Pigment Diversity in Freshwater Phytoplankton. I. A Comparison of Spectrophotometric and Paper Chromatographic Methods

Spectrophotometric and paper chromatographic analyses of the pigments in the phytoplankton were made from early spring till the end of summer in two small Dutch freshwater lakes. It was found that pigment diversity cannot be adequately estimated by MARGALEF'S pigment ratio nor by polychromatic spectrophotometric methods. The pigments detected with the paper chromatographic method were: chlorophyll-a, chlorophyll-b, chlorophyll-c, phaeophytin-a (traces), phaeophorbide-a, Mg-containing chlorophyll-derivatives, carotene, lutein, violaxanthin, neoxanthin (traces), fucoxanthin, diadinoxanthin, diatoxanthin (traces), peridinin and keto-carotenoids (traces). It is suggested to distinguish between a richness-component and an evenness-component of pigment diversity.     

PHOTOSYNTHETIC PIGMENT COMPOSITION IN THE PRIMITIVE GREEN ALGA MESOSTIGMA VIRIDE (PRASINOPHYCEAE): PHYLOGENETIC AND EVOLUTIONARY IMPLICATIONS1

The photosynthetic pigment composition of Mesostigma viride Lauterborn, a primitive green alga, was determined. This alga contained chl a and b, lycopene, lutein, siphonaxanthin, γ‐carotene, β‐carotene, antheraxanthin, violaxanthin, neoxanthin, and two novel carotenoid fatty acid esters, siphonaxanthin C12:0 ester and siphonaxanthin C14:0 ester. The esters were saturated, whereas all previously identified siphonaxanthin and loroxanthin esters have been mono‐unsaturated (trans‐Δ2). Neoxanthin was the all‐trans form. This is the first such case detected in the chloroplasts of green plants. The 9′‐cis form of neoxanthin is believed to be universally present in the chloroplasts of green plants (Streptophyta and Chlorophyta) and is a precursor of abscisic acid. However, the 9′‐cis form was not found in M. viride. Based on these results, we discuss the phylogenetic implications and early evolution of the antenna pigment system in green plants.     

A comparative study of carotenoids of Aschersonia aleyroides and Aspergillus giganteus

The orange-red sporodochium of Aschersonia aleyroides contains six carotenes with -carotene (87%) as the major pigment. In old cultures there is a decrease in total carotenoids, the disappearance of two trans-carotenes and the appearance of two cis-carotenes. In the case of Aspergillus giganteus and its mutant A. giganteus mut. alba the major carotene is also -carotene (80%) with six other carotenoids, including possibly the acid pigment asperxanthin. Until now this latter pigment has only been detected in Aspergillus and therefore it can be regarded as a criterion to discriminate between Apergillus and other fungi. Aschersonia and Aspergillus seem not to be closely related on the basis of pigment patterns, which is in agreement with distinct morphological differences.     

The relative proportions of the chloroplast pigments as influenced by different levels of iron and potassium supply

Summary 1. Potato plants were grown in pot sand cultures at different levels of iron and potassium supply. Plants grown at the lowest level of iron developed iron deficiency chlorosis and potassium deficiency symptoms when maintained at the lowest level of potassium but not at the highest level.2. Rapid procedures were developed for the extraction and estimation of chloroplast pigments from small samples of lamina—usually less than 1 g of fresh material.3. The expression of pigment content on an area basis gave results which were in better agreement with visual observations than those expressed on either fresh or dry weight basis.4. Both iron and potassium additions increased the chloroplast pigment content. Leaves exhibiting iron deficiency contained reduced quantities of all pigments per unit area of lamina.5. A linear relationship existed between chlorophyll and carotene, chlorophyll and xanthophyll, and carotene and xanthophyll contents.6. The results suggested that laminae completely deficient in chlorophyll would contain no carotene but might still contain xanthophyll.7. The relative proportions of chlorophyll, carotene and xanthophyll do not remain constant under varying conditions of iron status. When iron is deficient the proportion of xanthophyll increased in relation to the chlorophyll and carotene contents.     

The pigment composition of Phaeocystis antarctica (Haptophyceae) under various conditions of light,temperature, salinity,and iron

The pigment composition of Phaeocystis antarctica was monitored under various conditions of light, temperature, salinity, and iron. 19′‐Hexanoyloxyfucoxanthin (Hex‐fuco) always constituted the major light‐harvesting pigment, with remarkably stable ratios of Hex‐fuco‐to‐chl a under the various environmental conditions. Increased pigment‐to‐chl a ratios at low irradiance confirmed the light‐harvesting function of Fucoxanthin (Fuco), 19′‐Hexanoyloxy‐4‐ketofucoxanthin (Hex‐kfuco), 19′‐butanoyloxyfucoxanthin (But‐fuco), and chl c2 and c3. Increased pigment‐to‐chl a ratios at high irradiance, low iron concentrations, and to a lesser extent at high salinity confirmed the photoprotective function of diadinoxanthin, diatoxanthin, and ß,ß‐carotene. Pigment ratios were not always according to expectations. The consistent increase in But‐fuco/chl at high temperature, high salinity, and low iron suggests a role in photoprotection rather than in light harvesting. Low Hex‐kfuco/chl ratios at high salinity were consistent with a role as light harvester, but the high ratios at high temperature were not, leaving the function of Hex‐kfuco enigmatic. Dedicated experiments were performed to test whether or not the light‐harvesting pigment Fuco could be converted into its structural relative Hex‐fuco, and vice versa, in response to exposure to light shifts. Rapid conversions could not be confirmed, but long‐term conversions cannot be excluded. New pigment ratios are proposed for chemotaxonomic applications. The ratios will improve pigment‐based diagnosis of algal species in waters dominated by P. antarctica.     

Photovoltaic behavior of chlorophyll a and beta-carotene in Langmuir films

The photovoltaic properties of chlorophyll a and beta-carotene Langmuir films and Langmuir films of a mixure of chlorophyll a and beta-carotene at different molar ratios were investigated. SnO2-optically transparent electrodes were used as a support. It was shown that the film photovoltage value depends on surface pressure and the total film thickness (the number of layers deposited onto SnO2-optically transparent electrodes). Fifteen layers (SP = 35 mH/m) of chlorophyll and 7-10 layers (SP = 20 mH/m) of beta-carotene give rise to a maximal photovoltage of 140 and 270 mV (at a shunt resistance of 10(7) Ohm), correspondingly. It was found that the photovoltage values in films of the carotene-chlorophyll mixture increase with the carotene concentration. The photovoltage value of a film containing 80-90% of carotene exceeds that of single-component pigment films prepared under the same deposition conditions.     

Metabolic engineering of β‐carotene in orange fruit increases its in vivo antioxidant properties

Orange is a major crop and an important source of health‐promoting bioactive compounds. Increasing the levels of specific antioxidants in orange fruit through metabolic engineering could strengthen the fruit's health benefits. In this work, we have afforded enhancing the β‐carotene content of orange fruit through blocking by RNA interference the expression of an endogenous β‐carotene hydroxylase gene (Csβ‐CHX) that is involved in the conversion of β‐carotene into xanthophylls. Additionally, we have simultaneously overexpressed a key regulator gene of flowering transition, the FLOWERING LOCUS T from sweet orange (CsFT), in the transgenic juvenile plants, which allowed us to obtain fruit in an extremely short period of time. Silencing the Csβ‐CHX gene resulted in oranges with a deep yellow (‘golden’) phenotype and significant increases (up to 36‐fold) in β‐carotene content in the pulp. The capacity of β‐carotene‐enriched oranges for protection against oxidative stress in vivo was assessed using Caenorhabditis elegans as experimental animal model. Golden oranges induced a 20% higher antioxidant effect than the isogenic control. This is the first example of the successful metabolic engineering of the β‐carotene content (or the content of any other phytonutrient) in oranges and demonstrates the potential of genetic engineering for the nutritional enhancement of fruit tree crops.     

PIGMENT SIGNATURES AND PHYLOGENETIC RELATIONSHIPS OF THE PAVLOVOPHYCEAE (HAPTOPHYTA)1

Variations in the HPLC‐derived pigment composition of cultured Pavlovophyceae (Cavalier‐Smith) Green et Medlin were compared with phylogenetic relationships inferred from 18S rDNA sequencing, morphological characteristics, and current taxonomy. The four genera described for this haptophyte class (Diacronema Prauser emend. Green et Hibberd, Exanthemachrysis Lepailleur, Pavlova Butcher, and Rebecca Green) were represented by nine different species (one of which with data from GeneBank only). Chlorophylls a, c₁, c₂ and MgDVP (Mg‐[3,8‐divinyl]‐phytoporphyrin‐13²‐methylcarboxylate) and the carotenoids fucoxanthin, diadinoxanthin, diatoxanthin, and β,β‐carotene were detected in all cultures. Species only differed in the content of an unknown (diadinoxanthin‐like) xanthophyll and two polar chl c forms, identified as a monovinyl (chl c₁‐like) and a divinyl (chl c₂‐like) compound. This is the first observation of the monovinyl form in haptophytes. Based on distribution of these two chl c forms, species were separated into Pavlovophyceae pigment types A, B, and C. These pigment types crossed taxonomic boundaries at the generic level but were in complete accordance with species groupings based on molecular phylogenetic relationships and certain ultrastructural characteristics (position and nature of pyrenoid, stigma, and flagella). These results suggest that characterization of the pigment signature of unidentified culture strains of Pavlovophyceae can be used to predict their phylogenetic affinities and vice versa. Additional studies have been initiated to evaluate this possibility for the haptophyte class Prymnesiophyceae.     

Accumulation,degradation and biological effects of lindane on Scenedesmus obliquus (Turp.) Kütz

A study was made of accumulation, degradation and biological effects of lindane (r-BHC) on Scenedesmus obliquus (Turp.) Kütz. The work was conducted in a sewage reservoir in Han-Ku, Tianjin. Experimental results show that at 0.1–10 mgl⁻¹ concentration of lindane, the growth, chlorophyll and carotene content in S. obliquus do not differ much from the control. At concentrations of 50–100 mgl⁻¹, however, growth is inhibited, and chlorophyll and carotene content is low. From the beginning of the experiment until the fifth day, growth rate and pigment content increase constantly. On the seventh day, they start to decrease. Spectrographic analysis shows that pigment content decreases, but pigment structure is not changed even at high concentration of lindane. At 1–10 mgl⁻¹ of lindane, amino acids remain unchanged, with the exception of aspartine, arginine, methionine and phenylalanine. Scenedesmus obliquus (Turp.) Kütz possesses a certain accumulating capacity for r-BHC, which is higher at 1 mgl⁻¹ than at 10 mgl⁻¹. Accumulation is enhanced with time and is better under turbulent conditions. Gas chromatograms show that after treatment for 24 h, there are two more peaks at retention time 1′32″ and 1′53″, respectively, suggesting that a certain degree of lindane degradation occurs. The nature of these degradation products awaits further study. As a conclusion S. obliquus (Turp.) Kütz holds promise in the treatment of lindane-containing wastewaters in the range of 1–10 mgl⁻¹.     

Pigment composition and photoacclimation as keys to the ecological success of Gonyostomum semen (Raphidophyceae,Stramenopiles)

Aquatic habitats are usually structured by light attenuation with depth resulting in different microalgal communities, each one adapted to a certain light regime by their specific pigment composition. Several taxa contain pigments restricted to one phylogenetic group, making them useful as marker pigments in phytoplankton community studies. The nuisance and invasive freshwater microalga Gonyostomum semen (Raphidophyceae) is mainly found in brown water lakes with sharp vertical gradients in light intensity and color. However, its pigment composition and potential photoadaptations have not been comprehensively studied. We analyzed the photopigment composition of 12 genetically different strains of G. semen by high performance liquid chromatography after acclimation to different light conditions. We confirmed the pigments chl a, chl c1c2, diadinoxanthin, trans‐neoxanthin, cis‐neoxanthin, α and β carotene, which have already been reported for G. semen. In addition, we identified, for the first time, the pigments violaxan‐thin, zeaxanthin, and alloxanthin in this species. Alloxanthin has never been observed in raphidophytes before, suggesting differences in evolutionary plastid acquisition between freshwater lineages and the well‐described marine species. The amount of total chl a per cell generally decreased with increasing light intensity. In contrast, the increasing ratios of the prominent pigments diadinoxanthin and alloxanthin per chl a with light intensity suggest photoprotective functions. In addition, we found significant variation in cell‐specific pigment concentration among strains, grouped by lake of origin, which might correspond to genetic differences between strains and populations.     

Characterization of cyanobacterial β‐carotene ketolase and hydroxylase genes in Escherichia coli,and their application for astaxanthin biosynthesis

Carotenoid biosynthesis is highly conserved and well characterized up to the synthesis of β‐carotene. Conversely, the synthesis of astaxanthin from β‐carotene is less well characterized. Regardless, astaxanthin is a highly sought natural product, due to its various industrial applications and elevated antioxidant capacity. In this article, 12 β‐carotene ketolase and 4 β‐carotene hydroxylase genes, isolated from 5 cyanobacterial species, are investigated for their function, and potential for microbial astaxanthin synthesis. Further, this in vivo comparison identifies and applies the most promising genetic elements within a dual expression vector, which is maintained in Escherichia coli. Here, combined overexpression of individual β‐carotene ketolase and β‐carotene hydroxylase genes, within a β‐carotene accumulating host, enables a 23.5‐fold improvement in total carotenoid yield (1.99 mg g^?1), over the parental strain, with >90% astaxanthin. Biotechnol. Bioeng. 2009;103: 944–955. © 2009 Wiley Periodicals, Inc.     

Comparative studies on the nature and distribution of pigments from two thermophilic bacteria

Summary The pigments of two strains of extremely thermo-resistant bacteria were extracted and examined spectrophotometrically. Both strains contain -carotene as the main pigment according to the spectral characteristics of the pigment in various solvents. According to quantitative data Thermus aquaticus contains more than one hundred times as much of the pigment than is found in strain YT-G, representing almost 80% of the total lipid. The carotene of strain YT-G is destroyed by hot saponification, while that of T. aquaticus resists that procedure without alterations. T. aquaticus contains a small amount of a second pigment with absorption maxima at 370, 400 and 420 m.Solubilization of the membrane of spheroplasts derived from acetate-C¹⁴-labelled cells of T. aquaticus, followed by separation of the crude membrane fraction, showed that about 50% of the C¹⁴ is associated with the membrane fraction. More than 90% of the C¹⁴ is recovered in the yellow supernatant of the acetone-extracted crude membrane fraction, showing that a rapid incorporation of the label into the pigment occurs. The assumption that the pigment is associated with the membrane is supported by submicroscopical observations: T. aquaticus has a well-defined cytoplasmic membrane, and on top of this a sandwiched layer, which is embedded in a very electron-dense material, that we assume to consist of the pigment. Strain YT-G which contains only minute quantities of the same pigment, does not possess this kind of an outer membrane, but shows only a cytoplasmic membrane envelopped by the cell wall.The main experiments were done while on leave to the Ames Research Center, as a recipient of a Senior Post-doctoral Resident Research Associateship of the National Research Council, Washington, D.C., from the Dept. of Exobiology, University of Nijmegen, Driehuizerweg 200, Nijmegen, The Netherlands.     

Carotenes and Retinal in Phycomyces Mutants

Three different types of beta-carotene mutants of Phycomyces have been studied. In 2 mutants (Type I) beta-carotene is still the principal carotene but scaled down or up relative to wild type. The carotene mixture of 2 mutants (Type II) consists mainly of phytoene and phytofluene. In Type III (2 mutants) beta-carotene is replaced by lycopene.The examination of the mutants reveals that the receptor pigment is very likely neither beta-carotene nor retinal. Transmission spectra through the growing zone of live sporangiophores of 1 of these mutants which contains less than one-thousandth of the beta-carotene content of wild type show that the receptor pigment extinction is less than 0.003 at its maximum.     

Genetic variation in the β, β‐carotene‐9′, 10′‐dioxygenase gene and association with fat colour in bovine adipose tissue and milk

β, β‐carotene‐9′, 10′‐dioxygenase (BCO2) plays a role in cleaving β‐carotene eccentrically, and may be involved in the control of adipose and milk colour in cattle. The bovine BCO2 gene was sequenced as a potential candidate gene for a beef fat colour QTL on chromosome (BTA) 15. A single nucleotide base change located in exon 3 causes the substitution of a stop codon (encoded by the A allele) for tryptophan⁸⁰ (encoded by the G allele) (c. 240G>A, p.Trp80stop, referred to herein as SNP W80X). Association analysis showed significant differences in subcutaneous fat colour and beta‐carotene concentration amongst cattle with different BCO2 genotypes. Animals with the BCO2 AA genotype had more yellow beef fat and a higher beta‐carotene concentration in adipose tissues than those with the GA or GG genotype. QTL mapping analysis with the BCO2 SNP W80X fitted as a fixed effect confirmed that this SNP is likely to represent the quantitative trait nucleotide (QTN) for the fat colour‐related traits on BTA 15. Moreover, animals with the AA genotype had yellower milk colour and a higher concentration of beta‐carotene in the milk.     

Carotene production from waste cooking oil by Blakeslea trispora in a bubble column reactor: The role of oxidative stress

The oxidative stress induced by hydroperoxides and reactive oxygen species (ROS) during carotene production from waste cooking oil (WCO) and corn steep liquor (CSL) by the fungus Blakeslea trispora in a bubble column reactor was investigated. The specific activities of the intracellular enzymes superoxide dismutase (SOD) and catalase (CAT) as well as the micromorphology of the fungus were measured in order to study the response of the fungus to oxidative stress. The changes of the morphology of microorganism leaded to pellets formation and documented using a computerized image analysis system. As a consequence of the mild oxidative stress induced by hydroperoxides of WCO and ROS a significant increase in carotene production was obtained. The highest carotene concentration (980.0 mg/l or 51.5 mg/g dry biomass) was achieved in a medium consisted of CSL (80.0 g/L) and WCO (50.0 g/L) at an aeration rate of 5 vvm after 6 days of fermentation. In this case the carotenes produced consisted of β‐carotene (71%), γ‐carotene (26%), and lycopene (3%). The strong oxidative stress in the fungus caused a significant increase of γ‐carotene concentration. Bubble column reactor is a useful fermentation system for carotene production in industrial scale.     

Dissecting the mechanism of Solanum lycopersicum and Solanum chilense flower colour formation

Flowers are the defining feature of angiosperms, and function as indispensable organs for sexual reproduction. Flower colour typically plays an important role in attracting pollinators, and can show considerable variation, even between closely related species. For example, domesticated tomato (S. lycopersicum) has orange/yellow flowers, while the wild relative S. chilense (accession LA2405) has bright yellow flowers. In this study, the mechanism of flower colour formation in these two species was compared by evaluating the accumulation of carotenoids, assessing the expression genes related to carotenoid biosynthetic pathways and observing chromoplast ultrastructure. In S. chilense petals, genes associated with the lutein branch of the carotenoid biosynthetic pathway, phytoene desaturase (PDS), ζ‐carotene desaturase (ZDS), lycopene β‐cyclase (LCY‐B), β‐ring hydroxylase (CRTR‐B) and ε‐ring hydroxylase (CRTR‐E), were highly expressed, and this was correlated with high levels of lutein accumulation. In contrast, PDS, ZDS and CYC‐B from the neoxanthin biosynthetic branch were highly expressed in S. lycopersicum anthers, leading to increased β‐carotene accumulation and hence an orange/yellow colour. Changes in the size, amount and electron density of plastoglobules in chromoplasts provided further evidence of carotenoid accumulation and flower colour formation. Taken together, these results reveal the biochemical basis of differences in carotenoid pigment accumulation and colour between petals and anthers in tomato.     

The P450-type carotene hydroxylase PuCHY1 from Porphyra suggests the evolution of carotenoid metabolism in red algae

Carotene hydroxylases catalyze the hydroxylation of a-and b-carotene hydrocarbons into xanthophylls. In red algae, b-carotene is a ubiquitously distributed carotenoid, and hydroxylated carotenoids such...     

A lycopene β‐cyclase/lycopene ε‐cyclase/light‐harvesting complex‐fusion protein from the green alga Ostreococcus lucimarinus can be modified to produce α‐carotene and β‐carotene at different ratios

Biosynthesis of asymmetric carotenoids such as α‐carotene and lutein in plants and green algae involves the two enzymes lycopene β‐cyclase (LCYB) and lycopene ε‐cyclase (LCYE). The two cyclases are closely related and probably resulted from an ancient gene duplication. While in most plants investigated so far the two cyclases are encoded by separate genes, prasinophyte algae of the order Mamiellales contain a single gene encoding a fusion protein comprised of LCYB, LCYE and a C‐terminal light‐harvesting complex (LHC) domain. Here we show that the lycopene cyclase fusion protein from Ostreococcus lucimarinus catalyzed the simultaneous formation of α‐carotene and β‐carotene when heterologously expressed in Escherichia coli. The stoichiometry of the two products in E. coli could be altered by gradual truncation of the C‐terminus, suggesting that the LHC domain may be involved in modulating the relative activities of the two cyclase domains in the algae. Partial deletions of the linker region between the cyclase domains or replacement of one or both cyclase domains with the corresponding cyclases from the green alga Chlamydomonas reinhardtii resulted in pronounced shifts of the α‐carotene‐to‐β‐carotene ratio, indicating that both the relative activities of the cyclase domains and the overall structure of the fusion protein have a strong impact on the product stoichiometry. The possibility to tune the product ratio of the lycopene cyclase fusion protein from Mamiellales renders it useful for the biotechnological production of the asymmetric carotenoids α‐carotene or lutein in bacteria or fungi.     

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.