CAROTENOIDS OF SIPHONOUS GREEN ALGAE: A CHEMOTAXONOMICAL STUDY

The carotenoid pigments of 50 species of 9 siphonean orders were investigated. The algae of all orders contain the principal carotenoids known from other green algae: α- and β-carotene, lutein, lutein epoxide, violaxanthin, and neoxanthin. Additionally, in some Siphonodadales siphonaxanthin is present, in the Derbesiales, Codiales, and Caulerpales both siphonaxanthin and its ester siphonein are present, whereas in the Dichotomosiphonales only the ester siphonein can be found. The chemotaxonomical value of siphonaxanthin and siphonein is discussed.     

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.     

Refined carotenoid analysis of the major light-harvesting complex of Mantoniella squamata

The major light-harvesting complex (LHC) of the prasinophycean alga Mantoniella squamata is unique compared to other chlorophyll (Chl) a/b-binding LHC with respect to the primary protein structure and the pigmentation. Although the presence of Chl a, Chl b, a Chl c-type pigment and the xanthophylls neoxanthin, violaxanthin and prasinoxanthin was clearly determined, several carotenoids remained unidentified or were described controversially. We re-analysed the carotenoid composition and identified a new set of xanthophylls present in the LHC: uriolide, micromonol, micromonal and dihydrolutein. Additionally, one hydrophobic component was detected, presumably a xanthophyll. The pigment analysis in combination with quantitative protein determinations revealed a pigment-protein stoichiometry of 6 Chl a, 6 Chl b, 2 Chl c* and about 2 prasinoxanthin molecules per polypeptide. The other xanthophylls were present in sub-stoichiometric amounts. A comparison of results from LHC isolated either by sucrose density centrifugation or SDS-polyacryl gel electrophoresis revealed a decline in the amount of prasinoxanthin and a loss of violaxanthin using the latter preparation procedure, while the stoichiometric ratios of the other 6 xanthophylls remained constant. The fact that 8 different xanthophylls were found in the LHC of M. squamata can be explained best in terms of an oligomeric, presumably trimeric LHC organisation with subunits of heterogeneous pigmentation. Especially, the very stable assembly of most of the minor xanthophylls led to the assumption that these components play an important role in stabilisation and probably also in trunerisation of the LHC in vivo. This revised version was published online in August 2006 with corrections to the Cover Date.     

Changes in the chlorophylls and carotenoids of leaves of Nicotiana tabacum during senescence

In addition to chlorophylls a and b, β-carotene, lutein, violaxanthin and neoxanthin, leaves of tobacco (Nicotiana tabacum L. cv. Virginia Gold) contain antheraxanthin in some harvests. In lower leaves, chlorophylls decreased more rapidly than carotenoids during senescence, but both types of pigment decreased at equal rates in upper leaves. The chlorophyll a:b ratio decreased only in post-mature leaves. Total carotenoid decreased with age, with the relative proportion of β-carotene increasing in lower leaves. Seasonal influences rather than age of leaf determines whether antheraxanthin is present. No esterified xanthophylls were found in senescent leaves.     

PIGMENTS OF BATHYCOCCUS PRASINOS (PRASINOPHYCEAE): METHODOLOGICAL AND CHEMOSYSTEMATIC IMPLICATIONS1,2

Bathycoccus prasinos Eikrem et Throndsen exhibited a complex carotenoid distribution pattern including the carotenes β,β-carotene (0.8% of total carotenoids) and β, ° Carotene (0.4%) and several xanthophylls. These were prasinoxanthin (49% of total carotenoids), micromonal (16%), neoxanthin (14%), uriolide (7%), violaxanthin (0.8%), 3¹-dehydrouriolide (0.8%), dihydrolutein (0.1%), two partly characterized esterified carotenols (together 10%), and five minor unidentified carotenols (together 2%). The identifications were based on high-performance liquid chromatography (HPLC), thin-layer chromatography (TLC), visible spectroscopy (VIS), and mass spectra (MS) and in part on ¹H nuclear magnetic resonance (NMR), circular dichroism (CD), and chemical derivatization. The carotenoid composition of B. prasinos was related to that of other prasinoxanthin / uriolide / micromonal-producing prasinophytes (Mantoniella squamata, Micromonas pusilla, and Pseudoscourfieldia marina). The relative distribution of chlorophylls (w/w) were chlorophyll a (chl a; 63%), chl b (31%), and an unknown chl c-like chlorophyll (7%) with spectral characteristics similar to magnesium 2,4-divinylphaeoporphyrin a, monomethyl ester, compatible with other prasinophytes. The chemosystematic data and ultrastructural characteristics for the order Mamiellales are discussed. We conclude that HPLC studies alone are insufficient for the identification and characterization of the carotenoids, including the minor carotenoids essential for biosynthetic/chemosystematic considerations.     

A Yellow Marine Chiamydomonas: Morphology and Pigment Composition

A unicellular yellow marine microorganism was isolated fromwater samples collected in Hachinohe Harbor, on the northerncoast of Japan, and Off Tsushima Island, on the western coastof Japan, and its structure and pigment composition were investigated.Light and electron microscopy indicated that the alga belongsto the genus Chlamydomonas and it is identified as C. parkeae. Pigment analysis by high-performance liquid chromatography revealedthe presence of 2,4-divinylprotochlorophyllide (DVP) as a thirdchlorophyll in addition to chlorophylls a and b. Such a pigmentcomposition has been reported previously only for some prasinophytesamong autotrophically grown algae. With respect to carotenoids,the alga contains, in addition to the carotenoids of higherplants (neoxanthin, violaxanthin, zeaxanthin, lutein, ß-carotene),siphonaxanthin and siphonein (siphonaxanthin dodecenoate); thelatter have been detected previously only in marine benthiculvophycean algae and in some prasinophytes. However, the coexistenceof DVP, siphonein and siphonaxanthin in a single species hasnever been reported for either ulvophycean or prasinophyceanalgae. In addition to siphonaxanthin dodecenoate, the alga wasfound to contain two "siphoneins", siphonaxanthin decenate andsiphonaxanthin octanoate. ³ Present address: Nippon Roche Research Center, Kajiwara, Kamakura,Kanagawa, 247 Japan.     

Carotenoid binding sites in LHCIIb. Relative affinities towards major xanthophylls of higher plants.

The major light-harvesting complex of photosystem II can be reconstituted in vitro from its bacterially expressed apoprotein with chlorophylls a and b and neoxanthin, violaxanthin, lutein, or zeaxanthin as the only xanthophyll. Reconstitution of these one-carotenoid complexes requires low-stringency conditions during complex formation and isolation. Neoxanthin complexes (containing 30-50% of the all-trans isomer) disintegrate during electrophoresis, exhibit a largely reduced resistance against proteolytic attack; in addition, energy transfer from Chl b to Chl a is easily disrupted at elevated temperature. Complexes reconstituted in the presence of either zeaxanthin or lutein contain nearly two xanthophylls per 12 chlorophylls and are more resistant against trypsin. Lutein-LHCIIb also exhibits an intermediate maintenance of energy transfer at higher temperature. Violaxanthin complexes approach a xanthophyll/12 chlorophyll ratio of 3, similar to the ratio in recombinant LHCIIb containing all xanthophylls. On the other hand, violaxanthin-LHCIIb exhibits a low thermal stability like neoxanthin complexes, but an intermediate accessibility towards trypsin, similar to lutein-LHCIIb and zeaxanthin-LHCIIb. Binary competition experiments were performed with two xanthophylls at varying ratios in the reconstitution. Analysis of the xanthophyll contents in the reconstitution products yielded information about relative carotenoid affinities of three assumed binding sites. In lutein/neoxanthin competition experiments, two binding sites showed a strong preference (> 200-fold) for lutein, whereas the third binding site had a higher affinity (25-fold) to neoxanthin. Competition between lutein and violaxanthin gave a similar result, although the specificities were lower: two binding sites have a 36-fold preference for lutein and one has a fivefold preference for violaxanthin. The lowest selectivity was between lutein and zeaxanthin: two binding sites had a fivefold higher affinity for lutein and one has a threefold higher affinity to zeaxanthin.     

PHOTOSYNTHETIC LIGHT-HARVESTING FUNCTION OF VIOLAXANTHIN IN NANNOCHLOROPSIS SPP. (EUSTIGMATOPHYCEAE)1

Whole cell absorption spectra of the Eustigmatophycean algae Nannochloropsis salina Bourrelly and Nannochloropsis sp. reveal the presence of a distinct absorption peak at 490 nm. The lack of chlorophylls b and c in these species indicates that this peak must be attributed to carotenoid absorption. In vivo fluorescence excitation spectra for chlorophyll a emission show a corresponding maximum at 490 nm. This peak is more clearly resolved than carotenoid maxima in other algal classes due to the absence of accessory chlorophylls. The carotenoid composition of the two Nannochloropsis species shows that violaxanthin and vaucheriaxanthin are the main contributors to 490 nm absorption. Violaxanthin accounts for approximately 60% of the total carotenoid in both clones. We conclude that light absorption by violaxanthin, and possibly by vaucheriaxanthin, is coupled in energy transfer to chlorophyll a and that violaxanthin is the major light-harvesting pigment in the Eustigmatophyceae. This is the first report of the photosynthetic light-harvesting function of this carotenoid.     

Spectral and functional studies on siphonaxanthin-type light-harvesting complex of photosystem II from Bryopsis corticulans

Carotenoids with conjugated carbonyl groups possess special photophysical properties which have been studied in some water-soluble light-harvesting proteins (Polívka and Sundström, Chem Rev 104:2021–2071, 2004). However, siphonaxanthin-type light-harvesting complexes of photosystem II (LHCII) in siphonous green alga have received fewer studies. In the present study, we determined sequences of genes for several Bryopsis corticulans Lhcbm proteins, which showed that they belong to the group of major LHCII and diverged early from green algae and higher plants. Analysis of pigment composition indicated that this siphonaxanthin-type LHCII contained in total 3 siphonaxanthin and siphonein but no lutein and violaxanthin. In addition, 2 chlorophylls a in higher plant LHCII were replaced by chlorophyll b. These changes led to an increased absorption in green and blue-green light region compared with higher plant LHCII. The binding sites for chlorophylls, siphonaxanthin, and siphonein were suggested based on the structural comparison with that of higher plant LHCII. All of the ligands for the chlorophylls were completely conserved, suggesting that the two chlorophylls b were replaced by chlorophyll a without changing their binding sites in higher plant LHCII. Comparisons of the absorption spectra of isolated siphonaxanthin and siphonein in different organic solutions and the effect of heat treatment suggested that these pigments existed in a low hydrophobic protein environment, leading to an enhancement of light harvesting in the green light region. This low hydrophobic protein environment was maintained by the presence of more serine and threonine residues in B. corticulans LHCII. Finally, esterization of siphonein may also contribute to the enhanced harvesting of green light.     

Primary and Secondary Carotenoids of Spongiochloris typica

A qualitative and quantitative investigation was made of the pigments of Spongiochloris typica over an 8 week period. The pigments were chromatographed on thin layers of sucrose and measured spectrophotometrically. Pigments present after 1 week of growth were identified as chlorophylls a and b, β-carotene, lutein, zeaxanthin, violaxanthin, trollein, and neoxanthin. In cultures 2 weeks or more old, secondary carotenoids appeared. These were echinenone, canthaxanthin, astacene, and an unidentified ketocarotenoid. Carotenoids comprised nearly 100 percent of the total pigment composition on the 8th week. About 75 percent of the carotenoid fraction on the 8th week consisted of secondary carotenoids.     

Pigment analysis of chloroplast pigment-protein complexes in wheat

Pigment-protein complexes separated from wheat (Triticum aestivum L. selection ND96-25 by two gel electrophoresis techniques were analyzed by high-performance liquid chromatography for chlorophylls and carotenoids. The two techniques are compared, and pigment analyses are given for the major reaction centers and light-harvesting complexes. Reaction centers contain mostly chlorophyll a, carotene, and lutein, whereas light-harvesting complexes contain chlorophyll a, chlorophyll b, lutein, and neoxanthin. The amounts of violaxanthin are variable.     

Pigment Changes Associated with Application of Ethephon ((2-Chloroethyl)phosphonic Acid) to Fig (Ficus carica L.) Fruits

The application of (2-chloroethyl)phosphonic acid (Ethephon) to `Mission' fig fruits (Ficus carica L.) during late period II of their development stimulated ripening and change in color from green to bluish black within 8 days. Chlorophylls a and b decreased rapidly within 4 days after Ethephon treatment, and degradation continued at a decreasing rate for an additional 4 days, at which time the fruits had attained their maximum diameter and were considered fully ripe. Levels of β-carotene, lutein, violaxanthin, and neoxanthin decreased in a pattern similar to that of chlorophylls a and b. The rates of β-carotene and lutein degradation were initially greater than those of the xanthophyll pigments. Degradation rates of the various carotenoids were comparable 4 to 8 days after treatment.     

The xanthophyll cycle is induced by light irrespective of water status in field-grown lavender (Lavandula stoechas) plants

The water relations, the photosynthetic capacity and the pigment content of leaves, i.e. chlorophylls, carotenes and xanthophylls, were analysed during the summer drought and recovery after autumn rainfalls in lavender ( Lavandula stoechas L.) plants grown in Mediterranean field conditions. Summer drought caused photoinhibition of photosynthesis and significant decreases in chlorophylls (by ca 75%), β -carotene (by ca 65%), and lutein and neoxanthin (by ca 50%), although their contents remained unaltered between predawn and midday, suggesting a progressive decrease in response to drought. In contrast, the levels of violaxanthin decreased from predawn to midday, giving rise to enhanced formation of zeaxanthin and antheraxanthin in high light. Zeaxanthin and antheraxanthin formation was not induced by water deficit. Although the levels of photosynthetic pigments were severely affected by water deficit, carotenoids decreased less than chlorophylls, which resulted in increased levels of carotenoids per unit of chlorophyll. We conclude that the enhanced formation of zeaxanthin in high light and the increased levels of carotenoids per unit of chlorophyll observed in water-stressed plants may help to avoid photoinhibitory damage to the photosynthetic apparatus.     

The tomato mutation nxd1 reveals a gene necessary for neoxanthin biosynthesis and demonstrates that violaxanthin is a sufficient precursor for abscisic acid biosynthesis

Carotenoid pigments are indispensable for plant life. They are synthesized within plastids where they provide essential functions in photosynthesis. Carotenoids serve as precursors for the synthesis of the strigolactone phytohormones, which are made from β‐carotene, and of abscisic acid (ABA), which is produced from certain xanthophylls. Despite the significant progress that has been made in our understanding of the carotenoid biosynthesis pathway, the synthesis of the xanthophyll neoxanthin has remained unknown. We report here on the isolation of a tomato (Solanum lycopersicum) mutant, neoxanthin‐deficient 1 (nxd1), which lacks neoxanthin, and on the cloning of a gene that is necessary for neoxanthin synthesis in both tomato and Arabidopsis. The locus nxd1 encodes a gene of unknown function that is conserved in all higher plants. The activity of NXD1 is essential but cannot solely support neoxanthin synthesis. Lack of neoxanthin does not significantly reduce the fitness of tomato plants in cultivated field conditions and does not impair the synthesis of ABA, suggesting that in tomato violaxanthin is a sufficient precursor for ABA production in vivo.     

The current concepts of functional role of carotenoids in the eukaryotic chloroplasts

Current state of knowledge of functional role of carotenoids in algal and higher plant chloroplasts is reviewed. Basic functions of carotenoids are shown to be light-protective, light-absorbing, and structural, as well as participating in photochemical processes of photosystems I and II. Such xanthophylls as neoxanthin, fucoxanthin, peridinin and alloxanthin, which have allenic or acetylenic bond, mostly function as light-absorbers. They transmit absorbed energy to chlorophyll b. Other xanthophylls occupying certain strictly specified loci in light-absorbing chlorophyll-a/b-protein complexes of photosystems have either structural function (lutein) or light-protective function (zeaxanthin, antheraxanthin, violaxanthin). Carotenoids of xanthophyll cycles preserve chlorophylls and lipids of photosynthetic membranes from photodestruction at overlighting in the presence of oxygen. In eukaryotic chloroplasts, three types of xanthophyll cycles were found: violaxanthin, lutein-5,6-epoxide, and diadinoxanthin. The similarities and dissimilarities between epoxidation and de-epoxidation reactions of these cycles are discussed in detail in the present work. The pattern of occurrence of xanthophyll cycles among higher plants and freshwater and marine algae is outlined.     

Photodestruction of Pigments in Higher Plants by Herbicide Action: I. THE EFFECT OF DCMU (DIURON) ON ISOLATED CHLOROPLASTS

3-(3',4'-Dichlorophenyl)-1',1'-dimethyl urea (DCMU) inducedthe photobleaching of chlorophylls and carotenoids in isolatedchloroplasts of Hordeum vulgare. In chloroplasts illuminatedin both the absence and presence of DCMU (5.0 mmol m^–3),the destruction of carotenoid preceded that of the chlorophylls.The rate of photodestruction was accelerated by the presenceof DCMU. After only 2 h illumination the rates of loss of ß-caroteneand of the epoxyxanthophylls, neoxanthin and violaxanthin, weresimilar (approximately 40–50% loss in the presence of5–0 mmol m^–3 DCMU) but weremuch greater than thatof lutein (25% loss). Analysis of the individual pigment-proteincomplexes, isolated from chloroplasts following such treatment,showed that whilst pigment destruction had occurred in all complexes,the relative content of the LHCP2/CPa complexes (containingthe PSII core) had fallen to the greatest extent. Further illuminationof the chloroplasts, for up to 22 h, resulted in far greaterbleaching but showed a similar pattern of pigment loss, withDCMU again accelerating the rate at which this loss occurred.ß-Carotene-5,6-epoxide was identified as a productof such photo-oxidative conditions. Key words: DCMU, carotenoids, chlorophylls, photobleaching, ß-carotene-5,6-expoxide     

EST analysis of the scaly green flagellate Mesostigma viride (Streptophyta): Implications for the evolution of green plants (Viridiplantae)

Background  

The Viridiplantae (land plants and green algae) consist of two monophyletic lineages, the Chlorophyta and the Streptophyta. The Streptophyta include all embryophytes and a small but diverse group of freshwater algae traditionally known as the Charophyceae (e.g. Charales, Coleochaete and the Zygnematales). The only flagellate currently included in the Streptophyta is Mesostigma viride Lauterborn. To gain insight into the genome evolution in streptophytes, we have sequenced 10,395 ESTs from Mesostigma representing 3,300 independent contigs and compared the ESTs of Mesostigma with available plant genomes (Arabidopsis, Oryza, Chlamydomonas), with ESTs from the bryophyte Physcomitrella, the genome of the rhodophyte Cyanidioschyzon, the ESTs from the rhodophyte Porphyra, and the genome of the diatom Thalassiosira.     

BIOGENESIS OF CHLOROPLAST MEMBRANES : IV. Lipid and Pigment Changes during Synthesis of Chloroplast Membranes in a Mutant of Chlamydomonas reinhardi y-1

The glycolipid, phospholipid, pigment, and fatty acid content in whole y-1 cells during the greening process have been investigated. The time course of their changes indicates that phosphatidyl glycerol and glycolipids are the main lipids synthesized specifically during illumination of dark-grown cells, concomitant with an increase in the polyunsaturated C18:2 and C18:3 fatty acids. The pigment complex of light-grown cells consists mainly of chlorophylls a and b, lutein, β-carotene, violaxanthin, and neoxanthin. During the greening process, chlorophylls a and b are synthesized in constant proportions (ratio a/b equals 2.6), β-carotene and violaxanthin do not change significantly, and lutein and neoxanthin increase. The molar ratios of the different lipids and pigment to total chlorophyll during greening has been calculated. It was found that during the initial phase of greening when chlorophyll is synthesized at increasing rates, the molar ratios of various lipids and pigments to chlorophyll decrease and tend to become constant when chlorophyll and membrane synthesis proceed at constant rates. The implication of these findings with respect to the concept of membrane assembly through a spontaneous single step process is discussed     

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.