported the presence of the violaxanthin-antheraxanthin-zeaxanthin cycle in diatoms, and showed that violaxanthin is the putative precursor of both diadinoxanthin and fucoxanthin in the diatom Phaeodactylum tricornutum Bohlin (M. Lohr and C. Wilhelm, 1999, Proc. Natl. Acad. Sci. USA 96: 8784-8789). In the present study, two possible intermediates in the synthesis of violaxanthin from beta-carotene were identified in P. tricornutum, namely beta-cryptoxanthin and beta-cryptoxanthin epoxide. In low light, the latter pigment prevails, but in high light beta-cryptoxanthin accumulates, probably as the result of an increased activity of the xantophyll-cycle de-epoxidase. The apparent kinetics of several xanthophyll conversion steps were determined for P. tricornutum and Cyclotella meneghiniana Kuitzing. The experimentally determined conversion rates were used to evaluate the hypothetical pathway of xanthophyll synthesis in diatoms. For this purpose a mathematical model was developed which allows the calculation of theoretical rates of pigment conversion for microalgae under steady-state growth conditions. A comparison between measured and calculated conversion rates agreed well with the proposal of a sequential synthesis of fucoxanthin via violaxanthin and diadinoxanthin. The postulation of zeaxanthin as an obligatory intermediate in the synthesis of violaxanthin, however, resulted in large discrepancies between the measured and calculated rates of its epoxidation. Instead of zeaxanthin, beta-cryptoxanthin epoxide may be involved in the biosynthesis of violaxanthin in diatoms.

Topics: Antioxidants; beta Carotene; Carotenoids; Chromatography, High Pressure Liquid; Cryptoxanthins; Diatoms; Dithioerythritol; Epoxy Compounds; Herbicides; Light; Lutein; Models, Biological; Pigments, Biological; Pyridazines; Sulfhydryl Reagents; Xanthophylls; Zeaxanthins

Astaxanthin accumulation by green microalgae is a natural phenomenon known as red snows and blood rains. The fact that astaxanthin synthesis requires oxygen, NADPH and Fe(2+) led Cunningham and Gantt [Annu. Rev. Plant Physiol. Plant Mol. Biol. 49 (1998) 557-583] to propose that a cytochrome P450-dependent enzyme might be involved in the transformation of beta-carotene to astaxanthin. In Haematococcus only esterified astaxanthin molecules accumulate, but it is not determined whether a fatty acid synthesis should occur simultaneously to allow pigment accumulation. The aim of this contribution was to answer these two questions using specific inhibitors of beta-carotene (norflurazon) and fatty acid (cerulenin) synthesis, and of cytochrome P450 enzyme activity (ellipticine).

Topics: beta Carotene; Cerulenin; Chlorophyta; Cytochrome P-450 Enzyme System; Ellipticines; Enzyme Inhibitors; Fatty Acids; Light; Mixed Function Oxygenases; Pyridazines; Xanthophylls; Zeaxanthins

Inhibitors of the phytoene desaturase in carotene biosynthesis were tested in the enhanced rapid turnover of the D1 protein of photosystem II in high light exposure of Chlamydomonas reinhardtii cells. After 1 h high light on heterotrophically grown cells in the presence of norflurazon or fluridone, photosynthesis activity in vivo and PS II activity in vitro is lost. The D1 protein has disappeared. PS I activity is not affected, nor is the D2 protein. It is concluded that beta-carotene is essential for the assembly of the D1 protein into functional photosystem II. It is suspected that bleaching of beta-carotene in the reaction center of PS II by high light destabilizes the structure and triggers the degradation of the D1 protein.

Topics: Animals; beta Carotene; Chlamydomonas reinhardtii; Chlorophyll; Light; Light-Harvesting Protein Complexes; Oxidoreductases; Photosynthesis; Photosynthetic Reaction Center Complex Proteins; Photosystem II Protein Complex; Pyridazines; Pyridones

The photoreceptor that mediates blue-light-induced phototropism in dark-grown seedlings of higher plants has not been identified, although the carotenoid zeaxanthin has recently been proposed as the putative chromophore. In the experiments described in this paper, we analyzed phototropism and a blue-light-induced protein phosphorylation that has been genetically and physiologically implicated in phototropism in wild-type maize (Zea mays L.) seedlings and compared the results with those from seedlings that are either carotenoid deficient through a genetic lesion or have been chemically treated to block carotenoid biosynthesis. The blue-light-dependent phototropism and phosphorylation responses of seedlings deficient in carotenoids are the same as those of seedlings containing normal levels of carotenoids. These results and those in the literature make it unlikely that zeaxanthin or any other carotenoid is the chromophore of the blue-light photoreceptor for phototropism or the blue-light-induced phosphorylation related to phototropism.

Topics: beta Carotene; Carotenoids; Cotyledon; Darkness; Herbicides; Light; Phosphorylation; Photoreceptor Cells; Phototropism; Pyridazines; Xanthophylls; Zea mays; Zeaxanthins

Among the enzymes involved in carotenoid biosynthesis, phytoene desaturase is considered to be a rate-limiting enzyme in this pathway and is also the target of many bleaching herbicides. This enzyme shows diversity concerning its function and amino acid homology among various organisms. The phytoene desaturase gene crtl of Erwinia uredovora was expressed, the 5'-region of which was fused to the sequence for the transit peptide of a pea Rubisco small subunit, in tobacco plants under the control of the CaMV 35S promoter. This chimeric gene product was targeted into chloroplasts and processed in the transgenic plants. The production and processing of the corresponding protein could be demonstrated by Western blotting. Immunogold localization showed that the location of the gene product Crtl was preferentially in the thylakoids. A radioactive labeling study using the leaves demonstrated enhanced activity for the synthesis of beta-carotene. In addition, the transgenic tobacco acquired elevated resistance to the bleaching herbicide norflurazon.

Topics: Amino Acid Sequence; Base Sequence; beta Carotene; Carotenoids; Chloroplasts; Drug Resistance; Erwinia; Genes, Bacterial; Herbicides; Immunohistochemistry; Intracellular Membranes; Molecular Sequence Data; Nicotiana; Oxidoreductases; Plants, Genetically Modified; Plants, Toxic; Protein Precursors; Protein Sorting Signals; Pyridazines; Recombinant Fusion Proteins; Ribulose-Bisphosphate Carboxylase