Carotenoid changes in the peel of ripening persimmon (Diospyros kaki) cv Triumph

The changes of the carotenoid pigments in the peel of ripening persimmon (Diospyros kaki) cv Triumph were followed for an entire season. During ripening, the total carotenoid decreased until colour break, then increased gradually and drastically at the last ripening stages. The chloroplast carotenoid pattern of the unripe fruit changed into a chromoplast pattern in which cryptoxanthin was the predominant pigment, reaching a level between 40 and 50% of the total carotenoids. It accumulated continuously at a rate of approximately 10% at each 2 week interval, its percentage being characteristic for each ripening stage. Other major pigments at levels of approximately 10% of the total carotenoids were zeaxanthin, antheraxanthin and violaxanthin. In the fully ripe fruit, ripened both on and off the tree, lycopene which was not present before was found as the second major pigment. This unusual pattern change is discussed.     

Reversed-phase high-performance liquid chromatography of chlorophylls and carotenoids

Reversed-phase high-performance liquid chromatography with octadecyl- or octylsilylated silica gel as the stationary phase provides a powerful tool in the analysis of chloroplast pigments from higher plants and green algae. Chromatographic columns packed with 10 μm chemically bonded silica gel particles allow the simultaneous separation of chlorophylls a and b, chlorophyll isomers, pheophytins a and b, α-carotene, β-carotene, lutein, violaxanthin, lutein-5,6-epoxide, antheraxanthin, neoxanthin and several minor carotenoids from a single sample within a short analysis time. The quantitative analysis requires a minimum of 1–5 pmol for carotenoids and 5–10 pmol for chlorophylls. Pigment degradation products, formed on polar stationary phases, are not found in reversed-phase high-performance liquid chromatography due to the weak hydrophobic forces on which the separation mechanism is based. The production of altered pigments however, either induced by various treatments or generated during the isolation, can be monitored as the reversed-phase system is selective enough to separate cis-isomers and oxidation products from their parent compounds. The reproducibility of the individual retention time for each pigment is better than ±1.5% which facilitates the identification of unknown pigments. The method is applied to the analysis of the pigment composition of Chlorella fusca, spinach (Spinacia oleracea) chloroplasts, and to the rapid determination of the ratio of chlorophyll a to chlorophyll b.     

Near edge X-ray absorption fine structure spectroscopy (NEXAFS) of pigment–protein complexes: Peridinin–chlorophyll a protein (PCP) of Amphidinium carterae

Peridinin–chlorophyll a protein (PCP) is a unique water soluble antenna complex that employs the carotenoid peridinin as the main light-harvesting pigment. In the present study the near edge X-ray absorption fine structure (NEXAFS) spectrum of PCP was recorded at the carbon K-edge. Additionally, the NEXAFS spectra of the constituent pigments, chlorophyll a and peridinin, were measured. The energies of the lowest unoccupied molecular levels of these pigments appearing in the carbon NEXAFS spectrum were resolved. Individual contributions of the pigments and the protein to the measured NEXAFS spectrum of PCP were determined using a “building block” approach combining NEXAFS spectra of the pigments and the amino acids constituting the PCP apoprotein. The results suggest that absorption changes of the pigments in the carbon near K-edge region can be resolved following excitation using a suitable visible pump laser pulse. Consequently, it may be possible to study excitation energy transfer processes involving “optically dark” states of carotenoids in pigment–protein complexes by soft X-ray probe optical pump double resonance spectroscopy (XODR).     

Flower colours and pigments in Disa hybrid (Orchidaceae)

Flower colours and the composition of pigments in the perianths of five cultivars of Disa orchids were analyzed. Carotenoids were major pigment components in the orange-red flowers of ‘Dawn Angel’. We identified two types of pigment composition in the red flowered cultivars: ‘San Francisco’ contained more carotenoids and less anthocyanins, while ‘Marlene’ contained more anthocyanins than carotenoids. The red-purple flowered cultivars, only contained slight amounts of carotenoids, and the red-purple colour was attributed to the relatively high density of a cyanidin-based anthocyanin. The importance of the characterization of pigments in the perianths of orchid has been discussed in both breeding for flower colour improvement and chemotaxonomy.     

Near edge X-ray absorption fine structure spectroscopy (NEXAFS) of pigment-protein complexes: peridinin-chlorophyll a protein (PCP) of Amphidinium carterae

Peridinin-chlorophyll a protein (PCP) is a unique water soluble antenna complex that employs the carotenoid peridinin as the main light-harvesting pigment. In the present study the near edge X-ray absorption fine structure (NEXAFS) spectrum of PCP was recorded at the carbon K-edge. Additionally, the NEXAFS spectra of the constituent pigments, chlorophyll a and peridinin, were measured. The energies of the lowest unoccupied molecular levels of these pigments appearing in the carbon NEXAFS spectrum were resolved. Individual contributions of the pigments and the protein to the measured NEXAFS spectrum of PCP were determined using a “building block” approach combining NEXAFS spectra of the pigments and the amino acids constituting the PCP apoprotein. The results suggest that absorption changes of the pigments in the carbon near K-edge region can be resolved following excitation using a suitable visible pump laser pulse. Consequently, it may be possible to study excitation energy transfer processes involving “optically dark” states of carotenoids in pigment-protein complexes by soft X-ray probe optical pump double resonance spectroscopy (XODR).     

Chloroplast pigments and algal systematics

The chloroplast pigments of one typical representative (Pleurochloris magna) and two potential members (clone BSG Sticho and an isolate called Tunis) of the new class Eustigmatophyceae have been examined by modern methods including mass spectrometry. The three cultures all exhibited the same chloroplast pigments: Chlorophyll a, but no b or c, δ-carotene (I), canthaxanthin (II), violaxanthin (IIIa), and esterified vaucheriaxanthin (IVb) plus some free vaucheriaxanthin (IVa). Furanoid rearrangement of the epoxidic carotenoids complicated the analysis. The unique pigment complement hereby indicated for Eustigmatophyceae is clearly different from the pigment distribution patterns reported for Chlorophyceae and Xanthophyceae.     

Spectral characterization of the neuronal pigments of Aplysia juliana

Spectral analysis at liquid N₂ temperature of the circumesophageal ganglia of Aplysia juliana showed that carotenoids and a hemoglobin-like pigment are contained in concentrations of approx. 25 and 3 μM, respectively, in the whole ganglia. Microspectrophotometrical measurements of Aplysia neurons indicated that the carotenoids reside on lipochondria in a concentration of approx. 38 mM. In addition to lipochondria, two types of pigmented particulate having absorption maxima at about 512 and 525 nm, respectively, were found in the neurons. The neuronal carotenoids consist of violaxanthin, β-carotene and one minor component; among them the first occupies approx. 77% of total carotenoids. Two principal absorption maxima of the carotenoids, when existing in both ganglial homogenates and Triton X-100 extracts, show a red shift of 10 nm compared with those of free pigments in hexan. The red shift may be interpreted as due to the solvation of the carotenoids by surrounding lipids     

Applications of red pigments from psychrophilic Rhodonellum psychrophilum GL8 in health,food and antimicrobial finishes on textiles

A psychrophilic bacterium (named GL8) producing red pigments was isolated from the high altitude Pangong Tso Lake located in Leh Ladakh, India. Based on 16S rDNA sequencing amplicon of 1370 bp, the psychrophilic bacterium was identified as Rhodonellum psychrophilum (R. psychrophilum GL8) and deposited in Gen Bank under accession no. MH031708.1.The red colored pigments from R. psychrophilum GL8 showed antimicrobial activity against E. coli, S. aureus, C. albicans, (MTCC 277, ATCC 90028) and S. cerevisiae (H1086) with minimum inhibitory concentrations from 31.25 μg/mL to 500 μg/mL. Red colored pigments also showed synergistic antifungal activity when combined with fluconazole and amphotericin B against C. albicans (MTCC 277 and ATCC 90028) and S. cerevisiae; vancomycin and erythromycin against E. coli and S. aureus. The red pigments showed antioxidant activity with an IC₅₀ of 13.159 μg/mL, LC–MS/MS analysis demonstrated that red pigment extracts contained a mixture of 2-methyl-3-butyl-prodigine, Prodigiosin, 2-methyl-3hexyl-prodigine, 3, 4-Didehydrorhodopsin, anhydrorhodovibrin, alloxanthin and Tetradecanoyl-hexadecanoyl compounds. Red pigment extracts were used to develop antimicrobial fabrics and did not show any cytotoxicity to U87MG human glioblastoma cell line, but showed a marginal growth inhibition to A172 human glioblastoma cell lines. In contrast, the red pigment extracts showed significant growth stimulation on L929 mouse fibroblast cell lines.     

Comparative Study of the Carotenoid Composition of the Seeds of Ripening Momordica charantia and Tomatoes

The total carotenoid concentration of the seeds of Momordica charantia rose about 100-fold from the immature to the ripe stage. The massive increase was almost exclusively attributable to lycopene, which accounted for 96% of the carotenoids of the ripe seeds. The carotenoid pattern of the seed was found to be drastically different from that of the pericarp. The seed, which contained fewer carotenoids, had a total concentration 12 times greater than that in the pericarp at the ripe stage. The acyclic lycopene selectively accumulated in the seed, whereas the cyclic carotenoids, cryptoxanthin, zeaxanthin and β-carotene, were the principal pigments of the ripe pericarp. The seed of ripe tomatoes reflected the qualitative pattern of the whole fruit. The total carotenoid concentration was, however, much lower and the lycopene content was particularly low. β-Carotene, having a comparatively high concentration, emerged as the major pigment of the seed.     

Role of carotenoids in the phototropic response of corn seedlings

The herbicide, 4 chloro-5-(methylamino)-2-(α,α,α,-trifluoro-m-tolyl)-3 (2H)-pyridazinone (SAN 9789), which blocks the synthesis in higher plants of colored carotenoids but not of flavins, was used to examine the involvement of carotenoids in corn seedling phototropism. It was concluded that “bulk” carotenoids are not the photoreceptor pigment based on the results that increasing concentrations of SAN 9789 (up to 100 micromolar) did not alter the phototropic sensitivity to 380 nanometers light (using geotropism as a control) and did not increase the threshold intensities of fluence response curves for both 380 and 450 nanometers light even though carotenoid content was reduced to 1 to 2% of normal. SAN 9789 treatment, however, did reduce seedling sensitivity toward 450 nanometers light indicating that carotenoids are involved in phototropism. Carotenoids, which are located mainly in the primary leaves, may act in phototropism as an internal screen, enhancing the light intensity gradient across the seedling and thus contributing to the ability of the seedling to perceive light direction. These results indicate that the action spectra for phototropic responses can be significantly affected by the absorbance of screening pigments in vivo thus altering its shape from the in vitro absorption spectrum of the photoreceptor pigment.     

Phytoplankton of an eutrophic tropical reservoir: comparison of biomass estimated from counts with chlorophyll-a biomass from HPLC measurements

The seasonal variation of phytoplankton in an eutrophic tropical reservoir was evaluated through photosynthetic pigments analyzed by HPLC. The contributions of algal classes to total chlorophyll a (TChl-a) were estimated by two procedures. The first one used fixed marker pigment/chlorophyll a ratio available from culture studies of the major species of each class. In the second procedure, a matrix factorization program (CHEMTAX) was used to analyze the pigment data. The pigment data were compared with carbon biomass estimated from microscope analysis. A significant correlation between total chlorophyll a (measured by HPLC) and total biomass was obtained, indicating only a slight variation in the content of algal chlorophyll a when compared to its fluctuations in carbon biomass. The interpretation of pigment data with CHEMTAX resulted in a good agreement with biomass. Although displaying some differences, the general pattern of the phytoplankton community dynamics and the major shifts in composition, biomass and the cyanobacterial bloom were evidenced. In contrast, Chl-a biomass estimates from fixed Xan/Chl-a ratios presented poor agreement with microscope data and did not register the principal changes in phytoplankton. Our results also highlighted the needs of better understanding of the relationships between marker pigments, chlorophyll-a and algal biomass.     

Pigmented basidiomycetous yeasts are a promising source of carotenoids and ubiquinone Q<Subscript>10</Subscript>

Strains of basidiomycetous yeasts isolated from different sources were studied in order to determine the content of carotenoid pigments and ubiquinone Q₁₀ for subsequent selection work to obtain producers of these substances. The high specific productivity of carotenoids (600–700 mg/g) was revealed in the representatives of the following species: Cystofilobasidium capitatum, Rhodosporidium diobovatum, R. sphaerocarpum, Rhodotorula glutinis, Rh. minuta, and Sporobolomyces roseus. The ratio of the major pigments (torulene, torularhodine, and β-carotene) in the representatives of different species was studied. Certain specific features of pigment formation in relation to the taxonomic position of the yeasts were determined. Eurybiont species with substantial ecological lability are the most active producers of carotenoids and ubiquinone Q₁₀ among the epiphytes. It is the first time a comparative analysis of the coenzyme Q₁₀ content in different taxa has been performed using several strains of the same species. The maximal coenzyme Q₁₀ production (1.84 mg/g of dry biomass) was found in the yeast species R. sphaerocarpum.     

Phototrophic pigment production with microalgae: biological constraints and opportunities

There is increasing interest in naturally produced colorants, and microalgae represent a bio‐technologically interesting source due to their wide range of colored pigments, including chlorophylls (green), carotenoids (red, orange and yellow), and phycobiliproteins (red and blue). However, the concentration of these pigments, under optimal growth conditions, is often too low to make microalgal‐based pigment production economically feasible. In some Chlorophyta (green algae), specific process conditions such as oversaturating light intensities or a high salt concentration induce the overproduction of secondary carotenoids (β‐carotene in Dunaliella salina (Dunal) Teodoresco and astaxanthin in Haematococcus pluvialis (Flotow)). Overproduction of all other pigments (including lutein, fucoxanthin, and phycocyanin) requires modification in gene expression or enzyme activity, most likely combined with the creation of storage space outside of the photosystems. The success of such modification strategies depends on an adequate understanding of the metabolic pathways and the functional roles of all the pigments involved. In this review, the distribution of commercially interesting pigments across the most common microalgal groups, the roles of these pigments in vivo and their biosynthesis routes are reviewed, and constraints and opportunities for overproduction of both primary and secondary pigments are presented.     

Pigment profiles and bacterial communities from Thailand thermal mats

Differently colored layers of freshwater hot spring mats at Boekleung (Western Thailand) were studied. Temperatures ranged from over 50 up to 57°C. Two mats were characterized: a laminated mat with a green and a red layers, and a monolayer, greenish-yellow mat. Bacterial communities in green, red, and yellow layers were investigated using molecular, culturing and pigment analysis methods. Pigment profiles covered a wide spectrum from chlorophylls to carotenoids. A green mat layer showed higher relative content of chlorophyll than yellow and red layers which presented higher proportion of carotenoids. Cyanobacterial isolates grow up to 55–56°C and their pigment profiles showed a relatively high content of chlorophylls suggesting the importance of other bacterial groups in the mat pigment profiles. Bacterial communities were analyzed by 16S rDNA surveys showing Cyanobacteria and Chloroflexi as the mayor components of the community. Other significant members were Candidate Division OP10, Bacteroidetes, Planctomycetes and Actinobacteria. These results highlight a major participation of Cyanobacteria and Chloroflexi in thermal mat communities, and the preferential presence of Candidate Division OP10 in green mat layers. Differently colored mat layers showed characteristic bacterial communities which could be discriminated from pigment profiles and molecular surveys.     

A reduction in temperature induces bioactive red pigment production in a psychrotolerant Penicillium sp. GEU_37 isolated from Himalayan soil

Filamentous fungi are being globally explored for the production of industrially important bioactive compounds including pigments. In the present study, a cold and pH tolerant fungus strain Penicillium sp (GEU_37), isolated from the soil of Indian Himalaya, is characterized for the production of natural pigments as influenced by varying temperature conditions. The fungal strain produces a higher sporulation, exudation, and red diffusible pigment in Potato Dextrose (PD) at 15 °C as compared to 25 °C. In PD broth, a yellow pigment was observed at 25 °C. While measuring the effect of temperature and pH on red pigment production by GEU_37, 15 °C and pH 5, respectively, were observed to be the optimum conditions. Similarly, the effect of exogenous carbon and nitrogen sources and mineral salts on pigment production by GEU_37 was assessed in PD broth. However, no significant enhancement in pigmentation was observed. Chloroform extracted pigment was separated using thin layer chromatography (TLC) and column chromatography. The two separated fractions i.e., fractions I and II with Rf values 0.82 and 0.73, exhibited maximum light absorption, λ_max, at 360 nm and 510 nm, respectively. Characterization of pigments using GC–MS showed the presence of the compounds such as phenol, 2,4-bis (1,1-dimethylethyl) and eicosene from fraction I and derivatives of coumarine, friedooleanan, and stigmasterole in fraction II. However, LC-MS analysis detected the presence of derivatives of compound carotenoids from fraction II as well as derivative of chromenone and hydroxyquinoline as major compounds from both the fractions along with other numerous important bioactive compounds. The production of such bioactive pigments under low temperature conditions suggest their strategic role in ecological resilience by the fungal strain and may have biotechnological applications.     

In Vitro Reconstitution of Light-harvesting Complexes of Plants and Green Algae

In plants and green algae, light is captured by the light-harvesting complexes (LHCs), a family of integral membrane proteins that coordinate chlorophylls and carotenoids. In vivo, these proteins are folded with pigments to form complexes which are inserted in the thylakoid membrane of the chloroplast. The high similarity in the chemical and physical properties of the members of the family, together with the fact that they can easily lose pigments during isolation, makes their purification in a native state challenging. An alternative approach to obtain homogeneous preparations of LHCs was developed by Plumley and Schmidt in 1987¹, who showed that it was possible to reconstitute these complexes in vitro starting from purified pigments and unfolded apoproteins, resulting in complexes with properties very similar to that of native complexes. This opened the way to the use of bacterial expressed recombinant proteins for in vitro reconstitution. The reconstitution method is powerful for various reasons: (1) pure preparations of individual complexes can be obtained, (2) pigment composition can be controlled to assess their contribution to structure and function, (3) recombinant proteins can be mutated to study the functional role of the individual residues (e.g., pigment binding sites) or protein domain (e.g., protein-protein interaction, folding). This method has been optimized in several laboratories and applied to most of the light-harvesting complexes. The protocol described here details the method of reconstituting light-harvesting complexes in vitro currently used in our laboratory,and examples describing applications of the method are provided.     

Chlorophylle und Carotinoide der Chaetophorineae (Chlorophyceae; Ulotrichales)

In air-supplied inorganic liquid cultures, the highly differentiated green alga Fritschiella tuberosa forms only branched filaments with long slender cells. In nitrogen-deficient medium and with ageing of the cultures these cells become much shorter in length by subsequent formation of transverse walls. The chloroplasts of the slender cells contain the typical pigments of green algae. Together with the morphogenetic change to “short cells” secondary carotenoids are synthesized and stored in lipid droplets. Besides traces of lutein, violaxanthin and neoxanthin and reduced amounts of β-carotene, the following pigments have been demonstrated: Esters of astaxanthin (main pigment) and adonixanthin, canthaxanthin, echinenone and a Keto-α-carotinoid, which presumably is identical with α-doradexanthin, (3,3′-Dihydroxy-4-keto-α-Carotene) a pigment not known in plants until now. In nitrogen deficient cultures the chlorophylls are totally decomposed, the total-lipids increase by about 500%. By supplying nitrogen-deficient cultures with nitrogen and subsequent illumination regreening of the cells starts already 16 hrs later.     

Expression levels of the tetratricopeptide repeat protein gene ttc39b covary with carotenoid-based skin colour in cichlid fish

Carotenoid pigments play a major role in animal body colouration, generating strong interest in the genes involved in the metabolic processes that lead from their dietary uptake to their storage in the integument. Here, we used RNA sequencing (RNA-Seq) to test for differentially expressed genes in a taxonomically replicated design using three pairs of related cichlid fish taxa from the genera Tropheus and Aulonocara. Within each pair, taxa differed in terms of red and yellow body colouration, and high‐performance liquid chromatography (HPLC) analyses of skin extracts revealed different carotenoid profiles and concentrations across the studied taxa. Five genes were differentially expressed in all three yellow–red skin contrasts (dhrsx, nlrc3, tcaf2, urah and ttc39b), but only the tetratricopeptide repeat protein-coding gene ttc39b, whose gene product is linked to mammalian lipid metabolism, was consistently expressed more highly in the red skin samples. The RNA-Seq results were confirmed by quantitative PCR. We propose ttc39b as a compelling candidate gene for variation in animal carotenoid colouration. Since differential expression of ttc39b was correlated with the presence/absence of yellow carotenoids in a previous study, we suggest that ttc39b is more likely associated with the concentration of total carotenoids than with the metabolic formation of red carotenoids.     

The subcellular distribution of carotenoids in Neurospora crassa

The intracellular localization of carotenoids in the fungus Neurospora crassa was examined after completion of photoinduced biosynthesis of these pigments. Differential centrifugation of cell homogenates yielded subcellular fractions which were characterized by activities of several marker enzymes for cell constituents and in part purified by subsequent sucrose density gradient centrifugation. Most (ca 58%) of the carotenoids were found to be localized in lipid globules, but substantial amounts are also associated with two membrane fractions that were rich in membranes of the endoplasmic reticulum as indicated by high activities of NADPH- and NADH—cytochrome c reductase. These results, along with the coincidence in the distribution of both carotenoids and activities of specific marker enzymes in the sucrose density gradients, led to the conclusion that apart from lipid globules, carotenoids are also localized in membranes of the endoplasmic reticulum.     

Chlorosome antenna complexes from green photosynthetic bacteria

Chlorosomes are the distinguishing light-harvesting antenna complexes that are found in green photosynthetic bacteria. They contain bacteriochlorophyll (BChl) c, d, e in natural organisms, and recently through mutation, BChl f, as their principal light-harvesting pigments. In chlorosomes, these pigments self-assemble into large supramolecular structures that are enclosed inside a lipid monolayer to form an ellipsoid. The pigment assembly is dictated mostly by pigment–pigment interactions as opposed to protein–pigment interactions. On the bottom face of the chlorosome, the CsmA protein aggregates into a paracrystalline baseplate with BChl a, and serves as the interface to the next energy acceptor in the system. The exceptional light-harvesting ability at very low light conditions of chlorosomes has made them an attractive subject of study for both basic and applied science. This review, incorporating recent advancements, considers several important aspects of chlorosomes: pigment biosynthesis, organization of pigments and proteins, spectroscopic properties, and applications to bio-hybrid and bio-inspired devices.