beta-carotene and 2-(4-chlorophenylthio)triethylamine

Transgenic Solanum lycopersicum plants expressing an additional copy of the lycopene β-cyclase gene (LCYB) from Nicotiana tabacum, under the control of the Arabidopsis polyubiquitin promoter (UBQ3), have been generated. Expression of LCYB was increased some 10-fold in ripening fruit compared to vegetative tissues. The ripe fruit showed an orange pigmentation, due to increased levels (up to 5-fold) of β-carotene, with negligible changes to other carotenoids, including lycopene. Phenotypic changes in carotenoids were found in vegetative tissues, but levels of biosynthetically related isoprenoids such as tocopherols, ubiquinone and plastoquinone were barely altered. Transformants showed tolerance to the bleaching herbicide β-cyclase inhibitor, 2-(4-chlorophenylthio) triethylamine. The phenotype was inherited for at least three generations.

Topics: beta Carotene; Blotting, Northern; Carotenoids; Ethylamines; Fruit; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Plant; Herbicide Resistance; Intramolecular Lyases; Lycopene; Metabolic Engineering; Molecular Structure; Nicotiana; Plant Leaves; Plant Proteins; Plants, Genetically Modified; Solanum lycopersicum

The new bleaching herbicidal compound N,N-diethyl-N-(2-undecynyl)amine (NDUA) is identified here as an inhibitor of lycopene cyclase and is compared with the known cyclase inhibitors N,N-diethyl-N-[2-(4-chlorophenylthio)ethyl]amine (CPTA) and N,N-diethyl-N-[2-(4-methylphenoxy)ethyl]amine (MTPA). HPLC separation of chloroplast pigments shows lycopene accumulation in NDUA treated tissue. Variation in chain length of the undecynylamine moeity of NDUA from 7 to 21 C atoms reveals an optimum of 11 to 14 C atoms for herbicidal activity. A series of seven further analogues of NDUA and CPTA reveals the structural elements necessary for inhibition of lycopene cyclase. The effect of NDUA derivatives on photosynthesis has been studied in Chlamydomonas reinhardtii. Photosynthesis is highly sensitive, particularly towards the C14 and longer chain length analogues at nanomolar concentrations. It is shown that the breakdown of photosynthesis by NDUA is due to interference with the turnover of the D1 protein of the photosystem II reaction centre that requires the continuous biosynthesis of the two reaction-centre beta-carotene moieties in the reassembly phase. The D1 protein disappearance is most marked under strong light conditions. The depletion of photosystem II occurs before total pigment bleaching. This newly recognized mechanism in herbicidal activity is also the basis for the mode of action of other lycopene cyclase inhibitors as well as phytoene desaturase inhibitors.

Topics: Animals; beta Carotene; Carotenoids; Chlamydomonas reinhardtii; Diethylamines; Ethylamines; Herbicides; Immunoblotting; Intramolecular Lyases; Lycopene; Photosynthesis; Photosynthetic Reaction Center Complex Proteins; Photosystem II Protein Complex; Pigments, Biological; Plant Proteins; Structure-Activity Relationship

The carotenoid composition was investigated during enhanced D1 protein turnover in Chlamydomonas reinhardtii exposed to high light. After 2 h of high light there was no loss of the D1 protein yet. However, the beta-carotene content was significantly reduced. In parallel, an increase of the zeaxanthin content was found, which was higher than can be accounted for by the light-induced de-epoxidation of violaxanthin in the xanthophyll cycle reactions. We therefore assume that beta-carotene of photosystem II (PS II) is hydroxylated to zeaxanthin under high light stress. Inhibitors of carotene biosynthesis led to the loss of both PS II activity and D1 protein, indicating the requirement of beta-carotene synthesis for the reassembly of PS II in high light. Diuron blocked D1 protein as well as beta-carotene turnover. In the presence of chloramphenicol -- which allows just one turnover of the D1 protein -- 15% of the total beta-carotene was lost, calculated to be two beta-carotene.

Topics: Animals; beta Carotene; Chlamydomonas reinhardtii; Chloramphenicol; Diuron; Ethylamines; Light; Photosynthetic Reaction Center Complex Proteins; Photosystem II Protein Complex; Xanthophylls; Zeaxanthins

The existence and the mode of operation of certain enzyme aggregates may be established from the concentrations of intermediates measured in the presence of specific inhibitors. beta-Carotene, the most abundant carotenoid pigment in the fungus Phycomyces blakesleeanus, arises from ring formation at both ends of lycopene. The inhibitors nicotine, imidazole, alpha-picoline, and 2-(4-chlorophenylthio)triethylamine lead to the simultaneous accumulation of lycopene, beta-carotene, and the one-ring intermediate gamma-carotene. The quantitative analytical values obey precise mathematical relationships: those expected from the operation of an enzyme aggregate with two cyclases equally sensitive to the inhibitors. The intermediates lycopene and gamma-carotene rejected by chemically inhibited enzymes may be readmitted to other cyclases in the wild type but not in heterokaryons containing a carA mutation. We have calculated the fraction of inhibited cyclase under each condition, the affinity constant of each inhibitor for the cyclase, and the probability that a rejected intermediate molecule will be readmitted and further metabolized. The probabilities for lycopene and gamma-carotene are identical and independent of the inhibitor responsible for the rejection. Our calculations suggest that each rejected intermediate molecule is readmitted to the enzyme aggregates two or three times on the average.

Topics: beta Carotene; Carotenoids; Ethylamines; Imidazoles; Kinetics; Models, Theoretical; Multienzyme Complexes; Nicotine; Phycomyces; Picolines