LOW-TEMPERATURE-INDUCED SYNTHESIS OF α-CAROTENE IN THE MICROALGA DUNALIELLA SALINA (CHLOROPHYTA)

The microalga Dunaliella salina (Teo.) is well known as an accumulator of β-carotene (β,β-carotene) when subjected to growth-limiting conditions (e.g. exposure to high irradiances). In addition, the carotenoid α-carotene (β,ε-carotene) may also be synthesized and subsequently accumulated by this alga under specific growth conditions. The main factor in stimulating the synthesis of this carotene was determined to be exposure to lower than optimum temperatures for algal growth. A 7.5-fold increase in the levels of α-carotene was observed when the temperature was decreased from 34 to 17° C, whilst levels of β-carotene were unaltered. The accumulation of α-carotene was unaffected by irradiance, although its isomeric composition was greatly altered by light levels. The proportion of 9- cis α-carotene increased from 15% to 45% of total α-carotene when the irradiance was decreased from 260 to 50 μmol·m⁻²·s⁻¹. Exposure to higher irradiances had little influence on the isomeric composition of this carotenoid. A reduction in growth temperature did not influence the isomeric composition of α-carotene. Nutrient status (nitrogen and phosphate) had no effect on either the content or isomeric composition of α-carotene accumulated by D. salina.     

Carotenoids and carotenoid metabolism in Carcinus maenas (Crustacea: Decapoda)

The carotenoid pigments of the hepatopancreas, ovaries and epidermis of Carcinus maenas were investigated. The following pigments were identified: β-carotene, δ-carotene, echinenone, isocryptoxanthin, canthaxanthin, lutein, zeaxanthin, flavoxanthin and astacene.
The relative abundance of these pigments in the three tissues and the presence of possible hydroxy and keto intermediates suggest the metabolism of astaxanthin from β-carotene. The metabolic pathway in Carcinus is discussed in relation to recent studies on other invertebrates.     

Carotenoids and carotenoid metabolism in Carcinus maenas (Crustacea: Decapoda)

The carotenoid pigments of the hepatopancreas, ovaries and epidermis of Carcinus maenas were investigated. The following pigments were identified: β-carotene, δ-carotene, echinenone, isocryptoxanthin, canthaxanthin, lutein, zeaxanthin, flavoxanthin and astacene.
The relative abundance of these pigments in the three tissues and the presence of possible hydroxy and keto intermediates suggest the metabolism of astaxanthin from β-carotene. The metabolic pathway in Carcinus is discussed in relation to recent studies on other invertebrates.     

Lipid peroxidation in and photo-damage to a light-sensitive chlorescence pea mutant

Leaves of 10- to 12-day-old chlorescence lethal Pisum sativum L. mutant are similar to control plants with respect to the content of chlorophylls, carotenoids, fatty acids and α-tocopherol. Subsequent development of the mutant under high irradiation resulted in th destruction of the photosynthetic pigments, polyunsaturated fatty acids, α-tocopherol, and also in the accumulation of liposoluble fluorescent products. No increase in the level of malondialdehyde was observed. In chloroplasts isolated from mutant plants the contents of chlorophyll a and β-carotene were decreased to a greater extent than the more oxidized pigments (xanthophylls and chlorophyll b ). The data obtained are discussed with special reference to the role of lipid peroxidation in the injury of plant cells under the action of visible light and to the antioxidative mechanisms stabilizing photosynthetic membranes.     

Tissue-specific accumulation of carotenoids in carrot roots

Raman spectroscopy can be used for sensitive detection of carotenoids in living tissue and Raman mapping provides further information about their spatial distribution in the measured plant sample. In this work, the relative content and distribution of the main carrot (Daucus carota L.) root carotenoids, α-, β-carotene, lutein and lycopene were assessed using near-infrared Fourier transform Raman spectroscopy. The pigments were measured simultaneously in situ in root sections without any preliminary sample preparation. The Raman spectra obtained from carrots of different origin and root colour had intensive bands of carotenoids that could be assigned to β-carotene (1,520 cm⁻¹), lycopene (1,510 cm⁻¹) and α-carotene/lutein (1,527 cm⁻¹). The Raman mapping technique revealed detailed information regarding the relative content and distribution of these carotenoids. The level of β-carotene was heterogeneous across root sections of orange, yellow, red and purple roots, and in the secondary phloem increased gradually from periderm towards the core, but declined fast in cells close to the vascular cambium. α-carotene/lutein were deposited in younger cells with a higher rate than β-carotene while lycopene in red carrots accumulated throughout the whole secondary phloem at the same level. The results indicate developmental regulation of carotenoid genes in carrot root and that Raman spectroscopy can supply essential information on carotenogenesis useful for molecular investigations on gene expression and regulation.     

Molecular evolution of carotenoid biosynthesis from bacteria to plants

β-Carotene and derivatives are important pigments in plant photosynthesis. They are found not only in green plants but also accumulate in archea, prokaryotes and fungi. For β -carotene biosynthesis, enzymes are necessary to catalyse the formation of phytoene, several desaturation steps and cyclization reactions. This review is focused on the molecular phylogeny of the enzymes, the genes involved and their diversity. It outlines how genes and enzymes from prokaryotes and archea were modified to give rise to the corresponding plant constituents. In the cases of phytoene synthase, a direct line of evolution can be drawn. For other carotenogenic enzymes, new genes and enzymes have been acquired at certain stages of evolution. In addition, phytoene desaturases and lycopene cyclases are examples of convergent evolution of different types of enzymes, which are structurally completely unrelated but functionally identical. Finally, several gene duplications led to homologous enzymes with different catalytic functions including those involved in the synthesis of α -carotene.     

High-performance liquid chromatographic analysis of α-keto acids produced from amino acid metabolism in oral Bacteroides

Profiles of metabolic α-keto acids were determined by a high-performance liquid chromatographic method and applied to characterization of oral black-pigmented Bacteroides . Each bacterial strain was incubated with amino acids in a chemically defined medium. After production α-keto acids were purified by hydrazide gel column treatment and converted to u.v.-absorbing derivatives. They were analysed by reversed-phase ion-pair chromatography. Black-pigmented Bacteroides species were differentiated into two groups according to production of aromatic α-keto acids. Bacteroides gingivalis, B. endodontalis and B. loescheii produced both ρ-hydroxyphenylpyruvic and phenylpyruvic acids. However, no such α-keto acids were produced by B. levii, B. intermedius and B. denticola . In addition, production profiles of several aliphatic α-keto acids (α-ketoglutaric, pyruvic, α-ketobutyric, α-ketoisovaleric, α-ketoisocaproic, and α-keto-β-methylvaleric acids) separated each individual species in such groups. The present study offers useful chemotaxonomic information on amino acid metabolic activity of oral black-pigmented Bacteroides species.     

Irradiance levels affect growth parameters and carotenoid pigments in kale and spinach grown in a controlled environment

Carotenoids play critical roles in both light harvesting and energy dissipation for the protection of photosynthetic structures. However, limited research is available on the impact of irradiance on the production of secondary plant compounds, such as carotenoid pigments. Kale ( Brassica oleracea L.) and spinach ( Spinacia oleracea L.) are two leafy vegetables high in lutein and β-carotene carotenoids. The objectives of this study were to determine the effects of different irradiance levels on tissue biomass, elemental nutrient concentrations, and lutein β-carotene and chlorophyll (chl) pigment accumulation in the leaves of kale and spinach. 'Winterbor' kale and 'Melody' spinach were grown in nutrient solution culture in growth chambers at average irradiance levels of 125, 200, 335, 460, and 620 μmol m⁻² s⁻¹. Highest tissue lutein β-carotene and chls occurred at 335 μmol m⁻² s⁻¹ for kale, and 200 μmol m⁻² s⁻¹ for spinach. The accumulations of lutein and β-carotene were significantly different among irradiance levels for kale, but were not significantly different for spinach. However, lutein and β-carotene accumulation was significant for spinach when computed on a dry mass basis. Identifying effects of irradiance on carotenoid accumulation in kale and spinach is important information for growers producing these crops for dry capsule supplements and fresh markets.     

UV effects on photosynthesis and phycobiliprotein composition in the flagellate Cyanophora paradoxa

Abstract The effects of solar and artificial ultraviolet radiation on photosynthetic oxygen production and phycobiliprotein composition were investigated in the freshwater flagellate, Cyanophora paradoxa . The phycobiliproteins of the cell acting as photosynthetic accessory pigments were found to be readily affected by even short exposure to ultraviolet radiation, while the membrane-bound chlorophyll protein complexes were hardly impaired as demonstrated by SDS polyacrylamide gel electrophoresis, isoelectric focussing and fast protein liquid chromatography (FPLC). The phycobilisomes are dissembled from the outside inwards and go from higher molecular weight components to hexamers ( αβ )₆, trimers ( αβ )₃ and finally monomers (αβ). Fluorescence spectra indicated that the energy transfer from accessory pigments to the photosystems was impaired by ultraviolet radiation. Photosynthetic oxygen production was affected on a much faster timescale than changes in the absorption spectra or at the protein level.     

UV effects on photosynthesis and phycobiliprotein composition in the flagellate Cyanophora paradoxa

Abstract The effects of solar and artificial ultraviolet radiation on photosynthetic oxygen production and phycobiliprotein composition were investigated in the freshwater flagellate, Cyanophora paradoxa . The phycobiliproteins of the cell acting as photosynthetic accessory pigments were found to be readily affected by even short exposure to ultraviolet radiation, while the membrane-bound chlorophyll protein complexes were hardly impaired as demonstrated by SDS polyacrylamide gel electrophoresis, isoelectric focussing and fast protein liquid chromatography (FPLC). The phycobilisomes are dissembled from the outside inwards and go from higher molecular weight components to hexamers (αβ)₆, trimers (αβ)₃ and finally monomers (αβ). Fluorescence spectra indicated that the energy transfer from accessory pigments to the photosystems was impaired by ultraviolet radiation. Photosynthetic oxygen production was affected on a much faster timescale than changes in the absorption spectra or at the protein level.     

Expression in Escherichia coli and properties of the carotene ketolase from Haematococcus pluvialis

Abstract High level expression of the functional β-carotene ketolase gene bkt from Haematococcus pluvialis occurred in Escherichia coli transformants producing β-carotene or zeaxanthin as a result of the presence of additional carotenoid genes from Erwinia uredovora . Requirement of molecular oxygen for the insertion of the keto group was demonstrated. The final product of this two-step ketolase reaction from β-carotene is canthaxanthin (4,4'-diketo-β-carotene) with the 4-monoketo derivative echinenone as an intermediate. A reaction sequence for the formation of astaxanthin from β-carotene was established based on kinetic data on astaxanthin formation in E. coli transformants carrying the hydroxylase gene crtZ from Erwinia along with bkt . We conclude that the carotenoids zeaxanthin and adonixanthin which accumulate in addition to astaxanthin in this transformant are products of side reactions rather than direct precursors of astaxanthin. The possible mechanisms for the formation of the keto derivatives are discussed.     

Response of senescing cotyledons of clusterbean to water stress in moderate and low light: Possible photoprotective role of beta-carotene

Senescence of clusterbean ( Cyamopsis tetragonoloba L.) cotyledons in moderate light (12 W m⁻²) brings about a loss in the pigments, enhanced lipid peroxidation and a decline in PS II photochemical activity without any loss either in Dl protein or in the level of β -carotene. The senescence syndrome is aggravated in the cotyledons of water-stressed seedlings with an increase in thylakoid lipid peroxidation, a decline in the level of β -carotene and a quantitative loss in the Dl protein. Loss of the protein, however, is arrested in the seedlings experiencing water stress at low light (3 W m⁻²) intensity that correlates with the stability in the level of β - carotene and a slow rate of lipid peroxidation. Loss of the protein in moderate light is attributed to water-stress sensitized photoinhibitory damage. The data on changes in the components of xanthophyll cycle suggest the low activity of the cycle both during senescence and water stress. It is, therefore, concluded that β -carotene may contribute to the assembly and stability of the Dl protein during senescence and water stress in clusterbean cotyledons.     

Leaf senescence in a non-yellowing mutant of Festuca pratensis: Degradation of carotenoids

The loss of pigments was assessed in detached leaves of Festuca pratensis Huds. kept in permanent darkness. Two genotypes, a normal yellowing cultivar Rossa and a non-yellowing mutant Bf 993 were compared with each other. Analysis of individual pigments, chlorophylls. β-carotene, lutein, violaxanthin and neoxanthin was performed using HPLC. In the non-yellowing genotype the high retention of chlorophylls was associated with an equally high retention of total carotenoids. Although the two genotypes differ markedly with regard to the rate of pigment loss, the ratios of yellow to green pigments did not change significantly during dark-induced senescence. At the end of the senescence period β-carotene was retained to a higher degree than the xanthophylls, particularly in the yellowing genotype. In the mutant leaves the ratio of chlorophyll a to b remained nearly constant, whereas in leaves of the normal genotype a preferential retention of chlorophyll b was observed towards the end of the senescence period. It is concluded that the thylakoids of the non-yellowing genotype retain all the principal components of protein-pigment complexes, i.e. chlorophylls, carotenoids and apoproteins. Possible explanations for the stability of these complexes in the mutant are discussed.     

Lycopene cyclization inBlakeslea trispora

Fungi produce and accumulate various carotenoids. Mycelia of the ZygomyceteBlakeslea trispora contained β-carotene and its precursors γ-carotene and lycopene. When strains of opposite sex grew together, the β-carotene concentration increased fourfold, that of γ-carotene remained unchanged, and other intermediates practically disappeared. The inhibitors nicotine, 2-(4-chlorophenylthio)-triethylamine, α-picoline, and imidazole increased the concentrations of lycopene and γ-carotene and decreased those of β-carotene. From our quantitative results, we conclude thatBlakeslea has two pathways for lycopene metabolism, of which other fungi have only one or the other. The main one, two cyclizations from lycopene to β-carotene, is carried out by an enzyme dimer, is stimulated by sexual interaction, and is sensitive to the inhibitors. The other pathway, a cyclization to γ-carotene is not affected by mating or the inhibitors.     

Antioxidant compound responses to chilling stress in differentially sensitive inbred maize lines

Chilling temperatures increase the amounts of potentially lethal toxic oxygen compounds present within plants. These toxic oxygen compounds can be scavenged by antioxidant compounds such as ascorbate and β-carotene. Three developmental stages (first, third and fifth leaf) of four inbred lines of maize ( Zea mays L.) exhibiting differential sensitivity to chilling were examined in order to determine if the chilling-sensitive line had lower concentrations of antioxidant compounds than did the tolerant lines. Plants were exposed to one of three treatments: (1) control (25°C constant), (2) control treatment plus a short-term chilling exposure of 11°C one day prior to harvesting, and (3) long-term (11°C constant) chilling exposure. Total ascorbate, total glutathione, β-carotene, α-tocopherol and chlorophyll contents were quantified, and ratios of dehydroascorbate/ascorbate and reduced/oxidized glutathione were determined. Lower concentrations of β-carotene were found in the chilling-sensitive relative to those in the chilling-tolerant lines for the first-leaf stage under both short- and long-term chilling treatments. Concentrations of total ascorbate and glutathione and β-carotene in the chilling-sensitive line increased as the chilling treatment progressed and as the plants developed until they ultimately became either significantly higher or no different relative to the tolerant lines. Results suggest that this sensitive line became less sensitive to chilling-induced oxidative stress with development.     

Changes in carotenoids, tocopherols and diterpenes during drought and recovery, and the biological significance of chlorophyll loss in Rosmarinus officinalis plants

Two-year-old rosemary (Rosmarinus officinalis L.) plants were subjected to severe stress by exposure to prolonged drought during a Mediterranean summer. Severely stressed plants recovered completely after the autumn rainfalls although the relative water content remained below 35% for 3 months and the chlorophyll content of leaves was reduced by up to 85% during the drought. In severe stress: (i) α-tocopherol increased 9-fold per g dry weight and 20-fold per unit of chlorophyll; (ii) lutein and β-carotene contents decreased on a dry-weight basis, but an 80% increase in lutein and constant levels of β-carotene were observed on a chlorophyll basis; (iii) there were transient and sustained increases in the de-epoxidation state of the xanthophyll cycle; and (iv) the highly oxidised abietane diterpene isorosmanol increased 8-fold as a result of the oxidation of carnosic acid. With the autumn rainfalls, water status, α-tocopherol and violaxanthin recovered first and the levels of photosynthetic pigments and abietane diterpenes increased later. The photoprotection conferred by the xanthophyll cycle and the antioxidant function of tocopherols, lutein and diterpenes may help to avoid irreversible damage in severe drought, making possible the recovery of functional membranes after the autumn rainfalls. Besides, chlorophyll loss reduces the amount of photons absorbed by leaves, which enhances the photoprotective and antioxidant capacity of leaves per amount of photons absorbed, since the ratios of xanthophylls, α-tocopherol and abietane diterpenes to chlorophyll increase. Received: 12 July 1999 / Accepted: 25 November 1999     

Eco-physiological studies on the response of taxodiaceous conifers to shading with special reference to the behavior of leaf pigments

The effects of light intensity on the content and composition of leaf pigments, especially of carotenoids, were studied with mature current-year leaves of Taxodiaceous saplings grown under different grades of shade in summer. Both chlorophyll and total carotenoid contents increased with decreasing light intensity, maintaining approximately linear relations between each other, over a range of relative solar radiation of 100% to 7% of full daylight. The regression of total chlorophyll content on mean solar radiation could be well approximated by Shinozaki-Kira's reciprocal equation. The ratio of chlorophyll a to chlorophyll b was smaller in the shade than in the sun. The percentage of α-carotene and violaxanthin in the total carotenoid content tended to increase with increasing degree of shade, while those of β-carotene and lutein were reduced. The eco-physiological meanings of the pigments were considered based on this evidence. The order of shade tolerance among the four species tested is also discussed taking the responses of leaf weight and chlorophyll content to incident light intensity into consideration.     

Nicotine Enhances the Cyclic AMP-Dependent Protein Kinase-Mediated Phosphorylation of α4 Subunits of Neuronal Nicotinic Receptors

Abstract: Studies determined whether α4β2 or α3β2 neuronal nicotinic receptors expressed in Xenopus oocytes are substrates for cyclic AMP-dependent protein kinase (PKA) and whether nicotine affects receptor phosphorylation. The cRNAs for the subunits were coinjected into oocytes, and cells were incubated for 24 h in the absence or presence of nicotine (50 n M for α4β2 and 500 n M for α3β2 receptors). Nicotine did not interfere with the isolation of the receptors. When receptors isolated from oocytes expressing α4β2 receptors were incubated with [γ-³²P]ATP and the catalytic subunit of PKA, separated by electrophoresis, and visualized by autoradiography, a labeled phosphoprotein with the predicted molecular size of the α4 subunit was present. Phosphorylation of α4 subunits of α4β2 receptors increased within the first 5 min of incubation with nicotine and persisted for 24 h. In contrast, receptors isolated from oocytes expressing α3β2 receptors did not exhibit a labeled phosphoprotein corresponding to the size of the α3 subunit. Results suggest that the PKA-mediated phosphorylation of α4 and not α3 subunits may explain the differential inactivation by nicotine of these receptors subtypes expressed in oocytes.     

Sustained Nicotine Exposure Differentially Affects α3β2 and α4β2 Neuronal Nicotinic Receptors Expressed in Xenopus Oocytes

Abstract: To determine whether prolonged exposure to nicotine differentially affects α3β2 versus α4β2 nicotinic receptors expressed in Xenopus oocytes, oocytes were coinjected with subunit cRNAs, and peak responses to agonist, evoked by 0.7 or 7 µ M nicotine for α4β2 and α3β2 receptors, respectively, were determined before and following incubation for up to 48 h with nanomolar concentrations of nicotine. Agonist responses of α4β2 receptors decreased in a concentration-dependent manner with IC₅₀ values in the 10 n M range following incubation for 24 h and in the 1 n M range following incubation for 48 h. In contrast, responses of α3β2 receptors following incubation for 24–48 h with 1,000 n M nicotine decreased by only 50–60%, and total ablation of responses could not be achieved. Attenuation of responses occurred within the first 5 min of nicotine exposure and was a first-order process for both subtypes; half-lives for inactivation were 4.09 and 2.36 min for α4β2 and α3β2 receptors, respectively. Recovery was also first-order for both subtypes; half-lives for recovery were 21 and 7.5 h for α4β2 and α3β2 receptors, respectively. Thus, the responsiveness of both receptors decreased following sustained exposure to nicotine, but α4β2 receptors recovered much slower. Results may explain the differential effect of sustained nicotine exposure on nicotinic receptor-mediated neurotransmitter release.     

Scanning mutagenesis of regions in the Gα protein Gpa1 that are predicted to interact with yeast mating pheromone receptors

The mechanism by which receptors activate heterotrimeric G proteins was examined by scanning mutagenesis of the Saccharomyces cerevisiae pheromone-responsive Gα protein (Gpa1). The juxtaposition of high-resolution structures for rhodopsin and its cognate G protein transducin predicted that at least six regions of Gα are in close proximity to the receptor. Mutagenesis was targeted to residues in these domains in Gpa1, which included four loop regions (β2–β3, α2–β4, α3–β5, and α4–β6) as well as the N and C termini. The mutants displayed a range of phenotypes from nonsignaling to constitutive activation of the pheromone pathway. The constitutive activity of some mutants could be explained by decreased production of Gpa1, which permits unregulated signaling by Gβγ. However, the constitutive activity caused by the F344C and E335C mutations in the α2–β4 loop and F378C in the α3–β5 loop was not due to decreased protein levels, and was apparently due to defects in sequestering Gβγ. The strongest loss of the function mutant, which was not detectably induced by a pheromone, was caused by a K314C substitution in the β2–β3 loop. Several other mutations caused weak signaling phenotypes. Altogether, these results suggest that residues in different interface regions of Gα contribute to activation of signaling.