CHARACTERIZATION OF TWO UNIQUE CAROTENOID FATTY ACID ESTERS FROM PTEROSPERMA CRISTATUM (PRASINOPHYCEAE,CHLOROPHYTA)1

Carotenoid composition and spectroscopic characteristics were analyzed for Pterosperma cristatum Schiller, one of the most primitive known members of green algae. This alga contained a substantial amount of carotenoid esters, siphonaxanthin C14:1 trans‐Δ2 ester and 6′‐OH siphonaxanthin C14:1 trans‐Δ2 ester, but lacked lutein. This is the first report of carotenoid C14:1 trans‐Δ2 esters from phototrophic organisms. In vivo absorption spectra and excitation spectra of the cells revealed that these carotenoids absorbed blue‐green light and could transfer energy to chl a. These carotenoids were concluded to function as antenna pigments in P. cristatum.     

Light-dependent conformational change of neoxanthin in a siphonous green alga,Codium intricatum,revealed by Raman spectroscopy

Siphonous green algae, a type of deep-sea green algae, appear olive drab and utilize blue–green light for photosynthesis. A siphonous green alga, Codium (C.) intricatum, was isolated from Okinawa prefecture in Japan, and a clonal algal culture in filamentous form was established. The major light-harvesting antenna was analogous to the trimeric LHCII found in higher plants, but the C. intricatum complex contained an unusual carbonyl carotenoid siphonaxanthin. Culture conditions were optimized to achieve high siphonaxanthin content in intact lyophilized filamentous bodies. Interestingly, the carotenoid composition was different when cultured under high irradiance: all-trans neoxanthin was accumulated in addition to the normal 9′-cis form in whole cell extract. Resonance Raman spectra of intact filamentous bodies, cultured under high- and low-light conditions, confirmed the accumulation of all-trans neoxanthin under high irradiance conditions. A plausible function of the presence of all-trans neoxanthin will be discussed in relation to the regulation against high light stress.     

Diversity and evolution of photosynthetic antenna systems in green plants

Photosynthetic antenna systems are mainly involved in the absorption of light energy required for photosynthesis. The typical green plants arrange chlorophylls a and b and carotenoids, including lutein and 9′‐cis neoxanthin, in their antenna systems; such antenna systems have prospered on earth. Therefore, these antenna systems should be highly evolved and should adapt to the photoenvironments in which plants grow. However, little information is available on the diversity and evolution of antenna systems in green plants as a whole. To approach this, the present study focused on the antenna systems in the Prasinophyceae, an assemblage of early diverging lineages of green plants and analyzed their photosynthetic pigments in detail. In the present study, various novel blue–green light‐absorbing siphonaxanthin series were detected in the early diverging species of the Prasinophyceae and the distribution of these carotenoids was revealed. Additionally, to clarify the evolution of antenna systems in the Ulvophyceae, a highly developed green algal group that specializes in inhabiting various aquatic environments, members of the Cladophorales belonging to this class were selected and their carotenoid compositions were determined to compare them with the molecular phylogenetic tree constructed on the basis of the 18S rRNA gene sequences of the Cladophorales. In this review, these data will be summarized and the remarkable variation of photosynthetic pigments will be presented. A possible scenario detailing the evolution of antenna systems in green plants will be elucidated.     

VARIATION OF SIPHONAXANTHIN SERIES AMONG THE GENUS NEPHROSELMIS (PRASINOPHYCEAE,CHLOROPHYTA), INCLUDING A NOVEL PRIMARY METHOXY CAROTENOID1

Carotenoid compositions were analyzed for ten strains of Nephroselmis (Prasinophyceae) containing four described and three undescribed species. Based on the distribution pattern of the siphonaxanthin series, five carotenoid types were recognized in the examined strains/species: type I (N. astigmatica Inouye et Pienaar, N. pyriformis (N. Carter) Ettl, and Nephroselmis sp1. MBIC 11158) had siphonaxanthin C12:1 and C14:1 esters as well as 6′‐OH siphonaxanthin C12:1 and C14:1 esters, type II (Nephroselmis sp2. MBIC 11149) had siphonaxanthin C8:1 ester, type III (Nephroselmis sp3. NIES 486, NIES‐PS 535, and MBIC 10871) had 19‐methoxy siphonaxanthin and siphonaxanthin C12:1 and C14:1 esters, type IV (N. spinosa Suda) had only a small amount of siphonaxanthin C12:1 ester, and type V (N. olivacea Stein) had lutein as a major carotenoid but completely lacked the siphonaxanthin series. 19‐Methoxy siphonaxanthin was a novel and very unique carotenoid, that is, it contains a methoxy group and was found for the first time in photosynthetic eukaryotes. Additionally, carotenoids containing a primary methoxy group had previously never been found in any group of organisms. Siphonaxanthin C8:1 ester, which was only known as a trace carotenoid in Chlamydomonas parkeae Ettl, was first discovered as a major carotenoid in Nephroselmis sp2. (MBIC 11149). Based on these results and comparison of the phylogenetic relationships of the Nephroselmis species used, we discuss the taxonomic significance of the carotenoid types and evolutionary process of the photosynthetic antenna systems in green plants.     

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.     

CAROTENOID COMPOSITIONS OF CLADOPHORA BALLS (AEGAGROPILA LINNAEI) AND SOME MEMBERS OF THE CLADOPHORALES (ULVOPHYCEAE,CHLOROPHYTA): THEIR TAXONOMIC AND EVOLUTIONARY IMPLICATION1

Photosynthetic pigments were analyzed by HPLC for 27 samples of the Cladophorales (Ulvophyceae, Chlorophyta). The carotenoid compositions of the examined algae were classified into three types based on the final compound of biosynthesis of the α‐carotene branch: lutein type, characterized by containing lutein as a major carotenoid and lacking loroxanthin and siphonaxanthin; loroxanthin type, characterized by containing loroxanthin and lacking siphonaxanthin; and siphonaxanthin type, characterized by containing siphonaxanthin. We constructed molecular phylogenetic tree of the species examined in the present study using 18S rRNA gene sequences and mapped the carotenoid types of the species onto the tree. The molecular phylogenetic analysis divided the Cladophorales into two major clades, clade 1 and Aegagropila‐clade (clade 2), and divided clade 1 into subclade 1‐1 and subclade 1‐2. All the examined species positioned in the Aegagropila‐clade and those of the subclade 1‐1 belonged to the loroxanthin type, whereas both lutein type and siphonaxanthin type appeared only in the subclade 1‐2. The clades delineated by molecular phylogenetic analysis were congruent with distribution of the carotenoid types, indicating that the carotenoid types are of taxonomic significance in the Cladophorales. Considering the distribution pattern of these carotenoid types and minimum state changes in the Cladophorales, we concluded that the loroxanthin type was the primitive (plesiomorphic) state and the siphonaxanthin type and lutein type appeared in the subclade 1‐2 as advanced (apomorphic) state within this order and suggested that the cladophoralean siphonaxanthin type would have been secondarily acquired.     

PIGMENT SUITES AND TAXONOMIC GROUPS IN PRASINOPHYCEAE1

Pigment analysis performed on 30 Prasinophyceae strains revealed two main groups: the prasinoxanthin‐containing and prasinoxanthin‐less Prasinophyceae. Prasinoxanthin‐containing Prasinophyceae comprised the orders Mamiellales, Pseudoscourfieldiales (Pycnococcaceae), and Prasinococcales. For this group, classification with pigment composition showed a good agreement with molecular phylogeny. Mamiellales, except Crustomastix stigmatica, accumulated uriolide, micromonal, dihydrolutein, and the pigment Unidentified M1 as characteristic pigments. Prasinococcales and Pseudoscourfieldiales (Pycnococcaceae) lacked micromonal and Unidentified M1. In addition, Pseudoscourfieldiales (Pycnococcaceae) lacked uriolide. A chl c₃‐like pigment was present in prasinoxanthin‐containing strains isolated from the deep sea. Common green algae pigments, a loroxanthin derivative, and siphonaxanthin plus derivatives were found in the prasinoxanthin‐less Prasinophyceae, which included strains from Pyramimonadales, Pseudoscourfieldiales (Nephroselmidiaceae), Chlorodendrales, and a new order. Although some associations could be observed, the correspondence between pigments and molecular taxonomy was less clear for this group.     

Structural Confirmation of a Unique Carotenoid Lactoside,P457, in Symbiodinium sp. Strain nbrc 104787 Isolated from a Sea Anemone and its Distribution in Dinoflagellates and Various Marine Organisms

The molecular structure of the carotenoid lactoside P457, (3S,5R,6R,3′S,5′R,6′S)‐13′‐cis‐5,6‐epoxy‐3′,5′‐dihydroxy‐3‐(β‐d ‐galactosyl‐(1→4)‐β‐d ‐glucosyl)oxy‐6′,7′‐didehydro‐5,6,7,8,5′,6′‐hexahydro‐β,β‐caroten‐20‐al, was confirmed by spectroscopic methods using Symbiodinium sp. strain NBRC 104787 cells isolated from a sea anemone. Among various algae, cyanobacteria, land plants, and marine invertebrates, the distribution of this unique diglycosyl carotenoid was restricted to free‐living peridinin‐containing dinoflagellates and marine invertebrates that harbor peridinin‐containing zooxanthellae. Neoxanthin appeared to be a common precursor for biosynthesis of peridinin and P457, although neoxanthin was not found in peridinin‐containing dinoflagellates. Fucoxanthin‐containing dinoflagellates did not possess peridinin or P457; green dinoflagellates, which contain chlorophyll a and b, did not contain peridinin, fucoxanthin, or P457; and no unicellular algae containing both peridinin and P457, other than peridinin‐containing dinoflagellates, have been observed. Therefore, the biosynthetic pathways for peridinin and P457 may have been coestablished during the evolution of dinoflagellates after the host heterotrophic eukaryotic microorganism formed a symbiotic association with red alga that does not contain peridinin or P457.     

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.     

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.     

The individual variation in carotenoid content of the lichen species Dirinaria applanata (Fée) Awasthi

Column and thin‐layer chromatography revealed the presence of the following carotenoids in thalli of Dirinaria applanata from 13 different sites: α‐carotene, β‐carotene, β‐cryptoxanthin, lutein, 3′‐epilutein, zeaxanthin, antheraxanthin, canthaxanthin, astaxanthin, violaxanthin, mutatoxanthin, neoxanthin, capsochrome, fucoxanthinol, paracentrone and apo‐6′‐lycopenal. In the thalli of all 13 specimens of Dirinaria applanata β‐carotene, lutein, astaxanthin and violaxanthin were found as constant carotenoids. The total content of carotenoids ranged from 21.0 (from Mexico) to 54.9 μg g⁻¹ dry weight (from Antilles).     

Genetic modification of the fatty acid unsaturation of phosphatidylglycerol in chloroplasts alters the sensitivity of tobacco plants to cold stress

The cis‐unsaturated molecular species of phosphatidylglycerol (PG) in chloroplasts have been implicated in the chilling sensitivity of plants. Homozygous lines of transgenic tobacco (Nicotiana tabacum) that overexpressed the cDNA for glycerol‐3‐phosphate acyltransferase, a key enzyme in the determination of the extent of cis‐unsaturation of PG, were established from a chilling‐sensitive squash (Cucurbita moschata). In transgenic plants, the proportion of saturated plus trans‐monounsaturated molecular species of PG increased from 24 to 65%. However, this change did not affect the architecture of the chloroplasts. Chilling stress decreased the growth and biomass production of young seedlings of transgenic plants more severely than those of wild‐type plants, and this observation suggests that the changes in the proportion of cis‐unsaturated PG affected not only leaves but also developing plants. Chilling stress also damaged inflorescences. In particular, the abscission of flower buds and inflorescence meristems from transgenic plants occurred more frequently than that from wild‐type plants. Thus, it is likely that decreases in the proportion of cis‐unsaturated PG enhanced the sensitivity to chilling of reproductive organs.     

Membrane phospholipids and the dark side of vision

The key step in the visual pigment regeneration process is an enzyme-catalyzedtrans tocis retinoid isomerization reaction. This reaction is of substantial general interest, because it requires the input of metabolic energy. The energy is needed because the 11-cis-retinoid reaction products are approximately 4kcal/mol higher in energy than their all-trans congeners. In the retinal pigment epithelium a novel enzymatic system has been discovered which is capable of converting all-trans-retinol into all-trans retinyl esters, by means of a lecithin retinol acyl transferase (LRAT), followed by the direct processing of the ester into 11-cis-retinol. In this process the free energy of hydrolysis of a retinyl ester, estimated to be approximately –5kcal/mol, is coupled to the endothermic (+4kcal/mol) isomerization reaction, resulting in an overall exothermic process. The overall process is analogous to ATP-dependent group transfer reactions, but here the energy is provided by the membrane phospholipids. This process illustrates a new role for membranes: they can serve as an energy source.     

Optical purity of (3S,3'S)-astaxanthin from Haematococcus pluvialis

Haematococcus pluvialis cultivated in a N-deficient medium produced astaxanthin (1 % of total carotenoids), the monoester (76 %) and diester (7 %) of astaxanthin, β,β-carotene (1 %), an adonirubin ester (3 %), (3R,3′R,6′R)-lutein (7 %), violaxanthin (2 %) and neoxanthin (1 %). The CD values of the mono- and diesters of astaxanthin, the HPLC properties of astaxanthin monoester further esterified with (?)-camphanic acid and the optical purity of astaxanthin [determined by HPLC analysis of the diester of(?)-camphanic acid] produced by saponification of the natural mono- and diesters of astaxanthin in the absence of oxygen showed that this green alga synthesizes pure (3S,3′S)-astaxanthin esters.     

Secondary Carotenoids of Eremosphaera viridis De Bary (Chlorophyceae) Under Nitrogen Deficiency*

Under nitrogen deficiency the unicellular chlorococcalean green alga, Eremosphaera viridis De Bary, was able to synthesize secondary carotenoids (SC). Nine SC were identified as six astaxanthin esters, echinenone, canthaxanthin and a lutein ester, previously not described in green algae under nitrogen deficiency. These SC, jS-carotene and the main part of lutein were located in lipid bodies outside the chloroplasts in the cytosol. The synthesis of SC could be inhibited by the herbicides norflurazon and nicotine. This result supported the idea that SC in cells ot Eremosphaera viridis were synthesized de novo rather than derived from primary carotenoids.     

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.     

Nucleus‐encoded mRNAs for Chloroplast Proteins GapA,PetA, and PsbO are Trans‐spliced in the Flagellate Euglena gracilis Irrespective of Light and Plastid Function

Euglena gracilis is a fresh‐water flagellate possessing secondary chloroplasts of green algal origin. In contrast with organisms possessing primary plastids, mRNA levels of nucleus‐encoded genes for chloroplast proteins in E. gracilis depend on neither light nor plastid function. However, it remains unknown, if all these mRNAs are trans‐spliced and possess spliced leader sequence at the 5′‐end and if trans‐splicing depends on light or functional plastids. This study revealed that polyadenylated mRNAs encoding the chloroplast proteins glyceraldehyde‐3‐phosphate dehydrogenase (GapA), cytochrome f (PetA), and subunit O of photosystem II (PsbO) are trans‐spliced irrespective of light or plastid function.     

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.